U.S. patent application number 17/003952 was filed with the patent office on 2022-02-03 for prostate-specific membrane antigen antibody drug conjugates.
This patent application is currently assigned to Ambrx,Inc.. The applicant listed for this patent is Ambrx,Inc.. Invention is credited to Richard Barnett, Andrew Beck, Marco Gymnopoulos, Anna-Maria A. Hays Putnam, Nickolas Knudsen, Ying Sun, Feng Tian.
Application Number | 20220033518 17/003952 |
Document ID | / |
Family ID | 1000005883520 |
Filed Date | 2022-02-03 |
United States Patent
Application |
20220033518 |
Kind Code |
A1 |
Barnett; Richard ; et
al. |
February 3, 2022 |
PROSTATE-SPECIFIC MEMBRANE ANTIGEN ANTIBODY DRUG CONJUGATES
Abstract
This invention relates to prostate-specific membrane antigen
(PSMA) antibodies and antibody drug conjugates comprising at least
one non-naturally-encoded amino acid. Disclosed herein are
.alpha.PSMA antibodies with one or more non-naturally encoded amino
acids and further disclosed are antibody drug conjugates wherein
the .alpha.PSMA antibodies of the invention are conjugated to one
or more toxins. Also disclosed herein are non-natural amino acid
dolastatin analogs that are further modified post-translationally,
methods for effecting such modifications, and methods for purifying
such dolastatin analogs. Typically, the modified dolastatin analogs
include at least one oxime, carbonyl, dicarbonyl, and/or
hydroxylamine group. Further disclosed are methods for using such
non-natural amino acid antibody drug conjugates, dolastatin
analogs, and modified non-natural amino acid dolastatin analogs,
including therapeutic, diagnostic, and other biotechnology
uses.
Inventors: |
Barnett; Richard; (San
Marcos, CA) ; Tian; Feng; (San Diego, CA) ;
Hays Putnam; Anna-Maria A.; (San Diego, CA) ;
Gymnopoulos; Marco; (San Diego, CA) ; Knudsen;
Nickolas; (Escondido, CA) ; Beck; Andrew; (San
Diego, CA) ; Sun; Ying; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ambrx,Inc. |
La Jolla |
CA |
US |
|
|
Assignee: |
Ambrx,Inc.
La Jolla
CA
|
Family ID: |
1000005883520 |
Appl. No.: |
17/003952 |
Filed: |
August 26, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14406190 |
Dec 5, 2014 |
10800856 |
|
|
PCT/US2013/044850 |
Jun 7, 2013 |
|
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17003952 |
|
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61656883 |
Jun 7, 2012 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 5/0205 20130101;
A61K 47/6817 20170801; C07K 16/3069 20130101; C07K 16/40 20130101;
A61K 47/6869 20170801 |
International
Class: |
C07K 16/40 20060101
C07K016/40; C07K 16/30 20060101 C07K016/30; C07K 5/02 20060101
C07K005/02; A61K 47/68 20060101 A61K047/68 |
Claims
1. A compound comprising Formula (VIII) or (IX): ##STR00269##
wherein: A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; B is optional, and when present is a linker
selected from the group consisting of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene,
lower heteroalkylene, substituted lower heteroalkylene, --O--,
--O-(alkylene or substituted alkylene)-, --S--, --S-(alkylene or
substituted alkylene)-, --S(O).sub.k-- where k is 1, 2, or 3,
--S(O).sub.k(alkylene or substituted alkylene)-, --C(O)--,
--C(O)-(alkylene or substituted alkylene)-, --C(S)--,
--C(S)-(alkylene or substituted alkylene)-, --N(R')--,
--NR'-(alkylene or substituted alkylene)-, --C(O)N(R')--,
--CON(R')-(alkylene or substituted alkylene)-, --CSN(R')--,
--CSN(R')-(alkylene or substituted alkylene)-, --N(R')CO-(alkylene
or substituted alkylene)-, --N(R')C(O)O--, --S(O).sub.kN(R')--,
--N(R')C(O)N(R')--, --N(R')C(S)N(R')--, --N(R')S(O).sub.kN(R')--,
--N(R')--N.dbd., --C(R').dbd.N--, --C(R').dbd.N--N(R')--,
--C(R').dbd.N--N.dbd., --C(R').sub.2--N.dbd.N--, and
--C(R').sub.2--N(R')--N(R')--, where each R' is independently H,
alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl,
cycloalkyl, or substituted cycloalkyl; R.sub.1 is H, an amino
protecting group, resin, at least one amino acid, polypeptide, or
polynucleotide; R.sub.2 is OH, an ester protecting group, resin, at
least one amino acid, polypeptide, or polynucleotide; R.sub.3 and
R.sub.4 are each independently H, halogen, lower alkyl, or
substituted lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3
groups optionally form a cycloalkyl or a heterocycloalkyl; Z has
the structure of: ##STR00270## R.sub.5 is H, COR.sub.8,
C.sub.1-C.sub.6alkyl, or thiazole; R.sub.8 is OH; R.sub.6 is OH or
H; Ar is phenyl or pyridine; R.sub.7 is C.sub.1-C.sub.6alkyl or
hydrogen; L is a linker selected from the group consisting of
-alkylene-, -alkylene-C(O)--, -(alkylene-O).sub.n-alkylene -,
-(alkylene-O).sub.n-alkylene-C(O)--,
-(alkylene-O).sub.n--(CH.sub.2).sub.n'--NHC(O)--(CH.sub.2).sub.n''--C(Me)-
.sub.2-S--S--(CH.sub.2).sub.n''',
--NHC(O)-(alkylene-O).sub.n''''-alkylene-, -(alkylene-O).sub.n,
alkylene-W--, -alkylene-C(O)--W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--, and
-(alkylene-O).sub.n-alkylene-U-alkylene-; W has the structure of:
##STR00271## U has the structure of: ##STR00272## and each n, n',
n'', n''' and n'''' are independently integers greater than or
equal to one; or an active metabolite, or a pharmaceutically
acceptable prodrug or solvate thereof.
2. The compound of claim 1, wherein R.sub.1 is a polypeptide.
3. The compound of claim 2, wherein the polypeptide is an
antibody.
4. The compound of claim 3, wherein the antibody is an
anti-prostate-specific membrane antigen (.alpha.PSMA) antibody.
5. The compound of claim 4, wherein the .alpha.PSMA antibody
comprises one or more non-naturally encoded amino acids.
6. The compound of claim 4, wherein the .alpha.PSMA antibody
comprises at least one and no more than ten non-naturally encoded
amino acids.
7. The compound of claim 1, wherein R.sub.2 is a polypeptide.
8. The compound of claim 7, wherein the polypeptide is an
antibody.
9. The compound of claim 8, wherein the antibody is an .alpha.PSMA
antibody.
10. The compound of claim 9, wherein the .alpha.PSMA antibody
comprises one or more non-naturally encoded amino acids.
11. The compound of claim 9, wherein the .alpha.PSMA antibody
comprises at least one and no more than ten non-naturally encoded
amino acids.
12. The compound, or salt thereof, comprising Formula (X), (XI),
(XII) or (XIII): ##STR00273## ##STR00274## wherein: A is optional,
and when present is lower alkylene, substituted lower alkylene,
lower cycloalkylene, substituted lower cycloalkylene, lower
alkenylene, substituted lower alkenylene, alkynylene, lower
heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; B is optional, and when present is a linker
selected from the group consisting of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene,
lower heteroalkylene, substituted lower heteroalkylene, --O--,
--O-(alkylene or substituted alkylene)-, --S--, --S-(alkylene or
substituted alkylene)-, --S(O).sub.k-- where k is 1, 2, or 3,
--S(O).sub.k(alkylene or substituted alkylene)-, --C(O)--,
--C(O)-(alkylene or substituted alkylene)-, --C(S)--,
--C(S)-(alkylene or substituted alkylene)-, --N(R')--,
--NR'-(alkylene or substituted alkylene)-, --C(O)N(R')--,
--CON(R')-(alkylene or substituted alkylene)-, --CSN(R')--,
--CSN(R')-(alkylene or substituted alkylene)-, --N(R')CO-(alkylene
or substituted alkylene)-, --N(R')C(O)O--, --S(O).sub.kN(R')--,
--N(R')C(O)N(R')--, --N(R')C(S)N(R')--, --N(R')S(O).sub.kN(R')--,
--N(R')--N.dbd., --C(R').dbd.N--, --C(R').dbd.N--N(R')--,
--C(R').dbd.N--N.dbd., --C(R').sub.2--N.dbd.N--, and
--C(R').sub.2--N(R')--N(R')--, where each R' is independently H,
alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl,
cycloalkyl, or substituted cycloalkyl; R.sub.1 is H, an amino
protecting group, resin, at least one amino acid, polypeptide, or
polynucleotide; R.sub.2 is OH, an ester protecting group, resin, at
least one amino acid, polypeptide, or polynucleotide; R.sub.3 and
R.sub.4 are each independently H, halogen, lower alkyl, or
substituted lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3
groups optionally form a cycloalkyl or a heterocycloalkyl; Z has
the structure of: ##STR00275## R.sub.5 is H, CO.sub.2H,
C.sub.1-C.sub.6alkyl, or thiazole; R.sub.6 is OH or H; Ar is phenyl
or pyridine; R.sub.7 is C.sub.1-C.sub.6alkyl or hydrogen; L.sub.1,
L.sub.2, L.sub.3, and L.sub.4 are each linkers independently
selected from the group consisting of a bond, -alkylene-,
-(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n'-alkylene-J'-,
-(alkylene-O).sub.n-alkylene-J-alkylene'-, --W--, -alkylene-W--,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-alkylene-NMe-alkylene'-NMe-alkylene''-W--, and
-alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--; W has the
structure of: ##STR00276## each J and J' independently have the
structure of: ##STR00277## and each n and n' are independently
integers greater than or equal to one.
13. The compound of claim 12, wherein R.sub.1 is a polypeptide.
14. The compound of claim 13, wherein the polypeptide is an
antibody.
15. The compound of claim 14, wherein the antibody is an
.alpha.PSMA antibody.
16. The compound of claim 12, wherein R.sub.2 is a polypeptide.
17. The compound of claim 16, wherein the polypeptide is an
antibody.
18. The compound of claim 17, wherein the antibody is an
.alpha.PSMA antibody.
19. A method for derivatizing a dolastatin analog comprising
Formula (I), (III), (IV), (V), or (VI), the method comprising
contacting the dolastatin analog with a reagent of Formula
(XXXVII), wherein Formula (I), (III), (IV), (V), or (VI) correspond
to: ##STR00278## ##STR00279## wherein: Z has the structure of:
##STR00280## R.sub.5 is H, COR.sub.8, C.sub.1-C.sub.6alkyl, or
thiazole; R.sub.8 is OH or --NH-(alkylene-O).sub.n--NH.sub.2;
R.sub.6 is OH or H; Ar is phenyl or pyridine; R.sub.7 is
C.sub.1-C.sub.6 alkyl or hydrogen; Y is NH.sub.2--O-- or methyl; L,
L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each linkers selected
from the group consisting of a bond, -alkylene-, -alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-C(O)--,
-(alkcylene-O).sub.n--(CH.sub.2).sub.n'--HC(O)--(CH.sub.2).sub.n''--C(Me)-
.sub.2-S--S--(CH.sub.2).sub.n'''--NHC(O)-(alkylene-O).sub.n''''-alkylene-,
-(alkylene-O).sub.n-alkylene-W--, -alkylene-C(O)--W--,
-(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-alkylene',
J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n'-alkylene-J'-,
--W--, -alkylene-W--,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--, and
J-(alkylene-NMe).sub.n-alkylene-W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)-(alkylene-O).sub.n-alkylene--
U-alkylene-; -J-alkylene-NMe-alkylene'-NMe-alkylene''-W--, and
-alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--; W has the
structure of: ##STR00281## U has the structure of: ##STR00282##
each J and J' independently have the structure of: ##STR00283## or
L is absent, Y is methyl, R.sub.5 is COR.sub.8, and R.sub.8 is
--NH-(alkylene-O).sub.n--NH.sub.2; and each n, n', n'', n''' and
n'''' are independently integers greater than or equal to one;
wherein Formula (XXXVII) corresponds to: ##STR00284## wherein: A is
optional, and when present is lower alkylene, substituted lower
alkylene, lower alkenylene, substituted lower alkenylene, arylene,
substituted arylene, heteroarylene, substituted heteroarylene,
alkarylene, substituted alkarylene, aralkylene, or substituted
aralkylene; B is optional, and when present is a linker selected
from the group consisting of lower alkylene, substituted lower
alkylene, lower alkenylene, substituted lower alkenylene, --O--,
--O-(alkylene or substituted alkylene)-, --S--, --S-(alkylene or
substituted alkylene)-, --S(O).sub.k-- where k is 1, 2, or 3,
--S(O).sub.k(alkylene or substituted alkylene)-, --C(O)--,
--C(O)-(alkylene or substituted alkylene)-, --C(S)--,
--C(S)-(alkylene or substituted alkylene)-, --N(R')--,
--NR'-(alkylene or substituted alkylene)-, --C(O)N(R')--,
--CON(R')-(alkylene or substituted alkylene)-, --CSN(R')--,
--CSN(R')-(alkylene or substituted alkylene)-, --N(R')CO-(alkylene
or substituted alkylene)-, --N(R')C(O)O--, --S(O).sub.kN(R')--,
--N(R')C(O)N(R')--, --N(R')C(S)N(R')--, --N(R')S(O).sub.kN(R')--,
--N(R')--N.dbd., --C(R').dbd.N--, --C(R').dbd.N--N(R')--,
--C(R').dbd.N--N.dbd., --C(R').sub.2--N.dbd.N--, and
--C(R').sub.2--N(R')--N(R')--, where each R' is independently H,
alkyl, or substituted alkyl; each R' is independently H, alkyl, or
substituted alkyl; K is ##STR00285## R is H, alkyl, substituted
alkyl, cycloalkyl, or substituted cycloalkyl; R.sub.1 is H, an
amino protecting group, resin, at least one amino acid, or
polynucleotide; R.sub.2 is OH, an ester protecting group, resin, at
least one amino acid, or polynucleotide; and R.sub.3 and R.sub.4
are each independently H, halogen, lower alkyl, or substituted
lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3 groups
optionally form a cycloalkyl or a heterocycloalkyl.
20. The method of claim 19, wherein the derivatized dolastatin
analog comprises at least one oxime containing amino acid having
the structure of Formula (VIII), (IX), (X), (XI), (XII), or (XIII):
##STR00286## ##STR00287## ##STR00288##
21. The method of claim 19, wherein the dolastatin analog is
contacted with the reagent of Formula (XXXVII) in aqueous solution
under mildly acidic conditions.
22. A compound comprising Formula (XXV), (XXVI), (XXVII), (XXVIII),
(XXIX), or (XXX): ##STR00289## ##STR00290## wherein: Z has the
structure of: ##STR00291## R.sub.5 is H, CO.sub.2H,
C.sub.1-C.sub.6alkyl, or thiazole; R.sub.6 is OH or H; Ar is phenyl
or pyridine; R.sub.1 is H, an amino protecting group, resin, at
least one amino acid, polypeptide, or polynucleotide; R.sub.2 is
OH, an ester protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide; R.sub.4 is H, halogen, lower alkyl,
or substituted lower alkyl; R.sub.7 is C.sub.1-C.sub.6alkyl or
hydrogen; L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each
linkers selected from the group consisting of a bond, -alkylene-,
-alkylene-C(O)--, -alkylene-J-, -(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-C(O)--, -(alkylene-O).sub.n-J-,
-(alkylene-O).sub.n-J-alkylene-,
-(alkylene-O).sub.n--(CH.sub.2).sub.n
NHC(O)--(CH.sub.2).sub.n''--C(Me).sub.2-S--S--(CH.sub.2).sub.n'''--NHC(O)-
-(alkylene-O).sub.n''''-alkylene-, -(alkylene-O)i-alkylene-W--,
-alkylene-C(O)--W--, -(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-alkylene',
-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n'-alkylene-J'-,
--W--, -alkylene-W--,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-(alkylene-NMe).sub.n-alkylene-W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-U-alkylene-;
-J-alkylene-NMe-alkylene'-NMe-alkylene''-W--, and
-alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--; W has the
structure of: ##STR00292## U has the structure of: ##STR00293##
each J and J' independently have the structure of: ##STR00294##
each n and n' are independently integers greater than or equal to
one; and each R.sub.16 is independently selected from the group
consisting of hydrogen, halogen, alkyl, NO.sub.2, CN, and
substituted alkyl.
23. The compound of claim 22, wherein R.sub.1 is a polypeptide.
24. The compound of claim 23, wherein the polypeptide is an
antibody.
25. The compound of claim 24, wherein the antibody is an
.alpha.PSMA antibody.
26. The compound of claim 22, wherein R.sub.2 is a polypeptide.
27. The compound of claim 26, wherein the polypeptide is an
antibody.
28. The compound of claim 27, wherein the antibody is an
.alpha.PSMA antibody.
29. A compound comprising Formula (XXXI), (XXXII), (XXXIII),
(XXXIV), (XXXV), or (XXXVI): ##STR00295## ##STR00296## ##STR00297##
wherein: Z has the structure of: ##STR00298## R.sub.5 is H,
CO.sub.2H, C.sub.1-C.sub.6alkyl, or thiazole; R.sub.6 is OH or H;
Ar is phenyl or pyridine; R.sub.1 is H, an amino protecting group,
resin, at least one amino acid, polypeptide, or polynucleotide;
R.sub.2 is OH, an ester protecting group, resin, at least one amino
acid, polypeptide, or polynucleotide; R.sub.4 is H, halogen, lower
alkyl, or substituted lower alkyl; R.sub.7 is C.sub.1-C.sub.6alkyl
or hydrogen; L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each
linkers selected from the group consisting of a bond, -alkylene-,
-alkylene-C(O)--, -alkylene-J-, -(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-C(O)--, -(alkylene-O).sub.n-J-,
-(alkylene-O).sub.n-J-alkylene-,
-(alkylene-O).sub.n--(CH.sub.2).sub.n'--NHC(O)--(CH.sub.2).sub.n''--C(Me)-
.sub.2-S--S--(CH.sub.2).sub.n'''--NHC(O)-(alkylene-O).sub.n''''-alkylene,
-(alkylene-O).sub.n-alkylene-W--, -alkylene-C(O)--W--,
-(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-alkylene',
-J-(alkylene-O).sub.n-alkylene,
-(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n'-alkylene-J'-,
--W--, -alkylene-W--,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-(alkylene-NMe).sub.n-alkylene-W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-U-alkylene-;
-J-alkylene-NMe-alkylene'-NMe-alkylene''-W--, and -alkylene
-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--; ##STR00299## U has
the structure of: ##STR00300## each J and J independently have the
structure of: ##STR00301## each n and n' are independently integers
greater than or equal to one; D has the structure of: ##STR00302##
each R.sub.17 is independently selected from the group consisting
of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,
alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide,
substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl,
substituted aralkyl, -(alkylene or substituted
alkylene)-ON(R'').sub.2, -(alkylene or substituted
alkylene)-C(O)SR'', -(alkylene or substituted alkylene)-S--S-(aryl
or substituted aryl), --C(O)R'', --C(O).sub.2R'', or
--C(O)N(R'').sub.2, wherein each R'' is independently hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy,
substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl,
substituted alkaryl, aralkyl, or substituted aralkyl; each Z.sub.1
is a bond, CR.sub.17R.sub.17, O, S, NR',
CR.sub.17R.sub.17--CR.sub.17R.sub.17, CR.sub.17R.sub.17--O,
O--CR.sub.17R.sub.17, CR.sub.17R.sub.17--S, S--CR.sub.17R.sub.17,
CR.sub.17R.sub.17--NR', or NR'--CR.sub.17R.sub.17; each R' is H,
alkyl, or substituted alkyl; each Z.sub.2 is selected from the
group consisting of a bond, --C(O)--, --C(S)--, optionally
substituted C.sub.1-C.sub.3 alkylene, optionally substituted
C.sub.1-C.sub.3 alkenylene, and optionally substituted heteroalkyl;
each Z.sub.3 are independently selected from the group consisting
of a bond, optionally substituted C.sub.1-C.sub.4 alkylene,
optionally substituted C.sub.1-C.sub.4 alkenylene, optionally
substituted heteroalkyl, --O--, --S--, --C(O)--, --C(S)--, and
--N(R')--; each T.sub.3 is a bond, C(R'')(R''), O, or S; with the
proviso that when T.sub.3 is O or S, R'' cannot be halogen; each
R'' is H, halogen, alkyl, substituted alkyl, cycloalkyl, or
substituted cycloalkyl; m and p are 0, 1, 2, or 3, provided that at
least one of m or p is not 0; M.sub.2 is ##STR00303## where (a)
indicates bonding to the B group and (b) indicates bonding to
respective positions within the heterocycle group; M.sub.3 is
##STR00304## where (a) indicates bonding to the B group and (b)
indicates bonding to respective positions within the heterocycle
group; M.sub.4 is ##STR00305## where (a) indicates bonding to the B
group and (b) indicates bonding to respective positions within the
heterocycle group; each R.sub.19 is independently selected from the
group consisting of C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
ester, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl
ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl sulfonic
acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl,
nitrile, alkyl nitrite, and nitro; q is 0, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10 or 11; and each R.sub.16 is independently selected from the
group consisting of hydrogen, halogen, alkyl, NO.sub.2, CN, and
substituted alkyl.
30. The compound of claim 29, wherein R.sub.1 is a polypeptide.
31. The compound of claim 30, wherein the polypeptide is an
antibody.
32. The compound of claim 31, wherein the antibody is an PSMA
antibody.
33. The compound of claim 29, wherein R.sub.2 is a polypeptide.
34. The compound of claim 33, wherein the polypeptide is an
antibody.
35. The compound of claim 34, wherein the antibody is an
.alpha.PSMA antibody.
36. The compound of claim 29, comprising Formula (XXXI-A):
##STR00306##
37. A pharmaceutical composition comprising a compound of any of
claims 1-18 or 22-36 and a pharmaceutically acceptable carrier,
excipient, or binder.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Divisional Application of U.S.
application Ser. No. 14/406,190, filed Dec. 5, 2014, now U.S. Pat.
No. 10,800,856, which in turn is a U.S. National Stage entry under
35 U.S.C .sctn. 371 of International Application No.
PCT/US2013/44850, filed on Jun. 7, 2013, designating the United
States of America and published in English on Dec. 12, 2013, which
in turn claims priority to U.S. Provisional Application No.
61/656,883, filed on Jun. 7, 2012, each of which is hereby
incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. The ASCII copy, created
on Jun. 9, 2021, is named AMBX_0178_01_US_ST25.txt and is 41,660
bytes in size.
FIELD OF THE INVENTION
[0003] This invention relates to prostate-specific membrane antigen
(PSMA) antibody drug conjugates comprising at least one
non-naturally-encoded amino acid.
BACKGROUND OF THE INVENTION
[0004] Prostate cancer is the most commonly diagnosed non-skin
related malignancy in males in developed countries. It is estimated
that one in six males will be diagnosed with prostate cancer. The
diagnosis of prostate cancer has greatly improved following the use
of serum-based markers such as the prostate-specific antigen (PSA).
In addition, prostate tumor-associated antigens offer targets for
tumor imaging, diagnosis, and targeted therapies. The prostate
specific membrane antigen (PSMA), a prostate tumor associated
marker, is such a target.
[0005] PSMA is a glycoprotein highly restricted to prostate
secretory epithelial cell membranes. Its expression level has been
correlated with tumor aggressiveness. Various immunohistological
studies have demonstrated increased PSMA levels in virtually all
cases of prostatic carcinoma compared to those levels in benign
prostate epithelial cells. Intense PSMA staining is found in all
stages of the disease, including prostatic intraepithelial
neoplasia, late stage androgen-independent prostate cancer and
secondary prostate tumors localized to lymph nodes, bone, soft
tissue, and lungs.
[0006] PSMA forms a noncovalent homodimer that possesses glutamate
carboxypeptidase activity based on its ability to process the
neuropeptide N-acetylaspartylglutamate and glutamate-conjugated
folate derivatives. Although the precise biological role played by
PSMA in disease pathogenesis remains unknown, its overexpression in
prostate tumors is well known. It has been suggested that PSMA
performs multiple physiological functions related to cell survival
and migration.
[0007] Antibody-based therapeutics have emerged as important
components of therapies for an increasing number of human
malignancies in such fields as oncology, inflammatory and
infectious diseases. In most cases, the basis of the therapeutic
function is the high degree of specificity and affinity the
antibody-based drug has for its target antigen. Arming monoclonal
antibodies with drugs, toxins, or radionuclides is yet another
strategy by which mAbs may induce therapeutic effect. By combining
the exquisite targeting specificity of antibody with the tumor
killing power of toxic effector molecules, immunoconjugates permit
sensitive discrimination between target and normal tissue thereby
resulting in fewer side effects than most conventional
chemotherapeutic drugs.
[0008] Given the physical properties of PSMA and its expression
pattern in relation to prostate cancer progression PSMA is an
excellent target in the development of antibody-drug conjugates for
imaging, diagnostic and therapeutic uses. The first PSMA-specific
MAb reported, 7E11, was subsequently developed and commercialized
as a diagnostic agent for tumor imaging (ProstaScint, Cytogen,
Princeton, N.J.). However, this antibody recognizes an
intracellular epitope of PSMA which limits its usefulness as an
imaging agent for the detection of PSMA. More recently, MAbs such
as J591 that recognize the extracellular portion of PSMA have been
identified. Anti-PSMA antibody-drug conjugates that can be utilized
for imaging, diagnostic and/or therapeutic uses are therefore
needed. The present invention provides such antibody-drug
conjugates for use in prostate cancer.
SUMMARY OF THE INVENTION
[0009] Disclosed herein are PSMA antibodies linked to toxic
moieties through one or more non-natural amino acids with one or
more linker(s), and methods for making such non-natural amino acids
and polypeptides.
[0010] Some embodiments of the present invention describe a toxic
moiety, or salt thereof, comprising Formula (I):
##STR00001##
wherein: [0011] Z has the structure of:
[0011] ##STR00002## [0012] R.sub.5 is H, COR.sub.8,
C.sub.1-C.sub.6alkyl, or thiazole; [0013] R.sub.8 is OH or
--NH-(akylene-).sub.n--NH.sub.2; [0014] R.sub.6 is OH or H; [0015]
Ar is phenyl or pyridine; [0016] R.sub.7 is C.sub.1-C.sub.6alkyl or
hydrogen; [0017] Y is selected from the group consisting of an
hydroxylamine, methyl, aldehyde, protected aldehyde, ketone,
protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine,
imine, diamine, azide, keto-amine, keto-alkyne, alkyne,
cycloalkyne, and ene-dione; [0018] L is a linker selected from the
group consisting of -alkylene-, alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-C(O)--,
(alkylene-O).sub.n--(CH2).sub.n',
--NHC(O)--(CH.sub.2).sub.n''--C(Me).sub.2-S--S--(CH.sub.2).sub.n'''--NHC(-
O)-(alkylene-O).sub.n''''-alkylene-,
-(alkylene-O).sub.n-alkylene-W--, -alkylene-C(O)--W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--, and
-(alkylene-O).sub.n-alkylene-U-alkylene-; [0019] W has the
structure of:
[0019] ##STR00003## [0020] U has the structure of:
[0020] ##STR00004## [0021] or L is absent, Y is methyl, R.sub.5 is
COR.sub.8, and R.sub.8 is --NH-(alkylene-O).sub.n--NH.sub.2; and
[0022] each n, n', n'', n''' and n'''' are independently integers
greater than or equal to one.
[0023] In some embodiments, R.sub.5 is thiazole, In other
embodiments, R.sub.6 is H. In certain embodiments, Ar is phenyl. In
further or additional embodiments, R.sub.7 is methyl. In some
embodiments, n is an integer from 0 to 20, 0 to 10 or 0 to 5.
[0024] In some embodiments, a compound is described comprising
Formula (II):
##STR00005##
In certain embodiments, L is -(alkylene-O).sub.n-alkylene-. In
specific embodiments, each alkylene is CH.sub.2CH.sub.2--, n is
equal to 3, and R.sub.7 is methyl. In other embodiments, L is
-alkylene-. In specific embodiments, each alkylene is
--CH.sub.2CH.sub.2-- and R.sub.7 is methyl or hydrogen. In certain
embodiments, L is -(alkylene-O).sub.n-alkylene-C(O)--. In certain
specific embodiments, each alkylene is --CH.sub.2CH.sub.2--, n is
equal to 4, and R.sub.7 is methyl. In further or alternative
embodiments, L is
-(alkylene-O).sub.n--(CH.sub.2).sub.n'--NHC(O)--(CH.sub.2).sub.n''--C(Me)-
.sub.2-S--S--(CH.sub.2).sub.n'''--NHC(O)-(alkylene-O).sub.n''''-alkylene-.
In specific embodiments, each alkylene is --CH.sub.2CH.sub.2--, n
is equal to 1, n' is equal to 2, n'' is equal to 1, n''' is equal
to 2, n'''' is equal to 4, and R.sub.7 is methyl.
[0025] In some embodiments, Y is azide. In other embodiments, Y is
cyclooctyne. In specific embodiments, the cyclooctyne has a
structure of:
##STR00006## [0026] each R.sub.19 is independently selected from
the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkoxy, ester, ether, thioether, aminoalkyl, halogen, alkyl ester,
aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl
sulfonic acid, alkyl nitro, thioester, sulfonyl ester,
halosulfonyl, nitrile, alkyl nitrile, and nitro; and [0027] q is 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.
[0028] Some embodiments of the present invention describe a
compound, or salt thereof, comprising Formula (III), (IV), (V) or
(VI):
##STR00007## ##STR00008##
wherein: [0029] Z has the structure of:
[0029] ##STR00009## [0030] R.sub.5 is H, COR.sub.8,
C.sub.1-C.sub.6alkyl, or thiazole; [0031] R.sub.8 is OH; [0032]
R.sub.6 is OH or H; [0033] Ar is phenyl or pyridine; [0034] R.sub.7
is C.sub.1-C.sub.6alkyl or hydrogen; [0035] Y and V are each
selected from the group consisting of an hydroxylamine, methyl,
aldehyde, protected aldehyde, ketone, protected ketone, thioester,
ester, dicarbonyl, hydrazine, amidine, imine, diamine, azide,
keto-amine, keto-alkyne, alkyne, cycloalkyne, and ene-dione; [0036]
L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each linkers
independently selected from the group consisting of a bond,
-alkylene-, -(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n'-alkylene-J'-,
-(alkylene-O).sub.n-alkylene-J-alkylene'-, --W--, -alkylene-W--,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-alkylene-NMe-alkylene'-NMe-alkylene''-W--, and
-alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--; [0037] W
has the structure of:
[0037] ##STR00010## [0038] each J and F independently have the
structure of:
[0038] ##STR00011## and [0039] each n and n' are independently
integers greater than or equal to one.
[0040] In certain embodiments, a compound is described comprising
Formula (VII):
##STR00012##
[0041] In certain embodiments, L.sub.1 is
-(alkylene-O).sub.n-alkylene-J-, L.sub.2 is
-alkylene'-J'-(alkylene-O).sub.n'-alkylene-, L.sub.3 is
-J''-(alkylene-O).sub.n''-alkylene-, alkylene is
--CH.sub.2CH.sub.2--, alkylene' is --(CH.sub.2).sub.4--, n is 1, n'
and n'' are 3, J has the structure of H
##STR00013##
J' and J'' have the structure of
##STR00014##
and R.sub.7 is methyl. In other embodiments, L.sub.1 is
-J-(alkylene-O).sub.n-alkylene-, L.sub.2 is
-(alkylene-O).sub.n'-alkylene-J'-alkylene'-, L.sub.3 is
-(alkylene-O).sub.n''-alkylene-J''-, alkylene is
--CH.sub.2CH.sub.2--, alkylene' is --(CH.sub.2).sub.4--, n is 1, n'
and n'' are 4, and J, J' and J'' have the structure of
##STR00015##
[0042] In some embodiments, Y is azide. In other embodiments, Y is
cyclooctyne. In specific embodiments, the cyclooctyne has a
structure of:
##STR00016## [0043] each R.sub.19 is independently selected from
the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkoxy, ester, ether, thioether, aminoalkyl, halogen, alkyl ester,
aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl
sulfonic acid, alkyl nitro, thioester, sulfonyl ester,
halosulfonyl, nitrile, alkyl nitrile, and nitro; and [0044] q is 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.
[0045] Certain embodiments of the present invention describe a
compound comprising Formula (VIII) or (IX):
##STR00017## [0046] wherein: [0047] A is optional, and when present
is lower alkylene, substituted lower alkylene, lower cycloalkylene,
substituted lower cycloalkylene, lower alkenylene, substituted
lower alkenylene, alkynylene, lower heteroalkylene, substituted
heteroalkylene, lower heterocycloalkylene, substituted lower
heterocycloalkylene, arylene, substituted arylene, heteroarylene,
substituted heteroarylene, alkarylene, substituted alkarylene,
aralkylene, or substituted aralkylene; [0048] B is optional, and
when present is a linker selected from the group consisting of
lower alkylene, substituted lower alkylene, lower alkenylene,
substituted lower alkenylene, lower heteroalkylene, substituted
lower heteroalkylene, --O--, --O-(alkylene or substituted
alkylene)-, --S--, --S-(alkylene or substituted alkylene)-,
--S(O).sub.k-- where k is 1, 2, or 3, --S(O).sub.k(alkylene or
substituted alkylene)-, --C(O)--, --C(O)-(alkylene or substituted
alkylene)-, --C(S)--, --C(S)-(alkylene or substituted alkylene)-,
--N(R')--, --NR'-(alkylene or substituted alkylene)-,
--C(O)N(R')--, --CON(R')-(alkylene or substituted alkylene)-,
--CSN(R')--, --CSN(R')-(alkylene or substituted alkylene)-,
--N(R')CO-(alkylene or substituted alkylene)-, --N(R')C(O)O--,
--S(O).sub.kN(R')--, --N(R')C(O)N(R')--, --N(R')C(S)N(R')--,
--N(R')S(O).sub.kN(R')--, --N(R')--N.dbd., --C(R').dbd.N--,
--C(R').dbd.N--N(R')--, --C(R').dbd.N--N.dbd.,
--C(R').sub.2--N.dbd.N--, and --C(R').sub.2--N(R')--N(R')--, where
each R' is independently H, alkyl, or substituted alkyl; [0049] R
is H, alkyl, substituted alkyl, cycloalkyl, or substituted
cycloalkyl; [0050] R.sub.1 is H, an amino protecting group, resin,
at least one amino acid, polypeptide, or polynucleotide; [0051]
R.sub.2 is OH, an ester protecting group, resin, at least one amino
acid, polypeptide, or polynucleotide; [0052] R.sub.3 and R.sub.4
are each independently H, halogen, lower alkyl, or substituted
lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3 groups
optionally form a cycloalkyl or a heterocycloalkyl; [0053] Z has
the structure of:
[0053] ##STR00018## [0054] R.sub.5 is H, CO.sub.2H,
C.sub.1-C.sub.6alkyl, or thiazole; [0055] R.sub.6 is OH or H;
[0056] Ar is phenyl or pyridine; [0057] R.sub.7 is
C.sub.1-C.sub.6alkyl or hydrogen; [0058] L is a linker selected
from the group consisting of -alkylene-, -alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-C(O)--,
-(alkylene-O).sub.n--(CH.sub.2).sub.n'--NHC(O)--(CH.sub.2).sub.n''--C(Me)-
.sub.2-S--S--(CH.sub.2).sub.n'''--NHC(O)-(alkylene-O).sub.n''''-alkylene-,
-(alkylene-O).sub.n-alkylene-W--, -alkylene-C(O)--W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--, and
-(alkylene-O).sub.n-alkylene-U-alkylene-; [0059] W has the
structure of:
[0059] ##STR00019## [0060] U has the structure of:
[0060] ##STR00020## and [0061] each n, n', n'', n''' and n'''' are
independently integers greater than or equal to one; [0062] or an
active metabolite, or a pharmaceutically acceptable prodrug or
solvate thereof.
[0063] In some embodiments, R.sub.1 is a polypeptide. In specific
embodiments, the polypeptide is an antibody. In certain specific
embodiments, the antibody is a prostate-specific membrane antigen
antibody. In some embodiments, the antibody is .alpha.-PSMA. In
other embodiments, R.sub.2 is a polypeptide. In specific
embodiments, the polypeptide is an .alpha.-PSMA antibody. In
certain specific embodiments, the antibody is ARX-.alpha.PSMA (SEQ.
ID. NO.: 1).
[0064] Some embodiments of the present invention describe a
compound, or salt thereof, comprising Formula (X), (XI), (XII) or
(XIII):
##STR00021## ##STR00022## [0065] wherein: [0066] A is optional, and
when present is lower alkylene, substituted lower alkylene, lower
cycloalkylene, substituted lower cycloalkylene, lower alkenylene,
substituted lower alkenylene, alkynylene, lower heteroalkylene,
substituted heteroalkylene, lower heterocycloalkylene, substituted
lower heterocycloalkylene, arylene, substituted arylene,
heteroarylene, substituted heteroarylene, alkarylene, substituted
alkarylene, aralkylene, or substituted aralkylene; [0067] B is
optional, and when present is a linker selected from the group
consisting of lower alkylene, substituted lower alkylene, lower
alkenylene, substituted lower alkenylene, lower heteroalkylene,
substituted lower heteroalkylene, --O--, --O-(alkylene or
substituted alkylene)-, --S--, --S-(alkylene or substituted
alkylene)-, --S(O).sub.k-- where k is 1, 2, or 3,
--S(O).sub.k(alkylene or substituted alkylene)-, --C(O)--,
--C(O)-(alkylene or substituted alkylene)-, --C(S)--,
--C(S)-(alkylene or substituted alkylene)-, --N(R')--,
--NR'-(alkylene or substituted alkylene)-, --C(O)N(R')--,
--CON(R')-(alkylene or substituted alkylene)-, --CSN(R')--,
--CSN(R')-(alkylene or substituted alkylene)-, --N(R')CO-(alkylene
or substituted alkylene)-, --N(R')C(O)O--, --S(O).sub.kN(R')--,
--N(R')C(O)N(R')--, --N(R')C(S)N(R')--, --N(R')S(O).sub.kN(R')--,
--N(R')--N.dbd., --C(R').dbd.N--, --C(R').dbd.N--N(R')--,
--C(R').dbd.N--N.dbd., --C(R').sub.2--N.dbd.N--, and
--C(R').sub.2--N(R')--N(R')--, where each R' is independently H,
alkyl, or substituted alkyl; [0068] R is H, alkyl, substituted
alkyl, cycloalkyl, or substituted cycloalkyl; [0069] R.sub.1 is H,
an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide; [0070] R.sub.2 is OH, an ester
protecting group, resin, at least one amino acid, polypeptide, or
polynucleotide; [0071] R.sub.3 and R.sub.4 are each independently
H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and
R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a
heterocycloalkyl; [0072] Z has the structure of:
[0072] ##STR00023## [0073] R.sub.5 is H, CO.sub.2H,
C.sub.1-C.sub.6alkyl, or thiazole; [0074] R.sub.6 is OH or H;
[0075] Ar is phenyl or pyridine; [0076] R.sub.7 is
C.sub.1-C.sub.6alkyl or hydrogen; [0077] L.sub.1, L.sub.2, L.sub.3,
and L.sub.4 are each linkers independently selected from the group
consisting of a bond, -alkylene-, -(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.1-alkylene-J-(alkylene-O).sub.n'-alkylene-J'-,
-(alkylene-O).sub.n-alkylene-J-alkylene'-, --W--, -alkylene-W--,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-alkylene-NMe-alkylene'-NMe-alkylene''-W--, and
-alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--; [0078] W
has the structure of:
[0078] ##STR00024## [0079] each J and J' independently have the
structure of:
[0079] ##STR00025## and [0080] each n and n' are independently
integers greater than or equal to one.
[0081] In some embodiments, R.sub.1 is a polypeptide. In specific
embodiments, the polypeptide is an antibody. In certain specific
embodiments, the antibody is herceptin. In other embodiments,
R.sub.2 is a polypeptide. In specific embodiments, the polypeptide
is an antibody. In certain embodiments, the antibody is
.alpha.PSMA. In some embodiments, the antibody is derived from any
known .alpha.PSMA antibody. In certain specific embodiments, the
antibody is ARX-.alpha.PSMA.
[0082] In some embodiments, provided herein is a method for
derivatizing a dolastatin analog comprising Formula (I), (III),
(IV), (V), or (VI), the method comprising contacting the dolastatin
analog with a reagent of Formula (XXXVII), wherein Formula (I),
(III), (IV), (V), or (VI) correspond to:
##STR00026## ##STR00027## [0083] wherein: [0084] Z has the
structure of:
[0084] ##STR00028## [0085] R.sub.5 is H, COR.sub.8,
C.sub.1-C.sub.6alkyl, or thiazole; [0086] R.sub.8 is OH or
--NH-(alkylene-O).sub.n--NH.sub.2; [0087] R.sub.6 is OH or H;
[0088] Ar is phenyl or pyridine; [0089] R.sub.7 is C.sub.1-C.sub.6
alkyl or hydrogen; [0090] Y is NH.sub.2--O-- or methyl; [0091] L,
L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each linkers selected
from the group consisting of a bond, -alkylene-, -alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-C(O)--,
-(alkylene-O).sub.n--(CH.sub.2).sub.n'--NHC(O)--(CH.sub.2).sub.n''--C(Me)-
.sub.2-S--S--(CH.sub.2).sub.n'''--NHC(O)-(alkylene-O).sub.n''''-alkylene-,
-(alkylene-O).sub.n-alkylene-W--, -alkylene-C(O)--W--,
-(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-alkylene',
-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n'-alkylene-J'-,
--W--, -alkylene-W--, alkylene'-J-(alkylene-NMe)-alkylene-W--, and
J-(alkylene-NMe).sub.n-alkylene-W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-U-alkylene-;
-J-alkylene-NMe-alkylene'-NMe-alkylene''-W--, and
-alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--; [0092] W
has the structure of:
[0092] ##STR00029## [0093] U has the structure of:
[0093] ##STR00030## [0094] each J and J' independently have the
structure of:
[0094] ##STR00031## [0095] or L is absent, Y is methyl, R.sub.5 is
COR.sub.8, and R.sub.8 is --NH-(alkylene-O).sub.n--NH.sub.2; and
[0096] each n, n', n'', n''' and n'''' are independently integers
greater than or equal to one; wherein Formula (XXXVII) corresponds
to:
[0096] ##STR00032## [0097] wherein: [0098] A is optional, and when
present is lower alkylene, substituted lower alkylene, lower
alkenylene, substituted lower alkenylene, arylene, substituted
arylene, heteroarylene, substituted heteroarylene, alkarylene,
substituted alkarylene, aralkylene, or substituted aralkylene;
[0099] B is optional, and when present is a linker selected from
the group consisting of lower alkylene, substituted lower alkylene,
lower alkenylene, substituted lower alkenylene, --O--,
--O-(alkylene or substituted alkylene)-, --S--, --S-(alkylene or
substituted alkylene)-, --S(O).sub.k-- where k is 1, 2, or 3,
--S(O).sub.k(alkylene or substituted alkylene)-, --C(O)--,
--C(O)-(alkylene or substituted alkylene)-, --C(S)--,
--C(S)-(alkylene or substituted alkylene)-, --N(R')--,
--NR'-(alkylene or substituted alkylene)-, --C(O)N(R')--,
--CON(R')-(alkylene or substituted alkylene)-, --CSN(R')--,
--CSN(R')-(alkylene or substituted alkylene)-, --N(R')CO-(alkylene
or substituted alkylene)-, --N(R')C(O)O--, --S(O).sub.kN(R')--,
--N(R')C(O)N(R')--, --N(R')C(S)N(R')--, --N(R')S(O).sub.kN(R')--,
--N(R')--N.dbd., --C(R').dbd.N--, --C(R').dbd.N--N(R')--,
--C(R').dbd.N--N.dbd., --C(R').sub.2--N.dbd.N--, and
--C(R').sub.2--N(R')--N(R')--, where each R' is independently H,
alkyl, or substituted alkyl; [0100] each R' is independently H,
alkyl, or substituted alkyl; [0101] K is
[0101] ##STR00033## [0102] R is H, alkyl, substituted alkyl,
cycloalkyl, or substituted cycloalkyl; [0103] R.sub.1 is H, an
amino protecting group, resin, at least one amino acid, or
polynucleotide; [0104] R.sub.2 is OH, an ester protecting group,
resin, at least one amino acid, or polynucleotide; and [0105]
R.sub.3 and R.sub.4 are each independently H, halogen, lower alkyl,
or substituted lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3
groups optionally form a cycloalkyl or a heterocycloalkyl.
[0106] In some embodiments, the derivatized dolastatin analog
comprises at least one oxime containing amino acid having the
structure of Formula (VIII), (IX), (X), (XI), (XII), or (XIII):
##STR00034## ##STR00035##
In specific embodiments, the dolastatin analog is contacted with
the reagent of Formula (XXXVII) in aqueous solution under mildly
acidic conditions.
[0107] Certain embodiments of the present invention describe a
compound comprising Formula (XXV), (XXVI), (XXVII), (XXVIII),
(XXIX), or (XXX):
##STR00036## ##STR00037## [0108] wherein: [0109] Z has the
structure of:
[0109] ##STR00038## [0110] R.sub.5 is H, CO.sub.2H,
C.sub.1-C.sub.6alkyl, or thiazole; [0111] R.sub.6 is OH or H;
[0112] Ar is phenyl, or pyridine; [0113] R.sub.1 is H, an amino
protecting group, resin, at least one amino acid, polypeptide, or
polynucleotide; [0114] R.sub.2 is OH, an ester protecting group,
resin, at least one amino acid, polypeptide, or polynucleotide;
[0115] R.sub.4 is H, halogen, lower alkyl, or substituted lower
alkyl; [0116] R.sub.7 is C.sub.1-C.sub.6alkyl or hydrogen; [0117]
L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each linkers selected
from the group consisting of a bond, -alkylene-, -alkylene-C(O)--,
-alkylene-J-, -(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-C(O)--, -(alkylene-O).sub.n-J-,
-(alkylene-O).sub.n-J-alkylene-,
-(alkylene-O).sub.n--(CH.sub.2).sub.n'--NHC(O)--(CH.sub.2).sub.n''--C(Me)-
.sub.2-S--S--(CH.sub.2).sub.n'''--NHC(O)-(alkylene-O).sub.n''''-alkylene-,
-(alkylene-O).sub.n-alkylene-W--, -alkylene-C(O)--W--,
-(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-alkylene',
-J-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene
-J-(alkylene-O).sub.n'-alkylene-J'-, --W--, -alkylene-W--,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-(alkylene-NMe).sub.n-alkylene-W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-U-alkylene-;
-J-alkylene-NMe-alkylene'-NMe-alkylene''-W--, and
-alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--; [0118] W
has the structure of:
[0118] ##STR00039## [0119] U has the structure of:
[0119] ##STR00040## [0120] each J and J' independently have the
structure of:
[0120] ##STR00041## [0121] each n and n' are independently integers
greater than or equal to one; and [0122] each R.sub.16 is
independently selected from the group consisting of hydrogen,
halogen, alkyl, NO.sub.2, CN, and substituted alkyl.
[0123] In some embodiments, R.sub.1 is a polypeptide. In specific
embodiments, the polypeptide is an antibody. In certain specific
embodiments, the antibody is .alpha.PSMA. In other embodiments,
R.sub.2 is a polypeptide. In specific embodiments, the polypeptide
is an antibody. In certain specific embodiments, the antibody is
.alpha.PSMA.
[0124] Some embodiments of the present invention describe a
compound comprising Formula (XXXI), (XXXII), (XXXIII), (XXXIV),
(XXXV), or (XXXVI):
##STR00042## ##STR00043## [0125] wherein: [0126] Z has the
structure of:
[0126] ##STR00044## [0127] R.sub.5 is H, CO.sub.2H,
C.sub.1-C.sub.6alkyl, or thiazole; [0128] R.sub.6 is OH or H;
[0129] Ar is phenyl or pyridine; [0130] R.sub.1 is H, an amino
protecting group, resin, at least one amino acid, polypeptide, or
polynucleotide; [0131] R.sub.2 is OH, an ester protecting group,
resin, at least one amino acid, polypeptide, or polynucleotide;
[0132] R.sub.4 is H, halogen, lower alkyl, or substituted lower
alkyl; [0133] R.sub.1 is C.sub.1-C.sub.6alkyl or hydrogen; [0134]
L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each linkers selected
from the group consisting of a bond, -alkylene-, -alkylene-C(O)--,
-alkylene-J-, -(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-C(O)--, -(alkylene-O).sub.n-J-,
-(alkylene-O).sub.n-J-alkylene-,
-(alkylene-O).sub.n--(CH.sub.2).sub.n'--NHC(O)--(CH.sub.2).sub.n''--C(Me)-
.sub.2-S--S(CH.sub.2).sub.n'''--NHC(O)-(alkylene-O).sub.n''''-alkylene-,
-(alkylene-O).sub.n-alkylene-W--, -alkylene-C(O)--W--,
-(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J alkylene',
-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n'-alkylene-J'-,
--W--, -alkylene-W--,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-(alkylene-NMe).sub.n-alkylene-W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-U-alkylene-;
-J-alkylene-NMe-alkylene'-NMe-alkylene''-W--, and
-alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--;
[0134] ##STR00045## [0135] U has the structure of:
[0135] ##STR00046## [0136] each J and J' independently have the
structure of:
[0136] ##STR00047## [0137] each n and n' are independently integers
greater than or equal to one; [0138] D has the structure of:
[0138] ##STR00048## [0139] each R.sub.17 is independently selected
from the group consisting of H, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,
substituted alkoxy, alkylalkoxy, substituted alkylalkoxy,
polyalkylene oxide, substituted polyalkylene oxide, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, alkaryl,
substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or
substituted alkylene)-ON(R'').sub.2, -(alkylene or substituted
alkylene)-C(O)SR'', -(alkylene or substituted alkylene)-S--S-(aryl
or substituted aryl), --C(O)R'', --C(O).sub.2R'', or
--C(O)N(R'').sub.2, wherein each R'' is independently hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy,
substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl,
substituted alkaryl, aralkyl, or substituted aralkyl; [0140] each
Z.sub.1 is a bond, CR.sub.17R.sub.17, O, S, NR',
CR.sub.17R.sub.17--CR.sub.17R.sub.17, CR.sub.17--O,
O--CR.sub.17R.sub.17, CR.sub.17R.sub.17--S, S--CR.sub.17R.sub.17,
CR.sub.17R.sub.17--NR', or NR'--CR.sub.17R.sub.17; [0141] each R'
is H, alkyl, or substituted alkyl; [0142] each Z.sub.2 is selected
from the group consisting of a bond, --C(O)--, --C(S)--, optionally
substituted C.sub.1-C.sub.3 alkylene, optionally substituted
C.sub.1-C.sub.3 alkenylene, and optionally substituted heteroalkyl;
[0143] each Z.sub.3 are independently selected from the group
consisting of a bond, optionally substituted C.sub.1-C.sub.4
alkylene, optionally substituted C.sub.1-C.sub.4 alkenylene,
optionally substituted heteroalkyl, --O--, --S--, --C(O)--,
--C(S)--, and --N(R')--; [0144] each T.sub.3 is a bond,
C(R'')(R''), O, or S; with the proviso that when T.sub.3 is O or S,
R'' cannot be halogen; [0145] each R'' is H, halogen, alkyl,
substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0146] m
and p are 0, 1, 2, or 3, provided that at least one of m or p is
not 0; [0147] M.sub.2 is
##STR00049##
[0147] where (a) indicates bonding to the B group and (b) indicates
bonding to respective positions within the heterocycle group;
[0148] M.sub.3 is
[0148] ##STR00050## where (a) indicates bonding to the B group and
(b) indicates bonding to respective positions within the
heterocycle group; [0149] M.sub.4 is
[0149] ##STR00051## where (a) indicates bonding to the B group and
(b) indicates bonding to respective positions within the
heterocycle group; [0150] each R.sub.19 is independently selected
from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkoxy, ester, ether, thioether, aminoalkyl, halogen, alkyl ester,
aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl
sulfonic acid, alkyl nitro, thioester, sulfonyl ester,
halosulfonyl, nitrile, alkyl nitrile, and nitro; [0151] q is 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; and [0152] each R.sub.16 is
independently selected from the group consisting of hydrogen,
halogen, alkyl, NO.sub.2, CN, and substituted alkyl.
[0153] In some embodiments, R.sub.1 is a polypeptide. In specific
embodiments, the polypeptide is an antibody. In certain specific
embodiments, the antibody is .alpha.PSMA. In other embodiments,
R.sub.2 is a polypeptide. In specific embodiments, the polypeptide
is an antibody. In certain specific embodiments, the antibody is
.alpha.PSMA.
[0154] In some embodiments, a compound is described comprising
Formula (XXXI-A):
##STR00052##
[0155] In certain embodiments, a pharmaceutical composition is
provided comprising any of the compounds described and a
pharmaceutically acceptable carrier, excipient, or binder.
[0156] In further or alternative embodiments are methods for
detecting the presence of a polypeptide in a patient, the method
comprising administering a polypeptide comprising at least one
heterocycle-containing non-natural amino acid and the resulting
heterocycle-containing non-natural amino acid polypeptide modulates
the immunogenicity of the polypeptide relative to the homologous
naturally-occurring amino acid polypeptide.
[0157] It is to be understood that the methods and compositions
described herein are not limited to the particular methodology,
protocols, cell lines, constructs, and reagents described herein
and as such may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
methods and compositions described herein, which will be limited
only by the appended claims.
[0158] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly indicates otherwise.
[0159] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which the inventions described herein
belong. Although any methods, devices, and materials similar or
equivalent to those described herein can be used in the practice or
testing of the inventions described herein, the preferred methods,
devices and materials are now described.
[0160] All publications and patents mentioned herein are
incorporated herein by reference in their entirety for the purpose
of describing and disclosing, for example, the constructs and
methodologies that are described in the publications, which might
be used in connection with the presently described inventions. The
publications discussed herein are provided solely for their
disclosure prior to the filing date of the present application.
Nothing herein is to be construed as an admission that the
inventors described herein are not entitled to antedate such
disclosure by virtue of prior invention or for any other
reason.
[0161] The terms "aldol-based linkage" or "mixed aldol-based
linkage" refers to the acid- or base-catalyzed condensation of one
carbonyl compound with the enolate/enol of another carbonyl
compound, which may or may not be the same, to generate a p-hydroxy
carbonyl compound--an aldol.
[0162] The term "affinity label," as used herein, refers to a label
which reversibly or irreversibly binds another molecule, either to
modify it, destroy it, or form a compound with it. By way of
example, affinity labels include enzymes and their substrates, or
antibodies and their antigens.
[0163] The terms "alkoxy," "alkylamino" and "alkylthio" (or
thioalkoxy) are used in their conventional sense, and refer to
those alkyl groups linked to molecules via an oxygen atom, an amino
group, or a sulfur atom, respectively.
[0164] The term "alkyl," by itself or as part of another molecule
means, unless otherwise stated, a straight or branched chain, or
cyclic hydrocarbon radical, or combination thereof, which may be
fully saturated, mono- or polyunsaturated and can include di- and
multivalent radicals, having the number of carbon atoms designated
(i.e. C.sub.1-C.sub.10 means one to ten carbons). Examples of
saturated hydrocarbon radicals include, but are not limited to,
groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,
t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl,
cyclopropylmethyl, homologs and isomers of, for example, n-pentyl,
n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl
group is one having one or more double bonds or triple bonds.
Examples of unsaturated alkyl groups include, but are not limited
to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),
2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,
3-butynyl, and the higher homologs and isomers. The term "alkyl,"
unless otherwise noted, is also meant to include those derivatives
of alkyl defined in more detail herein, such as "heteroalkyl",
"haloalkyl" and "homoalkyl".
[0165] The term "alkylene" by itself or as part of another molecule
means a divalent radical derived from an alkane, as exemplified, by
(--CH.sub.2-).sub.n, wherein n may be 1 to about 24. By way of
example only, such groups include, but are not limited to, groups
having 10 or fewer carbon atoms such as the structures
--CH.sub.2CH.sub.2-- and --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. A
"lower alkyl" or "lower alkylene" is a shorter chain alkyl or
alkylene group, generally having eight or fewer carbon atoms. The
term "alkylene," unless otherwise noted, is also meant to include
those groups described herein as "heteroalkylene."
[0166] The term "amino acid" refers to naturally occurring and
non-natural amino acids, as well as amino acid analogs and amino
acid mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally encoded amino acids are the 20
common amino acids (alanine, arginine, asparagine, aspartic acid,
cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine, and valine) and pyrolysine and
selenocysteine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, by way of example only, an .alpha.-carbon that is bound to a
hydrogen, a carboxyl group, an amino group, and an R group. Such
analogs may have modified R groups (by way of example, norleucine)
or may have modified peptide backbones while still retaining the
same basic chemical structure as a naturally occurring amino acid.
Non-limiting examples of amino acid analogs include homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium.
[0167] Amino acids may be referred to herein by either their name,
their commonly known three letter symbols or by the one-letter
symbols recommended by the IUPAC-IUB Biochemical Nomenclature
Commission. Additionally, nucleotides, may be referred to by their
commonly accepted single-letter codes.
[0168] An "amino terminus modification group" refers to any
molecule that can be attached to a terminal amine group. By way of
example, such terminal amine groups may be at the end of polymeric
molecules, wherein such polymeric molecules include, but are not
limited to, polypeptides, polynucleotides, and polysaccharides.
Terminus modification groups include but are not limited to,
various water soluble polymers, peptides or proteins. By way of
example only, terminus modification groups include polyethylene
glycol or serum albumin. Terminus modification groups may be used
to modify therapeutic characteristics of the polymeric molecule,
including but not limited to increasing the serum half-life of
peptides.
[0169] By "antibody" herein is meant a protein consisting of one or
more polypeptides substantially encoded by all or part of the
antibody genes. The immunoglobulin genes include, but are not
limited to, the kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3, and
IgG4), delta, epsilon and mu constant region genes, as well as the
myriad immunoglobulin variable region genes. Antibody herein is
meant to include full-length antibodies and antibody fragments, and
include antibodies that exist naturally in any organism or are
engineered (e.g. are variants).
[0170] The term "antibody" refers to intact antibody, monoclonal or
polyclonal antibodies. The term "antibody" also encompasses,
multispecific antibodies such as bispecific antibodies. Human
antibodies are usually made of two light chains and two heavy
chains each comprising variable regions and constant regions. The
light chain variable region comprises 3 CDRs, identified herein as
CDRL1, CDRL2 and CDRL3 flanked by framework regions. The heavy
chain variable region comprises 3 CDRs, identified herein as CDRH1,
CDRH2 and CDRH3 flanked by framework regions.
[0171] Anti prostate-specific membrane antigen (.alpha.PSMA)
antibodies known in the art are suitable for use in the present
invention. For example, sequences for .alpha.PSMA J591 antibody are
given in U.S. Pat. No. 7,666,425; .alpha.PSMA antibodies and
antigen-binding fragments are given in U.S. Pat. No. 8,114,965;
each incorporated herein by reference. Other U.S. patents
disclosing .alpha.PSMA antibody sequences and/or PSMA binding
agents, all of which are herein incorporated by reference, include
U.S. Pat. Nos. 7,910,693; 7,875,278; 7,850,971; 7,514,078;
7,476,513; 7,381,407; 7,201,900; 7,192,586; 7,045,605; 6,962,981;
6,387,888; and 6,150,508.
[0172] The term "antigen-binding fragment", as used herein, refers
to one or more fragments of an antibody that retain the ability to
bind to an antigen. It has been shown that the antigen-binding
function of an antibody can be performed by fragments of an intact
antibody. Examples of binding fragments encompassed within the term
"antigen-binding fragment" of an antibody include (i) a Fab
fragment, a monovalent fragment consisting of the V.sub.L, V.sub.H,
C.sub.L and C.sub.H1 domains; (ii) a F(ab').sub.2 fragment, a
bivalent fragment comprising two Fab fragments linked by a
disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the V.sub.H and C.sub.H1 domains; (iv) a Fv fragment
consisting of the V.sub.L and V.sub.H domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546), which consists of a V.sub.H domain; (vi) an isolated
complementarity determining region (CDR), e.g., V.sub.H CDR3
comprising or not additional sequence (linker, framework region(s)
etc.) and (v) a combination of two to six isolated CDRs comprising
or not additional sequence (linker, framework region(s) etc.).
Furthermore, although the two domains of the Fv fragment, V.sub.L
and V.sub.H, are coded for by separate genes, they can be joined,
using recombinant methods, by a synthetic linker that enables them
to be made as a single polypeptide chain in which the V.sub.L and
V.sub.H regions pair to form monovalent molecules (known as single
chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
Such single chain antibodies are also intended to be encompassed
within the term "antigen-binding fragment" of an antibody.
Furthermore, the antigen-binding fragments include binding-domain
immunoglobulin fusion proteins comprising (i) a binding domain
polypeptide (such as a heavy chain variable region, a light chain
variable region, or a heavy chain variable region fused to a light
chain variable region via a linker peptide) that is fused to an
immunoglobulin hinge region polypeptide, (ii) an immunoglobulin
heavy chain CH2 constant region fused to the hinge region, and
(iii) an immunoglobulin heavy chain CH3 constant region fused to
the CH2 constant region. The hinge region may be modified by
replacing one or more cysteine residues with serine residues so as
to prevent dimerization. Such binding-domain immunoglobulin fusion
proteins are further disclosed in US 2003/0118592 and US
2003/0133939. These antibody fragments are obtained using
conventional techniques known to those with skill in the art, and
the fragments are screened for utility in the same manner as are
intact antibodies.
[0173] A typical antigen binding site is comprised of the variable
regions formed by the pairing of a light chain immunoglobulin and a
heavy chain immunoglobulin. The structure of the antibody variable
regions is very consistent and exhibits very similar structures.
These variable regions are typically comprised of relatively
homologous framework regions (FR) interspaced with three
hypervariable regions termed Complementarity Determining Regions
(CDRs). The overall binding activity of the antigen binding
fragment is often dictated by the sequence of the CDRs. The FRs
often play a role in the proper positioning and alignment in three
dimensions of the CDRs for optimal antigen binding.
[0174] In fact, because CDR sequences are responsible for most
antibody-antigen interactions, it is possible to express
recombinant antibodies that shows the properties of specific
naturally occurring antibodies by constructing expression vectors
that include CDR sequences from the specific naturally occurring
antibody grafted onto framework sequences from a different antibody
with different properties (see, e.g., Riechmann, L. et al., 1998,
Nature 332:323-327; Jones, P. et al., 1986, Nature 321:522-525; and
Queen, C. et al., 1989, Proc. Natl. Acad. See. U.S.A.
86:10029-10033). Such framework sequences can be obtained from
public DNA databases that include germline antibody gene sequences.
These germline sequences will differ from mature antibody gene
sequences because they will not include completely assembled
variable genes, which are formed by V(D)J joining during B cell
maturation. Germline gene sequences will also differ from the
sequences of a high affinity secondary repertoire antibody which
contains mutations throughout the variable gene but typically
clustered in the CDRs. For example, somatic mutations are
relatively infrequent in the amino terminal portion of framework
region 1 and in the carboxy-terminal portion of framework region 4.
Furthermore, many somatic mutations do not significantly alter the
binding properties of the antibody. For this reason, it is not
necessary to obtain the entire DNA sequence of a particular
antibody in order to recreate an intact recombinant antibody having
binding properties similar to those of the original antibody.
Partial heavy and light chain sequence spanning the CDR regions is
typically sufficient for this purpose. The partial sequence is used
to determine which germline variable and joining gene segments
contributed to the recombined antibody variable genes. The germline
sequence is then used to fill in missing portions of the variable
regions. Heavy and light chain leader sequences are cleaved during
protein maturation and do not contribute to the properties of the
final antibody. To add missing sequences, cloned cDNA sequences can
be combined with synthetic oligonucleotides by ligation or PCR
amplification. Alternatively, the entire variable region can be
synthesized to create an entirely synthetic variable region clone.
This process has certain advantages such as elimination or
inclusion of particular restriction sites, or optimization of
particular codons.
[0175] Of course, the totality or portions of the framework region
of the antibody described herein may be used in conjunction with
the CDRs in order to optimize the affinity, specificity or any
other desired properties of the antibody. By "antibody" herein is
meant a protein consisting of one or more polypeptides
substantially encoded by all or part of the antibody genes. The
immunoglobulin genes include, but are not limited to, the kappa,
lambda, alpha, gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon
and mu constant region genes, as well as the myriad immunoglobulin
variable region genes. Antibody herein is meant to include
full-length antibodies and antibody fragments, and include
antibodies that exist naturally in any organism or are engineered
(e.g. are variants).
[0176] By "antibody fragment" is meant any form of an antibody
other than the full-length form. Antibody fragments herein include
antibodies that are smaller components that exist within
full-length antibodies, and antibodies that have been engineered.
Antibody fragments include but are not limited to Fv, Fe, Fab, and
(Fab')2, single chain Fv (scFv), diabodies, triabodies,
tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3,
combinations of CDR's, variable regions, framework regions,
constant regions, heavy chains, light chains, and variable regions,
and alternative scaffold non-antibody molecules, bispecific
antibodies, and the like (Maynard & Georgiou, 2000, Annu. Rev.
Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol.
9:395-402). Another functional substructure is a single chain Fv
(scFv), comprised of the variable regions of the immunoglobulin
heavy and light chain, covalently connected by a peptide linker
(S-z Hu et al., 1996, Cancer Research, 56, 3055-3061). These small
(Mr 25,000) proteins generally retain specificity and affinity for
antigen in a single polypeptide and can provide a convenient
building block for larger, antigen-specific molecules. Unless
specifically noted otherwise, statements and claims that use the
term "antibody" or "antibodies" specifically includes "antibody
fragment" and "antibody fragments."
[0177] By "antibody-drug conjugate, or "ADC", as used herein,
refers to an antibody molecule, or fragment thereof, that is
covalently bonded to one or more biologically active molecule(s).
The biologically active molecule may be conjugated to the antibody
through a linker, polymer, or other covalent bond.
[0178] The term "aromatic" or "aryl", as used herein, refers to a
closed ring structure which has at least one ring having a
conjugated pi electron system and includes both carbocyclic aryl
and heterocyclic aryl (or "heteroaryl" or "heteroaromatic") groups.
The carbocyclic or heterocyclic aromatic group may contain from 5
to 20 ring atoms. The term includes monocyclic rings linked
covalently or fused-ring polycyclic (i.e., rings which share
adjacent pairs of carbon atoms) groups. An aromatic group can be
unsubstituted or substituted. Non-limiting examples of "aromatic"
or "aryl", groups include phenyl, 1-naphthyl, 2-naphthyl,
4-biphenyl, anthracenyl, and phenanthracenyl. Substituents for each
of the above noted aryl and heteroaryl ring systems are selected
from the group of acceptable substituents described herein.
[0179] For brevity, the term "aromatic" or "aryl" when used in
combination with other terms (including but not limited to,
aryloxy, arylthioxy, aralkyl) includes both aryl and heteroaryl
rings as defined above. Thus, the term "aralkyl" or "alkaryl" is
meant to include those radicals in which an aryl group is attached
to an alkyl group (including but not limited to, benzyl, phenethyl,
pyridylmethyl and the like) including those alkyl groups in which a
carbon atom (including but not limited to, a methylene group) has
been replaced by a heteroatom, by way of example only, by an oxygen
atom. Examples of such aryl groups include, but are not limited to,
phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the
like.
[0180] The term "arylene", as used herein, refers to a divalent
aryl radical. Non-limiting examples of "arylene" include phenylene,
pyridinylene, pyrimidinylene and thiophenylene. Substituents for
arylene groups are selected from the group of acceptable
substituents described herein.
[0181] A "bifunctional polymer", also referred to as a
"bifunctional linker", refers to a polymer comprising two
functional groups that are capable of reacting specifically with
other moieties to form covalent or non-covalent linkages. Such
moieties may include, but are not limited to, the side groups on
natural or non-natural amino acids or peptides which contain such
natural or non-natural amino acids. The other moieties that may be
linked to the bifunctional linker or bifunctional polymer may be
the same or different moieties. By way of example only, a
bifunctional linker may have a functional group reactive with a
group on a first peptide, and another functional group which is
reactive with a group on a second peptide, whereby forming a
conjugate that includes the first peptide, the bifunctional linker
and the second peptide. Many procedures and linker molecules for
attachment of various compounds to peptides are known. See, e.g.,
European Patent Application No. 188,256; U.S. Pat. Nos. 4,671,958,
4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789 which are
incorporated by reference herein in their entirety. A
"multi-functional polymer" also referred to as a "multi-functional
linker", refers to a polymer comprising two or more functional
groups that are capable of reacting with other moieties. Such
moieties may include, but are not limited to, the side groups on
natural or non-natural amino acids or peptides which contain such
natural or non-natural amino acids. (including but not limited to,
amino acid side groups) to form covalent or non-covalent linkages.
A bi-functional polymer or multi-functional polymer may be any
desired length or molecular weight, and may be selected to provide
a particular desired spacing or conformation between one or more
molecules linked to a compound and molecules it binds to or the
compound.
[0182] The term "bioavailability," as used herein, refers to the
rate and extent to which a substance or its active moiety is
delivered from a pharmaceutical dosage form and becomes available
at the site of action or in the general circulation. Increases in
bioavailability refers to increasing the rate and extent a
substance or its active moiety is delivered from a pharmaceutical
dosage form and becomes available at the site of action or in the
general circulation. By way of example, an increase in
bioavailability may be indicated as an increase in concentration of
the substance or its active moiety in the blood when compared to
other substances or active moieties. A non-limiting example of a
method to evaluate increases in bioavailability is given in
examples 21-25. This method may be used for evaluating the
bioavailability of any polypeptide.
[0183] The term "biologically active molecule", "biologically
active moiety" or "biologically active agent" when used herein
means any substance which can affect any physical or biochemical
properties of a biological system, pathway, molecule, or
interaction relating to an organism, including but not limited to,
viruses, bacteria, bacteriophage, transposon, prion, insects,
fungi, plants, animals, and humans. In particular, as used herein,
biologically active molecules include but are not limited to any
substance intended for diagnosis, cure, mitigation, treatment, or
prevention of disease in humans or other animals, or to otherwise
enhance physical or mental well-being of humans or animals.
Examples of biologically active molecules include, but are not
limited to, peptides, proteins, enzymes, small molecule drugs, hard
drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or
molecules, dyes, lipids, nucleosides, radionuclides,
oligonucleotides, toxins, cells, viruses, liposomes, microparticles
and micelles. Classes of biologically active agents that are
suitable for use with the methods and compositions described herein
include, but are not limited to, drugs, prodrugs, radionuclides,
imaging agents, polymers, antibiotics, fungicides, anti-viral
agents, anti-inflammatory agents, anti-tumor agents, cardiovascular
agents, anti-anxiety agents, hormones, growth factors, steroidal
agents, microbially derived toxins, and the like.
[0184] By "modulating biological activity" is meant increasing or
decreasing the reactivity of a polypeptide, altering the
selectivity of the polypeptide, enhancing or decreasing the
substrate selectivity of the polypeptide. Analysis of modified
biological activity can be performed by comparing the biological
activity of the non-natural polypeptide to that of the natural
polypeptide.
[0185] The term "biomaterial," as used herein, refers to a
biologically-derived material, including but not limited to
material obtained from bioreactors and/or from recombinant methods
and techniques.
[0186] The term "biophysical probe," as used herein, refers to
probes which can detect or monitor structural changes in molecules.
Such molecules include, but are not limited to, proteins and the
"biophysical probe" may be used to detect or monitor interaction of
proteins with other macromolecules. Examples of biophysical probes
include, but are not limited to, spin-labels, a fluorophores, and
photoactivatible groups.
[0187] The term "biosynthetically," as used herein, refers to any
method utilizing a translation system (cellular or non-cellular),
including use of at least one of the following components: a
polynucleotide, a codon, a tRNA, and a ribosome. By way of example,
non-natural amino acids may be "biosynthetically incorporated" into
non-natural amino acid polypeptides using the methods and
techniques described herein, "In vivo generation of polypeptides
comprising non-natural amino acids", and in the non-limiting
example 20. Additionally, the methods for the selection of useful
non-natural amino acids which may be "biosynthetically
incorporated" into non-natural amino acid polypeptides are
described in the non-limiting examples 20.
[0188] The term "biotin analogue," or also referred to as "biotin
mimic", as used herein, is any molecule, other than biotin, which
bind with high affinity to avidin and/or streptavidin.
[0189] The term "carbonyl" as used herein refers to a group
containing at a moiety selecting from the group consisting of
--C(O)--, --S(O)--, --S(O)2-, and --C(S)--, including, but not
limited to, groups containing a least one ketone group, and/or at
least one aldehyde groups, and/or at least one ester group, and/or
at least one carboxylic acid group, and/or at least one thioester
group. Such carbonyl groups include ketones, aldehydes, carboxylic
acids, esters, and thioesters. In addition, such groups may be part
of linear, branched, or cyclic molecules.
[0190] The term "carboxy terminus modification group" refers to any
molecule that can be attached to a terminal carboxy group. By way
of example, such terminal carboxy groups may be at the end of
polymeric molecules, wherein such polymeric molecules include, but
are not limited to, polypeptides, polynucleotides, and
polysaccharides. Terminus modification groups include but are not
limited to, various water soluble polymers, peptides or proteins.
By way of example only, terminus modification groups include
polyethylene glycol or serum albumin. Terminus modification groups
may be used to modify therapeutic characteristics of the polymeric
molecule, including but not limited to increasing the serum
half-life of peptides.
[0191] The term "chemically cleavable group," also referred to as
"chemically labile", as used herein, refers to a group which breaks
or cleaves upon exposure to acid, base, oxidizing agents, reducing
agents, chemical inititiators, or radical initiators.
[0192] The term "chemiluminescent group," as used herein, refers to
a group which emits light as a result of a chemical reaction
without the addition of heat. By way of example only, luminol
(5-amino-2,3-dihydro-1,4-phthalazinedione) reacts with oxidants
like hydrogen peroxide (H.sub.2O.sub.2) in the presence of a base
and a metal catalyst to produce an excited state product
(3-aminophthalate, 3-APA).
[0193] The term "chromophore," as used herein, refers to a molecule
which absorbs light of visible wavelengths, UV wavelengths or IR
wavelengths.
[0194] The term "cofactor," as used herein, refers to an atom or
molecule essential for the action of a large molecule. Cofactors
include, but are not limited to, inorganic ions, coenzymes,
proteins, or some other factor necessary for the activity of
enzymes. Examples include, heme in hemoglobin, magnesium in
chlorophyll, and metal ions for proteins.
[0195] "Cofolding," as used herein, refers to refolding processes,
reactions, or methods which employ at least two molecules which
interact with each other and result in the transformation of
unfolded or improperly folded molecules to properly folded
molecules. By way of example only, "cofolding," employ at least two
polypeptides which interact with each other and result in the
transformation of unfolded or improperly folded polypeptides to
native, properly folded polypeptides. Such polypeptides may contain
natural amino acids and/or at least one non-natural amino acid.
[0196] A "comparison window," as used herein, refers a segment of
any one of contiguous positions used to compare a sequence to a
reference sequence of the same number of contiguous positions after
the two sequences are optimally aligned. Such contiguous positions
include, but are not limited to a group consisting of from about 20
to about 600 sequential units, including about 50 to about 200
sequential units, and about 100 to about 150 sequential units. By
way of example only, such sequences include polypeptides and
polypeptides containing non-natural amino acids, with the
sequential units include, but are not limited to natural and
non-natural amino acids. In addition, by way of example only, such
sequences include polynucleotides with nucleotides being the
corresponding sequential units. Methods of alignment of sequences
for comparison are well-known in the art. Optimal alignment of
sequences for comparison can be conducted, including but not
limited to, by the local homology algorithm of Smith and Waterman
(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm
of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search
for similarity method of Pearson and Lipman (1988) Proc. Nat'l.
Acad. Sci. USA 85:2444, by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Ausubel et al., Current Protocols in Molecular Biology
(1995 supplement)).
[0197] By way of example, an algorithm which may be used to
determine percent sequence identity and sequence similarity are the
BLAST and BLAST 2.0 algorithms, which are described in Altschul et
at. (1997) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990)
J. Mol. Biol. 215:403-410, respectively. Software for performing
BLAST analyses is publicly available through the National Center
for Biotechnology Information. The BLAST algorithm parameters W, T,
and X determine the sensitivity and speed of the alignment. The
BLASTN program (for nucleotide sequences) uses as defaults a
wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a
comparison of both strands. For amino acid sequences, the BLASTP
program uses as defaults a wordlength of 3, and expectation (E) of
10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff
(1992) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50,
expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
The BLAST algorithm is typically performed with the "low
complexity" filter turned off.
[0198] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, or less than about
0.01, or less than about 0.001.
[0199] The term "conservatively modified variants" applies to both
natural and non-natural amino acid and natural and non-natural
nucleic acid sequences, and combinations thereof. With respect to
particular nucleic acid sequences, "conservatively modified
variants" refers to those natural and non-natural nucleic acids
which encode identical or essentially identical natural and
non-natural amino acid sequences, or where the natural and
non-natural nucleic acid does not encode a natural and non-natural
amino acid sequence, to essentially identical sequences. By way of
example, because of the degeneracy of the genetic code, a large
number of functionally identical nucleic acids encode any given
protein. For instance, the codons GCA, GCC, GCG and GCU all encode
the amino acid alanine. Thus, at every position where an alanine is
specified by a codon, the codon can be altered to any of the
corresponding codons described without altering the encoded
polypeptide. Such nucleic acid variations are "silent variations,"
which are one species of conservatively modified variations. Thus
by way of example every natural or non-natural nucleic acid
sequence herein which encodes a natural or non-natural polypeptide
also describes every possible silent variation of the natural or
non-natural nucleic acid. One of ordinary skill in the art will
recognize that each codon in a natural or non-natural nucleic acid
(except AUG, which is ordinarily the only codon for methionine, and
TGG, which is ordinarily the only codon for tryptophan) can be
modified to yield a functionally identical molecule. Accordingly,
each silent variation of a natural and non-natural nucleic acid
which encodes a natural and non-natural polypeptide is implicit in
each described sequence.
[0200] As to amino acid sequences, individual substitutions,
deletions or additions to a nucleic acid, peptide, polypeptide, or
protein sequence which alters, adds or deletes a single natural and
non-natural amino acid or a small percentage of natural and
non-natural amino acids in the encoded sequence is a
"conservatively modified variant" where the alteration results in
the deletion of an amino acid, addition of an amino acid, or
substitution of a natural and non-natural amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar natural amino acids are well known
in the art. Such conservatively modified variants are in addition
to and do not exclude polymorphic variants, interspecies homologs,
and alleles of the methods and compositions described herein.
[0201] Conservative substitution tables providing functionally
similar amino acids are known to those of ordinary skill in the
art. The following eight groups each contain amino acids that are
conservative substitutions for one another: [0202] 1) Alanine (A),
Glycine (G); [0203] 2) Aspartic acid (D), Glutamic acid (E); [0204]
3) Asparagine (N), Glutamine (Q); [0205] 4) Arginine (R), Lysine
(K); [0206] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine
(V); [0207] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
[0208] 7) Serine (S), Threonine (T); and [0209] 8) Cysteine (C),
Methionine (M) (see, e.g., Creighton, Proteins: Structures and
Molecular Properties (W H Freeman & Co.; 2nd edition (December
1993)
[0210] The terms "cycloalkyl" and "heterocycloalkyl", by themselves
or in combination with other terms, represent, unless otherwise
stated, cyclic versions of "alkyl" and "heteroalkyl", respectively.
Thus, a cycloalkyl or heterocycloalkyl include saturated, partially
unsaturated and fully unsaturated ring linkages. Additionally, for
heterocycloalkyl, a heteroatom can occupy the position at which the
heterocycle is attached to the remainder of the molecule. The
heteroatom may include, but is not limited to, oxygen, nitrogen or
sulfur. Examples of cycloalkyl include, but are not limited to,
cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl,
cycloheptyl, and the like. Examples of heterocycloalkyl include,
but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),
1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,
3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,
tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,
2-piperazinyl, and the like. Additionally, the term encompasses
multicyclic structures, including but not limited to, bicyclic and
tricyclic ring structures. Similarly, the term
"heterocycloalkylene" by itself or as part of another molecule
means a divalent radical derived from heterocycloalkyl, and the
term "cycloalkylene" by itself or as part of another molecule means
a divalent radical derived from cycloalkyl.
[0211] The term "cyclodextrin," as used herein, refers to cyclic
carbohydrates consisting of at least six to eight glucose molecules
in a ring formation. The outer part of the ring contains water
soluble groups; at the center of the ring is a relatively nonpolar
cavity able to accommodate small molecules.
[0212] The term "cytotoxic," as used herein, refers to a compound
which harms cells.
[0213] "Denaturing agent" or "denaturant," as used herein, refers
to any compound or material which will cause a reversible unfolding
of a polymer. By way of example only, "denaturing agent" or
"denaturants," may cause a reversible unfolding of a protein. The
strength of a denaturing agent or denaturant will be determined
both by the properties and the concentration of the particular
denaturing agent or denaturant. By way of example, denaturing
agents or denaturants include, but are not limited to, chaotropes,
detergents, organic, water miscible solvents, phospholipids, or a
combination thereof. Non-limiting examples of chaotropes include,
but are not limited to, urea, guanidine, and sodium thiocyanate.
Non-limiting examples of detergents may include, but are not
limited to, strong detergents such as sodium dodecyl sulfate, or
polyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl,
mild non-ionic detergents (e.g., digitonin), mild cationic
detergents such as
N->2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic
detergents (e.g. sodium cholate or sodium deoxycholate) or
zwitterionic detergents including, but not limited to,
sulfobetaines (Zwittergent),
3-(3-chlolamidopropyl)dimethylammonio-1-propane sulfate (CHAPS),
and 3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-1-propane
sulfonate (CHAPSO). Non-limiting examples of organic, water
miscible solvents include, but are not limited to, acetonitrile,
lower alkanols (especially C2-C4 alkanols such as ethanol or
isopropanol), or lower alkandiols (C2-C4 alkandiols such as
ethylene-glycol) may be used as denaturants. Non-limiting examples
of phospholipids include, but are not limited to, naturally
occurring phospholipids such as phosphatidylethanolamine,
phosphatidyleholine, phosphatidylserine, and phosphatidylinositol
or synthetic phospholipid derivatives or variants such as
dihexanoylphosphatidylcholine or
diheptanoylphosphatidylcholine.
[0214] The term "desired functionality" as used herein refers to
any group selected from a label; a dye; a polymer; a water-soluble
polymer; a derivative of polyethylene glycol; a photocrosslinker; a
cytotoxic compound; a drug; an affinity label; a photoaffnity
label; a reactive compound; a resin; a second protein or
polypeptide or polypeptide analog; an antibody or antibody
fragment; a metal chelator; a cofactor; a fatty acid; a
carbohydrate; a polynucleotide; a DNA; a RNA; an antisense
polynucleotide; a saccharide, a water-soluble dendrimer, a
cyclodextrin, a biomaterial; a nanoparticle; a spin label; a
fluorophore; a metal-containing moiety; a radioactive moiety; a
novel functional group; a group that covalently or noncovalently
interacts with other molecules; a photocaged moiety; an actinic
radiation excitable moiety; a ligand; a photoisomerizable moiety;
biotin; a biotin analogue; a moiety incorporating a heavy atom; a
chemically cleavable group; a photocleavable group; an elongated
side chain; a carbon-linked sugar; a redox-active agent; an amino
thioacid; a toxic moiety; an isotopically labeled moiety; a
biophysical probe; a phosphorescent group; a chemiluminescent
group; an electron dense group; a magnetic group; an intercalating
group; a chromophore; an energy transfer agent; a biologically
active agent (in which case, the biologically active agent can
include an agent with therapeutic activity and the non-natural
amino acid polypeptide or modified non-natural amino acid can serve
either as a co-therapeutic agent with the attached therapeutic
agent or as a means for delivery the therapeutic agent to a desired
site within an organism); a detectable label; a small molecule; an
inhibitory ribonucleic acid; a radionucleotide; a neutron-capture
agent; a derivative of biotin; quantum dot(s); a nanotransmitter; a
radiotransmitter; an abzyme, an activated complex activator, a
virus, an adjuvant, an aglycan, an allergan, an angiostatin, an
antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a
shuttle vector, a macromolecule, a mimotope, a receptor, a reverse
micelle, and any combination thereof.
[0215] The term "diamine," as used herein, refers to
groups/molecules comprising at least two amine functional groups,
including, but not limited to, a hydrazine group, an amidine group,
an imine group, a 1,1-diamine group, a 1,2-diamine group, a
1,3-diamine group, and a 1,4-diamine group. In addition, such
groups may be part of linear, branched, or cyclic molecules.
[0216] The term "detectable label," as used herein, refers to a
label which may be observable using analytical techniques
including, but not limited to, fluorescence, chemiluminescence,
electron-spin resonance, ultraviolet/visible absorbance
spectroscopy, mass spectrometry, nuclear magnetic resonance,
magnetic resonance, and electrochemical methods.
[0217] The term "dicarbonyl" as used herein refers to a group
containing at least two moieties selected from the group consisting
of --C(O)--, --S(O)--, --S(O).sub.2--, and --C(S)--, including, but
not limited to, 1,2-dicarbonyl groups, a 1,3-dicarbonyl groups, and
1,4-dicarbonyl groups, and groups containing a least one ketone
group, and/or at least one aldehyde groups, and/or at least one
ester group, and/or at least one carboxylic acid group, and/or at
least one thioester group. Such dicarbonyl groups include
diketones, ketoaldehydes, ketoacids, ketoesters, and
ketothioesters. In addition, such groups may be part of linear,
branched, or cyclic molecules. The two moieties in the dicarbonyl
group may be the same or different, and may include substituents
that would produce, by way of example only, an ester, a ketone, an
aldehyde, a thioester, or an amide, at either of the two
moieties.
[0218] The term "drug," as used herein, refers to any substance
used in the prevention, diagnosis, alleviation, treatment, or cure
of a disease or condition.
[0219] The term "dye," as used herein, refers to a soluble,
coloring substance which contains a chromophore.
[0220] The term "effective amount," as used herein, refers to a
sufficient amount of an agent or a compound being administered
which will relieve to some extent one or more of the symptoms of
the disease or condition being treated. The result can be reduction
and/or alleviation of the signs, symptoms, or causes of a disease,
or any other desired alteration of a biological system. By way of
example, an agent or a compound being administered includes, but is
not limited to, a natural amino acid polypeptide, non-natural amino
acid polypeptide, modified natural amino acid polypeptide, or
modified non-amino acid polypeptide. Compositions containing such
natural amino acid polypeptides, non-natural amino acid
polypeptides, modified natural amino acid polypeptides, or modified
non-natural amino acid polypeptides can be administered for
prophylactic, enhancing, and/or therapeutic treatments. An
appropriate "effective" amount in any individual case may be
determined using techniques, such as a dose escalation study.
[0221] The term "electron dense group," as used herein, refers to a
group which scatters electrons when irradiated with an electron
beam. Such groups include, but are not limited to, ammonium
molybdate, bismuth subnitrate cadmium iodide, 99%, carbohydrazide,
ferric chloride hexahydrate, hexamethylene tetramine, 98.5%, indium
trichloride anhydrous, lanthanum nitrate, lead acetate trihydrate,
lead citrate trihydrate, lead nitrate, periodic acid,
phosphomolybdic acid, phosphotungstic acid, potassium ferricyanide,
potassium ferrocyanide, ruthenium red, silver nitrate, silver
proteinate (Ag Assay: 8.0-8.5%) "Strong", silver tetraphenylporphin
(S-TPPS), sodium chloroaurate, sodium tungstate, thallium nitrate,
thiosemicarbazide (TSC), uranyl acetate, uranyl nitrate, and
vanadyl sulfate.
[0222] The term "energy transfer agent," as used herein, refers to
a molecule which can either donate or accept energy from another
molecule. By way of example only, fluorescence resonance energy
transfer (FRET) is a dipole-dipole coupling process by which the
excited-state energy of a fluorescence donor molecule is
non-radiatively transferred to an unexcited acceptor molecule which
then fluorescently emits the donated energy at a longer
wavelength.
[0223] The terms "enhance" or "enhancing" means to increase or
prolong either in potency or duration a desired effect. By way of
example, "enhancing" the effect of therapeutic agents refers to the
ability to increase or prolong, either in potency or duration, the
effect of therapeutic agents on during treatment of a disease,
disorder or condition. An "enhancing-effective amount," as used
herein, refers to an amount adequate to enhance the effect of a
therapeutic agent in the treatment of a disease, disorder or
condition. When used in a patient, amounts effective for this use
will depend on the severity and course of the disease, disorder or
condition, previous therapy, the patient's health status and
response to the drugs, and the judgment of the treating
physician.
[0224] As used herein, the term "eukaryote" refers to organisms
belonging to the phylogenetic domain Eucarya, including but not
limited to animals (including but not limited to, mammals, insects,
reptiles, birds, etc.), ciliates, plants (including but not limited
to, monocots, dicots, and algae), fungi, yeasts, flagellates,
microsporidia, and protists.
[0225] The term "fatty acid," as used herein, refers to carboxylic
acids with about C6 or longer hydrocarbon side chain.
[0226] The term "fluorophore," as used herein, refers to a molecule
which upon excitation emits photons and is thereby fluorescent.
[0227] The terms "functional group", "active moiety", "activating
group", "leaving group", "reactive site", "chemically reactive
group" and "chemically reactive moiety," as used herein, refer to
portions or units of a molecule at which chemical reactions occur.
The terms are somewhat synonymous in the chemical arts and are used
herein to indicate the portions of molecules that perform some
function or activity and are reactive with other molecules.
[0228] The term "halogen" includes fluorine, chlorine, iodine, and
bromine.
[0229] The term "haloacyl," as used herein, refers to acyl groups
which contain halogen moieties, including, but not limited to,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like.
[0230] The term "haloalkyl," as used herein, refers to alkyl groups
which contain halogen moieties, including, but not limited to,
--CF.sub.3 and --CH.sub.2CF.sub.3 and the like.
[0231] The term "heteroalkyl," as used herein, refers to straight
or branched chain, or cyclic hydrocarbon radicals, or combinations
thereof, consisting of an alkyl group and at least one heteroatom
selected from the group consisting of O, N, Si and S, and wherein
the nitrogen and sulfur atoms may optionally be oxidized and the
nitrogen heteroatom may optionally be quaternized. The
heteroatom(s) O, N and S and Si may be placed at any interior
position of the heteroalkyl group or at the position at which the
alkyl group is attached to the remainder of the molecule. Examples
include, but are not limited to, --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3, and
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3. In addition, up to two
heteroatoms may be consecutive, such as, by way of example,
--CH.sub.2--NH--OCH.sub.3 and
--CH.sub.2--O--Si(CH.sub.3).sub.3.
[0232] The terms "heterocyclic-based linkage" or "heterocycle
linkage" refers to a moiety formed from the reaction of a
dicarbonyl group with a diamine group. The resulting reaction
product is a heterocycle, including a heteroaryl group or a
heterocycloalkyl group. The resulting heterocycle group serves as a
chemical link between a non-natural amino acid or non-natural amino
acid polypeptide and another functional group. In one embodiment,
the heterocycle linkage includes a nitrogen-containing heterocycle
linkage, including by way of example only a pyrazole linkage, a
pyrrole linkage, an indole linkage, a benzodiazepine linkage, and a
pyrazalone linkage.
[0233] Similarly, the term "heteroalkylene" refers to a divalent
radical derived from heteroalkyl, as exemplified, but not limited
by, --CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, the same or different heteroatoms can also
occupy either or both of the chain termini (including but not
limited to, alkyleneoxy, alkylenedioxy, alkyleneamino,
alkylenediamino, aminooxyalkylene, and the like). Still further,
for alkylene and heteroalkylene linking groups, no orientation of
the linking group is implied by the direction in which the formula
of the linking group is written. By way of example, the formula
--C(O).sub.2R'-- represents both --C(O).sub.2R'-- and
--R'C(O).sub.2--.
[0234] The term "heteroaryl" or "heteroaromatic," as used herein,
refers to aryl groups which contain at least one heteroatom
selected from N, O, and S; wherein the nitrogen and sulfur atoms
may be optionally oxidized, and the nitrogen atom(s) may be
optionally quaternized. Heteroaryl groups may be substituted or
unsubstituted. A heteroaryl group may be attached to the remainder
of the molecule through a heteroatom. Non-limiting examples of
heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,
3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,
4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl,
4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,
2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl,
4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
[0235] The term "homoalkyl," as used herein refers to alkyl groups
which are hydrocarbon groups.
[0236] The term "identical," as used herein, refers to two or more
sequences or subsequences which are the same. In addition, the term
"substantially identical," as used herein, refers to two or more
sequences which have a percentage of sequential units which are the
same when compared and aligned for maximum correspondence over a
comparison window, or designated region as measured using
comparison algorithms or by manual alignment and visual inspection.
By way of example only, two or more sequences may be "substantially
identical" if the sequential units are about 60% identical, about
65% identical, about 70% identical, about 75% identical, about 80%
identical, about 85% identical, about 90% identical, or about 95%
identical over a specified region. Such percentages to describe the
"percent identity" of two or more sequences. The identity of a
sequence can exist over a region that is at least about 75-100
sequential units in length, over a region that is about 50
sequential units in length, or, where not specified, across the
entire sequence. This definition also refers to the complement of a
test sequence. By way of example only, two or more polypeptide
sequences are identical when the amino acid residues are the same,
while two or more polypeptide sequences are "substantially
identical" if the amino acid residues are about 60% identical,
about 65% identical, about 70% identical, about 75% identical,
about 80% identical, about 85% identical, about 90% identical, or
about 95% identical over a specified region. The identity can exist
over a region that is at least about 75 to about 100 amino acids in
length, over a region that is about 50 amino acids in length, or,
where not specified, across the entire sequence of a polypeptide
sequence. In addition, by way of example only, two or more
polynucleotide sequences are identical when the nucleic acid
residues are the same, while two or more polynucleotide sequences
are "substantially identical" if the nucleic acid residues are
about 60% identical, about 65% identical, about 70% identical,
about 75% identical, about 80% identical, about 85% identical,
about 90% identical, or about 95% identical over a specified
region. The identity can exist over a region that is at least about
75 to about 100 nucleic acids in length, over a region that is
about 50 nucleic acids in length, or, where not specified, across
the entire sequence of a polynucleotide sequence.
[0237] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0238] The term "immunogenicity," as used herein, refers to an
antibody response to administration of a therapeutic drug. The
immunogenicity toward therapeutic non-natural amino acid
polypeptides can be obtained using quantitative and qualitative
assays for detection of anti-non-natural amino acid polypeptides
antibodies in biological fluids. Such assays include, but are not
limited to, Radioimmunoassay (RIA), Enzyme-linked immunosorbent
assay (ELISA), luminescent immunoassay (LIA), and fluorescent
immunoassay (FIA). Analysis of immunogenicity toward therapeutic
non-natural amino acid polypeptides involves comparing the antibody
response upon administration of therapeutic non-natural amino acid
polypeptides to the antibody response upon administration of
therapeutic natural amino acid polypeptides.
[0239] The term "intercalating agent," also referred to as
"intercalating group," as used herein, refers to a chemical that
can insert into the intramolecular space of a molecule or the
intermolecular space between molecules. By way of example only an
intercalating agent or group may be a molecule which inserts into
the stacked bases of the DNA double helix.
[0240] The term "isolated," as used herein, refers to separating
and removing a component of interest from components not of
interest. Isolated substances can be in either a dry or semi-dry
state, or in solution, including but not limited to an aqueous
solution. The isolated component can be in a homogeneous state or
the isolated component can be a part of a pharmaceutical
composition that comprises additional pharmaceutically acceptable
carriers and/or excipients. Purity and homogeneity may be
determined using analytical chemistry techniques including, but not
limited to, polyacrylamide gel electrophoresis or high performance
liquid chromatography. In addition, when a component of interest is
isolated and is the predominant species present in a preparation,
the component is described herein as substantially purified. The
term "purified," as used herein, may refer to a component of
interest which is at least 85% pure, at least 90% pure, at least
95% pure, at least 99% or greater pure. By way of example only,
nucleic acids or proteins are "isolated" when such nucleic acids or
proteins are free of at least some of the cellular components with
which it is associated in the natural state, or that the nucleic
acid or protein has been concentrated to a level greater than the
concentration of its in vivo or in vitro production. Also, by way
of example, a gene is isolated when separated from open reading
frames which flank the gene and encode a protein other than the
gene of interest.
[0241] The term "label," as used herein, refers to a substance
which is incorporated into a compound and is readily detected,
whereby its physical distribution may be detected and/or
monitored.
[0242] The term "linkage," as used herein to refer to bonds or
chemical moiety formed from a chemical reaction between the
functional group of a linker and another molecule. Such bonds may
include, but are not limited to, covalent linkages and non-covalent
bonds, while such chemical moieties may include, but are not
limited to, esters, carbonates, imines phosphate esters,
hydrazones, acetals, orthoesters, peptide linkages, and
oligonucleotide linkages. Hydrolytically stable linkages means that
the linkages are substantially stable in water and do not react
with water at useful pH values, including but not limited to, under
physiological conditions for an extended period of time, perhaps
even indefinitely. Hydrolytically unstable or degradable linkages
means that the linkages are degradable in water or in aqueous
solutions, including for example, blood. Enzymatically unstable or
degradable linkages means that the linkage can be degraded by one
or more enzymes. By way of example only, PEG and related polymers
may include degradable linkages in the polymer backbone or in the
linker group between the polymer backbone and one or more of the
terminal functional groups of the polymer molecule. Such degradable
linkages include, but are not limited to, ester linkages formed by
the reaction of PEG carboxylic acids or activated PEG carboxylic
acids with alcohol groups on a biologically active agent, wherein
such ester groups generally hydrolyze under physiological
conditions to release the biologically active agent. Other
hydrolytically degradable linkages include but are not limited to
carbonate linkages; imine linkages resulted from reaction of an
amine and an aldehyde; phosphate ester linkages formed by reacting
an alcohol with a phosphate group; hydrazone linkages which are
reaction product of a hydrazide and an aldehyde; acetal linkages
that are the reaction product of an aldehyde and an alcohol;
orthoester linkages that are the reaction product of a formate and
an alcohol; peptide linkages formed by an amine group, including
but not limited to, at an end of a polymer such as PEG, and a
carboxyl group of a peptide; and oligonucleotide linkages formed by
a phosphoramidite group, including but not limited to, at the end
of a polymer, and a 5' hydroxyl group of an oligonucleotide.
[0243] The terms "medium" or "media," as used herein, refer to any
culture medium used to grow and harvest cells and/or products
expressed and/or secreted by such cells. Such "medium" or "media"
include, but are not limited to, solution, solid, semi-solid, or
rigid supports that may support or contain any host cell,
including, by way of example, bacterial host cells, yeast host
cells, insect host cells, plant host cells, eukaryotic host cells,
mammalian host cells, CHO cells, prokaryotic host cells, E. coli,
or Pseudomonas host cells, and cell contents. Such "medium" or
"media" includes, but is not limited to, medium or media in which
the host cell has been grown into which a polypeptide has been
secreted, including medium either before or after a proliferation
step. Such "medium" or "media" also includes, but is not limited
to, buffers or reagents that contain host cell lysates, by way of
example a polypeptide produced intracellularly and the host cells
are lysed or disrupted to release the polypeptide.
[0244] The term "metabolite," as used herein, refers to a
derivative of a compound, by way of example natural amino acid
polypeptide, a non-natural amino acid polypeptide, a modified
natural amino acid polypeptide, or a modified non-natural amino
acid polypeptide, that is formed when the compound, by way of
example natural amino acid polypeptide, non-natural amino acid
polypeptide, modified natural amino acid polypeptide, or modified
non-natural amino acid polypeptide, is metabolized. The term
"pharmaceutically active metabolite" or "active metabolite" refers
to a biologically active derivative of a compound, by way of
example natural amino acid polypeptide, a non-natural amino acid
polypeptide, a modified natural amino acid polypeptide, or a
modified non-natural amino acid polypeptide, that is formed when
such a compound, by way of example a natural amino acid
polypeptide, non-natural amino acid polypeptide, modified natural
amino acid polypeptide, or modified non-natural amino acid
polypeptide, is metabolized.
[0245] The term "metabolized," as used herein, refers to the sum of
the processes by which a particular substance is changed by an
organism. Such processes include, but are not limited to,
hydrolysis reactions and reactions catalyzed by enzymes. Further
information on metabolism may be obtained from The Pharmacological
Basis of Therapeutics, 9th Edition, McGraw-Hill (1996). By way of
example only, metabolites of natural amino acid polypeptides,
non-natural amino acid polypeptides, modified natural amino acid
polypeptides, or modified non-natural amino acid polypeptides may
be identified either by administration of the natural amino acid
polypeptides, non-natural amino acid polypeptides, modified natural
amino acid polypeptides, or modified non-natural amino acid
polypeptides to a host and analysis of tissue samples from the
host, or by incubation of natural amino acid polypeptides,
non-natural amino acid polypeptides, modified natural amino acid
polypeptides, or modified non-natural amino acid polypeptides with
hepatic cells in vitro and analysis of the resulting compounds.
[0246] The term "metal chelator," as used herein, refers to a
molecule which forms a metal complex with metal ions. By way of
example, such molecules may form two or more coordination bonds
with a central metal ion and may form ring structures.
[0247] The term "metal-containing moiety," as used herein, refers
to a group which contains a metal ion, atom or particle. Such
moieties include, but are not limited to, cisplatin, chelated
metals ions (such as nickel, iron, and platinum), and metal
nanoparticles (such as nickel, iron, and platinum).
[0248] The term "moiety incorporating a heavy atom," as used
herein, refers to a group which incorporates an ion of atom which
is usually heavier than carbon. Such ions or atoms include, but are
not limited to, silicon, tungsten, gold, lead, and uranium.
[0249] The term "modified," as used herein refers to the presence
of a change to a natural amino acid, a non-natural amino acid, a
natural amino acid polypeptide or a non-natural amino acid
polypeptide. Such changes, or modifications, may be obtained by
post synthesis modifications of natural amino acids, non-natural
amino acids, natural amino acid polypeptides or non-natural amino
acid polypeptides, or by co-translational, or by post-translational
modification of natural amino acids, non-natural amino acids,
natural amino acid polypeptides or non-natural amino acid
polypeptides. The form "modified or unmodified" means that the
natural amino acid, non-natural amino acid, natural amino acid
polypeptide or non-natural amino acid polypeptide being discussed
are optionally modified, that is, he natural amino acid,
non-natural amino acid, natural amino acid polypeptide or
non-natural amino acid polypeptide under discussion can be modified
or unmodified.
[0250] As used herein, the term "modulated serum half-life" refers
to positive or negative changes in the circulating half-life of a
modified biologically active molecule relative to its non-modified
form. By way of example, the modified biologically active molecules
include, but are not limited to, natural amino acid, non-natural
amino acid, natural amino acid polypeptide or non-natural amino
acid polypeptide. By way of example, serum half-life is measured by
taking blood samples at various time points after administration of
the biologically active molecule or modified biologically active
molecule, and determining the concentration of that molecule in
each sample. Correlation of the serum concentration with time
allows calculation of the serum half-life. By way of example,
modulated serum half-life may be an increased in serum half-life,
which may enable an improved dosing regimens or avoid toxic
effects. Such increases in serum may be at least about two fold, at
least about three-fold, at least about five-fold, or at least about
ten-fold. Methods for evaluating serum half-life are known in the
art and may be used for evaluating the serum half-life of
antibodies and antibody drug conjugates of the present
invention.
[0251] The term "modulated therapeutic half-life," as used herein,
refers to positive or negative change in the half-life of the
therapeutically effective amount of a modified biologically active
molecule, relative to its non-modified form. By way of example, the
modified biologically active molecules include, but are not limited
to, natural amino acid, non-natural amino acid, natural amino acid
polypeptide or non-natural amino acid polypeptide. By way of
example, therapeutic half-life is measured by measuring
pharmacokinetic and/or pharmacodynamic properties of the molecule
at various time points after administration. Increased therapeutic
half-life may enable a particular beneficial dosing regimen, a
particular beneficial total dose, or avoids an undesired effect. By
way of example, the increased therapeutic half-life may result from
increased potency, increased or decreased binding of the modified
molecule to its target, an increase or decrease in another
parameter or mechanism of action of the non-modified molecule, or
an increased or decreased breakdown of the molecules by enzymes
such as, by way of example only, proteases. Methods for evaluating
therapeutic half-life are known in the art and may be used for
evaluating the therapeutic half-life of antibodies and antibody
drug conjugates of the present invention.
[0252] The term "nanoparticle," as used herein, refers to a
particle which has a particle size between about 500 nm to about 1
nm.
[0253] The term "near-stoichiometric," as used herein, refers to
the ratio of the moles of compounds participating in a chemical
reaction being about 0.75 to about 1.5.
[0254] As used herein, the term "non-eukaryote" refers to
non-eukaryotic organisms. By way of example, a non-eukaryotic
organism may belong to the Eubacteria, (which includes but is not
limited to, Escherichia coli, Thermus thermophilus, or Bacillus
stearothermophilus, Pseudomonas fluorescens, Pseudomonas
aeruginosa, Pseudomonas putida), phylogenetic domain, or the
Archaea, which includes, but is not limited to, Methanococcus
jannaschii, Methanobacterium thermoautotrophicum, Archaeoglobus
fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum
pernix, or Halobacterium such as Haloferax volcanii and
Halobacterium species NRC-1, or phylogenetic domain.
[0255] A "non-natural amino acid" refers to an amino acid that is
not one of the 20 common amino acids or pyrolysine or
selenocysteine. Other terms that may be used synonymously with the
term "non-natural amino acid" is "non-naturally encoded amino
acid," "unnatural amino acid," "non-naturally-occurring amino
acid," and variously hyphenated and non-hyphenated versions
thereof. The term "non-natural amino acid" includes, but is not
limited to, amino acids which occur naturally by modification of a
naturally encoded amino acid (including but not limited to, the 20
common amino acids or pyrrolysine and selenocysteine) but are not
themselves incorporated into a growing polypeptide chain by the
translation complex. Examples of naturally-occurring amino acids
that are not naturally-encoded include, but are not limited to,
N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine,
and O-phosphotyrosine. Additionally, the term "non-natural amino
acid" includes, but is not limited to, amino acids which do not
occur naturally and may be obtained synthetically or may be
obtained by modification of non-natural amino acids.
[0256] The term "nucleic acid," as used herein, refers to
deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. By way of example only, such nucleic acids
and nucleic acid polymers include, but are not limited to, (i)
analogues of natural nucleotides which have similar binding
properties as a reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides; (ii)
oligonucleotide analogs including, but are not limited to, PNA
(peptidonucleic acid), analogs of DNA used in antisense technology
(phosphorothioates, phosphoroamidates, and the like); (iii)
conservatively modified variants thereof (including but not limited
to, degenerate codon substitutions) and complementary sequences and
sequence explicitly indicated. By way of example, degenerate codon
substitutions may be achieved by generating sequences in which the
third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol.
Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes
8:91-98 (1994)).
[0257] The term "oxidizing agent," as used herein, refers to a
compound or material which is capable of removing an electron from
a compound being oxidized. By way of example oxidizing agents
include, but are not limited to, oxidized glutathione, cystine,
cystamine, oxidized dithiothreitol, oxidized erythreitol, and
oxygen. A wide variety of oxidizing agents are suitable for use in
the methods and compositions described herein.
[0258] The term "pharmaceutically acceptable", as used herein,
refers to a material, including but not limited, to a salt, carrier
or diluent, which does not abrogate the biological activity or
properties of the compound, and is relatively nontoxic, i.e., the
material may be administered to an individual without causing
undesirable biological effects or interacting in a deleterious
manner with any of the components of the composition in which it is
contained.
[0259] The term "photoaffinity label," as used herein, refers to a
label with a group, which, upon exposure to light, forms a linkage
with a molecule for which the label has an affinity. By way of
example only, such a linkage may be covalent or non-covalent.
[0260] The term "photocaged moiety," as used herein, refers to a
group which, upon illumination at certain wavelengths, covalently
or non-covalently binds other ions or molecules.
[0261] The term "photocleavable group," as used herein, refers to a
group which breaks upon exposure to light.
[0262] The term "photocrosslinker," as used herein, refers to a
compound comprising two or more functional groups which, upon
exposure to light, are reactive and form a covalent or non-covalent
linkage with two or more monomeric or polymeric molecules.
[0263] The term "photoisomerizable moiety," as used herein, refers
to a group wherein upon illumination with light changes from one
isomeric form to another.
[0264] The term "polyalkylene glycol," as used herein, refers to
linear or branched polymeric polyether polyols. Such polyalkylene
glycols, including, but are not limited to, polyethylene glycol,
polypropylene glycol, polybutylene glycol, and derivatives thereof.
Other exemplary embodiments are listed, for example, in commercial
supplier catalogs, such as Shearwater Corporation's catalog
"Polyethylene Glycol and Derivatives for Biomedical Applications"
(2001). By way of example only, such polymeric polyether polyols
have average molecular weights between about 0.1 kDa to about 100
kDa. By way of example, such polymeric polyether polyols include,
but are not limited to, between about 100 Da and about 100,000 Da
or more. The molecular weight of the polymer may be between about
100 Da and about 100,000 Da, including but not limited to, about
100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da,
about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da,
about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da,
about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da,
about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da,
about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da,
about 4,000 Da, about 3,000 Da, about 2,000 Da, about 1,000 Da,
about 900 Da, about 800 Da, about 700 Da, about 600 Da, about 500
Da, 400 Da, about 300 Da, about 200 Da, and about 100 Da. In some
embodiments molecular weight of the polymer is between about 100 Da
and about 50,000 Da. In some embodiments, the molecular weight of
the polymer is between about 100 Da and about 40,000 Da. In some
embodiments, the molecular weight of the polymer is between about
1,000 Da and about 40,000 Da. In some embodiments, the molecular
weight of the polymer is between about 2,000 to about 50,000 Da. In
some embodiments, the molecular weight of the polymer is between
about 5,000 Da and about 40,000 Da. In some embodiments, the
molecular weight of the polymer is between about 10,000 Da and
about 40,000 Da. In some embodiments, the poly(ethylene glycol)
molecule is a branched polymer. The molecular weight of the
branched chain PEG may be between about 1,000 Da and about 100,000
Da, including but not limited to, about 100,000 Da, about 95,000
Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000
Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000
Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000
Da, about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000
Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000
Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da,
about 2,000 Da, and about 1,000 Da. In some embodiments, the
molecular weight of the branched chain PEG is between about 1,000
Da and about 50,000 Da. In some embodiments, the molecular weight
of the branched chain PEG is between about 1,000 Da and about
40,000 Da. In some embodiments, the molecular weight of the
branched chain PEG is between about 5,000 Da and about 40,000 Da.
In some embodiments, the molecular weight of the branched chain PEG
is between about 5,000 Da and about 20,000 Da. In other
embodiments, the molecular weight of the branched chain PEG is
between about 2,000 to about 50,000 Da.
[0265] The term "polymer," as used herein, refers to a molecule
composed of repeated subunits. Such molecules include, but are not
limited to, polypeptides, polynucleotides, or polysaccharides or
polyalkylene glycols.
[0266] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. That is, a description directed to a polypeptide applies
equally to a description of a peptide and a description of a
protein, and vice versa. The terms apply to naturally occurring
amino acid polymers as well as amino acid polymers in which one or
more amino acid residues is a non-natural amino acid. Additionally,
such "polypeptides," "peptides" and "proteins" include amino acid
chains of any length, including full length proteins, wherein the
amino acid residues are linked by covalent peptide bonds.
[0267] The term "post-translationally modified" refers to any
modification of a natural or non-natural amino acid which occurs
after such an amino acid has been translationally incorporated into
a polypeptide chain. Such modifications include, but are not
limited to, co-translational in vivo modifications,
co-translational in vitro modifications (such as in a cell-free
translation system), post-translational in vivo modifications, and
post-translational in vitro modifications.
[0268] The terms "prodrug" or "pharmaceutically acceptable
prodrug," as used herein, refers to an agent that is converted into
the parent drug in vivo or in vitro, wherein which does not
abrogate the biological activity or properties of the drug, and is
relatively nontoxic, i.e., the material may be administered to an
individual without causing undesirable biological effects or
interacting in a deleterious manner with any of the components of
the composition in which it is contained. Prodrugs are generally
drug precursors that, following administration to a subject and
subsequent absorption, are converted to an active, or a more active
species via some process, such as conversion by a metabolic
pathway. Some prodrugs have a chemical group present on the prodrug
that renders it less active and/or confers solubility or some other
property to the drug. Once the chemical group has been cleaved
and/or modified from the prodrug the active drug is generated.
Prodrugs are converted into active drug within the body through
enzymatic or non-enzymatic reactions. Prodrugs may provide improved
physiochemical properties such as better solubility, enhanced
delivery characteristics, such as specifically targeting a
particular cell, tissue, organ or ligand, and improved therapeutic
value of the drug. The benefits of such prodrugs include, but are
not limited to, (i) ease of administration compared with the parent
drug; (ii) the prodrug may be bioavailable by oral administration
whereas the parent is not; and (iii) the prodrug may also have
improved solubility in pharmaceutical compositions compared with
the parent drug. A pro-drug includes a pharmacologically inactive,
or reduced-activity, derivative of an active drug. Prodrugs may be
designed to modulate the amount of a drug or biologically active
molecule that reaches a desired site of action through the
manipulation of the properties of a drug, such as physiochemical,
biopharmaceutical, or pharmacokinetic properties. An example,
without limitation, of a prodrug would be a non-natural amino acid
polypeptide which is administered as an ester (the "prodrug") to
facilitate transmittal across a cell membrane where water
solubility is detrimental to mobility but which then is
metabolically hydrolyzed to the carboxylic acid, the active entity,
once inside the cell where water solubility is beneficial. Prodrugs
may be designed as reversible drug derivatives, for use as
modifiers to enhance drug transport to site-specific tissues.
[0269] The term "prophylactically effective amount," as used
herein, refers that amount of a composition containing at least one
non-natural amino acid polypeptide or at least one modified
non-natural amino acid polypeptide prophylactically applied to a
patient which will relieve to some extent one or more of the
symptoms of a disease, condition or disorder being treated. In such
prophylactic applications, such amounts may depend on the patient's
state of health, weight, and the like. It is considered well within
the skill of the art for one to determine such prophylactically
effective amounts by routine experimentation, including, but not
limited to, a dose escalation clinical trial.
[0270] The term "protected," as used herein, refers to the presence
of a "protecting group" or moiety that prevents reaction of the
chemically reactive functional group under certain reaction
conditions. The protecting group will vary depending on the type of
chemically reactive group being protected. By way of example only,
(i) if the chemically reactive group is an amine or a hydrazide,
the protecting group may be selected from tert-butyloxycarbonyl
(t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc); (ii) if the
chemically reactive group is a thiol, the protecting group may be
orthopyridyldisulfide; and (iii) if the chemically reactive group
is a carboxylic acid, such as butanoic or propionic acid, or a
hydroxyl group, the protecting group may be benzyl or an alkyl
group such as methyl, ethyl, or tert-butyl.
[0271] By way of example only, blocking/protecting groups may be
selected from:
##STR00053##
[0272] Additionally, protecting groups include, but are not limited
to, including photolabile groups such as Nvoc and MeNvoc and other
protecting groups known in the art. Other protecting groups are
described in Greene and Wuts, Protective Groups in Organic
Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y., 1999,
which is incorporated herein by reference in its entirety.
[0273] The term "radioactive moiety," as used herein, refers to a
group whose nuclei spontaneously give off nuclear radiation, such
as alpha, beta, or gamma particles; wherein, alpha particles are
helium nuclei, beta particles are electrons, and gamma particles
are high energy photons.
[0274] The term "reactive compound," as used herein, refers to a
compound which under appropriate conditions is reactive toward
another atom, molecule or compound.
[0275] The term "recombinant host cell," also referred to as "host
cell," refers to a cell which includes an exogenous polynucleotide,
wherein the methods used to insert the exogenous polynucleotide
into a cell include, but are not limited to, direct uptake,
transduction, f-mating, or other methods known in the art to create
recombinant host cells. By way of example only, such exogenous
polynucleotide may be a nonintegrated vector, including but not
limited to a plasmid, or may be integrated into the host
genome.
[0276] The term "redox-active agent," as used herein, refers to a
molecule which oxidizes or reduces another molecule, whereby the
redox active agent becomes reduced or oxidized. Examples of redox
active agent include, but are not limited to, ferrocene, quinones,
Ru.sup.2+/3+ complexes, Co.sup.2+/3+ complexes, and Os.sup.2+/3+
complexes.
[0277] The term "reducing agent," as used herein, refers to a
compound or material which is capable of adding an electron to a
compound being reduced. By way of example reducing agents include,
but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol,
dithioerythritol, cysteine, cysteamine (2-aminoethanethiol), and
reduced glutathione. Such reducing agents may be used, by way of
example only, to maintain sulfhydryl groups in the reduced state
and to reduce intra- or intermolecular disulfide bonds.
[0278] "Refolding," as used herein describes any process, reaction
or method which transforms an improperly folded or unfolded state
to a native or properly folded conformation. By way of example
only, refolding transforms disulfide bond containing polypeptides
from an improperly folded or unfolded state to a native or properly
folded conformation with respect to disulfide bonds. Such disulfide
bond containing polypeptides may be natural amino acid polypeptides
or non-natural amino acid polypeptides.
[0279] The term "resin," as used herein, refers to high molecular
weight, insoluble polymer beads. By way of example only, such beads
may be used as supports for solid phase peptide synthesis, or sites
for attachment of molecules prior to purification.
[0280] The term "saccharide," as used herein, refers to a series of
carbohydrates including but not limited to sugars, monosaccharides,
oligosaccharides, and polysaccharides.
[0281] The term "safety" or "safety profile," as used herein,
refers to side effects that might be related to administration of a
drug relative to the number of times the drug has been
administered. By way of example, a drug which has been administered
many times and produced only mild or no side effects is said to
have an excellent safety profile. A non-limiting example of a
method to evaluate the safety profile is given in example 26. This
method may be used for evaluating the safety profile of any
polypeptide.
[0282] The phrase "selectively hybridizes to" or "specifically
hybridizes to," as used herein, refers to the binding, duplexing,
or hybridizing of a molecule to a particular nucleotide sequence
under stringent hybridization conditions when that sequence is
present in a complex mixture including but not limited to, total
cellular or library DNA or RNA.
[0283] The term "spin label," as used herein, refers to molecules
which contain an atom or a group of atoms exhibiting an unpaired
electron spin (i.e. a stable paramagnetic group) that can be
detected by electron spin resonance spectroscopy and can be
attached to another molecule. Such spin-label molecules include,
but are not limited to, nitryl radicals and nitroxides, and may be
single spin-labels or double spin-labels.
[0284] The term "stoichiometric," as used herein, refers to the
ratio of the moles of compounds participating in a chemical
reaction being about 0.9 to about 1.1.
[0285] The term "stoichiometric-like," as used herein, refers to a
chemical reaction which becomes stoichiometric or
near-stoichiometric upon changes in reaction conditions or in the
presence of additives. Such changes in reaction conditions include,
but are not limited to, an increase in temperature or change in pH.
Such additives include, but are not limited to, accelerants.
[0286] The phrase "stringent hybridization conditions" refers to
hybridization of sequences of DNA, RNA, PNA or other nucleic acid
mimics, or combinations thereof, under conditions of low ionic
strength and high temperature. By way of example, under stringent
conditions a probe will hybridize to its target subsequence in a
complex mixture of nucleic acid (including but not limited to,
total cellular or library DNA or RNA) but does not hybridize to
other sequences in the complex mixture. Stringent conditions are
sequence-dependent and will be different in different
circumstances. By way of example, longer sequences hybridize
specifically at higher temperatures. Stringent hybridization
conditions include, but are not limited to, (i) about 5-10.degree.
C. lower than the thermal melting point (Tm) for the specific
sequence at a defined ionic strength and pH; (ii) the salt
concentration is about 0.01 M to about 1.0 M at about pH 7.0 to
about pH 8.3 and the temperature is at least about 30.degree. C.
for short probes (including but not limited to, about 10 to about
50 nucleotides) and at least about 60.degree. C. for long probes
(including but not limited to, greater than 50 nucleotides); (iii)
the addition of destabilizing agents including, but not limited to,
formamide, (iv) 50% formamide, 5.times.SSC, and 1% SDS, incubating
at 42.degree. C., or 5.times.SSC, about 1% SDS, incubating at
65.degree. C., with wash in 0.2.times.SSC, and about 0.1% SDS at
65.degree. C. for between about 5 minutes to about 120 minutes. By
way of example only, detection of selective or specific
hybridization, includes, but is not limited to, a positive signal
at least two times background. An extensive guide to the
hybridization of nucleic acids is found in Tijssen, Laboratory
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993).
[0287] The term "subject" as used herein, refers to an animal which
is the object of treatment, observation or experiment. By way of
example only, a subject may be, but is not limited to, a mammal
including, but not limited to, a human.
[0288] The term "substantially purified," as used herein, refers to
a component of interest that may be substantially or essentially
free of other components which normally accompany or interact with
the component of interest prior to purification. By way of example
only, a component of interest may be "substantially purified" when
the preparation of the component of interest contains less than
about 30%, less than about 25%, less than about 20%, less than
about 15%, less than about 10%, less than about 5%, less than about
4%, less than about 3%, less than about 2%, or less than about 1%
(by dry weight) of contaminating components. Thus, a "substantially
purified" component of interest may have a purity level of about
70%, about 75%, about 80%, about 85%, about 90%, about 95%, about
96%, about 97%, about 98%, about 99% or greater. By way of example
only, a natural amino acid polypeptide or a non-natural amino acid
polypeptide may be purified from a native cell, or host cell in the
case of recombinantly produced natural amino acid polypeptides or
non-natural amino acid polypeptides. By way of example a
preparation of a natural amino acid polypeptide or a non-natural
amino acid polypeptide may be "substantially purified" when the
preparation contains less than about 30%, less than about 25%, less
than about 20%, less than about 15%, less than about 10%, less than
about 5%, less than about 4%, less than about 3%, less than about
2%, or less than about 1% (by dry weight) of contaminating
material. By way of example when a natural amino acid polypeptide
or a non-natural amino acid polypeptide is recombinantly produced
by host cells, the natural amino acid polypeptide or non-natural
amino acid polypeptide may be present at about 30%, about 25%,
about 20%, about 15%, about 10%, about 5%, about 4%, about 3%,
about 2%, or about 1% or less of the dry weight of the cells. By
way of example when a natural amino acid polypeptide or a
non-natural amino acid polypeptide is recombinantly produced by
host cells, the natural amino acid polypeptide or non-natural amino
acid polypeptide may be present in the culture medium at about 5
g/L, about 4 g/L, about 3 g/L, about 2 g/L, about 1 g/L, about 750
mg/L, about 500 mg/L, about 250 mg/L, about 100 mg/L, about 50
mg/L, about 10 mg/L, or about 1 mg/L or less of the dry weight of
the cells. By way of example, "substantially purified" natural
amino acid polypeptides or non-natural amino acid polypeptides may
have a purity level of about 30%, about 35%, about 40%, about 45%,
about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,
about 80%, about 85%, about 90%, about 95%, about 99% or greater as
determined by appropriate methods, including, but not limited to,
SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.
[0289] The term "substituents" also referred to as "non-interfering
substituents" "refers to groups which may be used to replace
another group on a molecule. Such groups include, but are not
limited to, halo, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl,
C.sub.2-C.sub.10 alkynyl, C.sub.1-C.sub.10 alkoxy, C.sub.5-C.sub.12
aralkyl, C.sub.3-C.sub.12 cycloalkyl, C.sub.4-C.sub.12
cycloalkenyl, phenyl, substituted phenyl, toluolyl, xylenyl,
biphenyl, C.sub.2-C.sub.12 alkoxyalkyl, C.sub.5-C.sub.12
alkoxyaryl, C.sub.5-C.sub.12 aryloxyalkyl, C.sub.7-C.sub.2 oxyaryl,
C.sub.1-C.sub.6 alkylsulfinyl, C.sub.1-C.sub.10 alkylsulfonyl,
--(CH.sub.2).sub.m--O--(C.sub.1-C.sub.10 alkyl) wherein m is from 1
to 8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl,
heterocyclic radical, substituted heterocyclic radical, nitroalkyl,
--NO.sub.2, --CN, --NRC(O)--(C.sub.1-C.sub.10 alkyl),
--C(O)--(C.sub.1-C.sub.10 alkyl), C.sub.2-C.sub.10 alkthioalkyl,
--C(O)O--(C.sub.1-C.sub.10 alkyl), --OH, --SO.sub.2, .dbd.S,
--COOH, --NR.sub.2, carbonyl, --C(O)--(C.sub.1-C.sub.10
alkyl)-CF.sub.3, --C(O)--CF.sub.3, --C(O)NR.sub.2,
--(C.sub.1-C.sub.10 aryl)-S--(C.sub.6-C.sub.10 aryl),
--C(O)--(C.sub.6-C.sub.10 aryl),
--(CH.sub.2).sub.m--O--(CH.sub.2).sub.m--O--(C.sub.1-C.sub.10
alkyl) wherein each m is from 1 to 8, --C(O)NR.sub.2,
--C(S)NR.sub.2, --SO.sub.2NR.sub.2, --NRC(O)NR.sub.2,
--NRC(S)NR.sub.2, salts thereof, and the like. Each R group in the
preceding list includes, but is not limited to, H, alkyl or
substituted alkyl, aryl or substituted aryl, or alkaryl. Where
substituent groups are specified by their conventional chemical
formulas, written from left to right, they equally encompass the
chemically identical substituents that would result from writing
the structure from right to left; for example, --CH.sub.2O-- is
equivalent to --OCH.sub.2--.
[0290] By way of example only, substituents for alkyl and
heteroalkyl radicals (including those groups referred to as
alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl)
includes, but is not limited to: --OR, .dbd.O, .dbd.NR, .dbd.N--OR,
--NR.sub.2, --SR, -halogen, --SiR.sub.3, --OC(O)R, --C(O)R,
--CO.sub.2R, --CONR.sub.2, --OC(O)NR.sub.2, --NRC(O)R,
--NRC(O)NR.sub.2, --NR(O).sub.2R, --NR--C(NR.sub.2).dbd.NR,
--S(O)R, --S(O).sub.2R, --S(O).sub.2NR.sub.2, --NRSO.sub.2R, --CN
and --NO.sub.2. Each R group in the preceding list includes, but is
not limited to, hydrogen, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl, including but not limited to,
aryl substituted with 1-3 halogens, substituted or unsubstituted
alkyl, alkoxy or thioalkoxy groups, or aralkyl groups. When two R
groups are attached to the same nitrogen atom, they can be combined
with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For
example, --NR.sub.2 is meant to include, but not be limited to,
1-pyrrolidinyl and 4-morpholinyl.
[0291] By way of example, substituents for aryl and heteroaryl
groups include, but are not limited to, --OR, .dbd.O, .dbd.NR,
.dbd.N--OR, --NR.sub.2, --SR, -halogen, --SiR.sub.3, --OC(O)R,
--C(O)R, --CO.sub.2R, --CONR.sub.2, --OC(O)NR.sub.2, --NRC(O)R,
--NRC(O)NR.sub.2, --NR(O).sub.2R, --NR--C(NR.sub.2).dbd.NR,
--S(O)R, --S(O).sub.2R, --S(O).sub.2NR.sub.2, --NRSO.sub.2R, --CN,
--NO.sub.2, --R, --N.sub.3, --CH(Ph).sub.2,
fluoro(C.sub.1-C.sub.4)alkoxy, and fluoro(C.sub.1-C.sub.4)alkyl, in
a number ranging from zero to the total number of open valences on
the aromatic ring system; and where each R group in the preceding
list includes, but is not limited to, hydrogen, alkyl, heteroalkyl,
aryl and heteroaryl.
[0292] The term "therapeutically effective amount," as used herein,
refers to the amount of a composition containing at least one
non-natural amino acid polypeptide and/or at least one modified
non-natural amino acid polypeptide administered to a patient
already suffering from a disease, condition or disorder, sufficient
to cure or at least partially arrest, or relieve to some extent one
or more of the symptoms of the disease, disorder or condition being
treated. The effectiveness of such compositions depend conditions
including, but not limited to, the severity and course of the
disease, disorder or condition, previous therapy, the patient's
health status and response to the drugs, and the judgment of the
treating physician. By way of example only, therapeutically
effective amounts may be determined by routine experimentation,
including but not limited to a dose escalation clinical trial.
[0293] The term "thioalkoxy," as used herein, refers to sulfur
containing alkyl groups linked to molecules via an oxygen atom.
[0294] The term "thermal melting point" or Tm is the temperature
(under defined ionic strength, pH, and nucleic concentration) at
which 50% of probes complementary to a target hybridize to the
target sequence at equilibrium.
[0295] The term "toxic moiety" or "toxic group" as used herein,
refers to a compound which can cause harm, disturbances, or death.
Toxic moieties include, but are not limited to, NCA1, auristatin,
DNA minor groove binding agent, DNA minor groove alkylating agent,
enediyne, lexitropsin, duocarmycin, taxane, puromycin, dolastatin,
maytansinoid, vinca alkaloid, AFP, MMAF, MMAE, AEB, AEVB,
auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan,
morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,
dolastatin-10, echinomycin, combretatstatin, chalicheamicin,
maytansine, DM-1, netropsin, podophyllotoxin (e.g. etoposide,
teniposide, etc.), baccatin and its derivatives, anti-tubulin
agents, cryptophysin, combretastatin, auristatin E, vincristine,
vinblastine, vindesine, vinorelbine, VP-16, camptothecin,
epothilone A, epothilone B, nocodazole, colchicines, colcimid,
estramustine, cemadotin, discodermolide, maytansine, eleutherobin,
mechlorethamine, cyclophosphamide, melphalan, carmustine,
lomustine, semustine, streptozocin, chlorozotocin, uracil mustard,
chlormethine, ifosfamide, chlorambucil, pipobroman,
triethylenemelamine, triethylenethiophosphoramine, busulfan,
dacarbazine, and temozolomide, ytarabine, cytosine arabinoside,
fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine,
pentostatin, 5-fluorouracil, methotrexate,
10-propargyl-5,8-dideazafolate, 5,8-dideazatetrahydrofolic acid,
leucovorin, fludarabine phosphate, pentostatine, gemeitabine,
Ara-C, paclitaxel, docetaxel, deoxycoformycin, mitomycin-C,
L-asparaginase, azathioprine, brequinar, antibiotics (e.g.,
anthracycline, gentamicin, cefalotin, vancomycin, telavancin,
daptomycin, azithromycin, erythromycin, rocithromycin,
furazolidone, amoxicillin, ampicillin, carbenicillin,
flueloxacillin, methicillin, penicillin, ciprofloxacin,
moxifloxacin, ofloxacin, doxycycline, minocycline, oxytetracycline,
tetracycline, streptomycin, rifabutin, ethambutol, rifaximin,
etc.), antiviral drugs (e.g., abacavir, acyclovir, ampligen,
cidofovir, delavirdine, didanosine, efavirenz, entecavir, fosfonet,
ganciclovir, ibacitabine, imunovir, idoxuridine, inosine,
lopinavir, methisazone, nexavir, nevirapine, oseltamivir,
penciclovir, stavudine, trifluridine, truvada, valaciclovir,
zanamivir, etc.), daunorubicin hydrochloride, daunomycin,
rubidomycin, cerubidine, idarubicin, doxorubicin, epirubicin and
morpholino derivatives, phenoxizone biscyclopeptides (e.g.,
dactinomycin), basic glycopeptides (e.g., bleomycin), anthraquinone
glycosides (e.g., plicamycin, mithramycin), anthracenediones (e.g.,
mitoxantrone), azirinopyrrolo indolediones (e.g., mitomycin),
macrocyclic immunosuppressants (e.g., cyclosporine, FK-506,
tacrolimus, prograf, rapamycin etc.), navelbene, CPT-11,
anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide,
ifosamide, droloxafine, allocolchicine, Halichondrin B, colchicine,
colchicine derivatives, maytansine, rhizoxin, paclitaxel,
paclitaxel derivatives, docetaxel, thiocolchicine, trityl cysterin,
vinblastine sulfate, vineristine sulfate, cisplatin, carboplatin,
hydroxyurea, N-methylhydrazine, epidophyllotoxin, procarbazine,
mitoxantrone, leucovorin, and tegafur. "Taxanes" include
paclitaxel, as well as any active taxane derivative or pro-drug.
Chemotherapeutic agents such as erlotinib (TARCEVA.RTM.,
Genentech/OSI Pharm.), bortezomib (VELCADE.RTM., Millenium Pharm.),
fulvestrant (FASLODEX.RTM., AstraZeneca), sutent (SU11248, Pfizer),
letrozole (FEMARA.RTM., Novartis), imatinib mesylate (GLEEVEC.RTM.,
Novartis), PTK787/ZK 222584 (Novartis), oxaliplatin (Eloxatin.RTM.,
Sanofi), 5-FU (5-fluorouracil), leucovorin, Rapamycin (Sirolimus,
RAPAMUNE.RTM., Wyeth), lapatinib (TYKERB.RTM., GSK572016,
GlaxoSmithKline), lonafarnib (SCH 66336), sorafenib (BAY43-9006,
Bayer Labs.), and gefitinib (fRESSA.RTM., AstraZeneca), AG1478,
AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and
CYTOXAN.RTM. cyclosphosphamide; alkyl sulfonates such as busulfan,
improsulfan and piposulfan; antifolate antineoplastic such as
pemetrexed (ALIMTA.RTM. Eli Lilly), aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CBI-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosoureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics, calicheamicin, calicheamicin
gammall and calicheamicin omegall; dynemicin, including dynemicin
A; bisphosphonates, such as clodronate; an esperamicin; as well as
neocarzinostatin chromophore and related chromoprotein enediyne
antibiotic chromophores, aclacinomysins, actinomycin, anthramycin,
azaserine, bleomycins, cactinomycin, carabicin, caminomycin,
carzinophilin, chromomycinis, dactinomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM.
doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofuran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., paclitaxel (TAXOL.RTM., Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE.TM. Cremophor-free, albumin,
nanoparticle formulation of paclitaxel (American Pharmaceutical
Partners, Schaumberg, Ill.), and TAXOTERE.RTM. doxetaxel
(Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR.RTM.
gemeitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum
analogs such as cisplatin and carboplatin; vinblastine; platinum;
etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;
NAVELBINE.RTM. vinorelbine; novantrone; teniposide; edatrexate;
daunomycin; aminopterin; xeloda; ibandronate; CPT-1; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid; capecitabine; and pharmaceutically acceptable
salts, acids or derivatives of any of the above.
[0296] The terms "treat," "treating" or "treatment", as used
herein, include alleviating, abating or ameliorating a disease or
condition symptoms, preventing additional symptoms, ameliorating or
preventing the underlying metabolic causes of symptoms, inhibiting
the disease or condition, e.g., arresting the development of the
disease or condition, relieving the disease or condition, causing
regression of the disease or condition, relieving a condition
caused by the disease or condition, or stopping the symptoms of the
disease or condition. The terms "treat," "treating" or "treatment",
include, but are not limited to, prophylactic and/or therapeutic
treatments.
[0297] As used herein, the term "water soluble polymer" refers to
any polymer that is soluble in aqueous solvents. Such water soluble
polymers include, but are not limited to, polyethylene glycol,
polyethylene glycol propionaldehyde, mono C.sub.1-C.sub.10 alkoxy
or aryloxy derivatives thereof (described in U.S. Pat. No.
5,252,714 which is incorporated by reference herein),
monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinyl
alcohol, polyanino acids, divinylether maleic anhydride,
N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivatives
including dextran sulfate, polypropylene glycol, polypropylene
oxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin,
heparin fragments, polysaccharides, oligosaccharides, glycans,
cellulose and cellulose derivatives, including but not limited to
methylcellulose and carboxymethyl cellulose, serum albumin, starch
and starch derivatives, polypeptides, polyalkylene glycol and
derivatives thereof, copolymers of polyalkylene glycols and
derivatives thereof, polyvinyl ethyl ethers, and
alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or
mixtures thereof. By way of example only, coupling of such water
soluble polymers to natural amino acid polypeptides or non-natural
polypeptides may result in changes including, but not limited to,
increased water solubility, increased or modulated serum half-life,
increased or modulated therapeutic half-life relative to the
unmodified form, increased bioavailability, modulated biological
activity, extended circulation time, modulated immunogenicity,
modulated physical association characteristics including, but not
limited to, aggregation and multimer formation, altered receptor
binding, altered binding to one or more binding partners, and
altered receptor dimerization or multimerization. In addition, such
water soluble polymers may or may not have their own biological
activity.
[0298] Unless otherwise indicated, conventional methods of mass
spectroscopy, NMR, HPLC, protein chemistry, biochemistry,
recombinant DNA techniques and pharmacology, within the skill of
the art are employed.
[0299] Compounds, (including, but not limited to non-natural amino
acids, non-natural amino acid polypeptides, modified non-natural
amino acid polypeptides, and reagents for producing the
aforementioned compounds) presented herein include
isotopically-labeled compounds, which are identical to those
recited in the various formulas and structures presented herein,
but for the fact that one or more atoms are replaced by an atom
having an atomic mass or mass number different from the atomic mass
or mass number usually found in nature. Examples of isotopes that
can be incorporated into the present compounds include isotopes of
hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as
.sup.2H, .sup.3H, .sup.13C, .sup.14C, .sup.15N, .sup.18O, .sup.17O,
.sup.35S, .sup.18F, .sup.36Cl, respectively. Certain
isotopically-labeled compounds described herein, for example those
into which radioactive isotopes such as .sup.3H and .sup.4C are
incorporated, are useful in drug and/or substrate tissue
distribution assays. Further, substitution with isotopes such as
deuterium, i.e., .sup.2H, can afford certain therapeutic advantages
resulting from greater metabolic stability, for example increased
in vivo half-life or reduced dosage requirements.
[0300] Some of the compounds herein (including, but not limited to
non-natural amino acids, non-natural amino acid polypeptides and
modified non-natural amino acid polypeptides, and reagents for
producing the aforementioned compounds) have asymmetric carbon
atoms and can therefore exist as enantiomers or diastereomers.
Diasteromeric mixtures can be separated into their individual
diastereomers on the basis of their physical chemical differences
by methods known, for example, by chromatography and/or fractional
crystallization. Enantiomers can be separated by converting the
enantiomeric mixture into a diastereomeric mixture by reaction with
an appropriate optically active compound (e.g., alcohol),
separating the diastereomers and converting (e.g., hydrolyzing) the
individual diastereomers to the corresponding pure enantiomers. All
such isomers, including diastereomers, enantiomers, and mixtures
thereof are considered as part of the compositions described
herein.
[0301] In additional or further embodiments, the compounds
described herein (including, but not limited to non-natural amino
acids, non-natural amino acid polypeptides and modified non-natural
amino acid polypeptides, and reagents for producing the
aforementioned compounds) are used in the form of pro-drugs. In
additional or further embodiments, the compounds described herein
((including, but not limited to non-natural amino acids,
non-natural amino acid polypeptides and modified non-natural amino
acid polypeptides, and reagents for producing the aforementioned
compounds) are metabolized upon administration to an organism in
need to produce a metabolite that is then used to produce a desired
effect, including a desired therapeutic effect. In further or
additional embodiments are active metabolites of non-natural amino
acids and "modified or unmodified" non-natural amino acid
polypeptides.
[0302] The methods and formulations described herein include the
use of N-oxides, crystalline forms (also known as polymorphs), or
pharmaceutically acceptable salts of non-natural amino acids,
non-natural amino acid polypeptides and modified non-natural amino
acid polypeptides. In certain embodiments, non-natural amino acids,
non-natural amino acid polypeptides and modified non-natural amino
acid polypeptides may exist as tautomers. All tautomers are
included within the scope of the non-natural amino acids,
non-natural amino acid polypeptides and modified non-natural amino
acid polypeptides presented herein. In addition, the non-natural
amino acids, non-natural amino acid polypeptides and modified
non-natural amino acid polypeptides described herein can exist in
unsolvated as well as solvated forms with pharmaceutically
acceptable solvents such as water, ethanol, and the like. The
solvated forms of the non-natural amino acids, non-natural amino
acid polypeptides and modified non-natural amino acid polypeptides
presented herein are also considered to be disclosed herein.
[0303] Some of the compounds herein (including, but not limited to
non-natural amino acids, non-natural amino acid polypeptides and
modified non-natural amino acid polypeptides and reagents for
producing the aforementioned compounds) may exist in several
tautomeric forms. All such tautomeric forms are considered as part
of the compositions described herein. Also, for example all
enol-keto forms of any compounds (including, but not limited to
non-natural amino acids, non-natural amino acid polypeptides and
modified non-natural amino acid polypeptides and reagents for
producing the aforementioned compounds) herein are considered as
part of the compositions described herein.
[0304] Some of the compounds herein (including, but not limited to
non-natural amino acids, non-natural amino acid polypeptides and
modified non-natural amino acid polypeptides and reagents for
producing either of the aforementioned compounds) are acidic and
may form a salt with a pharmaceutically acceptable cation. Some of
the compounds herein (including, but not limited to non-natural
amino acids, non-natural amino acid polypeptides and modified
non-natural amino acid polypeptides and reagents for producing the
aforementioned compounds) can be basic and accordingly, may form a
salt with a pharmaceutically acceptable anion. All such salts,
including di-salts are within the scope of the compositions
described herein and they can be prepared by conventional methods.
For example, salts can be prepared by contacting the acidic and
basic entities, in either an aqueous, non-aqueous or partially
aqueous medium. The salts are recovered by using at least one of
the following techniques: filtration, precipitation with a
non-solvent followed by filtration, evaporation of the solvent, or,
in the case of aqueous solutions, lyophilization.
[0305] Pharmaceutically acceptable salts of the non-natural amino
acid polypeptides disclosed herein may be formed when an acidic
proton present in the parent non-natural amino acid polypeptides
either is replaced by a metal ion, by way of example an alkali
metal ion, an alkaline earth ion, or an aluminum ion; or
coordinates with an organic base. In addition, the salt forms of
the disclosed non-natural amino acid polypeptides can be prepared
using salts of the starting materials or intermediates. The
non-natural amino acid polypeptides described herein may be
prepared as a pharmaceutically acceptable acid addition salt (which
is a type of a pharmaceutically acceptable salt) by reacting the
free base form of non-natural amino acid polypeptides described
herein with a pharmaceutically acceptable inorganic or organic
acid. Alternatively, the non-natural amino acid polypeptides
described herein may be prepared as pharmaceutically acceptable
base addition salts (which are a type of a pharmaceutically
acceptable salt) by reacting the free acid form of non-natural
amino acid polypeptides described herein with a pharmaceutically
acceptable inorganic or organic base.
[0306] The type of pharmaceutical acceptable salts, include, but
are not limited to: (1) acid addition salts, formed with inorganic
acids such as hydrochloric acid, hydrobromic acid, sulfuric acid,
nitric acid, phosphoric acid, and the like; or formed with organic
acids such as acetic acid, propionic acid, hexanoic acid,
cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic
acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric
acid, tartaric acid, citric acid, benzoic acid,
3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic
acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,
2-naphthalenesulfonic acid,
4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic
acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid),
3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic
acid, lauryl sulfuric acid, gluconic acid, glutamic acid,
hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid,
and the like; (2) salts formed when an acidic proton present in the
parent compound either is replaced by a metal ion, e.g., an alkali
metal ion, an alkaline earth ion, or an aluminum ion; or
coordinates with an organic base. Acceptable organic bases include
ethanolamine, diethanolamine, triethanolamine, tromethamine,
N-methylglucamine, and the like. Acceptable inorganic bases include
aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium
carbonate, sodium hydroxide, and the like.
[0307] The corresponding counterions of the non-natural amino acid
polypeptide pharmaceutical acceptable salts may be analyzed and
identified using various methods including, but not limited to, ion
exchange chromatography, ion chromatography, capillary
electrophoresis, inductively coupled plasma, atomic absorption
spectroscopy, mass spectrometry, or any combination thereof. In
addition, the therapeutic activity of such non-natural amino acid
polypeptide pharmaceutical acceptable salts may be tested using the
techniques and methods described in examples 87-91.
[0308] It should be understood that a reference to a salt includes
the solvent addition forms or crystal forms thereof, particularly
solvates or polymorphs. Solvates contain either stoichiometric or
non-stoichiometric amounts of a solvent, and are often formed
during the process of crystallization with pharmaceutically
acceptable solvents such as water, ethanol, and the like. Hydrates
are formed when the solvent is water, or alcoholates are formed
when the solvent is alcohol. Polymorphs include the different
crystal packing arrangements of the same elemental composition of a
compound. Polymorphs usually have different X-ray diffraction
patterns, infrared spectra, melting points, density, hardness,
crystal shape, optical and electrical properties, stability, and
solubility. Various factors such as the recrystallization solvent,
rate of crystallization, and storage temperature may cause a single
crystal form to dominate.
[0309] The screening and characterization of non-natural amino acid
polypeptide pharmaceutical acceptable salts polymorphs and/or
solvates may be accomplished using a variety of techniques
including, but not limited to, thermal analysis, x-ray diffraction,
spectroscopy, vapor sorption, and microscopy. Thermal analysis
methods address thermo chemical degradation or thermo physical
processes including, but not limited to, polymorphic transitions,
and such methods are used to analyze the relationships between
polymorphic forms, determine weight loss, to find the glass
transition temperature, or for excipient compatibility studies.
Such methods include, but are not limited to, Differential scanning
calorimetry (DSC), Modulated Differential Scanning Calorimetry
(MDCS), Thermogravimetric analysis (TGA), and Thermogravi-metric
and Infrared analysis (TG/IR). X-ray diffraction methods include,
but are not limited to, single crystal and powder diffractometers
and synchrotron sources. The various spectroscopic techniques used
include, but are not limited to, Raman, FTIR, UVIS, and NMR (liquid
and solid state). The various microscopy techniques include, but
are not limited to, polarized light microscopy, Scanning Electron
Microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX),
Environmental Scanning Electron Microscopy with EDX (in gas or
water vapor atmosphere), IR microscopy, and Raman microscopy.
INCORPORATION BY REFERENCE
[0310] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0311] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which;
[0312] FIG. 1 presents a graphical illustration of anti-tumor
efficacy of ARX-.alpha.PSMA (10 mg/kg) and 1 mg/kg, 3 mg/kg, and 10
mg/kg doses of the antibody drug conjugate ARX-.alpha.PSMA-NCA1 in
the LNCaP prostate cancer model. A single injection of
PSMA-(591VK)-HA116-NCA1 at 10,3,1 mg/kg shrunk LNCaP (prostate
cancer) tumors. Wild type (unconjugated) had no anti-tumor
effect.
[0313] FIG. 2 presents a graphical illustration of body weight in
test subject groups treated intravenously with ARX-.alpha.PSMA (10
mg/kg) and 1 mg/kg, 3 mg/kg, and 10 mg/kg doses of the antibody
drug conjugate ARX-.alpha.PSMA-NCA in the LNCaP prostate cancer
model.
[0314] FIG. 3A presents a graphical illustration of the results
from the LNCaP cell viability assay described in Example 27.
[0315] FIG. 3B presents a graphical illustration of the results
from the MDA-PCa-2 cell viability assay described in Example
27.
[0316] FIG. 4 presents a graphical illustration of the PC-3 cell
viability assay used as a negative control in Example 27.
[0317] FIG. 5A presents a graphical illustration of the results
from the LNCaP cell viability assay described in Example 28.
[0318] FIG. 5B presents a graphical illustration of the results
from the MDA-PCa-2 cell viability assay described in Example
28.
[0319] FIG. 6 presents a graphical illustration of the PC-3 cell
viability assay used as a negative control in Example 28.
DETAILED DESCRIPTION OF THE INVENTION
[0320] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
I. Introduction
[0321] Recently, an entirely new technology in the protein sciences
has been reported, which promises to overcome many of the
limitations associated with site-specific modifications of
proteins. Specifically, new components have been added to the
protein biosynthetic machinery of the prokaryote Escherichia coli
(E. coli) (e.g., L. Wang, et al., (2001), Science 292:498-500) and
the eukaryote Saccharomyces cerevisiae (S. cerevisiae) (e.g., J.
Chin et al., Science 301:964-7 (2003)), which has enabled the
incorporation of non-natural amino acids to proteins in vivo. A
number of new amino acids with novel chemical, physical or
biological properties, including photoaffinity labels and
photoisomerizable amino acids, keto amino acids, and glycosylated
amino acids have been incorporated efficiently and with high
fidelity into proteins in E. coli and in yeast in response to the
amber codon, TAG, using this methodology. See, e.g., J. W. Chin et
al., (2002), Journal of the American Chemical Society 124:9026-9027
(incorporated by reference in its entirety); J. W. Chin, & P.
G. Schultz, (2002), ChemBioChem 3(11):1135-1137 (incorporated by
reference in its entirety); J. W. Chin, et al., (2002), PNAS United
States of America 99(17):11020-11024 (incorporated by reference in
its entirety); and, L. Wang, & P. G. Schultz, (2002), Chem.
Comm., 1-11 (incorporated by reference in its entirety). These
studies have demonstrated that it is possible to selectively and
routinely introduce chemical functional groups that are not found
in proteins, that are chemically inert to all of the functional
groups found in the 20 common, genetically-encoded amino acids and
that may be used to react efficiently and selectively to form
stable covalent linkages.
II. Overview
[0322] The present invention provides .alpha.PSMA antibodies having
a non-naturally encoded amino acid that facilitate antibody
conjugation to a drug (e.g. a drug, toxin, marker molecule). In one
embodiment, the ADC comprises an .alpha.PSMA antibody conjugated to
a drug wherein the conjugation occurs via a non-naturally encoded
amino acid in the antibody. In one embodiment, the ADC comprises an
.alpha.PSMA antibody conjugated to a drug wherein the conjugation
occurs via a non-naturally encoded amino acid in the heavy chain of
the antibody. In one embodiment, the ADC comprises an .alpha.PSMA
antibody conjugated to a drug wherein the conjugation occurs via a
non-naturally encoded amino acid in the light chain of the
antibody. In one embodiment, the ADC comprises a full-length
antibody conjugated to a drug wherein the conjugation occurs via a
non-naturally encoded amino acid in the antibody. In one
embodiment, the ADC comprises a full-length antibody conjugated to
a drug wherein the conjugation occurs via a non-naturally encoded
amino acid in the heavy chain of the antibody. In one embodiment,
the ADC comprises a full-length antibody conjugated to a drug
wherein the conjugation occurs via a non-naturally encoded amino
acid in the light chain of the antibody.
[0323] In some embodiments, the drug of the ADC is a cytotoxic
drug. In some aspects of the invention, the cytotoxic drug is
selected from the group consisting of an auristatin, a DNA minor
groove binding agent, a DNA minor groove alkylating agent, an
enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a
dolastatin, a maytansinoid, and a vinca alkaloid. In some aspects
of the invention, the cytotoxic drug is AFP, MMAF, MMAE, AEB, AEVB,
auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan,
morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,
dolastatin-10, echinomycin, combretatstatin, chalicheamicin,
maytansine, DM-1, or netropsin.
[0324] In some embodiments of the present invention, the cytotoxic
drug is
##STR00054##
[0325] In other embodiments of the present invention, the cytotoxic
drug is chosen from the group comprising:
##STR00055##
[0326] In some embodiments of the present invention, the cytotoxic
drug is chosen from the group consisting of
##STR00056##
[0327] In other aspects of the present invention, the cytotoxic
drug is chosen from the group consisting of:
##STR00057##
[0328] In other aspects of the present invention, the cytotoxic
drug is chosen from the group consisting of
##STR00058##
[0329] In some aspects of the invention, the cytotoxic drug is an
anti-tubulin agent. In some embodiments, the anti-tubulin agent is
an auristatin, a vinca alkaloid, a podophyllotoxin, a taxane, a
baccatin derivative, a cryptophysin, a maytansinoid, a
combretastatin, or a dolastatin. In other aspects of the invention,
the antitubulin agent is AFP, MMAF, MMAE, AEB, AEVB, auristatin E,
vineristine, vinblastine, vindesine, vinorelbine, VP-16,
camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B,
nocodazole, colchicines, colcimid, estramustine, cemadotin,
discodennolide, maytansine, DM-1, or eleutherobin.
[0330] In other aspects of the invention, the cytotoxic drug of the
ADC is gancyclovir, etanercept, cyclosporine, tacrolimus,
rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil,
methotrexate, cortisol, aldosterone, dexamethasone, a
cyclooxygenase inhibitor, a 5-ipoxygenase inhibitor, or a
leukotriene receptor antagonist.
[0331] In some embodiments of the invention, the antibody of the
ADC comprises a full length antibody that: (a) binds to PSMA, and
(b) is conjugated to a cytotoxic agent or an immunosuppressive
agent, wherein the antibody-drug conjugate exerts: (a) a cytotoxic
or cytostatic effect on a PSMA-expressing cancer cell line, or
[0032] (b) a cytotoxic, cytostatic, or immunosuppressive effect on
a PSMA-expressing immune cell, wherein the conjugation occurs at a
non-naturally encoded amino acid in the antibody.
[0332] At one level, described herein are the tools (methods,
compositions, techniques) for creating and using dolastatin linker
derivatives or analogs comprising at least one carbonyl,
dicarbonyl, oxime, hydroxylamine, aldehyde, protected aldehyde,
ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine,
azide, amidine, imine, diamine, keto-amine, keto-alkyne, alkyne,
cycloalkyne, or ene-dione. At another level, described herein are
the tools (methods, compositions, techniques) for creating and
using dolastatin linker derivatives or analogs comprising at least
one non-natural amino acid or modified non-natural amino acid with
an oxime, aromatic amine, heterocycle (e.g., indole, quinoxaline,
phenazine, pyrazole, triazole, etc.).
[0333] Such dolastatin linker derivatives comprising non-natural
amino acids may contain further functionality, including but not
limited to, a polymer; a water-soluble polymer; a derivative of
polyethylene glycol; a second protein or polypeptide or polypeptide
analog; an antibody or antibody fragment; and any combination
thereof. Note that the various aforementioned functionalities are
not meant to imply that the members of one functionality cannot be
classified as members of another functionality. Indeed, there will
be overlap depending upon the particular circumstances. By way of
example only, a water-soluble polymer overlaps in scope with a
derivative of polyethylene glycol, however the overlap is not
complete and thus both functionalities are cited above.
[0334] Provided herein in some embodiments, is a toxic group linker
derivative comprising a carbonyl, dicarbonyl, oxime, hydroxylamine,
aldehyde, protected aldehyde, ketone, protected ketone, thioester,
ester, dicarbonyl, hydrazine, azide, amidine, imine, diamine,
keto-amine, keto-alkyne, alkyne, cycloalkyne, or ene-dione. In some
embodiments, the toxic group derivative comprises any of the
linkers disclosed herein. In other embodiments, described herein
are the tools (methods, compositions, techniques) for creating and
using toxic group derivatives or analogs comprising at least one
non-natural amino acid or modified non-natural amino acid with an
oxime, aromatic amine, heterocycle (e.g., indole, quinoxaline,
phenazine, pyrazole, triazole, etc.).
[0335] In some embodiments, such toxic derivatives comprising
non-natural amino acids may contain further functionality,
including but not limited to, a polymer; a water-soluble polymer; a
derivative of polyethylene glycol; a second protein or polypeptide
or polypeptide analog; an antibody or antibody fragment; and any
combination thereof. In specific embodiments, the toxic group is a
tubulin inhibitor. In certain specific embodiments, the toxic group
is dolastatin or auristatin. In other specific embodiments, the
toxic group is dolastatin or auristatin derivative. Note that the
various aforementioned functionalities are not meant to imply that
the members of one functionality cannot be classified as members of
another functionality. Indeed, there will be overlap depending upon
the particular circumstances. By way of example only, a
water-soluble polymer overlaps in scope with a derivative of
polyethylene glycol, however the overlap is not complete and thus
both functionalities are cited above.
[0336] Certain embodiments of the present invention describe
preparations of certain toxic moieties with linkers that reduce the
toxicity of the moiety in vivo while the toxic moiety retains
pharmacological activity. In some embodiments, the toxicity of the
linked toxic group, when administered to an animal or human, is
reduced or eliminated compared to the free toxic group or toxic
group derivatives comprising labile linkages, while retaining
pharmacological activity. In some embodiments, increased doses of
the linked toxic group (e.g., dolastatin linker derivatives,
non-natural amino acid linked dolastatin derivatives) may be
administered to animals or humans with greater safety. In certain
embodiments, the non-natural amino acid polypeptides linked to a
toxic moiety (e.g., dolastatin derivative) provides in vitro and in
vivo stability. In some embodiments, the non-natural amino acid
polypeptides linked to a toxic moiety (e.g., tubulin inhibitor,
dolastatin-10 derivative) are efficacious and less toxic compared
to the free toxic moiety (e.g., tubulin inhibitor,
dolastatin-10).
III. Dolastatin Linker Derivatives
[0337] At one level, described herein are the tools (methods,
compositions, techniques) for creating and using a dolastatin
linker derivatives or analogs comprising at least one non-natural
amino acid or modified non-natural amino acid with a carbonyl,
dicarbonyl, oxime or hydroxylamine group. Such dolastatin linker
derivatives comprising non-natural amino acids may contain further
functionality, including but not limited to, a polymer; a
water-soluble polymer; a derivative of polyethylene glycol; a
second protein or polypeptide or polypeptide analog; an antibody or
antibody fragment; and any combination thereof. Note that the
various aforementioned functionalities are not meant to imply that
the members of one functionality cannot be classified as members of
another functionality. Indeed, there will be overlap depending upon
the particular circumstances. By way of example only, a
water-soluble polymer overlaps in scope with a derivative of
polyethylene glycol, however the overlap is not complete and thus
both functionalities are cited above.
[0338] In one aspect are methods for selecting and designing a
dolastatin linker derivative to be modified using the methods,
compositions and techniques described herein. The new dolastatin
linker derivative may be designed de novo, including by way of
example only, as part of high-throughput screening process (in
which case numerous polypeptides may be designed, synthesized,
characterized and/or tested) or based on the interests of the
researcher. The new dolastatin linker derivative may also be
designed based on the structure of a known or partially
characterized polypeptide. By way of example only, dolastatin has
been the subject of intense study by the scientific community; a
new compound may be designed based on the structure of dolastatin.
The principles for selecting which amino acid(s) to substitute
and/or modify are described separately herein. The choice of which
modification to employ is also described herein, and can be used to
meet the need of the experimenter or end user. Such needs may
include, but are not limited to, manipulating the therapeutic
effectiveness of the polypeptide, improving the safety profile of
the polypeptide, adjusting the pharmacokinetics, pharmacologics
and/or pharmacodynamics of the polypeptide, such as, by way of
example only, increasing water solubility, bioavailability,
increasing serum half-life, increasing therapeutic half-life,
modulating immunogenicity, modulating biological activity, or
extending the circulation time. In addition, such modifications
include, by way of example only, providing additional functionality
to the polypeptide, incorporating an antibody, and any combination
of the aforementioned modifications.
[0339] Also described herein are dolastatin linker derivatives that
have or can be modified to contain an oxime, carbonyl, dicarbonyl,
or hydroxylamine group. Included with this aspect are methods for
producing, purifying, characterizing and using such dolastatin
linker derivatives
[0340] The dolastatin linker derivative may contain at least one,
at least two, at least three, at least four, at least five, at
least six, at least seven, at least eight, at least nine, or ten or
more of a carbonyl or dicarbonyl group, oxime group, hydroxylamine
group, or protected forms thereof. The dolastatin linker derivative
can be the same or different, for example, there can be 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more
different sites in the derivative that comprise 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more
different reactive groups.
[0341] A. Structure and Synthesis of Dolastatin Linker Derivatives:
Electrophilic and Nucleophilic Groups
[0342] Dolastatin derivatives with linkers containing a
hydroxylamine (also called an aminooxy) group allow for reaction
with a variety of electrophilic groups to form conjugates
(including but not limited to, with PEG or other water soluble
polymers). Like hydrazines, hydrazides and semicarbazides, the
enhanced nucleophilicity of the aminooxy group permits it to react
efficiently and selectively with a variety of molecules that
contain carbonyl- or dicarbonyl-groups, including but not limited
to, ketones, aldehydes or other functional groups with similar
chemical reactivity. See, e.g., Shao, J. and Tam, J., J. Am, Chem.
Soc. 117:3893-3899 (1995); H. Hang and C. Bertozzi, Ace. Chem. Res.
34(9): 727-736 (2001). Whereas the result of reaction with a
hydrazine group is the corresponding hydrazone, however, an oxime
results generally from the reaction of an aminooxy group with a
carbonyl- or dicarbonyl-containing group such as, by way of
example, a ketones, aldehydes or other functional groups with
similar chemical reactivity. In some embodiments, dolastatin
derivatives with linkers comprising an azide, alkyne or cycloalkyne
allow for linking of molecules via cycloaddition reactions (e.g.,
1,3-dipolar cycloadditions, azide-alkyne Huisgen cycloaddition,
etc.). (Described in U.S. Pat. No. 7,807,619 which is incorporated
by reference herein to the extent relative to the reaction).
[0343] Thus, in certain embodiments described herein are dolastatin
derivatives with linkers comprising a hydroxylamine, aldehyde,
protected aldehyde, ketone, protected ketone, thioester, ester,
dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine,
keto-alkyne, and ene-dione hydroxylamine group, a
hydroxylamine-like group (which has reactivity similar to a
hydroxylamine group and is structurally similar to a hydroxylamine
group), a masked hydroxylamine group (which can be readily
converted into a hydroxylamine group), or a protected hydroxylamine
group (which has reactivity similar to a hydroxylamine group upon
deprotection). In some embodiments, the dolastatin derivatives with
linkers comprise azides, alkynes or cycloalkynes. Such dolastatin
linker derivatives include compounds having the structure of
Formula (I), (III), (IV), (V), and (VI):
##STR00059## ##STR00060## [0344] wherein: [0345] Z has the
structure of:
[0345] ##STR00061## [0346] R.sub.5 is H, COR.sub.8,
C.sub.1-C.sub.6alkyl, or thiazole; [0347] R.sub.8 is OH or
--NH-(alkylene-O).sub.n--NH.sub.2; [0348] R.sub.6 is OH or H;
[0349] Ar is phenyl or pyridine; [0350] R.sub.7 is
C.sub.1-C.sub.6alkyl or hydrogen; [0351] Y and V are each selected
from the group consisting of an hydroxylamine, methyl, aldehyde,
protected aldehyde, ketone, protected ketone, thioester, ester,
dicarbonyl, hydrazine, azide, amidine, imine, diamine, keto-amine,
keto-alkyne, alkyne, cycloalkyne, and ene-dione; [0352] L, L.sub.1,
L.sub.2, L.sub.3, and L.sub.4 are each linkers selected from the
group consisting of a bond, -alkylene-, -alkylene-C(O)--,
-alkylene-J-, -(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-C(O)--, -(alkylene-O).sub.n-J-,
-(alkylene-O).sub.n-J-alkylene-,
-(alkylene-O).sub.n--(CH.sub.2).sub.n'--NHC(O)--(CH.sub.2).sub.n''--C(Me)-
.sub.2-S--S--(CH.sub.2).sub.n'''--NHC(O)-(alkylene-O).sub.n''''-alkylene-,
-(alkylene-O).sub.n-alkylene-W--, -alkylene-C(O)--W--,
-(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-alkylene',
-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n'-alkylene-J'-,
--W--, -alkylene-W--,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-(alkylene-NMe).sub.n-alkylene-W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-U-alkylene-;
-J-alkylene-NMe-alkylene'-NMe-alkylene''-W--, and
-alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--; [0353] W
has the structure of:
[0353] ##STR00062## [0354] U has the structure of:
[0354] ##STR00063## [0355] each J and J' independently have the
structure of:
[0355] ##STR00064## [0356] each n, n' n'', n''' and n'''' are
independently integers greater than or equal to one; and [0357] or
L is absent, Y is methyl, R.sub.5 is COR.sub.8, and Ra is
--NIH-(alkylene-O).sub.n--NH.sub.2. Such dolastatin linker
derivatives may be in the form of a salt, or may be incorporated
into a non-natural amino acid polypeptide, polymer, polysaccharide,
or a polynucleotide and optionally post translationally
modified.
[0358] In certain embodiments of compounds of Formula (I), (III),
and (V), R.sub.5 is thiazole or carboxylic acid. In certain
embodiments of compounds of Formula (I), (III), and (V), R.sub.5 is
hydrogen. In certain embodiments of compounds of Formula (I),
(III), and (V), R.sub.5 is methyl, ethyl, propyl, iso-propyl,
butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In
certain embodiments of compounds of Formula (I), (III), and (V),
R.sub.5 is --NH-(alkylene-O).sub.n--NH.sub.2, wherein alkylene is
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.-
sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2--, or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2CH.sub.2--. In certain embodiments of compounds of
Formula (IV) and (VI), R.sub.5 is
--NH-(alkylene-O).sub.n--NH.sub.2, wherein n is 0, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100.
[0359] In some embodiments, Y is azide. In other embodiments, Y is
cycloalkyne. In specific embodiments, the cyclooctyne has a
structure of:
##STR00065## [0360] each R.sub.19 is independently selected from
the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkoxy, ester, ether, thioether, aminoalkyl, halogen, alkyl ester,
aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl
sulfonic acid, alkyl nitro, thioester, sulfonyl ester,
halosulfonyl, nitrile, alkyl nitrile, and nitro; and [0361] q is 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.
[0362] In certain embodiments of compounds of Formula (I), (III),
and (V), R.sub.6 is H. In some embodiments of compounds of Formula
(I), (III), and (V), R.sub.6 is hydroxy.
[0363] In certain embodiments of compounds of Formula (I), (III),
and (V), Ar is phenyl.
[0364] In certain embodiments of compounds of Formula (I), (III),
(IV), (V), and (VI), R.sub.7 is methyl, ethyl, propyl, iso-propyl,
butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In
certain embodiments of compounds of Formula (I), (III), (IV), (V),
and (VI), R.sub.7 is hydrogen.
[0365] In certain embodiments of compounds of Formula (I), (III),
and (V), Y is hydroxylamine, aldehyde, protected aldehyde, ketone,
protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine,
imine, diamine, keto-amine, keto-alkyne, or ene-dione.
[0366] In certain embodiments of compounds of Formula (IV) and
(VI), V is a hydroxylamine, methyl, aldehyde, protected aldehyde,
ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine,
amidine, imine, diamine, keto-amine, keto-alkyne, and
ene-dione.
[0367] In certain embodiments of compounds of Formula (I), (III),
(IV), (V), and (VI), each L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4
is independently a cleavable linker or non-cleavable linker. In
certain embodiments of compounds of Formula (I), (III), (IV), (V),
and (VI), each L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 is
independently a oligo(ethylene glycol) derivatized linker.
[0368] In certain embodiments of compounds of Formula (I), (III),
(IV), (V), and (VI), each alkylene, alkylene', alkylene'', and
alkylene''' independently is --CH.sub.2--, --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.-
sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2--, or
CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2C-
H.sub.2CH.sub.2CH.sub.2--. In certain embodiments of compounds of
Formula (XIV), (XV), (XVI), (XVII), and (XVIII), each n, n', n'',
n''', and n'''' is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99, or 100.
[0369] B. Structure and Synthesis of Dolastatin Linker Derivatives:
Hydroxylamine Groups
[0370] Thus, in certain embodiments described herein are dolastatin
derivatives with linkers comprising a hydroxylamine group, a
hydroxylamine-like group (which has reactivity similar to a
hydroxylamine group and is structurally similar to a hydroxylamine
group), a masked hydroxylamine group (which can be readily
converted into a hydroxylamine group), or a protected hydroxylamine
group (which has reactivity similar to a hydroxylamine group upon
deprotection). Such dolastatin linker derivatives include compounds
having the structure of Formula (I):
##STR00066##
wherein: [0371] Z has the structure of;
[0371] ##STR00067## [0372] R.sub.5 is H, COR.sub.8,
C.sub.1-C.sub.6alkyl, or thiazole; [0373] R.sub.8 is OH or
--NH-(alkylene-O).sub.n--NH.sub.2; [0374] R.sub.6 is OH or H;
[0375] Ar is phenyl or pyridine; [0376] R.sub.7 is
C.sub.1-C.sub.6alkyl or hydrogen; [0377] Y is NH.sub.2--O-- or
methyl; [0378] L is a linker selected from the group consisting of
-alkylene-, -alkylene-C(O)--, -(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-C(O)--,
-(alkylene-O).sub.n--(CH.sub.2).sub.n--NHC(O)--(CH.sub.2).sub.n''--C(Me).-
sub.2-S--S--(CH.sub.2).sub.n'''--NHC(O)-(alkylene-O).sub.n''''-alkylene-,
-(alkylene-O).sub.n-alkylene-W--, -alkylene-C(O)--W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--, and
-(alkylene-O).sub.n-alkylene-U-alkylene-; [0379] W has the
structure of:
[0379] ##STR00068## [0380] U has the structure of:
[0380] ##STR00069## [0381] or L is absent, Y is methyl, R.sub.5 is
COR.sub.8, and R.sub.8 is --NH(alkylene-O).sub.n--NH.sub.2; and
each n, n', n'', n''' and n'''' are independently integers greater
than or equal to one. Such dolastatin linker derivatives may be in
the form of a salt, or may be incorporated into a non-natural amino
acid polypeptide, polymer, polysaccharide, or a polynucleotide and
optionally post translationally modified.
[0382] In certain embodiments of compounds of Formula (I), R.sub.5
is thiazole. In certain embodiments of compounds of Formula (I),
R.sub.5 is hydrogen. In certain embodiments of compounds of Formula
(I), R.sub.5 is methyl, ethyl, propyl, iso-propyl, butyl,
iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In certain
embodiments of compounds of Formula (I), R.sub.5 is
--NH-(alkylene-O).sub.n--NH.sub.2, wherein alkylene is
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.-
sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2--, or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2CH.sub.2--. In certain embodiments of compounds of
Formula (I), R.sub.5 is --NH-(alkylene-O).sub.n--NH.sub.2, wherein
n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.
[0383] In certain embodiments of compounds of Formula (I), R.sub.6
is H. In some embodiments of compounds of Formula (I), R.sub.6 is
hydroxy.
[0384] In certain embodiments of compounds of Formula (I), Ar is
phenyl.
[0385] In certain embodiments of compounds of Formula (I), R.sub.7
is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl,
tert-butyl, pentyl, or hexyl. In certain embodiments of compounds
of Formula (I), R.sub.7 is hydrogen.
[0386] In certain embodiments of compounds of Formula (I), Y is
hydroxylamine, aldehyde, protected aldehyde, ketone, protected
ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine,
diamine, keto-amine, keto-alkyne, or ene-dione. In certain
embodiments of compounds of Formula (I), V is a hydroxylamine,
methyl, aldehyde, protected aldehyde, ketone, protected ketone,
thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine,
keto-amine, keto-alkyne, and ene-dione.
[0387] In certain embodiments of compounds of Formula (I), each L
is independently a cleavable linker or non-cleavable linker. In
certain embodiments of compounds of Formula (I), each L is
independently a oligo(ethylene glycol) derivatized linker.
[0388] In certain embodiments of compounds of Formula (I), alkylene
is --CH.sub.2--, --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.-
sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2--, or
CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2C-
H.sub.2CH.sub.2CH.sub.2--. In certain embodiments of compounds of
Formula (I), each n, n', n'', n''', and n'''' is 0, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100.
[0389] In certain embodiments, dolastatin linker derivatives
include compounds having the structure of Formula (II):
##STR00070##
In some embodiments of compounds of Formula (II), L is
-(alkylene-O).sub.n-alkylene-. In some embodiments, each alkylene
is --CH.sub.2CH.sub.2--, n is equal to 3, and R.sub.7 is methyl. In
some embodiments, L is -alkylene-. In some embodiments of compounds
of Formula (II), each alkylene is --CH.sub.2CH.sub.2-- and R.sub.7
is methyl or hydrogen. In some embodiments of compounds of Formula
(II), L is -(alkylene-O).sub.n-alkylene-C(O)--. In some embodiments
of compounds of Formula (II), each alkylene is
--CH.sub.2CH.sub.2--, n is equal to 4, and R.sub.7 is methyl. In
some embodiments of compounds of Formula (II), L is
-(alkylene-O).sub.n--(CH.sub.2).sub.n'--NHC(O)--(CH.sub.2).sub.n''--C(Me)-
.sub.2-S--S--(CH.sub.2).sub.n'''--NHC(O)-(alkylene-O).sub.n''''-alkylene-.
In some embodiments of compounds of Formula (II), each alkylene is
--CH.sub.2CH.sub.2--, n is equal to 1, n' is equal to 2, n'' is
equal to 1, n''' is equal to 2, n'''' is equal to 4, and R.sub.7 is
methyl. Such dolastatin linker derivatives may be in the form of a
salt, or may be incorporated into a non-natural amino acid
polypeptide, polymer, polysaccharide, or a polynucleotide and
optionally post translationally modified.
[0390] In certain embodiments of compounds of Formula (II), each L
is independently a cleavable linker or non-cleavable linker. In
certain embodiments of compounds of Formula (II), each L is
independently a oligo(ethylene glycol) derivatized linker.
[0391] In certain embodiments of compounds of Formula (II), R.sub.7
is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl,
tert-butyl, pentyl, or hexyl. In certain embodiments of compounds
of Formula (II), R.sub.7 is hydrogen.
[0392] In certain embodiments of compounds of Formula (II),
alkylene is --CH.sub.2--, --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.-
sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2--, or
CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2C-
H.sub.2CH.sub.2CH.sub.2--. In certain embodiments of compounds of
Formula (II), each n, n', n'', n''', and n'''' is 0, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100.
[0393] Such dolastatin linker derivatives include compounds having
the structure of Formula (III), (IV), (V) or (VI):
##STR00071## ##STR00072## [0394] wherein: [0395] Z has the
structure of:
[0395] ##STR00073## [0396] R.sub.5 is H, COR.sub.8,
C.sub.1-C.sub.6alkyl, or thiazole; [0397] R.sub.8 is OH; [0398]
R.sub.6 is OH or H; [0399] Ar is phenyl or pyridine; [0400] R.sub.7
is C.sub.1-C.sub.6alkyl or hydrogen; [0401] Y is NH.sub.2--O--;
[0402] V is --O--NH.sub.2 [0403] L.sub.1, L.sub.2, L.sub.3, and
L.sub.4 are each linkers independently selected from the group
consisting of a bond, -alkylene-, -(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n'-alkylene-J'-,
-(alkylene-O).sub.n-alkylene-J-alkylene'-, --W--, alkylene-,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-alkylene-NMe-alkylene'-NMe-alkylene''-W--, and
-alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--; W has the
structure of:
[0403] ##STR00074## [0404] each J and J' independently have the
structure of:
[0404] ##STR00075## and [0405] each n and n' are independently
integers greater than or equal to one, Such dolastatin linker
derivatives may be in the form of a salt, or may be incorporated
into a non-natural amino acid polypeptide, polymer, polysaccharide,
or a polynucleotide and optionally post translationally
modified.
[0406] In certain embodiments of compounds of Formula (III), (IV),
(V) or (VI), R.sub.5 is thiazole. In certain embodiments of
compounds of Formula (III), (IV), (V) or (VI), R.sub.6 is H. In
certain embodiments of compounds of Formula (III), (IV), (V) or
(VI), Ar is phenyl. In certain embodiments of compounds of Formula
(III), (IV), (V) or (VI), R.sub.7 is methyl. In certain embodiments
of compounds of Formula (III), (IV), (V) or (VI), n and n' are
integers from 0 to 20. In certain embodiments of compounds of
Formula (III), (IV), (V) or (VI), n and n' are integers from 0 to
10. In certain embodiments of compounds of Formula (III), (IV), (V)
or (VI), n and n' are integers from 0 to 5.
[0407] In certain embodiments of compounds of Formula (III) and
(V), R.sub.5 is thiazole or carboxylic acid. In certain embodiments
of compounds of Formula (III) and (V), R.sub.5 is hydrogen. In
certain embodiments of compounds of Formula (III) and (V), R.sub.5
is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl,
tert-butyl, pentyl, or hexyl. In certain embodiments of compounds
of Formula (III) and (V), R.sub.5 is
--NH-(alkylene-O).sub.n--NH.sub.2, wherein alkylene is
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.-
sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2--, or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2CH.sub.2--. In certain embodiments of compounds of
Formula (III) and (V), R.sub.5 is
--NH-(alkylene-O).sub.n--NH.sub.2, wherein n is 0, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100.
[0408] In certain embodiments of compounds of Formula (III), (IV),
(V) and (VI), R.sub.6 is H. In some embodiments of compounds of
Formula (III), (IV), (V) and (VI), R.sub.6 is hydroxy.
[0409] In certain embodiments of compounds of Formula (III), (IV),
(V) and (VI), Ar is phenyl.
[0410] In certain embodiments of compounds of Formula (III), (IV),
(V) and (VI), R.sub.7 is methyl, ethyl, propyl, iso-propyl, butyl,
sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In certain
embodiments of compounds of Formula (III), (IV), (V) and (VI),
R.sub.7 is hydrogen.
[0411] In certain embodiments of compounds of Formula (III) and
(V), Y is hydroxylamine, aldehyde, protected aldehyde, ketone,
protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine,
imine, diamine, keto-amine, keto-alkyne, or ene-dione. In certain
embodiments of compounds of Formula (IV) and (VI), V is a
hydroxylamine, methyl, aldehyde, protected aldehyde, ketone,
protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine,
imine, diamine, keto-amine, keto-alkyne, and ene-dione.
[0412] In certain embodiments of compounds of Formula (XIV), (XV),
(XVI), (XVII), and (XVIII), each L, L.sub.1, L.sub.2, L.sub.3, and
L.sub.4 is independently a cleavable linker or non-cleavable
linker. In certain embodiments of compounds of Formula (XIV), (XV),
(XVI), (XVII), and (XVIII), each L, L.sub.1, L.sub.2, L.sub.3, and
L.sub.4 is independently a oligo(ethylene glycol) derivatized
linker.
[0413] In certain embodiments of compounds of Formula (III), (IV),
(V) and (VI), each alkylene, alkylene', alkylene'', and alkylene'''
independently is --CH.sub.2--, --CH.sub.2CH.sub.2--,
--CH.sub.2C.sub.1H.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2Cl.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CF.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.-
sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2--, or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2CH.sub.2--. In certain embodiments of compounds of
Formula (III), (IV), (V) and (VI), alkylene is methylene, ethylene,
propylene, butylenes, pentylene, hexylene, or heptylene.
[0414] In certain embodiments of compounds of Formula (III), (IV),
(V) and (VI), each n and n' independently is 0, 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100.
[0415] In certain embodiments, dolastatin linker derivatives
include compounds having the structure of Formula (VII):
##STR00076##
[0416] In certain embodiments of compounds of Formula (VII),
L.sub.1 is -(alkylene-O).sub.n-alkylene-J-, L.sub.2 is
-alkylene'-J'-(alkylene-O).sub.n'-alkylene-, L.sub.3 is
-J''-(alkylene-O).sub.n''-alkylene-, alkylene is
--CH.sub.2CH.sub.2--, alkylene' is --(CH.sub.2).sub.4--, n is 1, n'
and n'' are 3, J has the structure of
##STR00077##
J' and J'' have the structure of
##STR00078##
and R.sub.7 is methyl. In certain embodiments of compounds of
Formula (VII), L.sub.1 is -J-(alkylene-O).sub.n-alkylene-, L.sub.2
is -(alkylene-O).sub.n-alkylene-J'-alkylene'-, L.sub.3 is
-(alkylene-O).sub.n''-alkylene-J''-, alkylene is
--CH.sub.2CH.sub.2--, alkylene' is --(CH.sub.2).sub.4--, n is 1, n'
and n'' are 4, and J, J' and J'' have the structure of
##STR00079##
Such dolastatin linker derivatives may be in the form of a salt, or
may be incorporated into a non-natural amino acid polypeptide,
polymer, polysaccharide, or a polynucleotide and optionally post
translationally modified.
[0417] In certain embodiments, compounds of Formula (I)-(VII) are
stable in aqueous solution for at least 1 month under mildly acidic
conditions. In certain embodiments, compounds of Formula (I)-(VII)
are stable for at least 2 weeks under mildly acidic conditions. In
certain embodiments, compound of Formula (I)-(VII) are stable for
at least 5 days under mildly acidic conditions. In certain
embodiments, such acidic conditions are pH 2 to 8.
[0418] The methods and compositions provided and described herein
include polypeptides comprising dolastatin linker derivative
containing at least one carbonyl or dicarbonyl group, oxime group,
hydroxylamine group, or protected or masked forms thereof.
Introduction of at least one reactive group into a dolastatin
linker derivative can allow for the application of conjugation
chemistries that involve specific chemical reactions, including,
but not limited to, with one or more dolastatin linker
derivative(s) while not reacting with the commonly occurring amino
acids. Once incorporated, the dolastatin linker derivative side
chains can also be modified by utilizing chemistry methodologies
described herein or suitable for the particular functional groups
or substituents present in the dolastatin linker derivative.
[0419] The dolastatin linker derivative methods and compositions
described herein provide conjugates of substances having a wide
variety of functional groups, substituents or moieties, with other
substances including but not limited to a polymer; a water-soluble
polymer; a derivative of polyethylene glycol; a second protein or
polypeptide or polypeptide analog; an antibody or antibody
fragment; and any combination thereof.
[0420] In certain embodiments, the dolastatin linker derivatives,
linkers and reagents described herein, including compounds of
Formulas (I)-(VII) are stable in aqueous solution under mildly
acidic conditions (including but not limited to pH 2 to 8). In
other embodiments, such compounds are stable for at least one month
under mildly acidic conditions. In other embodiments, such
compounds are stable for at least 2 weeks under mildly acidic
conditions. In other embodiments, such compounds are stable for at
least 5 days under mildly acidic conditions.
[0421] In another aspect of the compositions, methods, techniques
and strategies described herein are methods for studying or using
any of the aforementioned "modified or unmodified" non-natural
amino acid dolastatin linker derivatives. Included within this
aspect, by way of example only, are therapeutic, diagnostic,
assay-based, industrial, cosmetic, plant biology, environmental,
energy-production, consumer-products, and/or military uses which
would benefit from a dolastatin linker derivative comprising a
"modified or unmodified" non-natural amino acid polypeptide or
protein.
[0422] Non-limiting examples of dolastatin linker derivatives
include:
##STR00080## ##STR00081## ##STR00082## ##STR00083## ##STR00084##
##STR00085##
IV. Non-Natural Amino Acid Derivatives
[0423] The non-natural amino acids used in the methods and
compositions described herein have at least one of the following
four properties: (1) at least one functional group on the sidechain
of the non-natural amino acid has at least one characteristics
and/or activity and/or reactivity orthogonal to the chemical
reactivity of the 20 common, genetically-encoded amino acids (i.e.,
alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,
glutamic acid, glycine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, proline, serine, threonine, tryptophan,
tyrosine, and valine), or at least orthogonal to the chemical
reactivity of the naturally occurring amino acids present in the
polypeptide that includes the non-natural amino acid; (2) the
introduced non-natural amino acids are substantially chemically
inert toward the 20 common, genetically-encoded amino acids; (3)
the non-natural amino acid can be stably incorporated into a
polypeptide, preferably with the stability commensurate with the
naturally-occurring amino acids or under typical physiological
conditions, and further preferably such incorporation can occur via
an in vivo system; and (4) the non-natural amino acid includes an
oxime functional group or a functional group that can be
transformed into an oxime group by reacting with a reagent,
preferably under conditions that do not destroy the biological
properties of the polypeptide that includes the non-natural amino
acid (unless of course such a destruction of biological properties
is the purpose of the modification/transformation), or where the
transformation can occur under aqueous conditions at a pH between
about 4 and about 8, or where the reactive site on the non-natural
amino acid is an electrophilic site. Any number of non-natural
amino acids can be introduced into the polypeptide. Non-natural
amino acids may also include protected or masked oximes or
protected or masked groups that can be transformed into an oxime
group after deprotection of the protected group or unmasking of the
masked group. Non-natural amino acids may also include protected or
masked carbonyl or dicarbonyl groups, which can be transformed into
a carbonyl or dicarbonyl group after deprotection of the protected
group or unmasking of the masked group and thereby are available to
react with hydroxylamines or oximes to form oxime groups.
[0424] Non-natural amino acids that may be used in the methods and
compositions described herein include, but are not limited to,
amino acids comprising a amino acids with novel functional groups,
amino acids that covalently or noncovalently interact with other
molecules, glycosylated amino acids such as a sugar substituted
serine, other carbohydrate modified amino acids, keto-containing
amino acids, aldehyde-containing amino acids, amino acids
comprising polyethylene glycol or other polyethers, heavy atom
substituted amino acids, chemically cleavable and/or photocleavable
amino acids, amino acids with an elongated side chains as compared
to natural amino acids, including but not limited to, polyethers or
long chain hydrocarbons, including but not limited to, greater than
about 5 or greater than about 10 carbons, carbon-linked
sugar-containing amino acids, redox-active amino acids, amino
thioacid containing amino acids, and amino acids comprising one or
more toxic moiety.
[0425] In some embodiments, non-natural amino acids comprise a
saccharide moiety. Examples of such amino acids include
N-acetyl-L-glucosaminyl-L-serine,
N-acetyl-L-galactosaminyl-L-serine,
N-acetyl-L-glucosaminyl-L-threonine,
N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.
Examples of such amino acids also include examples where the
naturally-occurring N- or O-linkage between the amino acid and the
saccharide is replaced by a covalent linkage not commonly found in
nature--including but not limited to, an alkene, an oxime, a
thioether, an amide and the like. Examples of such amino acids also
include saccharides that are not commonly found in
naturally-occurring proteins such as 2-deoxy-glucose,
2-deoxygalactose and the like.
[0426] The chemical moieties incorporated into polypeptides via
incorporation of non-natural amino acids into such polypeptides
offer a variety of advantages and manipulations of polypeptides.
For example, the unique reactivity of a carbonyl or dicarbonyl
functional group (including a keto- or aldehyde-functional group)
allows selective modification of proteins with any of a number of
hydrazine- or hydroxylamine-containing reagents in vivo and in
vitro. A heavy atom non-natural amino acid, for example, can be
useful for phasing x-ray structure data. The site-specific
introduction of heavy atoms using non-natural amino acids also
provides selectivity and flexibility in choosing positions for
heavy atoms. Photoreactive non-natural amino acids (including but
not limited to, amino acids with benzophenone and arylazides
(including but not limited to, phenylazide) side chains), for
example, allow for efficient in vivo and in vitro photocrosslinking
of polypeptides. Examples of photoreactive non-natural amino acids
include, but are not limited to, p-azido-phenylalanine and
p-benzoyl-phenylalanine. The polypeptide with the photoreactive
non-natural amino acids may then be crosslinked at will by
excitation of the photoreactive group-providing temporal control.
In a non-limiting example, the methyl group of a non-natural amino
can be substituted with an isotopically labeled, including but not
limited to, with a methyl group, as a probe of local structure and
dynamics, including but not limited to, with the use of nuclear
magnetic resonance and vibrational spectroscopy.
[0427] A. Structure and Synthesis of Non-Natural Amino Acid
Derivatives: Carbonyl, Carbonyl like, Masked Carbonyl, and
Protected Carbonyl Groups
[0428] Amino acids with an electrophilic reactive group allow for a
variety of reactions to link molecules via various chemical
reactions, including, but not limited to, nucleophilic addition
reactions. Such electrophilic reactive groups include a carbonyl-
or dicarbonyl-group (including a keto- or aldehyde group), a
carbonyl-like- or dicarbonyl-like-group (which has reactivity
similar to a carbonyl- or dicarbonyl-group and is structurally
similar to a carbonyl- or dicarbonyl-group), a masked carbonyl- or
masked dicarbonyl-group (which can be readily converted into a
carbonyl- or dicarbonyl-group), or a protected carbonyl- or
protected dicarbonyl-group (which has reactivity similar to a
carbonyl- or dicarbonyl-group upon deprotection). Such amino acids
include amino acids having the structure of Formula (XXXVII):
##STR00086##
wherein; [0429] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0430] B is optional, and when present is a
linker selected from the group consisting of lower alkylene,
substituted lower alkylene, lower alkenylene, substituted lower
alkenylene, lower heteroalkylene, substituted lower heteroalkylene,
--O--, --O-(alkylene or substituted alkylene)-, --S--,
--S-(alkylene or substituted alkylene)-, --S(O).sub.k-- where k is
1, 2, or 3, --S(O).sub.k(alkylene or substituted alkylene)-,
--C(O)--, --NS(O).sub.2--, --OS(O).sub.2--, --C(O)-(alkylene or
substituted alkylene)-, --C(S)--, --C(S)-(alkylene or substituted
alkylene)-, --N(R')--, --NR'-(alkylene or substituted alkylene)-,
--C(O)N(R')--, --CON(R')-(alkylene or substituted alkylene)-,
--CSN(R')--, --CSN(R')-(alkylene or substituted alkylene)-,
--N(R')CO-(alkylene or substituted alkylene)-, --N(R')C(O)O--,
--S(O).sub.kN(R')--, --N(R')C(O)N(R')--, --N(R')C(S)N(R')--,
--N(R')S(O).sub.kN(R')--, --N(R')--N.dbd., --C(R').dbd.N--,
--C(R').dbd.N--N(R')--, --C(R').dbd.N--N.dbd.,
--C(R').sub.2--N.dbd.N--, and --C(R').sub.2--N(R')--N(R')--, where
each R' is independently H, alkyl, or substituted alkyl; [0431] K
is
[0431] ##STR00087## [0432] R is H, alkyl, substituted alkyl,
cycloalkyl, or substituted cycloalkyl; [0433] each R'' is
independently H, alkyl, substituted alkyl, or a protecting group,
or when more than one R'' group is present, two R'' optionally form
a heterocycloalkyl; [0434] R.sub.1 is H, an amino protecting group,
resin, amino acid, polypeptide, or polynucleotide; and [0435]
R.sub.2 is OH, an ester protecting group, resin, amino acid,
polypeptide, or polynucleotide; each of R.sub.3 and R.sub.4 is
independently H, halogen, lower alkyl, or substituted lower alkyl,
or R.sub.3 and R.sub.4 or two R.sub.3 groups optionally form a
cycloalkyl or a heterocycloalkyl; [0436] or the -A-B--K--R groups
together form a bicyclic or tricyclic cycloalkyl or
heterocycloalkyl comprising at least one carbonyl group, including
a dicarbonyl group, protected carbonyl group, including a protected
dicarbonyl group, or masked carbonyl group, including a masked
dicarbonyl group; [0437] or the --K--R group together forms a
monocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising at
least one carbonyl group, including a dicarbonyl group, protected
carbonyl group, including a protected dicarbonyl group, or masked
carbonyl group, including a masked dicarbonyl group; [0438] with a
proviso that when A is phenylene and each R.sub.3 is H, B is
present; and that when A is --(CH.sub.2).sub.4-- and each R.sub.3
is H, B is not --NHC(O)(CH.sub.2CH.sub.2)--; and that when A and B
are absent and each R.sub.3 is H, R is not methyl. Such non-natural
amino acids may be in the form of a salt, or may be incorporated
into a non-natural amino acid polypeptide, polymer, polysaccharide,
or a polynucleotide and optionally post translationally
modified.
[0439] In certain embodiments, compounds of Formula (XXXVII) are
stable in aqueous solution for at least 1 month under mildly acidic
conditions. In certain embodiments, compounds of Formula (XXXVII)
are stable for at least 2 weeks under mildly acidic conditions. In
certain embodiments, compound of Formula (XXXVII) are stable for at
least 5 days under mildly acidic conditions. In certain
embodiments, such acidic conditions are pH 2 to 8.
[0440] In certain embodiments of compounds of Formula (XXXVII), B
is lower alkylene, substituted lower alkylene, --O-(alkylene or
substituted alkylene)-, --C(R').dbd.N--N(R')--, --N(R')CO--,
--C(O)--, --C(R').dbd.N--, --C(O)-(alkylene or substituted
alkylene)-, --CON(R')-(alkylene or substituted alkylene)-,
--S(alkylene or substituted alkylene)-, --S(O)(alkylene or
substituted alkylene)-, or --S(O).sub.2(alkylene or substituted
alkylene)-. In certain embodiments of compounds of Formula
(XXXVII), B is --O(CH.sub.2)--, --CH.dbd.N--, --CH.dbd.N--NH--,
--NHCH.sub.2--, --NHCO--, --C(O)--, --C(O)--(CH.sub.2)--,
--CONH--(CH.sub.2)--, --SCH.sub.2--, --S(.dbd.O)CH.sub.2--, or
--S(O).sub.2CH.sub.2--. In certain embodiments of compounds of
Formula (XXXVII), R is C.sub.1-6 alkyl or cycloalkyl. In certain
embodiments of compounds of Formula (XXXVII) R is --CH.sub.3,
--CH(CH.sub.3).sub.2, or cyclopropyl. In certain embodiments of
compounds of Formula (XXXVII), R.sub.1 is H, tert-butyloxycarbonyl
(Boc), 9-Fluorenylmethoxycarbonyl (Fmoc), N-acetyl,
tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In certain
embodiments of compounds of Formula (XXXVII), R.sub.1 is a resin,
amino acid, polypeptide, antibody, or polynucleotide. In certain
embodiments of compounds of Formula (XXXVII), R.sub.2 is OH,
O-methyl, O-ethyl, or O-t-butyl. In certain embodiments of
compounds of Formula (XXXVII), R.sub.2 is a resin, amino acid,
polypeptide, antibody, or polynucleotide. In certain embodiments of
compounds of Formula (XXXVII), R.sub.2 is a polynucleotide. In
certain embodiments of compounds of Formula (XXXVII), R.sub.2 is
ribonucleic acid (RNA).
[0441] In certain embodiments of compounds of Formula (XXXVII),
##STR00088##
is selected from the group consisting of: [0442] (i) A is
substituted lower alkylene, C.sub.4-arylene, substituted arylene,
heteroarylene, substituted heteroarylene, alkarylene, substituted
alkarylene, aralkylene, or substituted aralkylene; [0443] B is
optional, and when present is a divalent linker selected from the
group consisting of lower alkylene, substituted lower alkylene,
lower alkenylene, substituted lower alkenylene, --O--,
--O-(alkylene or substituted alkylene)-, --S--, --S(O)--,
--S(O).sub.2--, --NS(O).sub.2--, --OS(O).sub.2--, --C(O)--,
--C(O)-(alkylene or substituted alkylene)-, --C(S)--, --N(R')--,
--C(O)N(R')--, --CON(R')-(alkylene or substituted alkylene)-,
--CSN(R')--, --N(R')CO-(alkylene or substituted alkylene)-,
--N(R')C(O)O--, --N(R')C(S)--, --S(O)N(R'), --S(O).sub.2N(R'),
--N(R')C(O)N(R')--, --N(R')C(S)N(R')--, --N(R')S(O)N(R')--,
--N(R')S(O).sub.2N(R')--, --N(R')--N.dbd., --C(R').dbd.N--N(R')--,
--C(R').dbd.N--N.dbd., --C(R').sub.2--N.dbd.N--, and
--C(R').sub.2--N(R')--N(R')--; [0444] (ii) A is optional, and when
present is substituted lower alkylene, C.sub.4-arylene, substituted
arylene, heteroarylene, substituted heteroarylene, alkarylene,
substituted alkarylene, aralkylene, or substituted aralkylene;
[0445] B is a divalent linker selected from the group consisting of
lower alkylene, substituted lower alkylene, lower alkenylene,
substituted lower alkenylene, --O--, --O-(alkylene or substituted
alkylene)-, --S--, --S(O)--, --S(O).sub.2--, --NS(O).sub.2--,
--OS(O).sub.2--, --C(O)--, --C(O)-(alkylene or substituted
alkylene)-, --C(S)--, --N(R')--, --C(O)N(R')--, --CON(R')-(alkylene
or substituted alkylene)-, --CSN(R')--, --N(R')CO-(alkylene or
substituted alkylene)-, --N(R')C(O)O--, --N(R')C(S)--, --S(O)N(R'),
--S(O).sub.2N(R'), --N(R')C(O)N(R')--, --N(R')C(S)N(R')--,
--N(R')S(O)N(R')--, --N(R')S(O).sub.2N(R')--, --N(R')--N.dbd.,
--C(R').dbd.N--N(R')--, --C(R').dbd.N--N.dbd.,
--C(R').sub.2--N.dbd.N--, and --C(R').sub.2--N(R')--N(R')--; [0446]
(iii) A is lower alkylene; [0447] B is optional, and when present
is a divalent linker selected from the group consisting of lower
alkylene, substituted lower alkylene, lower alkenylene, substituted
lower alkenylene, --O--, --O-(alkylene or substituted alkylene)-,
--S--, --S(O)--, --S(O).sub.2--, --NS(O).sub.2--, --OS(O).sub.2--,
--C(O)--, --C(O)-(alkylene or substituted alkylene)-, --C(S)--,
--N(R')--, --C(O)N(R')--, --CSN(R')--, --CON(R')-(alkylene or
substituted alkylene)-, --N(R')C(O)O--, --N(R')C(S)--, --S(O)N(R'),
--S(O).sub.2N(R'), --N(R')C(O)N(R')--, --N(R')C(S)N(R')--,
--N(R')S(O)N(R')--, --N(R')S(O).sub.2N(R')--, --N(R')--N.dbd.,
--C(R').dbd.N--N(R')--, --C(R').dbd.N--N.dbd.,
--C(R').sub.2--N.dbd.N--, and --C(R').sub.2--N(R')--N(R')--; and
[0448] (iv) A is phenylene; [0449] B is a divalent linker selected
from the group consisting of lower alkylene, substituted lower
alkylene, lower alkenylene, substituted lower alkenylene, --O--,
--O-(alkylene or substituted alkylene)-, --S--, --S(O)--,
--S(O).sub.2--, --NS(O).sub.2--, --OS(O).sub.2--, --C(O)--,
--C(O)-(alkylene or substituted alkylene)-, --C(S)--, --N(R')--,
--C(O)N(R')--, --CON(R')-(alkylene or substituted alkylene)-,
--CSN(R')--, --N(R')CO-(alkylene or substituted alkylene)-,
--N(R')C(O)O--, --N(R')C(S)--, --S(O)N(R'), --S(O).sub.2N(R'),
--N(R')C(O)N(R')--, --N(R')C(S)N(R')--, --N(R')S(O)N(R')--,
--N(R')S(O).sub.2N(R')--, --N(R')--N.dbd., --C(R').dbd.N--N(R')--,
--C(R').dbd.N--N.dbd., --C(R').sub.2--N.dbd.N--, and
--C(R').sub.2--N(R')--N(R')--; [0450] K is
[0450] ##STR00089## [0451] each R' is independently H, alkyl, or
substituted alkyl; [0452] R.sub.1 is optional, and when present, is
H, an amino protecting group, resin, amino acid, polypeptide, or
polynuelcotide; and [0453] R.sub.2 is optional, and when present,
is OH, an ester protecting group, resin, amino acid, polypeptide,
or polynucleotide; and [0454] each R.sub.3 and R.sub.4 is
independently H, halogen, lower alkyl, or substituted lower alkyl;
[0455] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted
cycloalkyl;
[0456] In addition, amino acids having the structure of Formula
(XXXVIII) are included:
##STR00090##
wherein: [0457] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0458] B is optional, and when present is a
linker selected from the group consisting of lower alkylene,
substituted lower alkylene, lower alkenylene, substituted lower
alkenylene, lower heteroalkylene, substituted lower heteroalkylene,
--O--, --O-(alkylene or substituted alkylene)-, --S--,
--S-(alkylene or substituted alkylene)-, --S(O).sub.k-- where k is
1, 2, or 3, --S(O).sub.k(alkylene or substituted alkylene)-,
--C(O)--, --NS(O).sub.2--, --OS(O).sub.2--, --C(O)-(alkylene or
substituted alkylene)-, --C(S)--, --C(S)-(alkylene or substituted
alkylene)-, --N(R')--, --NR'-(alkylene or substituted alkylene)-,
--C(O)N(R')--, --CON(R')-(alkylene or substituted alkylene)-,
--CSN(R')--, --CSN(R')-(alkylene or substituted alkylene)-,
--N(R')CO-(alkylene or substituted alkylene)-, --N(R')C(O)O--,
--S(O).sub.kN(R')--, --N(R')C(O)N(R')--, --N(R')C(S)N(R')--,
--N(R')S(O).sub.kN(R')--, --N(R')--N.dbd., --C(R').dbd.N--,
--C(R').dbd.N--N(R')--, --C(R').dbd.N--N.dbd.,
--C(R').sub.2--N.dbd.N--, and --C(R').sub.2--N(R')--N(R')--, where
each R' is independently H, alkyl, or substituted alkyl; [0459] R
is H, alkyl, substituted alkyl, cycloalkyl, or substituted
cycloalkyl; [0460] R.sub.1 is H, an amino protecting group, resin,
at least one amino acid, polypeptide, or polynucleotide; and [0461]
R.sub.2 is OH, an ester protecting group, resin, at least one amino
acid, polypeptide, or polynucleotide; with a proviso that when A is
phenylene, B is present; and that when A is --(CH.sub.2).sub.4--, B
is not --NHC(O)(CH.sub.2CH.sub.2)--; and that when A and B are
absent, R is not methyl. Such non-natural amino acids may be in the
form of a salt, or may be incorporated into a non-natural amino
acid polypeptide, polymer, polysaccharide, or a polynucleotide and
optionally post translationally modified.
[0462] In addition, amino acids having the structure of Formula
(XXXIX) are included:
##STR00091##
wherein: [0463] B is a linker selected from the group consisting of
lower alkylene, substituted lower alkylene, lower alkenylene,
substituted lower alkenylene, lower heteroalkylene, substituted
lower heteroalkylene, --O--, --O-(alkylene or substituted
alkylene)-, --S--, --S-(alkylene or substituted alkylene)-,
--S(O).sub.k-- where k is 1, 2, or 3, --S(O).sub.k(alkylene or
substituted alkylene)-, --C(O)--, --NS(O).sub.2--, --OS(O).sub.2--,
--C(O)-(alkylene or substituted alkylene)-, --C(S)--,
--C(S)-(alkylene or substituted alkylene)-, --N(R')--,
--NR'-(alkylene or substituted alkylene)-, --C(O)N(R')--,
--CON(R')-(alkylene or substituted alkylene)-, --CSN(R')--,
--CSN(R')-(alkylene or substituted alkylene)-, --N(R')CO-(alkylene
or substituted alkylene)-, --N(R')C(O)O--, --S(O).sub.kN(R')--,
--N(R')C(O)N(R')--, --N(R')C(S)N(R')--, --N(R')S(O).sub.kN(R')--,
--N(R')--N.dbd., --C(R').dbd.N--, --C(R').dbd.N--N(R')--,
--C(R').dbd.N--N.dbd., --C(R').sub.2--N.dbd.N--, and
--C(R').sub.2--N(R')--N(R')--, where each R' is independently H,
alkyl, or substituted alkyl; [0464] R is H, alkyl, substituted
alkyl, cycloalkyl, or substituted cycloalkyl; [0465] R.sub.1 is H,
an amino protecting group, resin, amino acid, polypeptide, or
polynucleotide; and [0466] R.sub.2 is OH, an ester protecting
group, resin, amino acid, polypeptide, or polynucleotide; each Ra
is independently selected from the group consisting of H, halogen,
alkyl, substituted alkyl, --N(R').sub.2, --C(O).sub.kR' where k is
1, 2, or 3, --C(O)N(R').sub.2, --OR', and --S(O).sub.kR', where
each R' is independently H, alkyl, or substituted alkyl. Such
non-natural amino acids may be in the form of a salt, or may be
incorporated into a non-natural amino acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally post
translationally modified.
[0467] In addition, the following amino acids are included:
##STR00092## ##STR00093##
Such non-natural amino acids may be are optionally amino protected
group, carboxyl protected and/or in the form of a salt, or may be
incorporated into a non-natural amino acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally post
translationally modified.
[0468] In addition, the following amino acids having the structure
of Formula (XXXX) are included:
##STR00094##
wherein [0469] --NS(O).sub.2--, --OS(O).sub.2--, optional, and when
present is a linker selected from the group consisting of lower
alkylene, substituted lower alkylene, lower alkenylene, substituted
lower alkenylene, lower heteroalkylene, substituted lower
heteroalkylene, --O--, --O-(alkylene or substituted alkylene)-,
--S--, --S-(alkylene or substituted alkylene)-, --S(O).sub.k--
where k is 1, 2, or 3, --S(O).sub.k(alkylene or substituted
alkylene)-, --C(O)--, --C(O)-(alkylene or substituted alkylene)-,
--C(S)--, --C(S)-(alkylene or substituted alkylene)-, --N(R')--,
--NR'-(alkylene or substituted alkylene)-, --C(O)N(R')--,
--CON(R')-(alkylene or substituted alkylene)-, --CSN(R')--,
--CSN(R')-(alkylene or substituted alkylene)-, --N(R')CO-(alkylene
or substituted alkylene)-, --N(R')C(O)O--, --S(O).sub.kN(R')--,
--N(R')C(O)N(R')--, --N(R')C(S)N(R')--, --N(R')S(O).sub.kN(R')--,
--N(R')--N.dbd., --C(R').dbd.N--, --C(R').dbd.N--N(R')--,
--C(R').dbd.N--N.dbd., --C(R').sub.2--N.dbd.N--, and
--C(R').sub.2--N(R')--N(R')--, where each R' is independently H,
alkyl, or substituted alkyl; [0470] R is H, alkyl, substituted
alkyl, cycloalkyl, or substituted cycloalkyl; [0471] R.sub.1 is H,
an amino protecting group, resin, amino acid, polypeptide, or
polynucleotide; and [0472] R.sub.2 is OH, an ester protecting
group, resin, amino acid, polypeptide, or polynucleotide; [0473]
each R.sub.a is independently selected from the group consisting of
H, halogen, alkyl, substituted alkyl, --N(R').sub.2, --C(O).sub.kR'
where k is 1, 2, or 3, --C(O)N(R').sub.2, --OR', and
--S(O).sub.kR', where each R' is independently H, alkyl, or
substituted alkyl; and n is 0 to 8; [0474] with a proviso that when
A is --(CH.sub.2).sub.4--, B is not --NHC(O)(CH.sub.2CH.sub.2)--.
Such non-natural amino acids may be in the form of a salt, or may
be incorporated into a non-natural amino acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally post
translationally modified.
[0475] In addition, the following amino acids are included:
##STR00095##
wherein such compounds are optionally amino protected, optionally
carboxyl protected, optionally amino protected and carboxyl
protected, or a salt thereof, or may be incorporated into a
non-natural amino acid polypeptide, polymer, polysaccharide, or a
polynucleotide and optionally post translationally modified.
[0476] In addition, the following amino acids having the structure
of Formula (XXXXI) are included:
##STR00096##
wherein, [0477] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0478] B is optional, and when present is a
linker selected from the group consisting of lower alkylene,
substituted lower alkylene, lower alkenylene, substituted lower
alkenylene, lower heteroalkylene, substituted lower heteroalkylene,
--O--, --O-(alkylene or substituted alkylene)-, --S--,
--S-(alkylene or substituted alkylene)-, --S(O).sub.k-- where k is
1, 2, or 3, --S(O).sub.k(alkylene or substituted alkylene)-,
--C(O)--, --NS(O).sub.2--, --OS(O).sub.2--, --C(O)-(alkylene or
substituted alkylene)-, --C(S)--, --C(S)-(alkylene or substituted
alkylene)-, --N(R')--, --NR'-(alkylene or substituted alkylene)-,
--C(O)N(R')--, --CON(R')-(alkylene or substituted alkylene)-,
--CSN(R')--, --CSN(R')-(alkylene or substituted alkylene)-,
--N(R')CO-(alkylene or substituted alkylene)-, --N(R')C(O)O--,
--S(O).sub.kN(R')--, --N(R')C(O)N(R')--, --N(R')C(S)N(R')--,
--N(R')S(O).sub.kN(R')--, --N(R')--N.dbd., --C(R').dbd.N--,
--C(R').dbd.N--N(R')--, --C(R').dbd.N--N.dbd.,
--C(R').sub.2--N.dbd.N--, and --C(R').sub.2--N(R')--N(R')--, where
each R' is independently H, alkyl, or substituted alkyl; [0479]
R.sub.1 is H, an amino protecting group, resin, amino acid,
polypeptide, or polynucleotide; and [0480] R.sub.2 is OH, an ester
protecting group, resin, amino acid, polypeptide, or
polynucleotide. Such non-natural amino acids may be in the form of
a salt, or may be incorporated into a non-natural amino acid
polypeptide, polymer, polysaccharide, or a polynucleotide and
optionally post translationally modified.
[0481] In addition, the following amino acids having the structure
of Formula (XXXXII) are included:
##STR00097##
wherein, [0482] B is optional, and when present is a linker
selected from the group consisting of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene,
lower heteroalkylene, substituted lower heteroalkylene, --O--,
--O-(alkylene or substituted alkylene)-, --S--, --S-(alkylene or
substituted alkylene)-, --S(O).sub.k-- where k is 1, 2, or 3,
--S(O).sub.k(alkylene or substituted alkylene)-, --C(O)--,
--NS(O).sub.2--, --OS(O).sub.2--, --C(O)-(alkylene or substituted
alkylene)-, --C(S)--, --C(S)-(alkylene or substituted alkylene)-,
--N(R')--, --NR'-(alkylene or substituted alkylene)-,
--C(O)N(R')--, --CON(R')-(alkylene or substituted alkylene)-,
--CSN(R')--, --CSN(R')-(alkylene or substituted alkylene)-,
--N(R')CO-(alkylene or substituted alkylene)-, --N(R')C(O)O--,
--S(O).sub.kN(R')--, --N(R')C(O)N(R')--, --N(R')C(S)N(R')--,
--N(R')S(O).sub.kN(R')--, --N(R')--N.dbd., --C(R').dbd.N--,
--C(R').dbd.N--N(R')--, --C(R').dbd.N--N.dbd.,
--C(R').sub.2--N.dbd.N--, and --C(R').sub.2--N(R')--N(R')--, where
each R' is independently H, alkyl, or substituted alkyl; [0483] R
is H, alkyl, substituted alkyl, cycloalkyl, or substituted
cycloalkyl; [0484] R.sub.1 is H, an amino protecting group, resin,
amino acid, polypeptide, or polynucleotide; and [0485] R.sub.2 is
OH, an ester protecting group, resin, amino acid, polypeptide, or
polynucleotide; wherein each R.sub.a is independently selected from
the group consisting of H, halogen, alkyl, substituted alkyl,
--N(R').sub.2, --C(O).sub.kR' where k is 1, 2, or 3,
--C(O)N(R').sub.2, --OR', and --S(O).sub.kR', where each R' is
independently H, alkyl, or substituted alkyl. Such non-natural
amino acids may be in the form of a salt, or may be incorporated
into a non-natural amino acid polypeptide, polymer, polysaccharide,
or a polynucleotide and optionally post translationally
modified.
[0486] In addition, the following amino acids are included:
##STR00098## ##STR00099##
wherein such compounds are optionally amino protected, optionally
carboxyl protected, optionally amino protected and carboxyl
protected, or a salt thereof, or may be incorporated into a
non-natural amino acid polypeptide, polymer, polysaccharide, or a
polynucleotide and optionally post translationally modified.
[0487] In addition, the following amino acids having the structure
of Formula (XXXXIV) are included:
##STR00100##
wherein, [0488] B is optional, and when present is a linker
selected from the group consisting of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene,
lower heteroalkylene, substituted lower heteroalkylene, --O--,
--O-(alkylene or substituted alkylene)-, --S--, --S-(alkylene or
substituted alkylene)-, --S(O).sub.k-- where k is 1, 2, or 3,
--S(O).sub.k(alkylene or substituted alkylene)-, --C(O)--,
--NS(O).sub.2--, --OS(O).sub.2--, --C(O)-(alkylene or substituted
alkylene)-, --C(S)--, --C(S)-(alkylene or substituted alkylene)-,
--N(R')--, --NR'-(alkylene or substituted alkylene)-,
--C(O)N(R')--, --CON(R')-(alkylene or substituted alkylene)-,
--CSN(R')--, --CSN(R')-(alkylene or substituted alkylene)-,
--N(R')CO-(alkylene or substituted alkylene)-, --N(R')C(O)O--,
--S(O).sub.kN(R')--, --N(R')C(O)N(R')--, --N(R')C(S)N(R')--,
--N(R')S(O).sub.kN(R')--, --N(R')--N.dbd., --C(R').dbd.N--,
--C(R').dbd.N--N(R')--, --C(R').dbd.N--N.dbd.,
--C(R').sub.2--N.dbd.N--, and --C(R').sub.2--N(R')--N(R')--, where
each R' is independently H, alkyl, or substituted alkyl; [0489] R
is H, alkyl, substituted alkyl, cycloalkyl, or substituted
cycloalkyl; [0490] R.sub.1 is H, an amino protecting group, resin,
amino acid, polypeptide, or polynucleotide; and [0491] R.sub.2 is
OH, an ester protecting group, resin, amino acid, polypeptide, or
polynucleotide; [0492] each R.sub.a is independently selected from
the group consisting of H, halogen, alkyl, substituted alkyl,
--N(R').sub.2, --C(O).sub.kR' where k is 1, 2, or 3,
--C(O)N(R').sub.2, --OR', and --S(O).sub.kR', where each R' is
independently H, alkyl, or substituted alkyl; and n is 0 to 8. Such
non-natural amino acids may be in the form of a salt, or may be
incorporated into a non-natural amino acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally post
translationally modified.
[0493] In addition, the following amino acids are included:
##STR00101##
wherein such compounds are optionally amino protected, optionally
carboxyl protected, optionally amino protected and carboxyl
protected, or a salt thereof, or may be incorporated into a
non-natural amino acid polypeptide, polymer, polysaccharide, or a
polynucleotide and optionally post translationally modified.
[0494] In addition to monocarbonyl structures, the non-natural
amino acids described herein may include groups such as dicarbonyl,
dicarbonyl like, masked dicarbonyl and protected dicarbonyl
groups.
For example, the following amino acids having the structure of
Formula (XXXXV) are included:
##STR00102##
wherein, [0495] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0496] B is optional, and when present is a
linker selected from the group consisting of lower alkylene,
substituted lower alkylene, lower alkenylene, substituted lower
alkenylene, lower heteroalkylene, substituted lower heteroalkylene,
--O--, --O-(alkylene or substituted alkylene)-, --S--,
--S-(alkylene or substituted alkylene)-, --S(O).sub.k-- where k is
1, 2, or 3, --S(O).sub.k(alkylene or substituted alkylene)-,
--C(O)--, --NS(O).sub.2--, --OS(O).sub.2--, --C(O)-(alkylene or
substituted alkylene)-, --C(S)--, --C(S)-(alkylene or substituted
alkylene)-, --N(R')--, --NR'-(alkylene or substituted alkylene)-,
--C(O)N(R')--, --CON(R')-(alkylene or substituted alkylene)-,
--CSN(R')--, --CSN(R')-(alkylene or substituted alkylene)-,
--N(R')CO-(alkylene or substituted alkylene)-, --N(R')C(O)O--,
--S(O).sub.kN(R')--, --N(R')C(O)N(R')--, --N(R')C(S)N(R')--,
--N(R')S(O).sub.kN(R')--, --N(R')--N.dbd., --C(R').dbd.N--,
--C(R').dbd.N--N(R')--, --C(R').dbd.N--N.dbd.,
--C(R').sub.2--N.dbd.N--, and --C(R').sub.2--N(R')--N(R')--, where
each R' is independently H, alkyl, or substituted alkyl; [0497] R
is H, alkyl, substituted alkyl, cycloalkyl, or substituted
cycloalkyl; [0498] R.sub.1 is H, an amino protecting group, resin,
amino acid, polypeptide, or polynucleotide; and [0499] R.sub.2 is
OH, an ester protecting group, resin, amino acid, polypeptide, or
polynucleotide. Such non-natural amino acids may be in the form of
a salt, or may be incorporated into a non-natural amino acid
polypeptide, polymer, polysaccharide, or a polynucleotide and
optionally post translationally modified.
[0500] In addition, the following amino acids having the structure
of Formula (XXXXVI) are included:
##STR00103##
wherein, [0501] B is optional, and when present is a linker
selected from the group consisting of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene,
lower heteroalkylene, substituted lower heteroalkylene, --O--,
--O-(alkylene or substituted alkylene)-, --S--, --S-(alkylene or
substituted alkylene)-, --S(O).sub.k-- where k is 1, 2, or 3,
--S(O).sub.k(alkylene or substituted alkylene)-, --C(O)--,
--NS(O).sub.2--, --OS(O).sub.2--, --C(O)-(alkylene or substituted
alkylene)-, --C(S)--, --C(S)-(alkylene or substituted alkylene)-,
--N(R')--, --NR'-(alkylene or substituted alkylene)-,
--C(O)N(R')--, --CON(R')-(alkylene or substituted alkylene)-,
--CSN(R')--, --CSN(R')-(alkylene or substituted alkylene)-,
--N(R')CO-(alkylene or substituted alkylene)-, --N(R')C(O)O--,
--S(O).sub.kN(R')--, --N(R')C(O)N(R')--, --N(R')C(S)N(R')--,
--N(R')S(O).sub.kN(R')--, --N(R')--N.dbd., --C(R').dbd.N--,
--C(R').dbd.N--N(R')--, --C(R').dbd.N--N.dbd.,
--C(R').sub.2--N.dbd.N--, and --C(R').sub.2--N(R')--N(R')--, where
each R' is independently H, alkyl, or substituted alkyl; [0502] R
is H, alkyl, substituted alkyl, cycloalkyl, or substituted
cycloalkyl; [0503] R.sub.1 is H, an amino protecting group, resin,
amino acid, polypeptide, or polynucleotide; and [0504] R.sub.2 is
OH, an ester protecting group, resin, amino acid, polypeptide, or
polynucleotide; wherein each R.sub.a is independently selected from
the group consisting of H, halogen, alkyl, substituted alkyl,
--N(R').sub.2, --C(O).sub.kR' where k is 1, 2, or 3,
--C(O)N(R').sub.2, --OR', and --S(O).sub.kR', where each R' is
independently H, alkyl, or substituted alkyl. Such non-natural
amino acids may be in the form of a salt, or may be incorporated
into a non-natural amino acid polypeptide, polymer, polysaccharide,
or a polynucleotide and optionally post translationally
modified.
[0505] In addition, the following amino acids are included:
##STR00104##
wherein such compounds are optionally amino protected and carboxyl
protected, or a salt thereof. Such non-natural amino acids may be
in the form of a salt, or may be incorporated into a non-natural
amino acid polypeptide, polymer, polysaccharide, or a
polynucleotide and optionally post translationally modified.
[0506] In addition, the following amino acids having the structure
of Formula (XXXXVII) are included:
##STR00105##
wherein, [0507] B is optional, and when present is a linker
selected from the group consisting of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene,
lower heteroalkylene, substituted lower heteroalkylene, --O--,
--O-(alkylene or substituted alkylene)-, --S--, --S-(alkylene or
substituted alkylene)-, --S(O).sub.k-- where k is 1, 2, or 3,
--S(O).sub.k(alkylene or substituted alkylene)-, --C(O)--,
--NS(O).sub.2--, --OS(O).sub.2--, --C(O)-(alkylene or substituted
alkylene)-, --C(S)--, --C(S)-(alkylene or substituted alkylene)-,
--N(R')--, --NR'-(alkylene or substituted alkylene)-,
--C(O)N(R')--, --CON(R')-(alkylene or substituted alkylene)-,
--CSN(R')--, --CSN(R')-(alkylene or substituted alkylene)-,
--N(R')CO-(alkylene or substituted alkylene)-, --N(R')C(O)O--,
--S(O).sub.kN(R')--, --N(R')C(O)N(R')--, --N(R')C(S)N(R')--,
--N(R')S(O).sub.kN(R')--, --N(R')--N.dbd., --C(R').dbd.N--,
--C(R').dbd.N--N(R')--, --C(R').dbd.N--N.dbd.,
--C(R').sub.2--N.dbd.N--, and --C(R').sub.2--N(R')--N(R')--, where
each R' is independently H, alkyl, or substituted alkyl; [0508] R
is H, alkyl, substituted alkyl, cycloalkyl, or substituted
cycloalkyl; [0509] R.sub.1 is H, an amino protecting group, resin,
amino acid, polypeptide, or polynucleotide; and [0510] R.sub.2 is
OH, an ester protecting group, resin, amino acid, polypeptide, or
polynucleotide; each R.sub.a is independently selected from the
group consisting of H, halogen, alkyl, substituted alkyl,
--N(R').sub.2, --C(O).sub.kR' where k is 1, 2, or 3,
--C(O)N(R').sub.2, --OR', and --S(O).sub.kR', where each R' is
independently H, alkyl, or substituted alkyl; and n is 0 to 8. Such
non-natural amino acids may be in the form of a salt, or may be
incorporated into a non-natural amino acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally post
translationally modified.
[0511] In addition, the following amino acids are included:
##STR00106##
wherein such compounds are optionally amino protected and carboxyl
protected, or a salt thereof, or may be incorporated into a
non-natural amino acid polypeptide, polymer, polysaccharide, or a
polynucleotide and optionally post translationally modified.
[0512] In addition, the following amino acids having the structure
of Formula (XXXXVIII) are included:
##STR00107##
wherein: [0513] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0514] R is H, alkyl, substituted alkyl,
cycloalkyl, or substituted cycloalkyl; [0515] R.sub.1 is H, an
amino protecting group, resin, amino acid, polypeptide, or
polynucleotide; and [0516] R.sub.2 is OH, an ester protecting
group, resin, amino acid, polypeptide, or polynucleotide; [0517]
X.sub.1 is C, S, or S(O); and L is alkylene, substituted alkylene,
N(R')(alkylene) or N(R')(substituted alkylene), where R' is H,
alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
Such non-natural amino acids may be in the form of a salt, or may
be incorporated into a non-natural amino acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally post
translationally modified.
[0518] In addition, the following amino acids having the structure
of Formula (XXXXIX) are included:
##STR00108##
wherein: [0519] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0520] R is H, alkyl, substituted alkyl,
cycloalkyl, or substituted cycloalkyl; [0521] R.sub.1 is H, an
amino protecting group, resin, amino acid, polypeptide, or
polynucleotide; and [0522] R.sub.2 is OH, an ester protecting
group, resin, amino acid, polypeptide, or polynucleotide; [0523] L
is alkylene, substituted alkylene, N(R')(alkylene) or
N(R')(substituted alkylene), where R' is H, alkyl, substituted
alkyl, cycloalkyl, or substituted cycloalkyl. Such non-natural
amino acids may be in the form of a salt, or may be incorporated
into a non-natural amino acid polypeptide, polymer, polysaccharide,
or a polynucleotide and optionally post translationally
modified.
[0524] In addition, the following amino acids having the structure
of Formula (XXXXX) are included:
##STR00109##
wherein: [0525] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0526] R is H, alkyl, substituted alkyl,
cycloalkyl, or substituted cycloalkyl; [0527] R.sub.1 is H, an
amino protecting group, resin, amino acid, polypeptide, or
polynucleotide; and [0528] R.sub.2 is OH, an ester protecting
group, resin, amino acid, polypeptide, or polynucleotide; [0529] L
is alkylene, substituted alkylene, N(R')(alkylene) or
N(R')(substituted alkylene), where R' is H, alkyl, substituted
alkyl, cycloalkyl, or substituted cycloalkyl. Such non-natural
amino acids may be in the form of a salt, or may be incorporated
into a non-natural amino acid polypeptide, polymer, polysaccharide,
or a polynucleotide and optionally post translationally
modified.
[0530] In addition, the following amino acids having the structure
of Formula (XXXXXI) are included:
##STR00110##
wherein: [0531] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0532] R is H, alkyl, substituted alkyl,
cycloalkyl, or substituted cycloalkyl; [0533] R.sub.1 is H, an
amino protecting group, resin, amino acid, polypeptide, or
polynucleotide; and [0534] R.sub.2 is OH, an ester protecting
group, resin, amino acid, polypeptide, or polynucleotide; [0535]
X.sub.1 is C, S, or S(O); and n is 0, 1, 2, 3, 4, or 5; and each
R.sub.8 and R.sup.9 on each CR.sup.8R.sup.9 group is independently
selected from the group consisting of H, alkoxy, alkylamine,
halogen, alkyl, aryl, or any R.sup.8 and R.sup.9 can together form
.dbd.O or a cycloalkyl, or any to adjacent R.sup.8 groups can
together form a cycloalkyl. Such non-natural amino acids may be in
the form of a salt, or may be incorporated into a non-natural amino
acid polypeptide, polymer, polysaccharide, or a polynucleotide and
optionally post translationally modified.
[0536] In addition, the following amino acids having the structure
of Formula (XXXXXII) are included:
##STR00111##
wherein: [0537] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0538] R is H, alkyl, substituted alkyl,
cycloalkyl, or substituted cycloalkyl; [0539] R.sub.1 is H, an
amino protecting group, resin, amino acid, polypeptide, or
polynucleotide; and [0540] R.sub.2 is OH, an ester protecting
group, resin, amino acid, polypeptide, or polynucleotide; [0541] n
is 0, 1, 2, 3, 4, or 5; and each R.sup.8 and R.sup.9 on each
CR.sup.8R.sup.9 group is independently selected from the group
consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any
R.sup.8 and R.sub.9 can together form .dbd.O or a cycloalkyl, or
any to adjacent R.sup.8 groups can together form a cycloalkyl. Such
non-natural amino acids may be in the form of a salt, or may be
incorporated into a non-natural amino acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally post
translationally modified.
[0542] In addition, the following amino acids having the structure
of Formula (XXXXXIII) are included:
##STR00112##
wherein: [0543] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0544] R is H, alkyl, substituted alkyl,
cycloalkyl, or substituted cycloalkyl; [0545] R.sub.1 is H, an
amino protecting group, resin, amino acid, polypeptide, or
polynucleotide; and [0546] R.sub.2 is OH, an ester protecting
group, resin, amino acid, polypeptide, or polynucleotide; [0547] n
is 0, 1, 2, 3, 4, or 5; and each R.sup.8 and R.sup.9 on each
CR.sup.8R.sup.9 group is independently selected from the group
consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any
R.sup.8 and R.sup.9 can together form .dbd.O or a cycloalkyl, or
any to adjacent R.sup.8 groups can together form a cycloalkyl. Such
non-natural amino acids may be in the form of a salt, or may be
incorporated into a non-natural amino acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally post
translationally modified.
[0548] In addition, the following amino acids having the structure
of Formula (XXXXXIV) are included:
##STR00113##
wherein: [0549] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0550] R is H, alkyl, substituted alkyl,
cycloalkyl, or substituted cycloalkyl; [0551] R.sub.1 is H, an
amino protecting group, resin, amino acid, polypeptide, or
polynucleotide; and [0552] R.sub.2 is OH, an ester protecting
group, resin, amino acid, polypeptide, or polynucleotide; [0553]
X.sub.1 is C, S, or S(O); and L is alkylene, substituted alkylene,
N(R')(alkylene) or N(R')(substituted alkylene), where R' is H,
alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
Such non-natural amino acids may be in the form of a salt, or may
be incorporated into a non-natural amino acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally post
translationally modified.
[0554] In addition, the following amino acids having the structure
of Formula (XXXXXV) are included:
##STR00114## [0555] wherein: [0556] A is optional, and when present
is lower alkylene, substituted lower alkylene, lower cycloalkylene,
substituted lower cycloalkylene, lower alkenylene, substituted
lower alkenylene, alkynylene, lower heteroalkylene, substituted
heteroalkylene, lower heterocycloalkylene, substituted lower
heterocycloalkylene, arylene, substituted arylene, heteroarylene,
substituted heteroarylene, alkarylene, substituted alkarylene,
aralkylene, or substituted aralkylene; [0557] R is H, alkyl,
substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0558]
R.sub.1 is H, an amino protecting group, resin, amino acid,
polypeptide, or polynucleotide; and [0559] R.sub.2 is OH, an ester
protecting group, resin, amino acid, polypeptide, or
polynucleotide; [0560] L is alkylene, substituted alkylene,
N(R')(alkylene) or N(R')(substituted alkylene), where R' is H,
alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
Such non-natural amino acids may be in the form of a salt, or may
be incorporated into a non-natural amino acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally post
translationally modified.
[0561] In addition, the following amino acids having the structure
of Formula (XXXXXVI) are included:
##STR00115##
wherein: [0562] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0563] R is H, alkyl, substituted alkyl,
cycloalkyl, or substituted cycloalkyl; [0564] R.sub.1 is H, an
amino protecting group, resin, amino acid, polypeptide, or
polynucleotide; and [0565] R.sub.2 is OH, an ester protecting
group, resin, amino acid, polypeptide, or polynucleotide; [0566] L
is alkylene, substituted alkylene, N(R')(alkylene) or
N(R')(substituted alkylene), where R' is H, alkyl, substituted
alkyl, cycloalkyl, or substituted cycloalkyl. Such non-natural
amino acids may be in the form of a salt, or may be incorporated
into a non-natural amino acid polypeptide, polymer, polysaccharide,
or a polynucleotide and optionally post translationally
modified.
[0567] In addition, amino acids having the structure of Formula
(XXXXXVII) are included:
##STR00116##
wherein: [0568] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0569] M is --C(R.sub.3)--,
##STR00117##
[0569] where (a) indicates bonding to the A group and (b) indicates
bonding to respective carbonyl groups, R.sub.3 and R.sub.4 are
independently chosen from H.sub.1, halogen, alkyl, substituted
alkyl, cycloalkyl, or substituted cycloalkyl, or R.sub.3 and
R.sub.4 or two R.sub.3 groups or two R.sub.4 groups optionally form
a cycloalkyl or a heterocycloalkyl; [0570] R is H, halogen, alkyl,
substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0571]
T.sub.3 is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl,
substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0572]
R.sub.1 is H, an amino protecting group, resin, amino acid,
polypeptide, or polynucleotide; and [0573] R.sub.2 is OH, an ester
protecting group, resin, amino acid, polypeptide, or
polynucleotide. Such non-natural amino acids may be in the form of
a salt, or may be incorporated into a non-natural amino acid
polypeptide, polymer, polysaccharide, or a polynucleotide and
optionally post translationally modified.
[0574] In addition, amino acids having the structure of Formula
(XXXXXVIII) are included:
##STR00118##
wherein: [0575] M is --C(R.sub.3)--,
##STR00119##
[0575] where (a) indicates bonding to the A group and (b) indicates
bonding to respective carbonyl groups, R.sub.3 and R.sub.4 are
independently chosen from H, halogen, alkyl, substituted alkyl,
cycloalkyl, or substituted cycloalkyl, or R.sub.3 and R.sub.4 or
two R.sub.3 groups or two R.sub.4 groups optionally form a
cycloalkyl or a heterocycloalkyl; [0576] R is H, halogen, alkyl,
substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0577]
T.sub.3 is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl,
substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0578]
R.sub.1 is H, an amino protecting group, resin, amino acid,
polypeptide, or polynucleotide; and [0579] R.sub.2 is OH, an ester
protecting group, resin, amino acid, polypeptide, or
polynucleotide; each R.sub.a is independently selected from the
group consisting of H, halogen, alkyl, substituted alkyl,
--N(R').sub.2, --C(O).sub.kR' where k is 1, 2, or 3,
--C(O)N(R').sub.2, --OR', and --S(O).sub.kR', where each R' is
independently H, alkyl, or substituted alkyl. Such non-natural
amino acids may be in the form of a salt, or may be incorporated
into a non-natural amino acid polypeptide, polymer, polysaccharide,
or a polynucleotide and optionally post translationally
modified.
[0580] In addition, amino acids having the structure of Formula
(XXXXXIX) are included:
##STR00120##
wherein: [0581] R is H, halogen, alkyl, substituted alkyl,
cycloalkyl, or substituted cycloalkyl; and [0582] T.sub.3 is O, or
S. Such non-natural amino acids may be in the form of a salt, or
may be incorporated into a non-natural amino acid polypeptide,
polymer, polysaccharide, or a polynucleotide and optionally post
translationally modified.
[0583] In addition, amino acids having the structure of Formula
(XXXXXX) are included:
##STR00121##
wherein: [0584] R is H, halogen, alkyl, substituted alkyl,
cycloalkyl, or substituted cycloalkyl.
[0585] In addition, the following amino acids having structures of
Formula (XXXXXX) are included:
##STR00122##
Such non-natural amino acids may be in the form of a salt, or may
be incorporated into a non-natural amino acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally post
translationally modified.
[0586] The carbonyl or dicarbonyl functionality can be reacted
selectively with a hydroxylamine-containing reagent under mild
conditions in aqueous solution to form the corresponding oxime
linkage that is stable under physiological conditions. See, e.g.,
Jencks, W. P., J. Am. Chem. Soc. 81, 475-481 (1959); Shao, J. and
Tam, J. P., J. Am. Chem. Soc. 117(14):3893-3899 (1995). Moreover,
the unique reactivity of the carbonyl or dicarbonyl group allows
for selective modification in the presence of the other amino acid
side chains. See, e.g., Cornish, V. W., et al., J. Am. Chem. Soc.
118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G.,
Bioconjug. Chem. 3:138-146 (1992); Mahal, L. K., et al., Science
276:1125-1128 (1997).
[0587] The synthesis of p-acetyl-(+/-)-phenylalanine and
m-acetyl-(+/-)-phenylalanine is described in Zhang, Z., et al.,
Biochemistry 42: 6735-6746 (2003), incorporated by reference. Other
carbonyl- or dicarbonyl-containing amino acids can be similarly
prepared.
[0588] In some embodiments, a polypeptide comprising a non-natural
amino acid is chemically modified to generate a reactive carbonyl
or dicarbonyl functional group. For instance, an aldehyde
functionality useful for conjugation reactions can be generated
from a functionality having adjacent amino and hydroxyl groups.
Where the biologically active molecule is a polypeptide, for
example, an N-terminal serine or threonine (which may be normally
present or may be exposed via chemical or enzymatic digestion) can
be used to generate an aldehyde functionality under mild oxidative
cleavage conditions using periodate. See, e.g., Gaertner, et. al.,
Bioconjug. Chem. 3: 262-268 (1992); Geoghegan, K. & Stroh, J.,
Bioconjug. Chem. 3:138-146 (1992); Gaertner et al., J. Biol. Chem.
269:7224-7230 (1994). However, methods known in the art are
restricted to the amino acid at the N-terminus of the peptide or
protein.
[0589] Additionally, by way of example a non-natural amino acid
bearing adjacent hydroxyl and amino groups can be incorporated into
a polypeptide as a "masked" aldehyde functionality. For example,
5-hydroxylysine bears a hydroxyl group adjacent to the epsilon
amine. Reaction conditions for generating the aldehyde typically
involve addition of molar excess of sodium metaperiodate under mild
conditions to avoid oxidation at other sites within the
polypeptide. The pH of the oxidation reaction is typically about
7.0. A typical reaction involves the addition of about 1.5 molar
excess of sodium meta periodate to a buffered solution of the
polypeptide, followed by incubation for about 10 minutes in the
dark. See, e.g. U.S. Pat. No. 6,423,685.
[0590] B. Structure and Synthesis of Non-Natural Amino Acids;
Dicarbonyl, Dicarbonyl-like, Masked Dicarbonyl, and Protected
Dicarbonyl Groups
[0591] Amino acids with an electrophilic reactive group allow for a
variety of reactions to link molecules via nucleophilic addition
reactions among others. Such electrophilic reactive groups include
a dicarbonyl group (including a diketone group, a ketoaldehyde
group, a ketoacid group, a ketoester group, and a ketothioester
group), a dicarbonyl-like group (which has reactivity similar to a
dicarbonyl group and is structurally similar to a dicarbonyl
group), a masked dicarbonyl group (which can be readily converted
into a dicarbonyl group), or a protected dicarbonyl group (which
has reactivity similar to a dicarbonyl group upon deprotection).
Such amino acids include amino acids having the structure of
Formula (XXXVII):
##STR00123##
wherein: [0592] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0593] B is optional, and when present is a
linker linked at one end to a diamine containing moiety, the linker
selected from the group consisting of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene,
lower heteroalkylene, substituted lower heteroalkylene,
--O-(alkylene or substituted alkylene)-, --S-(alkylene or
substituted alkylene)-, --C(O)R''--, --S(O).sub.k(alkylene or
substituted alkylene)-, where k is 1, 2, or 3, --C(O)-(alkylene or
substituted alkylene)-, --C(S)-(alkylene or substituted alkylene)-,
--NR''-(alkylene or substituted alkylene)-, --CON(R'')-(alkylene or
substituted alkylene)-, --CSN(R'')-(alkylene or substituted
alkylene)-, and --N(R'')CO-(alkylene or substituted alkylene)-,
where each R'' is independently H, alkyl, or substituted alkyl;
[0594] K is
##STR00124##
[0594] where, [0595] T.sub.1 is a bond, optionally substituted
C.sub.1-C.sub.4 alkylene, optionally substituted C.sub.1-C.sub.4
alkenylene, or optionally substituted heteroalkyl; [0596] wherein
each optional substituents is independently selected from lower
alkylene, substituted lower alkylene, lower cycloalkylene,
substituted lower cycloalkylene, lower alkenylene, substituted
lower alkenylene, alkynylene, lower heteroalkylene, substituted
heteroalkylene, lower heterocycloalkylene, substituted lower
heterocycloalkylene, arylene, substituted arylene, heteroarylene,
substituted heteroarylene, alkarylene, substituted alkarylene,
aralkylene, or substituted aralkylene; [0597] T.sub.2, is selected
from the group consisting of lower alkylene, substituted lower
alkylene, lower alkenylene, substituted lower alkenylene, lower
heteroalkylene, substituted lower heteroalkylene, --O--,
--O-(alkylene or substituted alkylene)-, --S--, --S-(alkylene or
substituted alkylene)-, --S(O).sub.k-- where k is 1, 2, or 3,
--S(O).sub.k(alkylene or substituted alkylene)-, --C(O)--,
--C(O)-(alkylene or substituted alkylene)-, --C(S)--,
--C(S)-(alkylene or substituted alkylene)-, --N(R')--,
--NR'-(alkylene or substituted alkylene)-, --C(O)N(R')--,
--CON(R')-(alkylene or substituted alkylene)-, --CSN(R')--,
--CSN(R')-(alkylene or substituted alkylene)-, --N(R')CO-(alkylene
or substituted alkylene)-, --N(R')C(O)O--, --S(O).sub.kN(R')--,
--N(R')C(O)N(R')--, --N(R')C(S)N(R')--, --N(R')S(O).sub.kN(R')--,
--N(R')--N.dbd., --C(R').dbd.N--, --C(R').dbd.N--N(R')--,
--C(R').dbd.N--N.dbd., --C(R').sub.2--N.dbd.N--, and
--C(R').sub.2--N(R')--N(R')--, where each R' is independently H,
alkyl, or substituted alkyl; [0598] T.sub.3 is
##STR00125##
[0598] where each X.sub.1 is independently selected from the group
consisting of --O--, --S--, --N(H)--, --N(R)--, --N(Ac)--, and
--N(OMe)-; X.sub.2 is --OR, --OAc, --SR, --N(R).sub.2, --N(R)(Ac),
--N(R)(OMe), or N.sub.3, and where each R' is independently H,
alkyl, or substituted alkyl; [0599] R is H, halogen, alkyl,
substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0600]
R.sub.1 is H, an amino protecting group, resin, amino acid,
polypeptide, or polynucleotide; and [0601] R.sub.2 is OH, an ester
protecting group, resin, amino acid, polypeptide, or
polynucleotide; [0602] or the -A-B--K--R groups together form a
bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at
least one carbonyl group, including a dicarbonyl group, protected
carbonyl group, including a protected dicarbonyl group, or masked
carbonyl group, including a masked dicarbonyl group; [0603] or the
--K--R group together forms a monocyclic or bicyclic cycloalkyl or
heterocycloalkyl comprising at least one carbonyl group, including
a dicarbonyl group, protected carbonyl group, including a protected
dicarbonyl group, or masked carbonyl group, including a masked
dicarbonyl group.
[0604] Non-limiting example of dicarbonyl amino acids having the
structure of Formula (XXXVII) include:
##STR00126## ##STR00127## ##STR00128## ##STR00129##
[0605] The following amino acids having structures of Formula
(XXXVII) are also included:
##STR00130## ##STR00131## ##STR00132##
Such non-natural amino acids may be in the form of a salt, or may
be incorporated into a non-natural amino acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally post
translationally modified.
[0606] C. Structure and Synthesis of Non-Natural Amino Acids:
Ketoalkyne, Ketoalkyne -like, Masked Ketoalkyne, Protected
Ketoalkyne Group, Alkyne, and Cycloalkyne Groups
[0607] Amino acids containing reactive groups with dicarbonyl-like
reactivity allow for the linking of molecules via nucleophilic
addition reactions. Such electrophilic reactive groups include a
ketoalkyne group, a ketoalkyne-like group (which has reactivity
similar to a ketoalkyne group and is structurally similar to a
ketoalkyne group), a masked ketoalkyne group (which can be readily
converted into a ketoalkyne group), or a protected ketoalkyne group
(which has reactivity similar to a ketoalkyne group upon
deprotection). In some embodiments, amino acids containing reactive
groups with a terminal alkyne, internal alkyne or cycloalkyne allow
for linking of molecules via cycloaddition reactions (e.g.,
1,3-dipolar cycloadditions, azide-alkyne Huisgen cycloaddition,
etc.) Such amino acids include amino acids having the structure of
Formula (XXXXXXI-A) or (XXXXXXI-B):
##STR00133##
wherein: [0608] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0609] B is optional, and when present is a
linker linked at one end to a diamine containing moiety, the linker
selected from the group consisting of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene,
lower heteroalkylene, substituted lower heteroalkylene,
--O-(alkylene or substituted alkylene)-, --S-(alkylene or
substituted alkylene)-, --C(O)R''--, --S(O).sub.k(alkylene or
substituted alkylene)-, where k is 1, 2, or 3, --C(O)-(alkylene or
substituted alkylene)-, --C(S)-(alkylene or substituted alkylene)-,
--NR''-(alkylene or substituted alkylene)-, --CON(R'')-(alkylene or
substituted alkylene)-, --CSN(R'')-(alkylene or substituted
alkylene)-, and --N(R'')CO-(alkylene or substituted alkylene)-,
where each R'' is independently H, alkyl, or substituted alkyl;
[0610] G is optional, and when present is
[0610] ##STR00134## [0611] T.sub.4 is a carbonyl protecting group
including, but not limited to,
##STR00135##
[0611] where each X.sub.1 is independently selected from the group
consisting of --O--, --S--, --N(H)--, --N(R)--, --N(Ac)--, and
--N(OMe)-; X.sub.2 is --OR, --OAc, --SR, --N(R).sub.2, --N(R)(Ac),
--N(R)(OMe), or N.sub.3, and where each R' is independently H,
alkyl, or substituted alkyl; [0612] R is H, halogen, alkyl,
substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0613]
R.sub.1 is H, an amino protecting group, resin, amino acid,
polypeptide, or polynucleotide; [0614] R.sub.2 is OH, an ester
protecting group, resin, amino acid, polypeptide, or
polynucleotide; [0615] each of R.sub.3 and R.sub.4 is independently
H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and
R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a
heterocycloalkyl; [0616] each R.sub.19 is independently selected
from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkoxy, ester, ether, thioether, aminoalkyl, halogen, alkyl ester,
aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl
sulfonic acid, alkyl nitro, thioester, sulfonyl ester,
halosulfonyl, nitrile, alkyl nitrile, and nitro; and [0617] q is 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.
[0618] D. Structure and Synthesis of Non-Natural Amino Acids:
Ketoamine, Ketoamine-like, Masked Ketoamine, and Protected
Ketoamine Groups
[0619] Amino acids containing reactive groups with dicarbonyl-like
reactivity allow for the linking of molecules via nucleophilic
addition reactions. Such reactive groups include a ketoamine group,
a ketoamine-like group (which has reactivity similar to a ketoamine
group and is structurally similar to a ketoamine group), a masked
ketoamine group (which can be readily converted into a ketoamine
group), or a protected ketoamine group (which has reactivity
similar to a ketoamine group upon deprotection), Such amino acids
include amino acids having the structure of Formula (XXXXXXII):
##STR00136##
wherein: [0620] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0621] B is optional, and when present is a
linker linked at one end to a diamine containing moiety, the linker
selected from the group consisting of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene,
lower heteroalkylene, substituted lower heteroalkylene,
--O-(alkylene or substituted alkylene)-, --S-(alkylene or
substituted alkylene)-, --C(O)R''--, --S(O).sub.k(alkylene or
substituted alkylene)-, where k is 1, 2, or 3, --C(O)-(alkylene or
substituted alkylene)-, --C(S)-(alkylene or substituted alkylene)-,
--NR''-(alkylene or substituted alkylene)-, --CON(R'')-(alkylene or
substituted alkylene)-, --CSN(R'')-(alkylene or substituted
alkylene)-, and --N(R'')CO-(alkylene or substituted alkylene)-,
where each R'' is independently H, alkyl, or substituted alkyl;
[0622] G is
[0622] ##STR00137## [0623] T.sub.1 is an optionally substituted
C.sub.1-C.sub.4 alkylene, an optionally substituted C.sub.1-C.sub.4
alkenylene, or an optionally substituted heteroalkyl; [0624]
T.sub.4 is a carbonyl protecting group including, but not limited
to,
##STR00138##
[0624] where each X.sub.1 is independently selected from the group
consisting of --O--, --S--, --N(H)--, --N(R')--, --N(Ac)--, and
--N(OMe)-; X.sub.2 is --OR, --OAc, --SR', --N(R').sub.2,
--N(R')(Ac), --N(R')(OMe), or N3, and where each R' is
independently H, alkyl, or substituted alkyl; [0625] R is H,
halogen, alkyl, substituted alkyl, cycloalkyl, or substituted
cycloalkyl; [0626] R.sub.1 is H, an amino protecting group, resin,
amino acid, polypeptide, or polynucleotide; and [0627] R.sub.2 is
OH, an ester protecting group, resin, amino acid, polypeptide, or
polynucleotide; [0628] each of R.sub.3 and R.sub.4 is independently
H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and
R.sub.4 or two i.sub.3 groups optionally form a cycloalkyl or a
heterocycloalkyl.
[0629] Amino acids having the structure of Formula (XXXXXXII)
include amino acids having the structure of Formula (XXXXXXIII) and
Formula (XXXXXXIV):
##STR00139## [0630] wherein each R.sub.a is independently selected
from the group consisting of H, halogen, alkyl, substituted alkyl,
--N(R').sub.2, --C(O).sub.kR' where k is 1, 2, or 3,
--C(O)N(R').sub.2, --OR', and --S(O).sub.kR', where each R' is
independently H, alkyl, or substituted alkyl.
[0631] E. Structure and Synthesis of Non-Natural Amino Acids:
Diamine, Diamine-like, Masked Diamine, Protected Amines and
Azides
[0632] Amino acids with a nucleophilic reactive group allow for a
variety of reactions to link molecules via electrophilic addition
reactions among others. Such nucleophilic reactive groups include a
diamine group (including a hydrazine group, an amidine group, an
imine group, a 1,1-diamine group, a 1,2-diamine group, a
1,3-diamine group, and a 1,4-diamine group), a diamine-like group
(which has reactivity similar to a diamine group and is
structurally similar to a diamine group), a masked diamine group
(which can be readily converted into a diamine group), or a
protected diamine group (which has reactivity similar to a diamine
group upon deprotection). In some embodiments, amino acids
containing reactive groups with azides allow for linking of
molecules via cycloaddition reactions (e.g., 1,3-dipolar
cycloadditions, azide-alkyne Huisgen cycloaddition, etc.).
[0633] In another aspect are methods for the chemical synthesis of
hydrazine-substituted molecules for the derivatization of
carbonyl-substituted dolastatin derivatives. In one embodiment, the
hydrazine-substituted molecule can dolastatin linked derivatives.
In one embodiment are methods for the preparation of
hydrazine-substituted molecules suitable for the derivatization of
carbonyl-containing non-natural amino acid polypeptides, including
by way of example only, ketone-, or aldehyde-containing non-natural
amino acid polypeptides. In a further or additional embodiment, the
non-natural amino acids are incorporated site-specifically during
the in vivo translation of proteins. In a further or additional
embodiment, the hydrazine-substituted dolastatin derivatives allow
for the site-specific derivatization of carbonyl-containing
non-natural amino acids via nucleophilic attack of each carbonyl
group to form a heterocycle-derivatized polypeptide, including a
nitrogen-containing heterocycle-derivatized polypeptide in a
site-specific fashion. In a further or additional embodiment, the
method for the preparation of hydrazine-substituted dolastatin
derivatives provides access to a wide variety of site-specifically
derivatized polypeptides. In a further or additional embodiment are
methods for synthesizing hydrazine-functionalized
polyethyleneglycol (PEG) linked dolastatin derivatives.
[0634] Such amino acids include amino acids having the structure of
Formula (XXXVII-A) or (XXXVII-B):
##STR00140##
wherein: [0635] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0636] B is optional, and when present is a
linker linked at one end to a diamine containing moiety, the linker
selected from the group consisting of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene,
lower heteroalkylene, substituted lower heteroalkylene,
--O-(alkylene or substituted alkylene)-, --S-(alkylene or
substituted alkylene)-, --C(O)R''--, --C(O)R''--,
--S(O).sub.k(alkylene or substituted alkylene)-, where k is 1, 2,
or 3, --C(O)-(alkylene or substituted alkylene)-, --C(S)-(alkylene
or substituted alkylene)-, --NR''-(alkylene or substituted
alkylene)-, --CON(R'')-(alkylene or substituted alkylene)-,
--CSN(R'')-(alkylene or substituted alkylene)-, and
--N(R'')CO-(alkylene or substituted alkylene)-, where each R'' is
independently H, alkyl, or substituted alkyl; [0637] K is
[0637] ##STR00141## where: [0638] R.sub.8 and R.sub.9 are
independently selected from H, alkyl, substituted alkyl,
cycloalkyl, substituted cycloalkyl, or amine protecting group;
[0639] T.sub.1 is a bond, optionally substituted C.sub.1-C.sub.4
alkylene, optionally substituted C.sub.1-C.sub.4 alkenylene, or
optionally substituted heteroalkyl; [0640] T.sub.2 is optionally
substituted C.sub.1-C.sub.4 alkylene, optionally substituted
C.sub.1-C.sub.4 alkenylene, optionally substituted heteroalkyl,
optionally substituted aryl, or optionally substituted heteroaryl;
[0641] wherein each optional substituents is independently selected
from lower alkyl, substituted lower alkyl, lower cycloalkyl,
substituted lower cycloalkyl, lower alkenyl, substituted lower
alkenyl, alkynyl, lower heteroalkyl, substituted heteroalkyl, lower
heterocycloalkyl, substituted lower heterocycloalkyl, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, alkaryl,
substituted alkaryl, aralkyl, or substituted aralkyl; [0642] R is
H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
[0643] R.sub.1 is H, an amino protecting group, resin, amino acid,
polypeptide, or polynucleotide; and [0644] R.sub.2 is OH, an ester
protecting group, resin, amino acid, polypeptide, or
polynucleotide; [0645] each of R.sub.3 and R.sub.4 is independently
H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and
R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a
heterocycloalkyl; [0646] or the -A-B--K--R groups together form a
bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at
least one diamine group, protected diamine group or masked diamine
group; [0647] or the --B--K--R groups together form a bicyclic or
tricyclic cycloalkyl or cycloaryl or heterocycloalkyl comprising at
least one diamine group, protected diamine group or masked diamine
group; [0648] or the --K--R group together forms a monocyclic or
bicyclic cycloalkyl or heterocycloalkyl comprising at least one
diamine group, protected diamine group or masked diamine group;
wherein at least one amine group on -A-B--K--R is optionally a
protected amine.
[0649] In one aspect are compounds comprising the structures 1 or
2:
##STR00142##
wherein: [0650] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0651] B is optional, and when present is a
linker linked at one end to a diamine containing moiety, the linker
selected from the group consisting of lower alkylene, substituted
lower alkylene, lower alkenylene, substituted lower alkenylene,
lower heteroalkylene, substituted lower heteroalkylene,
--O-(alkylene or substituted alkylene)-, --S-(alkylene or
substituted alkylene)-, --C(O)R''--, --S(O).sub.k(alkylene or
substituted alkylene)-, where k is 1, 2, or 3, --C(O)-(alkylene or
substituted alkylene)-, --C(S)-(alkylene or substituted alkylene)-,
--NR''-(alkylene or substituted alkylene)-, --CON(R'')-(alkylene or
substituted alkylene)-, --CSN(R'')-(alkylene or substituted
alkylene)-, and --N(R'')CO-(alkylene or substituted alkylene)-,
where each R'' is independently H, alkyl, or substituted alkyl;
[0652] T.sub.1 is a bond or CH.sub.2; and T.sub.2 is CH; [0653]
wherein each optional substituents is independently selected from
lower alkyl, substituted lower alkyl, lower cycloalkyl, substituted
lower cycloalkyl, lower alkenyl, substituted lower alkenyl,
alkynyl, lower heteroalkyl, substituted heteroalkyl, lower
heterocycloalkyl, substituted lower heterocycloalkyl, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, alkaryl,
substituted alkaryl, aralkyl, or substituted aralkyl; [0654]
R.sub.1 is H, an amino protecting group, resin, amino acid,
polypeptide, or polynucleotide; and [0655] R.sub.2 is OH, an ester
protecting group, resin, amino acid, polypeptide, or
polynucleotide; [0656] each of R.sub.3 and R.sub.4 is independently
H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and
R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a
heterocycloalkyl; [0657] or the -A-B-diamine containing moiety
together form a bicyclic cycloalkyl or heterocycloalkyl comprising
at least one diamine group, protected diamine group or masked
diamine group; [0658] or the --B-diamine containing moiety groups
together form a bicyclic or tricyclic cycloalkyl or cycloaryl or
heterocycloalkyl comprising at least one diamine group, protected
diamine group or masked diamine group; [0659] wherein at least one
amine group on -A-B-diamine containing moiety is optionally a
protected amine; [0660] or an active metabolite, salt, or a
pharmaceutically acceptable prodrug or solvate thereof.
[0661] The following non-limiting examples of amino acids having
the structure of Formula (XXXVII) are included:
##STR00143## ##STR00144##
Such non-natural amino acids may also be in the form of a salt or
may be incorporated into a non-natural amino acid polypeptide,
polymer, polysacebaride, or a polynucleotide and/or optionally post
translationally modified.
[0662] In certain embodiments, compounds of Formula (XXXVII) are
stable in aqueous solution for at least 1 month under mildly acidic
conditions. In certain embodiments, compounds of Formula (XXXVII)
are stable for at least 2 weeks under mildly acidic conditions. In
certain embodiments, compound of Formula (XXXVII) are stable for at
least 5 days under mildly acidic conditions. In certain
embodiments, such acidic conditions are pH about 2 to about 8.
[0663] In certain embodiments of compounds of Formula (XXXVII), B
is lower alkylene, substituted lower alkylene, O-(alkylene or
substituted alkylene)-, C(R').dbd.NN(R')--, --N(R')CO--, C(O)--,
--C(R').dbd.N--, C(O)-(alkylene or substituted alkylene)-,
CON(R')(alkylene or substituted alkylene)-, --S(alkylene or
substituted alkylene)-, --S(O)(alkylene or substituted alkylene)-,
or --S(O).sub.2(alkylene or substituted alkylene)-. In certain
embodiments of compounds of Formula (XXXVII), B is --O(CH.sub.2)--,
--CH.dbd.N--, CH.dbd.NNH--, --NHCH.sub.2--, --NHCO--, C(O)--,
C(O)(CH.sub.2)--, CONH(CH.sub.2)--, --SCH.sub.2--,
--S(.dbd.O)CH.sub.2--, or --S(O).sub.2CH.sub.2--. In certain
embodiments of compounds of Formula (XXXVII), R is C.sub.1-6 alkyl
or cycloalkyl. In certain embodiments of compounds of Formula
(XXXVII) R is --CH.sub.3, --CH(CH.sub.3).sub.2, or cyclopropyl. In
certain embodiments of compounds of Formula (XXXVII), R.sub.1 is H,
tert-butyloxycarbonyl (Boc), 9-Fluorenylmethoxycarbonyl (Fmoc),
N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In
certain embodiments of compounds of Formula (XXXVII), R.sub.1 is a
resin, amino acid, polypeptide, or polynucleotide. In certain
embodiments of compounds of Formula (XXXVII), R.sub.1 is an
antibody, antibody fragment or monoclonal antibody. In certain
embodiments of compounds of Formula (XXXVII), R.sub.2 is OH,
O-methyl, O-ethyl, or O-t-butyl. In certain embodiments of
compounds of Formula (XXXVII), R.sub.2 is a resin, at least one
amino acid, polypeptide, or polynucleotide. In certain embodiments
of compounds of Formula (XXXVII), R.sub.2 is an antibody, antibody
fragment or monoclonal antibody.
[0664] The following non-limiting examples of amino acids having
the structure of Formula (XXXVII) are also included:
##STR00145## ##STR00146## ##STR00147##
[0665] Non-limiting examples of protected amino acids having the
structure of Formula (XXXVII) include:
##STR00148##
[0666] F. Structure and Synthesis of Non-Natural Amino Acids:
Aromatic Amines
[0667] Non-natural amino acids with nucleophilic reactive groups,
such as, by way of example only, an aromatic amine group (including
secondary and tertiary amine groups), a masked aromatic amine group
(which can be readily converted into a aromatic amine group), or a
protected aromatic amine group (which has reactivity similar to an
aromatic amine group upon deprotection) allow for a variety of
reactions to link molecules via various reactions, including but
not limited to, reductive alkylation reactions with aldehyde
containing dolastatin linker derivatives. Such aromatic amine
containing non-natural amino acids include amino acids having the
structure of Formula (XXXXXXV):
##STR00149##
wherein:
##STR00150##
is selected from the group consisting of a monocyclic aryl ring, a
bicyclic aryl ring, a multicyclic aryl ring, a monocyclic
heteroaryl ring, a bicyclic heteroaryl ring, and a multicyclic
heteroaryl ring; [0668] A is independently CR.sub.a, or N; [0669] B
is independently CR.sub.a, N, O, or S; [0670] each R.sub.a is
independently selected from the group consisting of H, halogen,
alkyl, --NO.sub.2, --CN, substituted alkyl, --N(R').sub.2,
--C(O).sub.kR', --C(O)N(R').sub.2, --OR', and --S(O).sub.kR', where
k is 1, 2, or 3; and n is 0, 1, 2, 3, 4, 5, or 6; [0671] R.sub.1 is
H, an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide; and [0672] R.sub.2 is OH, an ester
protecting group, resin, at least one amino acid, polypeptide, or
polynucleotide; [0673] each of R.sub.3 and R.sub.4 is independently
H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and
R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a
heterocycloalkyl; [0674] M is H or --CH.sub.2R.sub.5; or the
M-N--C(R.sub.5) moiety may form a 4 to 7 membered ring structure;
[0675] R.sub.5 is alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,
alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide,
substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocycle, substituted heterocycle, alkaryl, substituted alkaryl,
aralkyl, substituted aralkyl, --C(O)R'', --C(O)OR'',
--C(O)N(R'').sub.2, --C(O)NHCH(R'').sub.2, -(alkylene or
substituted alkylene)-N(R'').sub.2, -(alkenylene or substituted
alkenylene)-N(R'').sub.2, -(alkylene or substituted alkylene)-(aryl
or substituted aryl), -(alkenylene or substituted alkenylene)-(aryl
or substituted aryl), -(alkylene or substituted
alkylene)-ON(R'').sub.2, -(alkylene or substituted alkylene)
C(O)SR'', -(alkylene or substituted alkylene)-S--S-(aryl or
substituted aryl), wherein each R'' is independently hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy,
substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocycle, substituted heterocycle, alkaryl,
substituted alkaryl, aralkyl, substituted aralkyl, or --C(O)OR';
[0676] or two R.sub.5 groups optionally form a cycloalkyl or a
heterocycloalkyl; [0677] or R.sub.5 and any R.sub.8 optionally form
a cycloalkyl or a heterocycloalkyl; and [0678] each R' is
independently H, alkyl, or substituted alkyl. Such non-natural
amino acids may also be in the form of a salt, or may be
incorporated into a non-natural amino acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally reductively
alkylated. The structure
##STR00151##
[0678] (as presented in all examples herein) does not present the
relative orientations of "A," "B," "NH-M" and "R.sub.a"; rather
these four features of this structure may be oriented in any
chemically-sound manner (along with other features of this
structure), as illustrated by example herein.
[0679] Non-natural amino acids containing an aromatic amine moiety
having the structure of Formula (A) include non-natural amino acids
having the structures:
##STR00152##
wherein, each A' is independently selected from CR.sub.a, N, or
##STR00153##
and [0680] up to two A' may be
##STR00154##
[0680] with the remaining A' selected from CR.sub.a, or N. Such
non-natural amino acids may also be in the form of a salt, or may
be incorporated into a non-natural amino acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally reductively
alkylated.
[0681] Non-limiting examples of non-natural amino acids containing
an aromatic amine moiety having the structure of Formula (XXXXXXV)
include non-natural amino acids having the structure of Formula
(XXXXXXVI), and Formula (XXXXXXVII),
##STR00155##
wherein; G is an amine protecting group, including, but not limited
to,
##STR00156##
Such non-natural amino acids may be in the form of a salt, or may
be incorporated into a non-natural amino acid polypeptide, polymer,
polysaccharide, or a polynucleotide and optionally reductively
alkylated.
[0682] Non-natural amino acids containing an aromatic amine moiety
have the following structures:
##STR00157## [0683] wherein each R.sub.a is independently selected
from the group consisting of H, halogen, alkyl, --NO.sub.2, --CN,
substituted alkyl, --N(R').sub.2, --C(O).sub.kR',
--C(O)N(R').sub.2, --OR', and --S(O).sub.kR', where k is 1, 2, or
3; [0684] M is H or --CH.sub.2R.sub.5; or the M-N--C(R.sub.5)
moiety may form a 4 to 7 membered ring structure; [0685] R.sub.1 is
H, an amino protecting group, resin, amino acid, polypeptide, or
polynucleotide; [0686] R.sub.2 is OH, an ester protecting group,
resin, amino acid, polypeptide, or polynucleotide; [0687] R.sub.5
is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy,
substituted alkylalkoxy, polyalkylene oxide, substituted
polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, heterocycle,
substituted heterocycle, alkaryl, substituted alkaryl, aralkyl,
substituted aralkyl, --C(O)R'', --C(O)OR'', --C(O)N(R'').sub.2,
--C(O)NHCH(R'').sub.2, -(alkylene or substituted
alkylene)-N(R'').sub.2, -(alkenylene or substituted
alkenylene)-N(R'').sub.2, -(alkylene or substituted alkylene)-(aryl
or substituted aryl), -(alkenylene or substituted alkenylene)-(aryl
or substituted aryl), -(alkylene or substituted
alkylene)-ON(R'').sub.2, -(alkylene or substituted
alkylene)-C(O)SR'', -(alkylene or substituted alkylene)-S--S-(aryl
or substituted aryl), wherein each R'' is independently hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy,
substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocycle, substituted heterocycle, alkaryl,
substituted alkaryl, aralkyl, substituted aralkyl, or --C(O)OR';
[0688] or R.sub.5 and any R.sub.8 optionally form a cycloalkyl or a
heterocycloalkyl; and [0689] each R' is independently H, alkyl, or
substituted alkyl. Such non-natural amino acids may also be in the
form of a salt, or may be incorporated into a non-natural amino
acid polypeptide, polymer, polysaccharide, or a polynucleotide.
[0690] Such non-natural amino acids of Formula (XXXXXXV) may be
formed by reduction of protected or masked amine moieties on the
aromatic moiety of a non-natural amino acid. Such protected or
masked amine moieties include, but are not limited to, imines,
hydrazines, nitro, or azide substituents. The reducing agents used
to reduce such protected or masked amine moieties include, but are
not limited to, TCEP, Na.sub.2S, Na.sub.2S.sub.2O.sub.4,
LiAlH.sub.4, NaBH.sub.4 or NaBCNH.sub.3.
V. Non-Natural Amino Acid Linked Dolastatin Derivatives
[0691] In another aspect described herein are methods, strategies
and techniques for incorporating at least one such dolastatin
linker derivatives into a non-natural amino acid. Also included
with this aspect are methods for producing, purifying,
characterizing and using such dolastatin linker derivatives
containing at least one such non-natural amino acid. Also included
with this aspect are compositions of and methods for producing,
purifying, characterizing and using oligonucleotides (including DNA
and RNA) that can be used to produce, at least in part, a
dolastatin linker derivative containing at least one non-natural
amino acid. Also included with this aspect are compositions of and
methods for producing, purifying, characterizing and using cells
that can express such oligonucleotides that can be used to produce,
at least in part, a dolastatin linker derivative containing at
least one non-natural amino acid.
[0692] Thus, dolastatin linker derivatives comprising at least one
non-natural amino acid or modified non-natural amino acid with a
carbonyl, dicarbonyl, alkyne, cycloalkyne, azide, oxime or
hydroxylamine group are provided and described herein. In certain
embodiments, dolastatin linker derivatives with at least one
non-natural amino acid or modified non-natural amino acid with a
carbonyl, dicarbonyl, alkyne, cycloalkyne, azide, oxime or
hydroxylamine group include at least one post-translational
modification at some position on the polypeptide. In some
embodiments the co-translational or post-translational modification
occurs via the cellular machinery (e.g., glycosylation,
acetylation, acylation, lipid-modification, palmitoylation,
palmitate addition, phosphorylation, glycolipid-linkage
modification, and the like), in many instances, such
cellular-machinery-based co-translational or post-translational
modifications occur at the naturally occurring amino acid sites on
the polypeptide, however, in certain embodiments, the
cellular-machinery-based co-translational or post-translational
modifications occur on the non-natural amino acid site(s) on the
polypeptide.
[0693] In other embodiments, the post-translational modification
does not utilize the cellular machinery, but the functionality is
instead provided by attachment of a molecule (a polymer; a
water-soluble polymer; a derivative of polyethylene glycol; a
second protein or polypeptide or polypeptide analog; an antibody or
antibody fragment; and any combination thereof) comprising a second
reactive group to the at least one non-natural amino acid
comprising a first reactive group (including but not limited to,
non-natural amino acid containing a ketone, aldehyde, acetal,
hemiacetal, alkyne, cycloalkyne, azide, oxime, or hydroxylamine
functional group) utilizing chemistry methodology described herein,
or others suitable for the particular reactive groups. In certain
embodiments, the co-translational or post-translational
modification is made in vivo in a eukaryotic cell or in a
non-eukaryotic cell. In certain embodiments, the post-translational
modification is made in vitro not utilizing the cellular machinery.
Also included with this aspect are methods for producing,
purifying, characterizing and using such dolastatin linker
derivatives containing at least one such co-translationally or
post-translationally modified non-natural amino acids.
[0694] Also included within the scope of the methods, compositions,
strategies and techniques described herein are reagents capable of
reacting with a dolastatin linker derivative (containing a carbonyl
or dicarbonyl group, oxime group, alkyne, cycloalkyne, azide,
hydroxylamine group, or masked or protected forms thereof) that is
part of a polypeptide so as to produce any of the aforementioned
post-translational modifications. In certain embodiments, the
resulting post-translationally modified dolastatin linker
derivative will contain at least one oxime group; the resulting
modified oxime-containing dolastatin linker derivative may undergo
subsequent modification reactions. Also included with this aspect
are methods for producing, purifying, characterizing and using such
reagents that are capable of any such post-translational
modifications of such dolastatin linker derivative(s).
[0695] In certain embodiments, the polypeptide or non-natural amino
acid linked dolastatin derivative includes at least one
co-translational or post-translational modification that is made in
vivo by one host cell, where the post-translational modification is
not normally made by another host cell type. In certain
embodiments, the polypeptide includes at least one co-translational
or post-translational modification that is made in vivo by a
eukaryotic cell, where the co-translational or post-translational
modification is not normally made by a non-eukaryotic cell.
Examples of such co-translational or post-translational
modifications include, but are not limited to, glycosylation,
acetylation, acylation, lipid-modification, palmitoylation,
palmitate addition, phosphorylation, glycolipid-linkage
modification, and the like. In one embodiment, the co-translational
or post-translational modification comprises attachment of an
oligosaccharide to an asparagine by a GlcNAc-asparagine linkage
(including but not limited to, where the oligosaccharide comprises
(GlcNAc-Man).sub.2-Man-GlcNAc-GlcNAc, and the like). In another
embodiment, the co-translational or post-translational modification
comprises attachment of an oligosaccharide (including but not
limited to, Gal-GalNAc, Gal-GlcNAc, etc.) to a serine or threonine
by a GalNAc-serine, a GalNAc-threonine, a GlcNAc-serine, or a
GlcNAc-threonine linkage. In certain embodiments, a protein or
polypeptide can comprise a secretion or localization sequence, an
epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, and/or
the like. Also included with this aspect are methods for producing,
purifying, characterizing and using such polypeptides containing at
least one such co-translational or post-translational modification.
In other embodiments, the glycosylated non-natural amino acid
polypeptide is produced in a non-glycosylated form. Such a
non-glycosylated form of a glycosylated non-natural amino acid may
be produced by methods that include chemical or enzymatic removal
of oligosaccharide groups from an isolated or substantially
purified or unpurified glycosylated non-natural amino acid
polypeptide; production of the non-natural amino acid in a host
that does not glycosylate such a non-natural amino acid polypeptide
(such a host including, prokaryotes or eukaryotes engineered or
mutated to not glycosylate such a polypeptide), the introduction of
a glycosylation inhibitor into the cell culture medium in which
such a non-natural amino acid polypeptide is being produced by a
eukaryote that normally would glycosylate such a polypeptide, or a
combination of any such methods. Also described herein are such
non-glycosylated forms of normally-glycosylated non-natural amino
acid polypeptides (by normally-glycosylated is meant a polypeptide
that would be glycosylated when produced under conditions in which
naturally-occurring polypeptides are glycosylated). Of course, such
non-glycosylated forms of normally-glycosylated non-natural amino
acid polypeptides (or indeed any polypeptide described herein) may
be in an unpurified form, a substantially purified form, or in an
isolated form.
[0696] In certain embodiments, the non-natural amino acid
polypeptide includes at least one post-translational modification
that is made in the presence of an accelerant, wherein the
post-translational modification is stoichiometric,
stoichiometric-like, or near-stoichiometric. In other embodiments
the polypeptide is contacted with a reagent of Formula (XIX) in the
presence of an accelerant. In other embodiments the accelerant is
selected from the group consisting of:
##STR00158##
[0697] A. Chemical Synthesis of Non-Natural Amino Acid Linked
Dolastatin Derivatives: Oxime-Containing Linked Dolastatin
Derivatives
[0698] Non-natural amino acid dolastatin linked derivatives
containing an oxime group allow for reaction with a variety of
reagents that contain certain reactive carbonyl- or
dicarbonyl-groups (including but not limited to, ketones,
aldehydes, or other groups with similar reactivity) to form new
non-natural amino acids comprising a new oxime group. Such an oxime
exchange reaction allows for the further functionalization of
dolastatin linked derivatives. Further, the original dolastatin
linked derivative containing an oxime group may be useful in their
own right as long as the oxime linkage is stable under conditions
necessary to incorporate the amino acid into a polypeptide (e.g.,
the in vivo, in vitro and chemical synthetic methods described
herein).
[0699] Thus, in certain embodiments described herein are
non-natural amino acid dolastatin linked derivatives with
sidechains comprising an oxime group, an oxime-like group (which
has reactivity similar to an oxime group and is structurally
similar to an oxime group), a masked oxime group (which can be
readily converted into an oxime group), or a protected oxime group
(which has reactivity similar to an oxime group upon
deprotection).
[0700] Such non-natural amino acid dolastatin linked derivatives
include dolastatin linked derivatives having the structure of
Formula (VIII) or (IX):
##STR00159##
wherein: [0701] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, Lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0702] B is optional, and when present is a
linker selected from the group consisting of lower alkylene,
substituted lower alkylene, lower alkenylene, substituted lower
alkenylene, lower heteroalkylene, substituted lower heteroalkylene,
--O--, --O-(alkylene or substituted alkylene)-, --S--,
--S-(alkylene or substituted alkylene)-, --S(O).sub.k-- where k is
1, 2, or 3, --S(O).sub.k(alkylene or substituted alkylene)-,
--C(O)--, --C(O)-(alkylene or substituted alkylene)-, --C(S)--,
--C(S)-(alkylene or substituted alkylene)-, --N(R')--,
--NR'-(alkylene or substituted alkylene)-, --C(O)N(R')--,
--CON(R')-(alkylene or substituted alkylene)-, --CSN(R')--,
--CSN(R')-(alkylene or substituted alkylene)-, --N(R')CO-(alkylene
or substituted alkylene)-, --N(R')C(O)O--, --S(O).sub.kN(R')--,
--N(R')C(O)N(R')--, --N(R')C(S)N(R')--, --N(R')S(O).sub.kN(R')--,
--N(R')--N.dbd., --C(R').dbd.N--, --C(R').dbd.N--N(R')--,
--C(R').dbd.N--N.dbd., --C(R').sub.2--N.dbd.N--, and
--C(R').sub.2--N(R')--N(R')--, where each R' is independently H,
alkyl, or substituted alkyl; [0703] R is H, alkyl, substituted
alkyl, cycloalkyl, or substituted cycloalkyl; [0704] R.sub.1 is H,
an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide; [0705] R.sub.2 is OH, an ester
protecting group, resin, at least one amino acid, polypeptide, or
polynucleotide; [0706] R.sub.3 and R.sub.4 are each independently
H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and
R.sub.4 or two R groups optionally form a cycloalkyl or a
heterocycloalkyl; [0707] Z has the structure of:
[0707] ##STR00160## [0708] R.sub.5 is H, COR.sub.8,
C.sub.1-C.sub.6alkyl, or thiazole; [0709] R.sub.8 is OH [0710]
R.sub.6 is OH or H; [0711] Ar is phenyl or pyridine; [0712] R.sub.7
is C.sub.1-C.sub.6alkyl or hydrogen; [0713] L is a linker selected
from the group consisting of -alkylene-, -alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-C(O)--,
-(alkylene-O).sub.n--(CH.sub.2).sub.n'--NHC(O)--(CH.sub.2).sub.n''--C(Me)-
.sub.2-S--S--(CH.sub.2).sub.n'''-- NHC(O)-(alkylene-O).sub.n''''--,
-alkylene-, -(alkylene-O).sub.n-alkylene-W--, -alkylene-C(O)--W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--, and
-(alkylene-O).sub.n-alkylene-U-alkylene-; [0714] W has the
structure of:
[0714] ##STR00161## [0715] U has the structure of
[0715] ##STR00162## and [0716] each n, n', n'', n''' and n'''' are
independently integers greater than or equal to one; [0717] or an
active metabolite, or a pharmaceutically acceptable prodrug or
solvate thereof.
[0718] In certain embodiments of compounds of Formula (VIII) and
(IX), R.sub.5 is thiazole. In certain embodiments of compounds of
Formula (VIII) and (IX), R.sub.6 is H. In certain embodiments of
compounds of Formula (VIII) and (IX), Ar is phenyl. In certain
embodiments of compounds of Formula (VIII) and (IX), R.sub.7 is
methyl. In certain embodiments of compounds of Formula (VIII) and
(IX), n is an integer from 0 to 20. In certain embodiments of
compounds of Formula (VIII) and (IX), n is an integer from 0 to 10.
In certain embodiments of compounds of Formula (VIII) and (IX), n
is an integer from 0 to 5.
[0719] In certain embodiments of compounds of Formula (VIII) and
(IX), R.sub.5 is thiazole. In certain embodiments of compounds of
Formula (VIII) and (IX), R.sub.5 is hydrogen. In certain
embodiments of compounds of Formula (VIII) and (IX), R.sub.5 is
methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl,
tert-butyl, pentyl, or hexyl. In certain embodiments of compounds
of Formula (VIII) and (IX), R.sub.5 is
--NH-(alkylene-O).sub.n--NH.sub.2, wherein alkylene is
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.-
sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2--, or
CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2C-
H.sub.2CH.sub.2CH.sub.2--. In certain embodiments of Formula (VIII)
and (IX), alkylene is methylene, ethylene, propylene, butylenes,
pentylene, hexylene, or heptylene.
[0720] In certain embodiments of compounds of Formula (VIII) and
(IX), R.sub.5 is --NH-(alkylene-O).sub.n--NH.sub.2, wherein n is 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.
[0721] In certain embodiments of compounds of Formula (VIII) and
(IX), R.sub.6 is H.
[0722] In certain embodiments of compounds of Formula (VIII) and
(IX), Ar is phenyl.
[0723] In certain embodiments of compounds of Formula (VIII) and
(IX), R.sub.2 is methyl, ethyl, propyl, iso-propyl, butyl,
sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In certain
embodiments of compounds of Formula (VIII) and (IX), R.sub.7 is
hydrogen.
[0724] In certain embodiments of compounds of Formula (VIII) and
(IX), each L is independently a cleavable linker or non-cleavable
linker. In certain embodiments of compounds of Formula (VIII) and
(IX), each L is independently a oligo(ethylene glycol) derivatized
linker.
[0725] In certain embodiments of compounds of Formula (VIII) and
(IX), each alkylene, alkylene', alkylene'', and alkylene'''
independently is --CH.sub.2--, --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.-
sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2, or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2CH.sub.2--. In certain embodiments of compounds of
Formula (VIII) and (IX), alkylene is methylene, ethylene,
propylene, butylenes, pentylene, hexylene, or heptylene.
[0726] In certain embodiments of compounds of Formula (VIII) and
(IX), each n, n', n'', n''', and n'''' independently is 0, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.
[0727] In certain embodiments of compounds of Formula (VIII) or
(IX), R.sub.1 is a polypeptide. In certain embodiments of compounds
of Formula (VIII) or (IX), R.sub.2 is a polypeptide. In certain
embodiments of compounds of Formula (VIII) or (IX), the polypeptide
is an antibody. In certain embodiments of compounds of Formula
(VIII) or (IX), the antibody is herceptin.
[0728] Such non-natural amino acid dolastatin linked derivatives
include dolastatin linked derivatives having the structure of
Formula (X), (XI), (XII) or (XIII):
##STR00163## ##STR00164##
wherein: [0729] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0730] B is optional, and when present is a
linker selected from the group consisting of lower alkylene,
substituted lower alkylene, lower alkenylene, substituted lower
alkenylene, lower heteroalkylene, substituted lower heteroalkylene,
--O--, --O-(alkylene or substituted alkylene)-, --S--,
--S-(alkylene or substituted alkylene)-, --S(O).sub.k-- where k is
1, 2, or 3, --S(O).sub.k(alkylene or substituted alkylene)-,
--C(O)--, --C(O)-(alkylene or substituted alkylene)-, --C(S)--,
--C(S)-(alkylene or substituted alkylene)-, --N(R')--,
--NR'-(alkylene or substituted alkylene)-, --C(O)N(R')--,
--CON(R')-(alkylene or substituted alkylene)-, --CSN(R')--,
--CSN(R')-(alkylene or substituted alkylene)-, --N(R')CO-(alkylene
or substituted alkylene)-, --N(R')C(O)O--, --S(O).sub.kN(R')--,
--N(R')C(O)N(R')--, --N(R')C(S)N(R')--, --N(R')S(O).sub.kN(R')--,
--N(R')--N.dbd., --C(R').dbd.N--, --C(R').dbd.N--N(R')--,
--C(R').dbd.N--N.dbd., --C(R').sub.2--N.dbd.N--, and
--C(R').sub.2--N(R')--N(R')--, where each R' is independently H,
alkyl, or substituted alkyl; [0731] R is H, alkyl, substituted
alkyl, cycloalkyl, or substituted cycloalkyl; [0732] R.sub.1 is H,
an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide; [0733] R.sub.2 is OH, an ester
protecting group, resin, at least one amino acid, polypeptide, or
polynucleotide; [0734] R.sub.3 and R.sub.4 are each independently
H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and
R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a
heterocycloalkyl; [0735] Z has the structure of:
[0735] ##STR00165## [0736] R.sub.5 is H, CO.sub.2H,
C.sub.1-C.sub.6alkyl, or thiazole; [0737] R.sub.6 is OH or H;
[0738] Ar is phenyl or pyridine; [0739] R.sub.7 is
C.sub.1-C.sub.6alkyl or hydrogen; [0740] L.sub.1, L.sub.2, L.sub.3,
and L.sub.4 are each linkers independently selected from the group
consisting of a bond, -alkylene-, -(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n'-alkylene-J'-,
-(alkylene-O).sub.n-alkylene-J-alkylene'-, --W--, -alkylene-W--,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-alkylene-NMe-alkylene'-NMe-alkylene''-W--, and
-alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--; [0741] W
has the structure of:
[0741] ##STR00166## [0742] each J and J' independently have the
structure of:
[0742] ##STR00167## and [0743] each n and n' are independently
integers greater than or equal to one.
[0744] In certain embodiments of compounds of Formula (X), (XI),
(XII) or (XIII), R.sub.5 is thiazole or carboxylic acid. In certain
embodiments of compounds of Formula (X), (XI), (XII) or (XIII),
R.sub.6 is H. In certain embodiments of compounds of Formula (X),
(XI), (XII) or (XIII), Ar is phenyl. In certain embodiments of
compounds of Formula (X), (XI), (XII) or (XIII), R.sub.7 is methyl.
In certain embodiments of compounds of Formula (X), (XI), (XII) or
(XIII), n and n' are integers from 0 to 20. In certain embodiments
of compounds of Formula (X), (XI), (XII) or (XIII), n and n' are
integers from 0 to 10. In certain embodiments of compounds of
Formula (X), (XI), (XII) or (XIII), n and n' are integers from 0 to
5.
[0745] In certain embodiments of compounds of Formula (X), (XI),
(XII) or (XIII), R.sub.5 is thiazole. In certain embodiments of
compounds of Formula (X), (XI), (XII) or (XIII), R.sub.5 is
hydrogen. In certain embodiments of compounds of Formula (X), (XI),
(XII) or (XIII), R.sub.5 is methyl, ethyl, propyl, iso-propyl,
butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In
certain embodiments of compounds of Formula (X), (XI), (XII) or
(XIII), R.sub.5 is --NH-(alkylene-O).sub.n--NH.sub.2, wherein
alkylene is --CH.sub.2--, --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2C.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2-
--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.s-
ub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2--, or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2CH.sub.2--. In certain embodiments of Formula (X),
(XI), (XII) or (XIII), alkylene is methylene, ethylene, propylene,
butylenes, pentylene, hexylene, or heptylene.
[0746] In certain embodiments of compounds of Formula (X), (XI),
(XII) or (XIII), R.sub.5 is --NH-(alkylene-O).sub.n--NH.sub.2,
wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100.
[0747] In certain embodiments of compounds of Formula (X), (XI),
(XII) or (XIII), R.sub.6 is H. In some embodiments of compounds of
Formula (X), (XI), (XII) or (XIII), R.sub.6 is hydroxy.
[0748] In certain embodiments of compounds of Formula (X), (XI),
(XII) or (XIII), Ar is phenyl.
[0749] In certain embodiments of compounds of Formula (X), (XI),
(XII) or (XIII), R.sub.7 is methyl, ethyl, propyl, iso-propyl,
butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In
certain embodiments of compounds of Formula (X), (XI), (XII) or
(XIII), R.sub.7 is hydrogen.
[0750] In certain embodiments of compounds of Formula (X), (XI),
(XII) or (XIII), each L.sub.1, L.sub.2, L.sub.3, and L.sub.4 is
independently a cleavable linker or non-cleavable linker. In
certain embodiments of compounds of Formula (X), (XI), (XII) or
(XIII), each L.sub.1, L.sub.2, L.sub.3, and L.sub.4 is
independently a oligo(ethylene glycol) derivatized linker.
[0751] In certain embodiments of compounds of Formula (X), (XI),
(XII) or (XIII), each alkylene, alkylene', alkylene'', and
alkylene''' independently is --CH.sub.2--, --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.-
sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2--, or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2CH.sub.2--. In certain embodiments of compounds of
Formula (X), (XI), (XII) or (XIII), alkylene is methylene,
ethylene, propylene, butylenes, pentylene, hexylene, or
heptylene.
[0752] In certain embodiments of compounds of Formula (X), (XI),
(XII) or (XIII), each n and n' independently is 0, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100.
[0753] In certain embodiments of compounds of Formula (X), (XI),
(XII) or (XIII), R.sub.1 is a polypeptide. In certain embodiments
of compounds of Formula (X), (XI), (XII) or (XIII), R.sub.2 is a
polypeptide. In certain embodiments of compounds of Formula (X),
(XI), (XII) or (XIII), the polypeptide is an antibody. In certain
embodiments of compounds of Formula (X), (XI), (XII) or (XIII), the
antibody is herceptin.
[0754] In certain embodiments, compounds of Formula (X), (XI),
(XII) or (XIII) are stable in aqueous solution for at least 1 month
under mildly acidic conditions. In certain embodiments, compounds
of Formula (X), (XI), (XII) or (XIII) are stable for at least 2
weeks under mildly acidic conditions. In certain embodiments,
compound of Formula (X), (XI), (XII) or (XIII) are stable for at
least 5 days under mildly acidic conditions. In certain
embodiments, such acidic conditions are pH 2 to 8. Such non-natural
amino acids may be in the form of a salt, or may be incorporated
into a non-natural amino acid polypeptide, polymer, polysaccharide,
or a polynucleotide and optionally post translationally
modified.
[0755] Oxime-based non-natural amino acids may be synthesized by
methods already described in the art, or by methods described
herein, including: (a) reaction of a hydroxylamine-containing
non-natural amino acid with a carbonyl- or dicarbonyl-containing
reagent; (b) reaction of a carbonyl- or dicarbonyl-containing
non-natural amino acid with a hydroxylamine-containing reagent; or
(c) reaction of an oxime-containing non-natural amino acid with
certain carbonyl- or dicarbonyl-containing reagents.
[0756] B. Chemical Structure and Synthesis of Non-Natural Amino
Acid Linked Dolastatin Derivatives: Alkylated Aromatic Amine Linked
Dolastatin Derivatives
[0757] In one aspect are dolastatin linker derivatives for the
chemical derivatization of non-natural amino acids based upon the
reactivity of an aromatic amine group. In further or additional
embodiments, at least one of the aforementioned non-natural amino
acids is incorporated into a dolastatin linker derivative, that is,
such embodiments are non-natural amino acid linked dolastatin
derivatives. In further or additional embodiments, the dolastatin
linker derivatives are functionalized on their sidechains such that
their reaction with a derivatizing non-natural amino acid generates
an amine linkage. In further or additional embodiments, the
dolastatin linker derivatives are selected from dolastatin linker
derivatives having aromatic amine sidechains. In further or
additional embodiments, the dolastatin linker derivatives comprise
a masked sidechain, including a masked aromatic amine group. In
further or additional embodiments, the non-natural amino acids are
selected from amino acids having aromatic amine sidechains. In
further or additional embodiments, the non-natural amino acids
comprise a masked sidechain, including a masked aromatic amine
group.
[0758] In another aspect are carbonyl-substituted dolastatin linker
derivatives such as, by way of example, aldehydes, and ketones, for
the production of derivatized non-natural amino acid polypeptides
based upon an amine linkage. In a further embodiment are
aldehyde-substituted dolastatin linker derivatives used to
derivative aromatic amine-containing non-natural amino acid
polypeptides via the formation of an amine linkage between the
derivatizing dolastatin linker and the aromatic amine-containing
non-natural amino acid polypeptide.
[0759] In further or additional embodiments, the non-natural amino
acids comprise aromatic amine sidechains where the aromatic amine
is selected from an aryl amine or a heteroaryl amine. In a further
or additional embodiment, the non-natural amino acids resemble a
natural amino acid in structure but contain aromatic amine groups.
In another or further embodiment the non-natural amino acids
resemble phenylalanine or tyrosine (aromatic amino acids). In one
embodiment, the non-natural amino acids have properties that are
distinct from those of the natural amino acids. In one embodiment,
such distinct properties are the chemical reactivity of the
sidechain; in a further embodiment this distinct chemical
reactivity permits the sidechain of the non-natural amino acid to
undergo a reaction while being a unit of a polypeptide even though
the sidechains of the naturally-occurring amino acid units in the
same polypeptide do not undergo the aforementioned reaction. In a
further embodiment, the sidechain of the non-natural amino acid has
a chemistry orthogonal to those of the naturally-occurring amino
acids. In a further embodiment, the sidechain of the non-natural
amino acid comprises a nucleophile-containing moiety; in a further
embodiment, the nucleophile-containing moiety on the sidechain of
the non-natural amino acid can undergo a reaction to generate an
amine-linked derivatized dolastatin. In a further embodiment, the
sidechain of the non-natural amino acid comprises an
electrophile-containing moiety; in a further embodiment, the
electrophile-containing moiety on the sidechain of the non-natural
amino acid can undergo nucleophilic attack to generate an
amine-linked derivatized dolastatin. In any of the aforementioned
embodiments in this paragraph, the non-natural amino acid may exist
as a separate molecule or may be incorporated into a polypeptide of
any length; if the latter, then the polypeptide may further
incorporate naturally-occurring or non-natural amino acids.
[0760] Modification of non-natural amino acids described herein
using reductive alkylation or reductive amination reactions have
any or all of the following advantages. First, aromatic amines can
be reductively alkylated with carbonyl-containing compounds,
including aldehydes, and ketones, in a pH range of about 4 to about
10 (and in certain embodiments in a pH range of about 4 to about 7)
to generate substituted amine, including secondary and tertiary
amine, linkages. Second, under these reaction conditions the
chemistry is selective for non-natural amino acids as the
sidechains of naturally occurring amino acids are unreactive. This
allows for site-specific derivatization of polypeptides which have
incorporated non-natural amino acids containing aromatic amine
moieties or protected aldehyde moieties, including, by way of
example, recombinant proteins. Such derivatized polypeptides and
proteins can thereby be prepared as defined homogeneous products.
Third, the mild conditions needed to effect the reaction of an
aromatic amine moiety on an amino acid, which has been incorporated
into a polypeptide, with an aldehyde-containing reagent generally
do not irreversibly destroy the tertiary structure of the
polypeptide (excepting, of course, where the purpose of the
reaction is to destroy such tertiary structure). Similarly, the
mild conditions needed to effect the reaction of an aldehyde moiety
on an amino acid, which has been incorporated into a polypeptide
and deprotected, with an aromatic amine-containing reagent
generally do not irreversibly destroy the tertiary structure of the
polypeptide (excepting, of course, where the purpose of the
reaction is to destroy such tertiary structure). Fourth, the
reaction occurs rapidly at room temperature, which allows the use
of many types of polypeptides or reagents that would otherwise be
unstable at higher temperatures. Fifth, the reaction occurs readily
is aqueous conditions, again allowing use of polypeptides and
reagents incompatible (to any extent) with non-aqueous solutions.
Six, the reaction occurs readily even when the ratio of polypeptide
or amino acid to reagent is stoichiometric, stoichiometric-like, or
near-stoichiometric, so that it is unnecessary to add excess
reagent or polypeptide to obtain a useful amount of reaction
product. Seventh, the resulting amine can be produced
regioselectively and/or regiospecifically, depending upon the
design of the amine and carbonyl portions of the reactants.
Finally, the reductive alkylation of aromatic amines with
aldehyde-containing reagents, and the reductive amination of
aldehydes with aromatic amine containing reagents, generates amine,
including secondary and tertiary amine, linkages which are stable
under biological conditions.
[0761] Non-natural amino acids with nucleophilic reactive groups,
such as, by way of example only, an aromatic amine group (including
secondary and tertiary amine groups), a masked aromatic amine group
(which can be readily converted into a aromatic amine group), or a
protected aromatic amine group (which has reactivity similar to a
aromatic amine group upon deprotection) allow for a variety of
reactions to link molecules via various reactions, including but
not limited to, reductive alkylation reactions with aldehyde
containing dolastatin linked derivatives. Such alkylated
non-natural amino acid linked dolastatin derivatives include amino
acids having the structure of Formula (XXV), (XXVI), (XXVII),
(XXVIII), (XXIX), or (XXX):
##STR00168## ##STR00169## [0762] wherein: [0763] Z has the
structure of:
[0763] ##STR00170## [0764] R.sub.5 is H, CO.sub.2H,
C.sub.1-C.sub.6alkyl, or thiazole; [0765] R.sub.6 is OH or H;
[0766] Ar is phenyl or pyridine; [0767] R.sub.1 is H, an amino
protecting group, resin, at least one amino acid, polypeptide, or
polynucleotide; [0768] R.sub.2 is OH, an ester protecting group,
resin, at least one amino acid, polypeptide, or polynucleotide;
[0769] R.sub.4 is H, halogen, lower alkyl, or substituted lower
alkyl; [0770] R.sub.7 is C.sub.1-C.sub.6alkyl or hydrogen; [0771]
L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each linkers selected
from the group consisting of a bond, -alkylene-, -alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-C(O)--,
-(alkylene-O).sub.n--(CH.sub.2).sub.n'--NHC(O)--(CH.sub.2).sub.n''--C(Me)-
.sub.2-S--(CH.sub.2).sub.n'''--NHC(O)-(alkylene-O).sub.n''''-alkylene-,
-(alkylene-O).sub.n-alkylene-W--, -alkylene-C(O)--W--,
-(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-alkylene',
-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n'-alkylene-J'-,
--W--, -alkylene-W--,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--, and
J-(alkylene-NMe).sub.n-alkylene-W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-U-alkylene-;
-J-alkylene-NMe-alkylene'-NMe-alkylene''-W--, and
-alkylene-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--; [0772] W
has the structure of:
[0772] ##STR00171## [0773] U has the structure of:
[0773] ##STR00172## [0774] each J and J' independently have the
structure of:
[0774] ##STR00173## [0775] each n and n' are independently integers
greater than or equal to one; and [0776] each R.sub.16 is
independently selected from the group consisting of hydrogen,
halogen, alkyl, NO.sub.2, CN, and substituted alkyl. Such alkylated
non-natural amino acid linked dolastatin derivatives may also be in
the form of a salt, or may be incorporated into a non-natural amino
acid polypeptide, polymer, polysaccharide, or a polynucleotide and
optionally reductively alkylated.
[0777] In certain embodiments of compounds of Formula (XXV),
(XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.5 is thiazole or
carboxylic acid. In certain embodiments of compounds of Formula
(XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.6 is H.
In certain embodiments of compounds of Formula (XXV), (XXVI),
(XXVII), (XXVIII), (XXIX), or (XXX), Ar is phenyl. In certain
embodiments of compounds of Formula (XXV), (XXVI), (XXVII),
(XXVIII), (XXIX), or (XXX), R.sub.7 is methyl. In certain
embodiments of compounds of Formula (XXV), (XXVI), (XXVII),
(XXVIII), (XXIX), or (XXX), n is an integer from 0 to 20. In
certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII),
(XXVIII), (XXIX), or (XXX), n is an integer from 0 to 10. In
certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII),
(XXVIII), (XXIX), or (XXX) or (XXIV), n is an integer from 0 to
5.
[0778] In certain embodiments of compounds of Formula (XXV),
(XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.5 is thiazole or
carboxylic acid. In certain embodiments of compounds of Formula
(XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.5 is
hydrogen. In certain embodiments of compounds of Formula (XXV),
(XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.5 is methyl,
ethyl, propyl, iso-propyl, butyl, iso-butyl, see-butyl, tert-butyl,
pentyl, or hexyl. In certain embodiments of compounds of Formula
((XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.5 is
--NH-(alkylene-O).sub.n--NH.sub.2, wherein alkylene is
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.-
sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2--, or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2CH.sub.2--. In certain embodiments of Formula
(XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), alkylene is
methylene, ethylene, propylene, butylenes, pentylene, hexylene, or
heptylene.
[0779] In certain embodiments of compounds of Formula (XXV),
(XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.5 is
--NH.sub.4-(alkylene-O).sub.n--NH.sub.2, wherein n is 0, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.
[0780] In certain embodiments of compounds of Formula (XXV),
(XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.6 is H. In some
embodiments of compounds of Formula (XXV), (XXVI), (XXVII),
(XXVIII), (XXIX), or (XXX), R.sub.6 is hydroxy.
[0781] In certain embodiments of compounds of Formula (XXV),
(XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), Ar is phenyl.
[0782] In certain embodiments of compounds of Formula (XXV),
(XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.7 is methyl,
ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl,
pentyl, or hexyl. In certain embodiments of compounds of Formula
(XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.7 is
hydrogen.
[0783] In certain embodiments of compounds of Formula (XXV),
(XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), each L, L.sub.1,
L.sub.2, L.sub.3, and L.sub.4 is independently a cleavable linker
or non-cleavable linker. In certain embodiments of compounds of
Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), each L,
L.sub.1, L.sub.2, L.sub.3, and L.sub.4 is independently a
oligo(ethylene glycol) derivatized linker.
[0784] In certain embodiments of compounds of Formula (XXV),
(XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), each alkylene,
alkylene', alkylene'', and alkylene''' independently is
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.-
sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2--, or
CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2C-
H.sub.2CH.sub.2CH.sub.2--, In certain embodiments of compounds of
Formula (XIX), (XX), (XXI), (XXII), (XXIII) or (XXIV), alkylene is
methylene, ethylene, propylene, butylenes, pentylene, hexylene, or
heptylene.
[0785] In certain embodiments of compounds of Formula (XXV),
(XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), each n, n', n'', n''',
and n'''' independently is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100.
[0786] In certain embodiments of compounds of Formula (XXV),
(XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.1 is a
polypeptide. In certain embodiments of compounds of Formula (XXV),
(XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.2 is a
polypeptide. In certain embodiments of compounds of Formula (XXV),
(XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), the polypeptide is an
antibody. In certain embodiments of compounds of Formula (XXV),
(XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), the antibody is
herceptin.
[0787] Compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII),
(XXIX), or (XXX) may be formed by the reductive alkylation of
aromatic amine compounds with carbonyl containing reagents such as,
by way of example, ketones, esters, thioesters, and aldehydes.
[0788] In some embodiments, the masked amine moieties of
non-natural amino acids contained in polypeptides are initially
reduced to give non-natural amino acids containing aromatic amine
moieties incorporated into non-natural amino acid polypeptides.
Such aromatic amine moieties are then reductive alkylated with
carbonyl-containing reagents described above to give polypeptides
containing non-natural amino acids of Formula (XXV), (XXVI),
(XXVII), (XXVIII), (XXIX), or (XXX). Such reactions may also be
applied to non-natural amino acids incorporated into synthetic
polymers, polysaccharides, or polynucleotides. Additionally, such
reactions may be applied to non-incorporated non-natural amino
acids. By way of example the reducing agent used to reduce masked
amine moieties includes, but is not limited to, TCEP, Na.sub.2S,
Na.sub.2S.sub.2O.sub.4, LiAlH.sub.4, B.sub.2H.sub.6, and
NaBH.sub.4. By way of example only, reductive alkylation may occur
in aqueous buffers with a pH of about 4 to about 7 and using a mild
reducing agent, such as, by way of example only, sodium
cyanoborohydride (NaBCNH.sub.3). In addition, other reducing agents
may be used for reductive alkylation including, but not limited to,
TCEP, Na.sub.2S, Na.sub.2S.sub.2O.sub.4, LiAlH.sub.4,
B.sub.2H.sub.6, and NaBH.sub.4.
[0789] A non-limiting exemplary syntheses of non-natural amino acid
polypeptides containing amino acids of Formula (XXV), (XXVI),
(XXVII), (XXVIII), (XXIX), or (XXX) by reductive alkylation of
secondary aromatic amine moieties, contained in non-natural amino
acids, with carbonyl-containing reagents described above. Such
reductive alkylations give polypeptides containing non-natural
amino acids with tertiary aromatic amine moieties. Such reactions
may also be applied to non-natural amino acids incorporated into
synthetic polymers, polysaccharides, or polynucleotides.
Additionally, such reactions may be applied to non-incorporated
non-natural amino acids. By way of example only, reductive
alkylation may occur in aqueous buffers with a pH of about 4 to
about 7 and using a mild reducing agent, such as, by way of example
only, sodium cyanoborohydride (NaBCNH.sub.3). In addition, other
reducing agents may be used for reductive alkylation including, but
not limited to, TCEP, Na.sub.2S, Na.sub.2S.sub.2O.sub.4,
LiAlH.sub.4, B.sub.2H.sub.6, and NaBH.sub.4.
[0790] C. Chemical Synthesis of Non-Natural Amino Acid Linked
Dolastatin Derivatives: Heteroaryl-Containing Linked Dolastatin
Derivatives
[0791] In one aspect are non-natural amino acids for the chemical
derivatization of dolastatin linked derivatives based upon the
reactivity of a dicarbonyl group, including a group containing at
least one ketone group, and/or at least one aldehyde groups, and/or
at least one ester group, and/or at least one carboxylic acid,
and/or at least one thioester group, and wherein the dicarbonyl
group can be a 1,2-dicarbonyl group, a 1,3-dicarbonyl group, or a
1,4-dicarbonyl group. In further or additional aspects are
non-natural amino acids for the chemical derivatization of
dolastatin linked derivatives based upon the reactivity of a
diamine group, including a hydrazine group, an amidine group, an
imine group, a 1,1-diamine group, a 1,2-diamine group, a
1,3-diamine group, and a 1,4-diamine group. In further or
additional embodiments, at least one of the aforementioned
non-natural amino acids is incorporated into a dolastatin linked
derivative, that is, such embodiments are non-natural amino acid
linked dolastatin derivatives. In further or additional
embodiments, the non-natural amino acids are functionalized on
their sidechains such that their reaction with a derivatizing
molecule generates a linkage, including a heterocyclic-based
linkage, including a nitrogen-containing heterocycle, and/or an
aldol-based linkage. In further or additional embodiments are
non-natural amino acid polypeptides that can react with a
derivatizing dolastatin linker to generate a non-natural amino acid
linked dolastatin derivatives containing a linkage, including a
heterocyclic-based linkage, including a nitrogen-containing
heterocycle, and/or an aldol-based linkage. In further or
additional embodiments, the non-natural amino acids are selected
from amino acids having dicarbonyl and/or diamine sidechains. In
further or additional embodiments, the non-natural amino acids
comprise a masked sidechain, including a masked diamine group
and/or a masked dicarbonyl group. In further or additional
embodiments, the non-natural amino acids comprise a group selected
from: keto-amine (i.e., a group containing both a ketone and an
amine); keto-alkyne (i.e., a group containing both a ketone and an
alkyne); and an ene-dione (i.e., a group containing a dicarbonyl
group and an alkene).
[0792] In further or additional embodiments, the non-natural amino
acids comprise dicarbonyl sidechains where the carbonyl is selected
from a ketone, an aldehyde, a carboxylic acid, or an ester,
including a thioester. In another embodiment are non-natural amino
acids containing a functional group that is capable of forming a
heterocycle, including a nitrogen-containing heterocycle, upon
treatment with an appropriately functionalized reagent. In a
further or additional embodiment, the non-natural amino acids
resemble a natural amino acid in structure but contain one of the
aforementioned functional groups. In another or further embodiment
the non-natural amino acids resemble phenylalanine or tyrosine
(aromatic amino acids); while in a separate embodiment, the
non-natural amino acids resemble alanine and leucine (hydrophobic
amino acids). In one embodiment, the non-natural amino acids have
properties that are distinct from those of the natural amino acids.
In one embodiment, such distinct properties are the chemical
reactivity of the sidechain, in a further embodiment this distinct
chemical reactivity permits the sidechain of the non-natural amino
acid to undergo a reaction while being a unit of a polypeptide even
though the sidechains of the naturally-occurring amino acid units
in the same polypeptide do not undergo the aforementioned reaction.
In a further embodiment, the sidechain of the non-natural amino
acid has a chemistry orthogonal to those of the naturally-occurring
amino acids. In a further embodiment, the sidechain of the
non-natural amino acid comprises an electrophile-containing moiety;
in a further embodiment, the electrophile-containing moiety on the
sidechain of the non-natural amino acid can undergo nucleophilic
attack to generate a heterocycle-derivatized protein, including a
nitrogen-containing heterocycle-derivatized protein. In any of the
aforementioned embodiments in this paragraph, the non-natural amino
acid may exist as a separate molecule or may be incorporated into a
polypeptide of any length; if the latter, then the polypeptide may
further incorporate naturally-occurring or non-natural amino
acids.
[0793] In another aspect are diamine-substituted molecules, wherein
the diamine group is selected from a hydrazine, an amidine, an
imine, a 1,1-diamine, a 1,2-diamine, a 1,3-diamine and a
1,4-diamine group, for the production of derivatized non-natural
amino acid linked dolastatin derivatives based upon a heterocycle,
including a nitrogen-containing heterocycle, linkage. In a further
embodiment are diamine-substituted dolastatin derivatives used to
derivatize dicarbonyl-containing non-natural amino acid
polypeptides via the formation of a heterocycle, including a
nitrogen-containing heterocycle, linkage between the derivatizing
molecule and the dicarbonyl-containing non-natural amino acid
polypeptide. In further embodiments the aforementioned
dicarbonyl-containing non-natural amino acid polypeptides are
diketone-containing non-natural amino acid polypeptides. In further
or additional embodiments, the dicarbonyl-containing non-natural
amino acids comprise sidechains where the carbonyl is selected from
a ketone, an aldehyde, a carboxylic acid, or an ester, including a
thioester. In further or additional embodiments, the
diamine-substituted molecules comprise a group selected from a
desired functionality. In a further embodiment, the sidechain of
the non-natural amino acid has a chemistry orthogonal to those of
the naturally-occurring amino acids that allows the non-natural
amino acid to react selectively with the diamine-substituted
molecules. In a further embodiment, the sidechain of the
non-natural amino acid comprises an electrophile-containing moiety
that reacts selectively with the diamine-containing molecule; in a
further embodiment, the electrophile-containing moiety on the
sidechain of the non-natural amino acid can undergo nucleophilic
attack to generate a heterocycle-derivatized protein, including a
nitrogen-containing heterocycle-derivatized protein. In a further
aspect related to the embodiments described in this paragraph are
the modified non-natural amino acid polypeptides that result from
the reaction of the derivatizing molecule with the non-natural
amino acid polypeptides. Further embodiments include any further
modifications of the already modified non-natural amino acid
polypeptides.
[0794] In another aspect are dicarbonyl-substituted molecules for
the production of derivatized non-natural amino acid polypeptides
based upon a heterocycle, including a nitrogen-containing
heterocycle, linkage. In a further embodiment are
dicarbonyl-substituted molecules used to derivatize
diamine-containing non-natural amino acid polypeptides via the
formation of a heterocycle, including a nitrogen-containing
heterocycle group. In a further embodiment are
dicarbonyl-substituted molecules that can form such heterocycle,
including a nitrogen-containing heterocycle groups with a
diamine-containing non-natural amino acid polypeptide in a pH range
between about 4 and about 8. In a further embodiment are
dicarbonyl-substituted molecules used to derivatize
diamine-containing non-natural amino acid polypeptides via the
formation of a heterocycle, including a nitrogen-containing
heterocycle, linkage between the derivatizing molecule and the
diamine-containing non-natural amino acid polypeptides. In a
further embodiment the dicarbonyl-substituted molecules are
diketone-substituted molecules, in other aspects
ketoaldehyde-substituted molecules, in other aspects
ketoacid-substituted molecules, in other aspects
ketoester-substituted molecules, including
ketothioester-substituted molecules. In further embodiments, the
dicarbonyl-substituted molecules comprise a group selected from a
desired functionality. In further or additional embodiments, the
aldehyde-substituted molecules are aldehyde-substituted
polyethylene glycol (PEG) molecules. In a further embodiment, the
sidechain of the non-natural amino acid has a chemistry orthogonal
to those of the naturally-occurring amino acids that allows the
non-natural amino acid to react selectively with the
carbonyl-substituted molecules. In a further embodiment, the
sidechain of the non-natural amino acid comprises a moiety (e.g.,
diamine group) that reacts selectively with the
dicarbonyl-containing molecule; in a further embodiment, the
nucleophilic moiety on the sidechain of the non-natural amino acid
can undergo electrophilic attack to generate a
heterocyclic-derivatized protein, including a nitrogen-containing
heterocycle-derivatized protein. In a further aspect related to the
embodiments described in this paragraph are the modified
non-natural amino acid polypeptides that result from the reaction
of the derivatizing molecule with the non-natural amino acid
polypeptides. Further embodiments include any further modifications
of the already modified non-natural amino acid polypeptides.
[0795] In one aspect are methods to derivatize proteins via the
reaction of carbonyl and hydrazine reactants to generate a
heterocycle-derivatized protein, including a nitrogen-containing
heterocycle-derivatized dolastatin. Included within this aspect are
methods for the derivatization of dolastatin linker derivatives
based upon the condensation of carbonyl- and hydrazine-containing
reactants to generate a heterocycle-derivatized dolastatin,
including a nitrogen-containing heterocycle-derivatized dolastatin.
In additional or further embodiments are methods to derivatize
ketone-containing dolastatin derivatives or aldehyde-containing
dolastatin derivatives with hydrazine-functionalized non-natural
amino acids. In yet additional or further aspects, the
hydrazine-substituted molecule can include proteins, other
polymers, and small molecules.
[0796] In another aspect are methods for the chemical synthesis of
hydrazine-substituted molecules for the derivatization of
carbonyl-substituted dolastatin derivatives. In one embodiment, the
hydrazine-substituted molecule is a dolastatin linked derivative
suitable for the derivatization of carbonyl-containing non-natural
amino acid polypeptides, including by way of example only, ketone-,
or aldehyde-containing non-natural amino acid polypeptides.
[0797] In one aspect are non-natural amino acids for the chemical
derivatization of dolastatin analogs based upon a quinoxaline or
phenazine linkage. In further or additional embodiments, the
non-natural amino acids are functionalized on their sidechains such
that their reaction with a derivatizing dolastatin linker generates
a quinoxaline or phenazine linkage. In further or additional
embodiments, the non-natural amino acids are selected from amino
acids having 1,2-dicarbonyl or 1,2-aryldiamine sidechains. In
further or additional embodiments, the non-natural amino acids are
selected from amino acids having protected or masked 1,2-dicarbonyl
or 1,2-aryldiamine sidechains. Further included are equivalents to
1,2-dicarbonyl sidechains, or protected or masked equivalents to
1,2-dicarbonyl sidechains.
[0798] In another aspect are derivatizing molecules for the
production of derivatized non-natural amino acid polypeptides based
upon quinoxaline or phenazine linkages. In one embodiment are
1,2-dicarbonyl substituted dolastatin linker derivatives used to
derivatize 1,2-aryldiamine containing non-natural amino acid
polypeptides to form quinoxaline or phenazine linkages. In another
embodiment are 1,2-aryldiamine substituted dolastatin linker
derivatives used to derivatize 1,2-dicarbonyl containing
non-natural amino acid polypeptides to form quinoxaline or
phenazine linkages. In a further aspect related to the above
embodiments are the modified non-natural amino acid polypeptides
that result from the reaction of the derivatizing dolastatin linker
with the non-natural amino acid polypeptides. In one embodiment are
1,2-aryldiamine containing non-natural amino acid polypeptides
derivatized with 1,2-dicarbonyl substituted dolastatin linker
derivative to form quinoxaline or phenazine linkages. In another
embodiment are 1,2-dicarbonyl containing non-natural amino acid
polypeptides derivatized with 1,2-aryldiamine substituted
dolastatin linker derivatives to form quinoxaline or phenazine
linkages.
[0799] Provided herein in certain embodiments are derivatizing
molecules for the production of toxic compounds comprising
non-natural amino acid polypeptides based upon triazole linkages.
In some embodiments, the reaction between the first and second
reactive groups can proceed via a dipolarophile reaction. In
certain embodiments, the first reactive group can be an azide and
the second reactive group can be an alkyne. In further or
alternative embodiments, the first reactive group can be an alkyne
and the second reactive group can be an azide. In some embodiments,
the Huisgen cycloaddition reaction (see, e.g., Huisgen, in
1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), p.
1-176) provides for the incorporation of non-naturally encoded
amino acids bearing azide and alkyne-containing side chains permits
the resultant polypeptides to be modified with extremely high
selectivity. In certain embodiments, both the azide and the alkyne
functional groups are inert toward the twenty common amino acids
found in naturally-occurring polypeptides. When brought into close
proximity, however, the "spring-loaded" nature of the azide and
alkyne groups is revealed and they react selectively and
efficiently via Huisgen [3 2] cycloaddition reaction to generate
the corresponding triazole. See, e.g., Chin et al., Science
301:964-7 (2003); Wang et al., J. Am. Chem. Soc., 125, 3192-3193
(2003); Chin et al., J. Am. Chem. Soc., 124:9026-9027 (2002).
Cycloaddition reaction involving azide or alkyne-containing
polypeptides can be carried out at room temperature under aqueous
conditions by the addition of Cu(II) (e.g., in the form of a
catalytic amount of CuSO.sub.4) in the presence of a reducing agent
for reducing Cu(II) to Cu(I), in situ, in catalytic amount. See,
e.g., Wang et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe
et al., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev, Angew. Chem.
Int. Ed. 41:2596-2599 (2002). Preferred reducing agents include
ascorbate, metallic copper, quinine, hydroquinone, vitamin K,
glutathione, cysteine, Fe.sup.2, Co.sup.2, and an applied electric
potential.
[0800] Such non-natural amino acid heteroaryl-linked dolastatin
derivatives include amino acids having the structure of Formula
(XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI):
##STR00174## [0801] wherein: [0802] Z has the structure of:
[0802] ##STR00175## [0803] R.sub.5 is H, CO.sub.2H,
C.sub.1-C.sub.6alkyl, or thiazole; [0804] R.sub.6 is OH or H;
[0805] Ar is phenyl or pyridine; [0806] R.sub.1 is, an amino
protecting group, resin, at least one amino acid, polypeptide, or
polynucleotide; [0807] R.sub.2 is OH, an ester protecting group,
resin, at least one amino acid, polypeptide, or polynucleotide;
[0808] R.sub.4 is H, halogen, lower alkyl, or substituted lower
alkyl; [0809] R.sub.7 is C.sub.1-C.sub.6alkyl or hydrogen; [0810]
L, L.sub.1, L.sub.2, La, and L.sub.4 are each linkers selected from
the group consisting of a bond, -alkylene-alkylene-C(O)--,
-alkylene-J-, -(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-C(O)--, -(alkylene-O).sub.n-J-,
-(alkylene-O).sub.n-J-alkylene-,
-(alkylene-O).sub.n--(CH.sub.2).sub.n'--NHC(O)--(CH.sub.2).sub.n''--C(Me)-
.sub.2-S--S--(CH.sub.2).sub.n'''--NHC(O)-(alkylene-O).sub.n''''-alkylene-,
-(alkylene-O).sub.n-alkylene-W--, -alkylene-C(O)--W--,
-(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-alkylene',
-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n-alkylene-J'-,
--W--, -alkylene-W--,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-(alkylene-NMe).sub.n-alkylene-W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-U-alkylene-;
-J-alkylene-NMe-alkylene'-NMe-alkylene''-W--, and
-alkylene-J-alkylene'-NMe-alkylene''-NMe-allkylene'''-W--; [0811] W
has the structure of:
[0811] ##STR00176## [0812] U has the structure of:
[0812] ##STR00177## [0813] each J and J' independently have the
structure of:
[0813] ##STR00178## [0814] each n and n' are independently integers
greater than or equal to one; [0815] D has the structure of:
[0815] ##STR00179## [0816] each R.sub.17 is independently selected
from the group consisting of H, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,
substituted alkoxy, alkylalkoxy, substituted alkylalkoxy,
polyalkylene oxide, substituted polyalkylene oxide, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, alkaryl,
substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or
substituted alkylene)-ON(R'').sub.2, -(alkylene or substituted
alkylene)-C(O)SR'', -(alkylene or substituted alkylene)-S--S-(aryl
or substituted aryl), --C(O)R'', --C(O).sub.2R'', or
--C(O)N(R'').sub.2, wherein each R'' is independently hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy,
substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl,
substituted alkaryl, aralkyl, or substituted aralkyl; [0817] each
Z.sub.1 is a bond, CR.sub.17R.sub.17, O, S, NR',
CR.sub.17R.sub.17--CR.sub.17R.sub.17, CR.sub.17R.sub.17--O,
O--CR.sub.17R.sub.17, CR.sub.17R.sub.17--S, S--CR.sub.17R.sub.17,
CR.sub.17R.sub.17--NR', or NR'--CR.sub.17R.sub.17; each R' is H,
alkyl, or substituted alkyl; [0818] each Z.sub.2 is selected from
the group consisting of a bond, --C(O)--, --C(S)--, optionally
substituted C.sub.1-C.sub.3 alkylene, optionally substituted
C.sub.1-C.sub.3 alkenylene, and optionally substituted heteroalkyl;
[0819] each Z.sub.3 are independently selected from the group
consisting of a bond, optionally substituted C.sub.1-C.sub.4
alkylene, optionally substituted C.sub.1-C.sub.4 alkenylene,
optionally substituted heteroalkyl, --O--, --S--, --C(O)--,
--C(S)--, and --N(R')--; [0820] each T.sub.3 is a bond,
C(R'')(R''), O, or S; with the proviso that when T.sub.3 is O or S,
R'' cannot be halogen; [0821] each R'' is H, halogen, alkyl,
substituted alkyl, cycloalkyl, or substituted cycloalkyl; m and p
are 0, 1, 2, or 3, provided that at least one of m or p is not
0;
[0821] ##STR00180## where (a) indicates bonding to the B group and
(b) indicates bonding to respective positions within the
heterocycle group; [0822] M.sub.3 is
[0822] ##STR00181## where (a) indicates bonding to the B group and
(b) indicates bonding to respective positions within the
heterocycle group;
##STR00182## [0823] M.sub.4 is where (a) indicates bonding to the B
group and (b) indicates bonding to respective positions within the
heterocycle group; [0824] each R.sub.19 is independently selected
from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkoxy, ester, ether, thioether, aminoalkyl, halogen, alkyl ester,
aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl
sulfonic acid, alkyl nitro, thioester, sulfonyl ester,
halosulfonyl, nitrile, alkyl nitrite, and nitro; [0825] q is 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; and [0826] each R.sub.16 is
independently selected from the group consisting of hydrogen,
halogen, alkyl, NO.sub.2, CN, and substituted alkyl.
[0827] In some embodiments, the compound of Formula (XXXI) include
compounds having the structure of Formula (XXXI-A):
##STR00183##
[0828] In certain embodiments of compounds of Formula (XXXI),
(XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.5 is thiazole
or carboxylic acid. In certain embodiments of compounds of Formula
(XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.6 is
H. In certain embodiments of compounds of Formula (XXXI), (XXXII),
(XXXIII), (XXXIV), (XXXV), or (XXXVI), Ar is phenyl. In certain
embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII),
(XXXIV), (XXXV), or (XXXVI), R.sub.7 is methyl. In certain
embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII),
(XXXIV), (XXXV), or (XXXVI), n is an integer from 0 to 20. In
certain embodiments of compounds of Formula (XXXI), (XXXII),
(XXXIII), (XXXIV), (XXXV), or (XXXVI), n is an integer from 0 to
10. In certain embodiments of compounds of Formula (XXXI), (XXXII),
(XXXIII), (XXXIV), (XXXV), or (XXXVI), n is an integer from 0 to
5.
[0829] In certain embodiments of compounds of Formula (XXXI),
(XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.5 is thiazole
or carboxylic acid. In certain embodiments of compounds of Formula
(XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.5 is
hydrogen. In certain embodiments of compounds of Formula (XXXI),
(XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.5 is methyl,
ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl,
pentyl, or hexyl. In certain embodiments of compounds of Formula
(XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.5 is
--NH-(alkylene-O).sub.n--NH.sub.2, wherein alkylene is
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.-
sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2--, or
CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2C-
H.sub.2CH.sub.2CH.sub.2--. In certain embodiments of Formula
(XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), alkylene is
methylene, ethylene, propylene, butylenes, pentylene, hexylene, or
heptylene.
[0830] In certain embodiments of compounds of Formula (XXXI),
(XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.5 is
--NH-(alkylene-O).sub.n--NH.sub.2, wherein n is 0, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100.
[0831] In certain embodiments of compounds of Formula (XXXI),
(XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.6 is H. In
some embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII),
(XXXIV), (XXXV), or (XXXVI), R.sub.6 is hydroxy.
[0832] In certain embodiments of compounds of Formula (XXXI),
(XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), Ar is phenyl.
[0833] In certain embodiments of compounds of Formula (XXXI),
(XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.7 is methyl,
ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl,
pentyl, or hexyl. In certain embodiments of compounds of Formula
(XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.7 is
hydrogen.
[0834] In certain embodiments of compounds of Formula (XXXI),
(XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), each L, L.sub.1,
L.sub.2, L.sub.3, and L.sub.4 is independently a cleavable linker
or non-cleavable linker. In certain embodiments of compounds of
Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI),
each L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 is independently a
oligo(ethylene glycol) derivatized linker.
[0835] In certain embodiments of compounds of Formula (XXXI),
(XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), each alkylene,
alkylene', alkylene'', and alkylene''' independently is
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.-
sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2--, or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2CH.sub.2CH.sub.2--. In certain embodiments of compounds of
Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI),
alkylene is methylene, ethylene, propylene, butylenes, pentylene,
hexylene, or heptylene.
[0836] In certain embodiments of compounds of Formula (XXXI),
(XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), each n, n', n'',
n''', and n'''' independently is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,
96, 97, 98, 99, or 100.
[0837] In certain embodiments of compounds of Formula (XXXI),
(XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.1 is a
polypeptide. In certain embodiments of compounds of Formula (XXXI),
(XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.2 is a
polypeptide. In certain embodiments of compounds of Formula (XXXI),
(XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), the polypeptide is
an antibody. In certain embodiments of compounds of Formula (XXXI),
(XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), the antibody is
.alpha.PSMA.
[0838] Compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV),
(XXXV), or (XXXVI) may be formed by the reductive alkylation of
aromatic amine compounds with carbonyl containing reagents such as,
by way of example, ketones, esters, thioesters, and aldehydes.
[0839] The formation of such non-natural amino acid
heterocycle-linked dolastatin derivatives having the structure of
Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI)
includes, but is not limited to, (i) reactions of
diamine-containing non-natural amino acids with
dicarbonyl-containing dolastatin linked derivatives or reactions of
diamine-containing non-natural amino acids with
ketoalkyne-containing dolastatin linked derivatives, (ii) reactions
of dicarbonyl-containing non-natural amino acids with either
diamine-containing dolastatin linked derivatives or reactions of
dicarbonyl-containing non-natural amino acids with
ketoamine-containing dolastatin linked derivatives, (iii) reactions
of ketoalkyne-containing non-natural amino acids with
diamine-containing dolastatin linked derivatives, or (iv) reactions
of ketoamine-containing non-natural amino acids with
dicarbonyl-containing v.
[0840] Modification of dolastatin linked derivatives described
herein with such reactions have any or all of the following
advantages. First, diamines undergo condensation with
dicarbonyl-containing compounds in a pH range of about 5 to about 8
(and in further embodiments in a pH range of about 4 to about 10,
in other embodiments in a pH range of about 3 to about 8, in other
embodiments in a pH range of about 4 to about 9, and in further
embodiments a pH range of about 4 to about 9, in other embodiments
a pH of about 4, and in yet another embodiment a pH of about 8) to
generate heterocycle, including a nitrogen-containing heterocycle,
linkages. Under these conditions, the sidechains of the naturally
occurring amino acids are unreactive. Second, such selective
chemistry makes possible the site-specific derivatization of
recombinant proteins: derivatized proteins can now be prepared as
defined homogeneous products. Third, the mild conditions needed to
effect the reaction of the diamines described herein with the
dicarbonyl-containing polypeptides described herein generally do
not irreversibly destroy the tertiary structure of the polypeptide
(excepting, of course, where the purpose of the reaction is to
destroy such tertiary structure). Fourth, the reaction occurs
rapidly at room temperature, which allows the use of many types of
polypeptides or reagents that would be unstable at higher
temperatures. Fifth, the reaction occurs readily is aqueous
conditions, again allowing use of polypeptides and reagents
incompatible (to any extent) with non-aqueous solutions. Six, the
reaction occurs readily even when the ratio of polypeptide or amino
acid to reagent is stoichiometric, near stoichiometric, or
stoichiometric-like, so that it is unnecessary to add excess
reagent or polypeptide to obtain a useful amount of reaction
product. Seventh, the resulting heterocycle can be produced
regioselectively and/or regiospecifically, depending upon the
design of the diamine and dicarbonyl portions of the reactants.
Finally, the condensation of diamines with dicarbonyl-containing
molecules generates heterocycle, including a nitrogen-containing
heterocycle, linkages which are stable under biological
conditions.
VI. Location of Non-Natural Amino Acids in Dolastatin Linker
Derivatives
[0841] The methods and compositions described herein include
incorporation of one or more non-natural amino acids into a
dolastatin linker derivative. One or more non-natural amino acids
may be incorporated at one or more particular positions which do
not disrupt activity of the dolastatin linker derivative. This can
be achieved by making "conservative" substitutions, including but
not limited to, substituting hydrophobic amino acids with
non-natural or natural hydrophobic amino acids, bulky amino acids
with non-natural or natural bulky amino acids, hydrophilic amino
acids with non-natural or natural hydrophilic amino acids) and/or
inserting the non-natural amino acid in a location that is not
required for activity.
[0842] A variety of biochemical and structural approaches can be
employed to select the desired sites for substitution with a
non-natural amino acid within the dolastatin linker derivative. In
some embodiments, the non-natural amino acid is linked at the
C-terminus of the dolastatin derivative. In other embodiments, the
non-natural amino acid is linked at the N-terminus of the
dolastatin derivative. Any position of the dolastatin linker
derivative is suitable for selection to incorporate a non-natural
amino acid, and selection may be based on rational design or by
random selection for any or no particular desired purpose.
Selection of desired sites may be based on producing a non-natural
amino acid polypeptide (which may be further modified or remain
unmodified) having any desired property or activity, including but
not limited to a receptor binding modulators, receptor activity
modulators, modulators of binding to binder partners, binding
partner activity modulators, binding partner conformation
modulators, dimer or multimer formation, no change to activity or
property compared to the native molecule, or manipulating any
physical or chemical property of the polypeptide such as
solubility, aggregation, or stability. Alternatively, the sites
identified as critical to biological activity may also be good
candidates for substitution with a non-natural amino acid, again
depending on the desired activity sought for the polypeptide.
Another alternative would be to simply make serial substitutions in
each position on the polypeptide chain with a non-natural amino
acid and observe the effect on the activities of the polypeptide.
Any means, technique, or method for selecting a position for
substitution with a non-natural amino acid into any polypeptide is
suitable for use in the methods, techniques and compositions
described herein.
[0843] The structure and activity of naturally-occurring mutants of
a polypeptide that contain deletions can also be examined to
determine regions of the protein that are likely to be tolerant of
substitution with a non-natural amino acid. Once residues that are
likely to be intolerant to substitution with non-natural amino
acids have been eliminated, the impact of proposed substitutions at
each of the remaining positions can be examined using methods
including, but not limited to, the three-dimensional structure of
the relevant polypeptide, and any associated ligands or binding
proteins. X-ray crystallographic and NMR structures of many
polypeptides are available in the Protein Data Bank (PDB,
www.rcsb.org), a centralized database containing three-dimensional
structural data of large molecules of proteins and nucleic acids,
one can be used to identify amino acid positions that can be
substituted with non-natural amino acids. In addition, models may
be made investigating the secondary and tertiary structure of
polypeptides, if three-dimensional structural data is not
available. Thus, the identity of amino acid positions that can be
substituted with non-natural amino acids can be readily
obtained.
[0844] Exemplary sites of incorporation of a non-natural amino acid
include, but are not limited to, those that are excluded from
potential receptor binding regions, or regions for binding to
binding proteins or ligands may be fully or partially solvent
exposed, have minimal or no hydrogen-bonding interactions with
nearby residues, may be minimally exposed to nearby reactive
residues, and/or may be in regions that are highly flexible as
predicted by the three-dimensional crystal structure of a
particular polypeptide with its associated receptor, ligand or
binding proteins.
[0845] A wide variety of non-natural amino acids can be substituted
for, or incorporated into, a given position in a polypeptide. By
way of example, a particular non-natural amino acid may be selected
for incorporation based on an examination of the three dimensional
crystal structure of a polypeptide with its associated ligand,
receptor and/or binding proteins, a preference for conservative
substitutions
[0846] In one embodiment, the methods described herein include
incorporating into the dolastatin linker derivative, where the
dolastatin linker derivative comprises a first reactive group; and
contacting the dolastatin linker derivative with a molecule
(including but not limited to a second protein or polypeptide or
polypeptide analog; an antibody or antibody fragment; and any
combination thereof) that comprises a second reactive group. In
certain embodiments, the first reactive group is a hydroxylamine
moiety and the second reactive group is a carbonyl or dicarbonyl
moiety, whereby an oxime linkage is formed. In certain embodiments,
the first reactive group is a carbonyl or dicarbonyl moiety and the
second reactive group is a hydroxylamine moiety, whereby an oxime
linkage is formed. In certain embodiments, the first reactive group
is a carbonyl or dicarbonyl moiety and the second reactive group is
an oxime moiety, whereby an oxime exchange reaction occurs. In
certain embodiments, the first reactive group is an oxime moiety
and the second reactive group is carbonyl or dicarbonyl moiety,
whereby an oxime exchange reaction occurs.
[0847] In some cases, the dolastatin linker derivative
incorporation(s) will be combined with other additions,
substitutions, or deletions within the polypeptide to affect other
chemical, physical, pharmacologic and/or biological traits. In some
cases, the other additions, substitutions or deletions may increase
the stability (including but not limited to, resistance to
proteolytic degradation) of the polypeptide or increase affinity of
the polypeptide for its appropriate receptor, ligand and/or binding
proteins. In some cases, the other additions, substitutions or
deletions may increase the solubility (including but not limited
to, when expressed in E. coli or other host cells) of the
polypeptide. In some embodiments sites are selected for
substitution with a naturally encoded or non-natural amino acid in
addition to another site for incorporation of a non-natural amino
acid for the purpose of increasing the polypeptide solubility
following expression in E. coli, or other recombinant host cells.
In some embodiments, the polypeptides comprise another addition,
substitution, or deletion that modulates affinity for the
associated ligand, binding proteins, and/or receptor, modulates
(including but not limited to, increases or decreases) receptor
dimerization, stabilizes receptor dimers, modulates circulating
half-life, modulates release or bio-availability, facilitates
purification, or improves or alters a particular route of
administration. Similarly, the non-natural amino acid polypeptide
can comprise chemical or enzyme cleavage sequences, protease
cleavage sequences, reactive groups, antibody-binding domains
(including but not limited to, FLAG or poly-His) or other affinity
based sequences (including but not limited to, FLAG, poly-His, GST,
etc.) or linked molecules (including but not limited to, biotin)
that improve detection (including but not limited to, GFP),
purification, transport thru tissues or cell membranes, prodrug
release or activation, size reduction, or other traits of the
polypeptide.
VII. PSMA Gene as Exemplar
[0848] The methods, compositions, strategies and techniques
described herein are not limited to a particular type, class or
family of polypeptides or proteins. Indeed, virtually any
polypeptides may be designed or modified to include at least one
"modified or unmodified" non-natural amino acids containing
dolastatin linker derivative described herein. By way of example
only, the polypeptide can be homologous to a therapeutic protein
selected from the group consisting of: alpha-1 antitrypsin,
angiostatin, antihemolytic factor, antibody, antibody fragment,
monoclonal antibody (e.g., bevacizumab, cetuximab, panitumumab,
infliximab, adalimumab, basiliximab, daclizumab, omalizumab,
ustekinumab, etanercept, gemtuzumab, alemtuzumab, rituximab,
trastuzumab, nimotuzumab, palivizumab, and abeiximab),
apolipoprotein, apoprotein, atrial natriuretic factor, atrial
natriuretic polypeptide, atrial peptide, C--X--C chemokine, T39765,
NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1,
PF4, MIG, calcitonin, c-kit ligand, cytokine, CC chemokine,
monocyte chemoattractant protein-1, monocyte chemoattractant
protein-2, monocyte chemoattractant protein-3, monocyte
inflammatory protein-1 alpha, monocyte inflammatory protein-i beta,
RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40,
CD40 ligand, c-kit ligand, collagen, colony stimulating factor
(CSF), complement factor 5a, complement inhibitor, complement
receptor 1, cytokine, epithelial neutrophil activating peptide-78,
MIP-16, MCP-1, epidermal growth factor (EGF), epithelial neutrophil
activating peptide, erythropoietin (EPO), exfoliating toxin, Factor
IX, Factor VII, Factor VIII, Factor X, fibroblast growth factor
(FGF), fibrinogen, fibronectin, four-helical bundle protein, G-CSF,
glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor,
growth factor receptor, grf, hedgehog protein, hemoglobin,
hepatocyte growth factor (hGF), hirudin, human growth hormone
(hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1
receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II,
interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL),
IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemia
inhibitory factor, luciferase, neurturin, neutrophil inhibitory
factor (NIF), oncostatin M, osteogenic protein, oncogene product,
paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone,
pleiotropin, protein A, protein G, pth, pyrogenic exotoxin A,
pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxin, renin,
SCF, small biosynthetic protein, soluble complement receptor 1,
soluble I-CAM 1, soluble interleukin receptor, soluble TNF
receptor, somatomedin, somatostatin, somatotropin, streptokinase,
superantigens, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2,
SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase,
toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen
activator, tumor growth factor (TGF), tumor necrosis factor, tumor
necrosis factor alpha, tumor necrosis factor beta, tumor necrosis
factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular
endothelial growth factor (VEGF), urokinase, mos, ras, raf, met,
p53, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone
receptor, testosterone receptor, aldosterone receptor, LDL
receptor, and corticosterone.
[0849] Thus, the following description of ARX-.alpha.PSMA is
provided for illustrative purposes and by way of example only, and
not as a limit on the scope of the methods, compositions,
strategies and techniques described herein. Further, reference to
trastuzumab in this application is intended to use the generic term
as an example of any antibody. Thus, it is understood that the
modifications and chemistries described herein with reference to
trastuzumab can be equally applied to any antibody or monoclonal
antibody, including those specifically listed herein.
[0850] ARX-.alpha.PSMA is a humanized monoclonal antibody that
binds to the extracellular domain of prostate specific membrane
antigen. Prostate specific membrane antigen is a type II membrane
protein that is highly expressed, for example, in prostatic
intraepithelial neoplasia (PIN), primary prostate cancers, and
metastatic prostate cancers. For purposes of this invention, it is
possible for the .alpha.PSMA antibody to be any known PSMA antibody
with one non-naturally encoded amino acid. For purposes of
illustration, ARX-.alpha.PSMA antibodies are described in Table
1.
[0851] In one embodiment of the present invention, the antibody
comprises the .alpha.PSMA antibody of SEQ ID NO: 1 and an
.alpha.PSMA antibody heavy chain selected from SEQ ID NO.s 2 and
9-14. In other embodiments of the present invention, the
.alpha.PSMA antibody comprises SEQ ID NO. 2 wherein the heavy chain
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-naturally encoded
amino acids. In other embodiments of the present invention, the
.alpha.PSMA antibody comprises SEQ ID NO. 1 wherein the heavy chain
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-naturally encoded
amino acids. In another embodiment of the present invention, the
antibody comprises an .alpha.PSMA antibody with one or more
non-naturally encoded amino acids substituted at one or more
positions of SEQ ID NO: 2. In another embodiment of the present
invention, the antibody comprises a heavy and light chain as
presented in Table 1 (SEQ ID NO.s 1 and 2). In some embodiments of
the present invention, the antibody is an .alpha.PSMAantibody
comprising the light chain from SEQ ID NO: 1 with a non-naturally
encoded amino acid substituted at a position with high surface
accessibility and a site that will be charge neutral to the
antibody. In other embodiments of the present invention, the
antibody is an .alpha.PSMAantibody comprising the heavy chain from
SEQ ID NO: 2 with a non-naturally encoded amino acid substituted at
a position with high surface accessibility and a site that will be
charge neutral to the antibody. In other embodiments of the present
invention, the antibody is an .alpha.PSMAantibody comprising an
amino acid sequence with one or more non-naturally encoded amino
acids and 90% homology to SEQ ID NO: 2. In other embodiments of the
present invention, the antibody is an .alpha.PSMAantibody
comprising an amino acid sequence a non-naturally encoded amino
acids and 90% homology to SEQ ID NO: 2. In other embodiments of the
present invention, the antibody is an .alpha.PSMAantibody
comprising an amino acid sequence with one or more non-naturally
encoded amino acids and 95% homology to SEQ ID NO: 2. In other
embodiments of the present invention, the antibody is an
.alpha.PSMAantibody comprising an amino acid sequence a
non-naturally encoded amino acids and 95% homology to SEQ ID NO: 2.
In other embodiments of the present invention, the antibody is an
.alpha.PSMAantibody comprising an amino acid sequence with one or
more non-naturally encoded amino acids and 96% homology to SEQ ID
NO: 2. In other embodiments of the present invention, the antibody
is an .alpha.PSMAantibody comprising an amino acid sequence a
non-naturally encoded amino acids and 96% homology to SEQ ID NO: 2.
In other embodiments of the present invention, the antibody is an
.alpha.PSMAantibody comprising an amino acid sequence with one or
more non-naturally encoded amino acids and 97% homology to SEQ ID
NO: 2. In other embodiments of the present invention, the antibody
is an .alpha.PSMAantibody comprising an amino acid sequence a
non-naturally encoded amino acids and 97% homology to SEQ ID NO: 2.
In other embodiments of the present invention, the antibody is an
.alpha.PSMAantibody comprising an amino acid sequence with one or
more non-naturally encoded amino acids and 98% homology to SEQ ID
NO: 2. In other embodiments of the present invention, the antibody
is an .alpha.PSMAantibody comprising an amino acid sequence a
non-naturally encoded amino acids and 98% homology to SEQ ID NO: 2.
In other embodiments of the present invention, the antibody is an
.alpha.PSMAantibody comprising an amino acid sequence with one or
more non-naturally encoded amino acids and at least 98% homology to
SEQ ID NO: 2. In other embodiments of the present invention, the
antibody is an .alpha.PSMAantibody comprising an amino acid
sequence a non-naturally encoded amino acids and at least 98%
homology to SEQ ID NO: 2. In other embodiments of the present
invention, the antibody is an .alpha.PSMAantibody comprising an
amino acid sequence with one or more non-naturally encoded amino
acids and 99% homology to SEQ ID NO: 2. In other embodiments of the
present invention, the antibody is an .alpha.PSMAantibody
comprising an amino acid sequence a non-naturally encoded amino
acids and 99% homology to SEQ ID NO: 2. In other embodiments of the
present invention, the antibody is an .alpha.PSMAantibody
comprising an amino acid sequence with one or more non-naturally
encoded amino acids and at least 99% homology to SEQ ID NO: 2. In
other embodiments of the present invention, the antibody is an
.alpha.PSMAantibody comprising an amino acid sequence a
non-naturally encoded amino acids and at least 99% homology to SEQ
ID NO: 2. Non-naturally encoded amino acid site selection is
described within this specification, and as it specifically
pertains to .alpha.-PSMA antibodies, sites were selected based on
surface exposure/site accessibility within the antibody and
hydrophobic/neutral amino acid sites were selected to maintain the
charge on the antibody.
TABLE-US-00001 TABLE 1 ARX-.alpha.PSMA(A116) .alpha.PSMA light
chain (SEQ. ID. NO.: 1) 1 DIVMTQSHKF MSTSVGDRVS IICKASQDVG
TAVDWYQQKP GQSPKLLIYW 51 ASTRHTGVPD RFTGSGSGTD FTLTITNVQS
EDLADYFCQQ YNSYPLTFGA 101 GTMLDLKRTV AAPSVFIFPP SDEQLKSGTA
SVVCLLNNFY PREAKVQWKV 151 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT
LSKADYEKHK VYACEVTHQG 201 LSSPVTKSFN RGEC .alpha.PSMA heavy chain
(SEQ. ID. NO.: 2) 1 EVQLQQSGPE LVKPGTSVRI SCKTSGYTFT EYTIHWVKQS
HGKSLEWIGN 51 INPNNGGTTY NQKFEDKATL TVDKSSSTAY MELRSLTSED
SAVYYCAAGW 101 NFDYWGQGTT LTVSSASTKG PSVFPLAPSS KSTSGGTAAL
GCLVKDYFPE 151 PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS
LGTQTYICNV 201 NHKPSNTKVD KKVEPKSCDK THTCPPCPAP ELLGGPSVFL
FPPKPKDTLM 251 ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR
EEQYNSTYRV 301 VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ
PREPQVYTLP 351 PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK
TTPPVLDSDG 401 SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPG
ARX-.alpha.PSMA(S7) light chain (SEQ. ID. NO.: 3) 1 DIVMTQ*HKF
MSTSVGDRVS IICKASQDVG TAVDWYQQKP GQSPKLLIYW 51 ASTRHTGVPD
RFTGSGSGTD FTLTITNVQS EDLADYFCQQ YNSYPLTFGA 101 GTMLDLKRTV
AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 151 DNALQSGNSQ
ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 201 LSSPVTKSFN RGEC
ARX-.alpha.PSMA(R18) light chain (SEQ. ID. NO.: 4) 1 DIVMTQSHKF
MSTSVGD*VS IICKASQDVG TAVDWYQQKP GQSPKLLIYW 51 ASTRHTGVPD
RFTGSGSGTD FTLTITNVQS EDLADYFCQQ YNSYPLTFGA 101 GTMLDLKRTV
AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 151 DNALQSGNSQ
ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 201 LSSPVTKSFN RGEC
ARX-.alpha.PSMA(Q79) light chain (SEQ. ID. NO.: 5) 1 DIVMTQSHKF
MSTSVGDRVS IICKASQDVG TAVDWYQQKP GQSPKLLTYW 51 ASTRHTGVPD
RFTGSGSGTD FTLTITNV*S EDLADYFCQQ YNSYPLTFGA 101 GTMLDLKRTV
AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 151 DNALQSGNSQ
ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 201 LSSPVTKSFN RGEC
ARX-.alpha.PSMA(T109) light chain (SEQ. ID. NO.: 6) 1 DIVMTQSHKF
MSTSVGDRVS IICKASQDVG TAVDWYQQKP GQSPKLLIYW 51 ASTRHTGVPD
RFTGSGSGTD FTLTITNVQS EDLADYFCQQ YNSYPLTFGA 101 GTMLDLKR*V
AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 151 DNALQSGNSQ
ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 201 LSSPVTKSFN RGEC
ARX-.alpha.PSMA(Q155) light chain (SEQ. ID. NO.: 7) 1 DIVMTQSHKF
MSTSVGDRVS IICKASQDVG TAVDWYQQKP GQSPKLLIYW 51 ASTRHTGVPD
RFTGSGSGTD FTLTITNVQS EDLADYFCQQ YNSYPLTFGA 101 GTMLDLKRTV
AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 151 DNAL*SGNSQ
ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 201 LSSPVTKSFN RGEC
ARX-.alpha.PSMA(S202) light chain (SEQ. ID. NO.: 8) 1 DIVMTQSHKF
MSTSVGDRVS IICKASQDVG TAVDWYQQKP GQSPKLLIYW 51 ASTRHTGVPD
RFTGSGSGTD FTLTITNVQS EDLADYFCQQ YNSYPLTFGA 101 GTMLDLKRTV
AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 151 DNALQSGNSQ
ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 201 L*SPVTKSFN RGEC
ARX-.alpha.PSMA(A116) heavy chain (SEQ. ID. NO.: 9) 1 EVQLQQSGPE
LVKPGTSVRI SCKTSGYTFT EYTIHWVKQS HGKSLEWIGN 51 INPNNGGTTY
NQKFEDKATL TVDKSSSTAY MELRSLTSED SAVYYCAAGW 101 NFDYWGQGTT
LTVSS*STKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE 151 PVTVSWNSGA
LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV 201 NHKPSNTKVD
KKVEPKSCDK THTCPPCPAP ELLGGPSVFL FPPKPKDTLM 251 ISRTPEVTCV
VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV 301 VSVLTVLHQD
WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP 351 PSRDELTKNQ
VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG 401 SFFLYSKLTV
DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPG ARX-.alpha.PSMA(V280) heavy
chain (SEQ. ID. NO.: 10) 1 EVQLQQSGFE LVKPGTSVRI SCKTSGYTFT
EYTIHWVKQS HGKSLEWIGN 51 INPNNGGTTY NQKFEDKATL TVDKSSSTAY
MELRSLTSED SAVYYCAAGW 101 NFDYWGQGTT LTVSSASTKG PSVFPLAPSS
KSTSGGTAAL GCLVKDYFPE 151 PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL
SSVVTVPSSS LGTQTYICNV 201 NHKPSNTKVD KKVEPKSCDK THTCPPCPAP
ELLGGPSVFL FPPKPKDTLM 251 ISRTPEVTCV VVDVSHEDPE VKFNWYVDG*
EVHNAKTKPR EEQYNSTYRV 301 VSVLTVLHQD WLNGKEYKCK VSNKALPAPI
EKTISKAKGQ PREPQVYTLA 351 PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE
SNGQPENNYK TTPPVLDSDG 401 SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL
HNHYTQKSLS LSPG ARX-.alpha.PSMA(Y294) heavy chain (SEQ. ID. NO.:
11) 1 EVQLQQSGPE LVKPGTSVRI SCKTSGYTFT EYTIHWVKQS HGKSLEWIGN 51
INPNNGGTTY NQKFEDKATL TVDKSSSTAY MELRSLTSED SAVYYCAAGN 101
NEDIWGQGTT LTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE 151
PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV 201
NHKpSNTKVD KKVEPKSCDK THTCPPCPAP ELLGGPSVFL FPPKPKDTLM 251
ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQ*NSTYRV 301
VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP 351
PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG 401
SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPG
ARX-.alpha.PSMA(Q340) heavy chain (SEQ. ID. NO.: 12) 1 EVQLQQSGPE
LVKPGTSVRI SCKTSGYTFT EYTIHWVKQS HGKSLEWIGN 51 INPNNGGTTY
NQKFEDKATL TVDKSSSTAY MELRSLTSED SAVYYCAAGW 101 NEDYWGQGTT
LTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE 151 PVTVSWNSGA
LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV 201 NHKPSNTKVD
KKVEPKSCDK THTCPPCPAP ELLGGPSVFL FPPKPKDTLM 251 TSRTPEVTCV
VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV 301 VSVLTVLHQD
WLNGKEYKCK VSNKALPAPI EKTISKAKG* PREPQVYTLP 351 PSRDELTKNQ
VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG 401 SFFLYSKLTV
DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPG ARX-.alpha.PSMA(N359) heavy
chain (SEQ. ID. NO.: 13) 1 EVQLQQSGPE LVKPGTSVRI SCKTSGYTFT
EYTIHWVKQS HGKSLEWIGN 51 INPNNGGTTY NQKFEDKATL TVDKSSSTAY
MELRSLTSED SAVYYCAAGW 101 NFDYWGQGTT LTVSSASTKG PSVFPLAPSS
KSTSGGTAAL GCLVKDYFPE 151 PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL
SSVVTVPSSS LGTQTYICNV 201 NHKPSNTKVD KKVEPKSCDK THTCPPCPAP
ELLGGPSVFL FPPKPKDTLM 251 ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV
EVHNAKTKPR EEQYNSTYRV 301 VSVLTVLHQD WLNGKEYKCK VSNKALPAPI
EKTISKAKGQ PREPQVYTLP 351 PSRDELTK*Q VSLTCLVKGF YPSDIAVEWE
SNGQPENNYK TTPPVLDSDG 401 SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL
HNHYTQKSLS LSPG ARX-.alpha.PSMA(Q417) heavy chain (SEQ ID. NO.: 14)
1 EVQLQQSGPE LVKPGTSVRI SCKTSGYTFT EYTIHWVKQS HGKSLEWIGN 51
INPNNGGTTY NQKFEDKATL TVDKSSSTAY MELRSLTSED SAVYYCAAGW 101
NFDYWGQGTT LTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE 151
PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV 201
NHKPSNTKVD KKVEPKSCDK THTCPPCPAP ELLGGPSVFL FPPKPKDTLM 251
ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV 301
VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP 351
PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG 401
SFFLYSKLTV DKSRWQ*GNV FSCSVMHEAL HNHYTQKSLS LSPG
[0852] In one embodiment, the present invention provides an
.alpha.PSMA antibody drug conjugate (ADC or an .alpha.PSMA-ADC)
wherein the antibody is an .alpha.PSMA comprising a light and heavy
chain. In another embodiment of the present invention is provided
an .alpha.PSMA-ADC comprising a light (SEQ. ID. NO.: 1) and heavy
chain (SEQ. ID. NO.: 2). In some embodiments of the present
invention, the antibody comprises an .alpha.PSMA antibody with one
or more non-naturally encoded amino acids. In another embodiment of
the present invention, the antibody comprises an .alpha.PSMA
antibody with one or more non-naturally encoded amino acids
substituted at one or more positions of SEQ ID NO: 1. In another
embodiment of the present invention, the antibody comprises an
.alpha.PSMA antibody with one or more non-naturally encoded amino
acids substituted at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of
SEQ ID NO: 1. In another embodiment of the present invention, the
antibody comprises an .alpha.PSMA antibody with one or more
non-naturally encoded amino acids substituted at 1, 2, 3, 4, or 5
positions of SEQ ID NO: 1. In another embodiment of the present
invention, the antibody comprises an .alpha.PSMA antibody with one
or more non-naturally encoded amino acids substituted at 1, 2, or 3
positions of SEQ ID NO: 1. In another embodiment of the present
invention, the antibody comprises an .alpha.PSMA antibody with one
or more non-naturally encoded amino acids substituted at 1 or 2
positions of SEQ ID NO: 1. In another embodiment of the present
invention, the antibody comprises an .alpha.PSMA antibody with two
non-naturally encoded amino acids substituted at 2 positions of SEQ
ID NO: 1. In another embodiment of the present invention, the
antibody comprises an .alpha.PSMA antibody with one non-naturally
encoded amino acids substituted at one positions of SEQ ID NO: 1.
In another embodiment of the present invention, the antibody
comprises the .alpha.PSMA antibody of SEQ ID NO: 3. In another
embodiment of the present invention, the antibody comprises the
.alpha.PSMA antibody of SEQ ID NO: 4. In another embodiment of the
present invention, the antibody comprises the .alpha.PSMA antibody
of SEQ ID NO: 5. In another embodiment of the present invention,
the antibody comprises the .alpha.PSMA antibody of SEQ ID NO: 6. In
another embodiment of the present invention, the antibody comprises
the .alpha.PSMA antibody of SEQ ID NO: 7. In another embodiment of
the present invention, the antibody comprises the .alpha.PSMA
antibody of SEQ ID NO: 8. In another embodiment of the present
invention, the antibody comprises the .alpha.PSMA antibody of SEQ
ID NO: 1 and an .alpha.PSMA antibody heavy chain. In another
embodiment of the present invention, the antibody comprises the
.alpha.PSMA antibody of SEQ ID NO: 1 and an .alpha.PSMA antibody
heavy chain of SEQ ID NO. 2 wherein the heavy chain comprises one
or more non-naturally encoded amino acids. In another embodiment of
the present invention, the antibody comprises the .alpha.PSMA
antibody of SEQ ID NO: 1 and an .alpha.PSMA antibody heavy chain of
SEQ ID NO. 2 wherein the heavy chain comprises 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 non-naturally encoded amino acids. In another
embodiment of the present invention, the antibody comprises the
.alpha.PSMA antibody of SEQ ID NO: 1 and an .alpha.PSMA antibody
heavy chain of SEQ ID NO. 2 wherein the heavy chain comprises 1, 2,
3, 4, or 5 non-naturally encoded amino acids. In another embodiment
of the present invention, the antibody comprises the .alpha.PSMA
antibody of SEQ ID NO: 1 and an .alpha.PSMA antibody heavy chain of
SEQ ID NO. 2 wherein the heavy chain comprises 1, 2, or 3
non-naturally encoded amino acids. In another embodiment of the
present invention, the antibody comprises the .alpha.PSMA antibody
of SEQ ID NO: 1 and an .alpha.PSMA antibody heavy chain of SEQ ID
NO. 2 wherein the heavy chain comprises two non-naturally encoded
amino acids. In another embodiment of the present invention, the
antibody comprises the .alpha.PSMA antibody of SEQ ID NO: 1 and an
.alpha.PSMA antibody heavy chain of SEQ ID NO. 2 wherein the heavy
chain comprises one non-naturally encoded amino acid. In another
embodiment, the present invention provides an .alpha.PSMA-ADC
comprising SEQ ID NO: 1 and SEQ ID NO: 9. In another embodiment,
the present invention provides an .alpha.PSMA-ADC comprising SEQ ID
NO: 1 and SEQ ID NO: 10. In another embodiment, the present
invention provides an .alpha.PSMA-ADC comprising SEQ ID NO: 1 and
SEQ ID NO: 11. In another embodiment, the present invention
provides an .alpha.PSMA-ADC comprising SEQ ID NO: 1 and SEQ ID NO:
12. In another embodiment, the present invention provides an
.alpha.PSMA-ADC comprising SEQ ID NO: 1 and SEQ ID NO: 13. In
another embodiment, the present invention provides an
.alpha.PSMA-ADC comprising SEQ ID NO: 1 and SEQ ID NO: 14.
[0853] In another embodiment of the present invention, the antibody
comprises an .alpha.PSMA antibody with one or more non-naturally
encoded amino acids substituted at one or more positions of SEQ ID
NO: 2. In another embodiment of the present invention, the antibody
comprises an .alpha.PSMA antibody with one or more non-naturally
encoded amino acids substituted at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
positions of SEQ ID NO: 2. In another embodiment of the present
invention, the antibody comprises an .alpha.PSMA antibody with one
or more non-naturally encoded amino acids substituted at 1, 2, 3,
4, or 5 positions of SEQ ID NO: 2. In another embodiment of the
present invention, the antibody comprises an .alpha.PSMA antibody
with one or more non-naturally encoded amino acids substituted at
1, 2, or 3 positions of SEQ ID NO: 2. In another embodiment of the
present invention, the antibody comprises an .alpha.PSMA antibody
with one or more non-naturally encoded amino acids substituted at 1
or 2 positions of SEQ ID NO: 2. In another embodiment of the
present invention, the antibody comprises an .alpha.PSMA antibody
with two non-naturally encoded amino acids substituted at 2
positions of SEQ ID NO: 2. In another embodiment of the present
invention, the antibody comprises an .alpha.PSMA antibody with one
non-naturally encoded amino acids substituted at one positions of
SEQ ID NO: 2. In another embodiment of the present invention, the
antibody comprises the .alpha.PSMA antibody of SEQ ID NO: 9. In
another embodiment of the present invention, the antibody comprises
the .alpha.PSMA antibody of SEQ ID NO: 10. In another embodiment of
the present invention, the antibody comprises the .alpha.PSMA
antibody of SEQ ID NO: 11. In another embodiment of the present
invention, the antibody comprises the .alpha.PSMA antibody of SEQ
ID NO: 12. In another embodiment of the present invention, the
antibody comprises the .alpha.PSMA antibody of SEQ ID NO: 13. In
another embodiment of the present invention, the antibody comprises
the .alpha.PSMA antibody of SEQ ID NO: 14. In another embodiment of
the present invention, the antibody comprises the .alpha.PSMA
antibody of SEQ ID NO: 2 and an .alpha.PSMA antibody light chain.
In another embodiment of the present invention, the antibody
comprises the .alpha.PSMA antibody of SEQ ID NO: 9 and an
.alpha.PSMA antibody light chain. In another embodiment of the
present invention, the antibody comprises the .alpha.PSMA antibody
of SEQ ID NO: 10 and an .alpha.PSMA antibody light chain. In
another embodiment of the present invention, the antibody comprises
the .alpha.PSMA antibody of SEQ ID NO: 11 and an .alpha.PSMA
antibody light chain. In another embodiment of the present
invention, the antibody comprises the .alpha.PSMA antibody of SEQ
ID NO: 12 and an .alpha.PSMA antibody light chain. In another
embodiment of the present invention, the antibody comprises the
.alpha.PSMA antibody of SEQ ID NO: 13 and an .alpha.PSMA antibody
light chain. In another embodiment of the present invention, the
antibody comprises the .alpha.PSMA antibody of SEQ ID NO: 14 and an
.alpha.PSMA antibody light chain.
[0854] As an additional, non-limiting example, the following
description of HER2 antibody trastuzumab is provided for
illustrative purposes and by way of example only, and not as a
limit on the scope of the methods, compositions, strategies and
techniques described herein. Further, reference to trastuzumab in
this application is intended to use the generic term as an example
of any antibody. Thus, it is understood that the modifications and
chemistries described herein with reference to trastuzumab can be
equally applied to any antibody or monoclonal antibody, including
those specifically listed herein.
[0855] Trastuzumab is a humanized monoclonal antibody that binds to
the domain IV of the extracellular segment of the HER2/neu
receptor. The HER2 gene (also known as HER2/neu and ErbB2 gene) is
amplified in 20-30% of early-stage breast cancers, which makes it
overexpressed. Also, in cancer, HER2 may send signals without
mitogens arriving and binding to any receptor, making it
overactive.
[0856] HER2 extends through the cell membrane, and carries signals
from outside the cell to the inside. In healthy people, signaling
compounds called mitogens arrive at the cell membrane, and bind to
the outside part of other members of the HER family of receptors.
Those bound receptors then link (dimerize) with HER2, activating
it. HER2 then sends a signal to the inside of the cell. The signal
passes through different biochemical pathways. This includes the
PI3K/Akt pathway and the MAPK pathway. These signals promote
invasion, survival and growth of blood vessels (angiogenesis) of
cells.
[0857] Cells treated with trastuzumab undergo arrest during the GI
phase of the cell cycle so there is reduced proliferation. It has
been suggested that trastuzumab induces some of its effect by
downregulation of HER2/neu leading to disruption of receptor
dimerization and signaling through the downstream PI3K cascade.
P27Kip1 is then not phosphorylated and is able to enter the nucleus
and inhibit cdk2 activity, causing cell cycle arrest. Also,
trastuzumab suppresses angiogenesis by both induction of
antiangiogenic factors and repression of proangiogenic factors. It
is thought that a contribution to the unregulated growth observed
in cancer could be due to proteolytic cleavage of HER2/neu that
results in the release of the extracellular domain. Trastuzumab has
been shown to inhibit HER2/neu ectodomain cleavage in breast cancer
cells.
VII. Cellular Uptake of Non-Natural Amino Acids
[0858] Non-natural amino acid uptake by a eukaryotic cell is one
issue that is typically considered when designing and selecting
non-natural amino acids, including but not limited to, for
incorporation into a protein. For example, the high charge density
of .alpha.-amino acids suggests that these compounds are unlikely
to be cell permeable. Natural amino acids are taken up into the
eukaryotic cell via a collection of protein-based transport
systems. A rapid screen can be done which assesses which
non-natural amino acids, if any, are taken up by cells (examples 15
& 16 herein illustrate non-limiting examples of tests which can
be done on non-natural amino acids). See, e.g., the toxicity assays
in, e.g., the U.S. Patent Publication No. 2004/198637 entitled
"Protein Arrays," which is herein incorporated by reference in its
entirety, and Liu, D. R. & Schultz, P. G. (1999) Progress
toward the evolution of an organism with an expanded genetic code.
PNAS United States 96:4780-4785. Although uptake is easily analyzed
with various assays, an alternative to designing non-natural amino
acids that are amenable to cellular uptake pathways is to provide
biosynthetic pathways to create amino acids in vivo.
[0859] Typically, the non-natural amino acid produced via cellular
uptake as described herein is produced in a concentration
sufficient for efficient protein biosynthesis, including but not
limited to, a natural cellular amount, but not to such a degree as
to affect the concentration of the other amino acids or exhaust
cellular resources. Typical concentrations produced in this manner
are about 10 mM to about 0.05 mM.
IX. Biosynthesis of Non-Natural Amino Acids
[0860] Many biosynthetic pathways already exist in cells for the
production of amino acids and other compounds. While a biosynthetic
method for a particular non-natural amino acid may not exist in
nature, including but not limited to, in a cell, the methods and
compositions described herein provide such methods. For example,
biosynthetic pathways for non-natural amino acids can be generated
in host cell by adding new enzymes or modifying existing host cell
pathways. Additional new enzymes include naturally occurring
enzymes or artificially evolved enzymes. For example, the
biosynthesis of p-aminophenylalanine (as presented in an example in
WO 2002/085923 entitled "In vivo incorporation of unnatural amino
acids") relies on the addition of a combination of known enzymes
from other organisms. The genes for these enzymes can be introduced
into a eukaryotic cell by transforming the cell with a plasmid
comprising the genes. The genes, when expressed in the cell,
provide an enzymatic pathway to synthesize the desired compound.
Examples of the types of enzymes that are optionally added are
provided herein. Additional enzymes sequences are found, for
example, in Genbank. Artificially evolved enzymes can be added into
a cell in the same manner. In this manner, the cellular machinery
and resources of a cell are manipulated to produce non-natural
amino acids.
[0861] A variety of methods are available for producing novel
enzymes for use in biosynthetic pathways or for evolution of
existing pathways. For example, recursive recombination, including
but not limited to, as developed by Maxygen, Inc. (available on the
world wide web at www.maxygen.com), can be used to develop novel
enzymes and pathways. See, e.g., Stemmer (1994), Rapid evolution of
a protein in vitro by DNA shuffling, Nature 370(4):389-391; and,
Stemmer, (1994), DNA shuffling by random fragmentation and
reassembly: In vitro recombination for molecular evolution, Proc.
Natl. Acad. Sci. USA., 91:10747-10751. Similarly DesignPath.TM.,
developed by Genencor (available on the world wide web at
genencor.com) is optionally used for metabolic pathway engineering,
including but not limited to, to engineer a pathway to create a
non-natural amino acid in a cell. This technology reconstructs
existing pathways in host organisms using a combination of new
genes, including but not limited to those identified through
functional genomics, molecular evolution and design. Diversa
Corporation (available on the world wide web at diversa.com) also
provides technology for rapidly screening libraries of genes and
gene pathways, including but not limited to, to create new pathways
for biosynthetically producing non-natural amino acids.
[0862] Typically, the non-natural amino acid produced with an
engineered biosynthetic pathway as described herein is produced in
a concentration sufficient for efficient protein biosynthesis,
including but not limited to, a natural cellular amount, but not to
such a degree as to affect the concentration of the other amino
acids or exhaust cellular resources. Typical concentrations
produced in vivo in this manner are about 10 mM to about 0.05 mM.
Once a cell is transformed with a plasmid comprising the genes used
to produce enzymes desired for a specific pathway and a non-natural
amino acid is generated, in vivo selections are optionally used to
further optimize the production of the non-natural amino acid for
both ribosomal protein synthesis and cell growth.
X. Additional Synthetic Methodology
[0863] The non-natural amino acids described herein may be
synthesized using methodologies described in the art or using the
techniques described herein or by a combination thereof. As an aid,
the following table provides various starting electrophiles and
nucleophiles which may be combined to create a desired functional
group. The information provided is meant to be illustrative and not
limiting to the synthetic techniques described herein.
TABLE-US-00002 TABLE 2 Examples of Covalent Linkages and Precursors
Thereof Covalent Linkage Product Electrophile Nucleophile
Carboxamides Activated esters amines/anilines Carboxamides acyl
azides amines/anilines Carboxamides acyl halides amines/anilines
Esters acyl halides alcohols/phenols Esters acyl nitriles
alcohols/phenols Carboxamides acyl nitriles amines/anilines Imines
Aldehydes amines/anilines Hydrazones aldehydes or ketones
Hydrazines Oximes aldehydes or ketones Hydroxylamines Alkyl amines
alkyl halides amines/anilines Esters alkyl halides carboxylic acids
Thioethers alkyl halides Thiols Ethers alkyl halides
alcohols/phenols Thioethers alkyl sulfonates Thiols Esters alkyl
sulfonates carboxylic acids Ethers alkyl sulfonates
alcohols/phenols Esters Anhydrides alcohols/phenols Carboxamides
Anhydrides amines/anilines Thiophenols aryl halides Thiols Aryl
amines aryl halides Amines Thioethers Azindines Thiols Boronate
esters Boronates Glycols Carboxamides carboxylic acids
amines/anilines Esters carboxylic acids Alcohols hydrazines
Hydrazides carboxylic acids N-acylureas or Anhydrides carbodiimides
carboxylic acids Esters diazoalkanes carboxylic acids Thioethers
Epoxides Thiols Thioethers haloacetamides Thiols Ammotriazines
halotriazines amines/anilines Triazinyl ethers halotriazines
alcohols/phenols Amidines imido esters amines/anilines Ureas
Isocyanates amines/anilines Urethanes Isocyanates alcohols/phenols
Thioureas isothiocyanates amines/anilines Thioethers Maleimides
Thiols Phosphite esters phosphoramidites Alcohols Silyl ethers
silyl halides Alcohols Alkyl amines sulfonate esters
amines/anilines Thioethers sulfonate esters Thiols Esters sulfonate
esters carboxylic acids Ethers sulfonate esters Alcohols
Sulfonamides sulfonyl halides amines/anilines Sulfonate esters
sulfonyl halides phenols/alcohols
[0864] In general, carbon electrophiles are susceptible to attack
by complementary nucleophiles, including carbon nucleophiles,
wherein an attacking nucleophile brings an electron pair to the
carbon electrophile in order to form a new bond between the
nucleophile and the carbon electrophile.
[0865] Non-limiting examples of carbon nucleophiles include, but
are not limited to alkyl, alkenyl, aryl and alkynyl Grignard,
organolithium, organozinc, alkyl-, alkenyl, aryl- and alkynyl-tin
reagents (organostannanes), alkyl-, alkenyl-, aryl- and
alkynyl-borane reagents (organoboranes and organoboronates); these
carbon nucleophiles have the advantage of being kinetically stable
in water or polar organic solvents. Other non-limiting examples of
carbon nucleophiles include phosphorus ylids, enol and enolate
reagents; these carbon nucleophiles have the advantage of being
relatively easy to generate from precursors well known to those
skilled in the art of synthetic organic chemistry. Carbon
nucleophiles, when used in conjunction with carbon electrophiles,
engender new carbon-carbon bonds between the carbon nucleophile and
carbon electrophile.
[0866] Non-limiting examples of non-carbon nucleophiles suitable
for coupling to carbon electrophiles include but are not limited to
primary and secondary amines, thiols, thiolates, and thioethers,
alcohols, alkoxides, azides, semicarbazides, and the like. These
non-carbon nucleophiles, when used in conjunction with carbon
electrophiles, typically generate heteroatom linkages (C--X--C),
wherein X is a hetereoatom, including, but not limited to, oxygen,
sulfur, or nitrogen.
[0867] An additional embodiment of the present invention is a
compound comprising Formula (VIII) or (IX):
##STR00184##
wherein: [0868] A is optional, and when present is lower alkylene,
substituted lower alkylene, lower cycloalkylene, substituted lower
cycloalkylene, lower alkenylene, substituted lower alkenylene,
alkynylene, lower heteroalkylene, substituted heteroalkylene, lower
heterocycloalkylene, substituted lower heterocycloalkylene,
arylene, substituted arylene, heteroarylene, substituted
heteroarylene, alkarylene, substituted alkarylene, aralkylene, or
substituted aralkylene; [0869] B is optional, and when present is a
linker selected from the group consisting of lower alkylene,
substituted lower alkylene, lower alkenylene, substituted lower
alkenylene, lower heteroalkylene, substituted lower heteroalkylene,
--O--, --O-(alkylene or substituted alkylene)-, --S--,
--S-(alkylene or substituted alkylene)-, --S(O).sub.k-- where k is
1, 2, or 3, --S(O).sub.k(alkylene or substituted alkylene)-,
--C(O)--, --C(O)-(alkylene or substituted alkylene)-, --C(S)--,
--C(S)-(alkylene or substituted alkylene)-, --N(R')--,
--NR'-(alkylene or substituted alkylene)-, --C(O)N(R')--,
--CON(R')-(alkylene or substituted alkylene)-, --CSN(R')--,
--CSN(R')-(alkylene or substituted alkylene)-, --N(R')CO-(alkylene
or substituted alkylene)-, --N(R')C(O)O--, --S(O).sub.kN(R')--,
--N(R')C(O)N(R')--, --N(R')C(S)N(R')--, --N(R')S(O).sub.kN(R')--,
--N(R')--N.dbd., --C(R').dbd.N--, --C(R').dbd.N--N(R')--,
--C(R').dbd.N--N.dbd., --C(R').sub.2--N.dbd.N--, and
--C(R').sub.2--N(R')--N(R')--, where each R' is independently H,
alkyl, or substituted alkyl; [0870] R is H, alkyl, substituted
alkyl, cycloalkyl, or substituted cycloalkyl; [0871] R.sub.1 is H,
an amino protecting group, resin, at least one amino acid,
polypeptide, or polynucleotide; [0872] R.sub.2 is OH, an ester
protecting group, resin, at least one amino acid, polypeptide, or
polynucleotide; [0873] R.sub.3 and R.sub.4 are each independently
H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and
R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a
heterocycloalkyl; [0874] Z has the structure of:
[0874] ##STR00185## [0875] R.sub.5 is H, COR.sub.8,
C.sub.1-C.sub.6alkyl, or thiazole; [0876] R.sub.8 is OH; [0877]
R.sub.6 is OH or H; [0878] Ar is phenyl or pyridine; [0879] R.sub.7
is C.sub.1-C.sub.6alkyl or hydrogen; [0880] L is a linker selected
from the group consisting of -alkylene-, -alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-C(O)--,
-(alkylene-O).sub.n--(CH.sub.2).sub.n'--NHC(O)--(CH.sub.2).sub.n''--C(Me)-
.sub.2-S--S--(CH.sub.2).sub.n'''--NHC(O)-(alkylene-O).sub.n''''-alkylene-,
-(alkylene-O).sub.n-alkylene-W--, -alkylene-C(O)--W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--, and
-(alkylene-O).sub.n-alkylene-U-alkylene-; [0881] W has the
structure of:
[0881] ##STR00186## [0882] U has the structure of:
[0882] ##STR00187## [0883] and each n, n', n'', n''' and n'''' are
independently integers greater than or equal to one; or an active
metabolite, or a pharmaceutically acceptable prodrug or solvate
thereof. Additional embodiments of the present invention include
the compound wherein R.sub.1 is a polypeptide. In other embodiments
of the present invention R.sub.1 is an antibody. In another
embodiment of the present invention, R.sub.1 is an
anti-prostate-specific membrane antigen (.alpha.PSMA) antibody. In
other embodiments of the present invention, the .alpha.PSMA
antibody comprises a non-naturally encoded amino acid. In other
embodiments of the present invention, the .alpha.PSMA antibody
comprises more than one non-naturally encoded amino acid. In other
embodiments of the present invention, the .alpha.PSMA antibody
comprises at least one and no more than ten non-naturally encoded
amino acids. In other embodiments of the present invention, R.sub.2
is a polypeptide. In some embodiments of the present invention,
R.sub.2 is an antibody. In other embodiments of the present
invention, R.sub.2 is an .alpha.PSMA antibody. In some embodiments
of the present invention, R.sub.2 is an .alpha.PSMA antibody that
comprises a non-naturally encoded amino acid. In some embodiments
of the present invention, R.sub.2 is an .alpha.PSMA antibody that
comprises one or more non-naturally encoded amino acids. In some
embodiments of the present invention, R.sub.2 is an .alpha.PSMA
antibody that comprises at least one and no more than ten
non-naturally encoded amino acids.
[0884] In other embodiments the present invention includes a
compound, or salt thereof, comprising Formula (X), (XI), (XII) or
(XIII):
##STR00188## [0885] wherein: [0886] A is optional, and when present
is lower alkylene, substituted lower alkylene, lower cycloalkylene,
substituted lower cycloalkylene, lower alkenylene, substituted
lower alkenylene, alkynylene, lower heteroalkylene, substituted
heteroalkylene, lower heterocycloalkylene, substituted lower
heterocycloalkylene, arylene, substituted arylene, heteroarylene,
substituted heteroarylene, alkarylene, substituted alkarylene,
aralkylene, or substituted aralkylene; [0887] B is optional, and
when present is a linker selected from the group consisting of
lower alkylene, substituted lower alkylene, lower alkenylene,
substituted lower alkenylene, lower heteroalkylene, substituted
lower heteroalkylene, --O--, --O-(alkylene or substituted
alkylene)-, --S--, --S-(alkylene or substituted alkylene)-,
--S(O).sub.k-- where k is 1, 2, or 3, --S(O).sub.k(alkylene or
substituted alkylene)-, --C(O)--, --C(O)-(alkylene or substituted
alkylene)-, --C(S)--, --C(S)-(alkylene or substituted alkylene)-,
--N(R')--, --NR'-(alkylene or substituted alkylene)-,
--C(O)N(R')--, --CON(R')-(alkylene or substituted alkylene)-,
--CSN(R')--, --CSN(R')-(alkylene or substituted alkylene)-,
--N(R')CO-(alkylene or substituted alkylene)-, --N(R')C(O)O--,
--S(O).sub.kN(R')--, --N(R')C(O)N(R')--, --N(R')C(S)N(R')--,
--N(R')S(O).sub.kN(R')--, --N(R')--N.dbd., --C(R').dbd.N--,
--C(R').dbd.N--N(R')--, --C(R').dbd.N--N.dbd.,
--C(R').sub.2--N.dbd.N--, and --C(R').sub.2--N(R')--N(R')--, where
each R' is independently H, alkyl, or substituted alkyl; [0888] R
is H, alkyl, substituted alkyl, cycloalkyl, or substituted
cycloalkyl; [0889] R.sub.1 is H, an amino protecting group, resin,
at least one amino acid, polypeptide, or polynucleotide; [0890]
R.sub.2 is OH, an ester protecting group, resin, at least one amino
acid, polypeptide, or polynucleotide; [0891] R.sub.3 and R.sub.4
are each independently H, halogen, lower alkyl, or substituted
lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3 groups
optionally form a cycloalkyl or a heterocycloalkyl; [0892] Z has
the structure of:
[0892] ##STR00189## [0893] R.sub.5 is H, CO.sub.2H,
C.sub.1-C.sub.6alkyl, or thiazole; [0894] R.sub.6 is OH or H;
[0895] Ar is phenyl or pyridine; [0896] R.sub.7 is
C.sub.1-C.sub.6alkyl or hydrogen; [0897] L.sub.1, L.sub.2, L.sub.3,
and L.sub.4 are each linkers independently selected from the group
consisting of a bond, -alkylene-, -(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-J-(alkylene-O)).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n'-alkylene-J'-,
-(alkylene-O).sub.n-alkylene-J-alkylene'-, --W--, -alkylene-W--,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-alkylene-NMe).sub.n-alkylene-W--,
-J-alkylene-NMe-alkylene'-NMe-alkylene''-W--, and
-alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--; [0898] W
has the structure of:
[0898] ##STR00190## [0899] each J and J' independently have the
structure of:
[0899] ##STR00191## and [0900] each n and n' are independently
integers greater than or equal to one. In some embodiments, R.sub.1
is a polypeptide. In other embodiments, R.sub.1 is an antibody. In
other embodiments, R.sub.1 is an .alpha.PSMA antibody. In some
embodiments, R.sub.2 is a polypeptide. In other embodiments,
R.sub.2 is an antibody. In other embodiments, R.sub.2 is an
.alpha.PSMA antibody.
[0901] The present invention also provides methods for derivatizing
a dolastatin analog comprising Formula (I), (III), (IV), (V), or
(VI), one method comprising contacting the dolastatin analog with a
reagent of Formula (XXXVII), wherein Formula (I), (III), (IV), (V),
or (VI) correspond to:
##STR00192## [0902] wherein: [0903] Z has the structure of:
[0903] ##STR00193## [0904] R.sub.5 is H, COR.sub.8,
C.sub.1-C.sub.6alkyl, or thiazole; [0905] R.sub.8 is OH or
--NH-(alkylene-O).sub.n--NH.sub.2; [0906] R.sub.6 is OH or H;
[0907] Ar is phenyl or pyridine; [0908] R.sub.7 is C.sub.1-C.sub.6
alkyl or hydrogen; [0909] Y is NH.sub.2--O-- or methyl; [0910] L,
L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each linkers selected
from the group consisting of a bond, -alkylene-, -alkylene-C(O)--,
-(alkylene-O).sub.1-alkylene-, -(alkylene-O).sub.n-alkylene-C(O)--,
-(alkylene-O).sub.n--(CH.sub.2).sub.n--NHC(O)--(CH.sub.2).sub.n''--C(Me).-
sub.2-S--S--(CH.sub.2).sub.n'''--
NHC(O)-(alkylene-O).sub.n''''-alkylene-,
-(alkylene-O).sub.n-alkylene-W--, -alkylene-C(O)--W--,
-(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-alkylene',
-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n'-alkylene-J'-,
--W--, -alkylene-W--,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--, and
J-(alkylene-NMe).sub.n-alkylene-W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-U-alkylene-;
-J-alkylene-NMe-alkylene'-NMe-alkylene''-W--, and
-alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--; [0911] W
has the structure of:
[0911] ##STR00194## [0912] U has the structure of:
[0912] ##STR00195## [0913] each J and J' independently have the
structure of:
[0913] ##STR00196## [0914] or L is absent, Y is methyl, R.sub.5 is
COR.sub.8, and R.sub.8 is --NH-(alkylene-O).sub.n--NH.sub.2; and
[0915] each n, n', n'', n''' and n'''' are independently integers
greater than or equal to one; wherein Formula (XXXVII) corresponds
to:
[0915] ##STR00197## [0916] wherein: [0917] A is optional, and when
present is lower alkylene, substituted lower alkylene, lower
alkenylene, substituted lower alkenylene, arylene, substituted
arylene, heteroarylene, substituted heteroarylene, alkarylene,
substituted alkarylene, aralkylene, or substituted aralkylene;
[0918] B is optional, and when present is a linker selected from
the group consisting of lower alkylene, substituted lower alkylene,
lower alkenylene, substituted lower alkenylene, --O--,
--O-(alkylene or substituted alkylene)-, --S--, --S-(alkylene or
substituted alkylene)-, --S(O).sub.k-- where k is 1, 2, or 3,
--S(O).sub.k(alkylene or substituted alkylene)-, --C(O)--,
--C(O)-(alkylene or substituted alkylene)-, --C(S)--,
--C(S)-(alkylene or substituted alkylene)-, --N(R')--,
--NR'-(alkylene or substituted alkylene)-, --C(O)N(R')--,
--CON(R')-(alkylene or substituted alkylene)-, --CSN(R')--,
--CSN(R')-(alkylene or substituted alkylene)-, --N(R')CO-(alkylene
or substituted alkylene)-, --N(R')C(O)O--, --S(O).sub.kN(R')--,
--N(R')C(O)N(R')--, --N(R')C(S)N(R')--, --N(R')S(O).sub.kN(R')--,
--N(R')--N.dbd., --C(R').dbd.N--, --C(R').dbd.N--N(R')--,
--C(R').dbd.N--N.dbd., --C(R').sub.2--N.dbd.N--, and
--C(R').sub.2--N(R')--N(R')--, where each R' is independently H,
alkyl, or substituted alkyl; [0919] each R' is independently H,
alkyl, or substituted alkyl; [0920] K is
[0920] ##STR00198## [0921] R is H, alkyl, substituted alkyl,
cycloalkyl, or substituted cycloalkyl; [0922] R.sub.1 is H, an
amino protecting group, resin, at least one amino acid, or
polynucleotide; [0923] R.sub.2 is OH, an ester protecting group,
resin, at least one amino acid, or polynucleotide; and [0924]
R.sub.3 and R.sub.4 are each independently H, halogen, lower alkyl,
or substituted lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3
groups optionally form a cycloalkyl or a heterocycloalkyl.
[0925] Another embodiment of the present invention provides a
method for derivatizing a dolastatin analog comprising at least one
oxime containing amino acid having the structure of Formula (VIII),
(IX), (X), (XI), (XII), or (XIII):
##STR00199##
[0926] In another embodiment of the present invention, the
dolastatin analog is contacted with the reagent of Formula (XXXVII)
in aqueous solution under mildly acidic conditions. In another
embodiment, the present invention provides a compound comprising
Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX):
##STR00200## [0927] wherein: [0928] Z has the structure of:
[0928] ##STR00201## [0929] R.sub.5 is H, CO.sub.2H,
C.sub.1-C.sub.6alkyl, or thiazole; [0930] R.sub.6 is OH or H;
[0931] Ar is phenyl or pyridine; [0932] R.sub.1 is H, an amino
protecting group, resin, at least one amino acid, polypeptide, or
polynucleotide; [0933] R.sub.2 is OH, an ester protecting group,
resin, at least one amino acid, polypeptide, or polynucleotide;
[0934] R.sub.4 is H, halogen, lower alkyl, or substituted lower
alkyl; [0935] R.sub.7 is C.sub.1-C.sub.6alkyl or hydrogen; [0936]
L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each linkers selected
from the group consisting of a bond, -alkylene-, -alkylene-C(O)--,
-alkylene-J-, -(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-C(O)--, -(alkylene-O).sub.n-J-,
-(alkylene-O).sub.n-J-alkylene-,
-(alkylene-O).sub.n--(CH.sub.2).sub.n'--NHC(O)--(CH.sub.2).sub.n''--C(Me)-
.sub.2-S--S--(CH.sub.2).sub.n''',
--NHC(O)-(alkylene-O).sub.n''''-alkylene-,
-(alkylene-O).sub.n-alkylene-W--, -alkylene-C(O)--W--,
-(alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-alkylene',
-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n'-alkylene-J'-,
--W--, -alkylene-W--,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-(alkylene-NMe).sub.n-alkylene-W--,
-(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--, -(alkylene-O).sub.n
alkylene-U-alkylene-; -J-alkylene-NMe-alkylene'-NMe-alkylene''-W--,
and -alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--;
[0937] W has the structure of:
[0937] ##STR00202## [0938] U has the structure of:
[0938] ##STR00203## [0939] each J and J' independently have the
structure of:
[0939] ##STR00204## [0940] each n and n' are independently integers
greater than or equal to one; and [0941] each R.sub.16 is
independently selected from the group consisting of hydrogen,
halogen, alkyl, NO.sub.2, CN, and substituted alkyl. In other
embodiments of the present invention, R.sub.1 is a polypeptide. In
other embodiments of the present invention, R.sub.1 is an antibody.
In still other embodiments, R.sub.1 is an .alpha.PSMA antibody. In
other embodiments of the present invention, R.sub.2 is a
polypeptide. In other embodiments of the present invention, R.sub.2
is an antibody. In still other embodiments, R.sub.2 is an
.alpha.PSMA antibody. [0942] 1. Other embodiments of the present
invention provide a compound comprising Formula (XXXI), (XXXII),
(XXXIII), (XXXIV), (XXXV), or (XXXVI):
[0942] ##STR00205## [0943] wherein [0944] Z has the structure
of:
[0944] ##STR00206## [0945] R.sub.5 is H, CO.sub.2H,
C.sub.1-C.sub.6alkyl, or thiazole; [0946] R.sub.6 is OH or H;
[0947] Ar is phenyl or pyridine; [0948] R.sub.1 is H, an amino
protecting group, resin, at least one amino acid, polypeptide, or
polynucleotide; [0949] R.sub.2 is OH, an ester protecting group,
resin, at least one amino acid, polypeptide, or polynucleotide;
[0950] R.sub.4 is H, halogen, lower alkyl, or substituted lower
alkyl; [0951] R.sub.7 is C.sub.1-C.sub.6alkyl or hydrogen; [0952]
L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each linkers selected
from the group consisting of a bond, -alkylene-, -alkylene-C(O)--,
-alkylene-J-, -(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-C(O)--, -(alkylene-O).sub.n-J-,
-(alkylene-O).sub.n-J-alkylene-,
-(alkylene-O).sub.n--(CH.sub.2).sub.n'--NHC(O)--(CH.sub.2).sub.n''--C(Me)-
.sub.2-S--S--(CH.sub.2).sub.n'''--NHC(O)-(alkylene-O).sub.n''''-alkylene-,
-(alkylene-O).sub.n-alkylene-W--, -alkylene-C(O)--W--,
-alkylene-O).sub.n-alkylene-J-,
-alkylene'-J-(alkylene-O)-alkylene-,
-(alkylene-O).sub.n-alkylene-J-alkylene',
-J-(alkylene-O).sub.n-alkylene-,
-(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n'-alkylene-J'-,
--W--, -alkylene-W--,
alkylene'-J-(alkylene-NMe).sub.n-alkylene-W--,
-J-(alkylene-NMe).sub.n-alkylene-W--,
(alkylene-O).sub.n-alkylene-U-alkylene-C(O)--,
-(alkylene-O).sub.n-alkylene-U-alkylene-;
-J-alkylene-NMe-allylene'-NMe-alkylene''-W--, and
-alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W--;
[0952] ##STR00207## [0953] U has the structure of:
[0953] ##STR00208## [0954] each J and J' independently have the
structure of:
[0954] ##STR00209## [0955] each n and n' are independently integers
greater than or equal to one; [0956] D has the structure of:
[0956] ##STR00210## [0957] each R.sub.17 is independently selected
from the group consisting of H, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,
substituted alkoxy, alkylalkoxy, substituted alkylalkoxy,
polyalkylene oxide, substituted polyalkylene oxide, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, alkaryl,
substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or
substituted alkylene)-ON(R'').sub.2, -(alkylene or substituted
alkylene)-C(O)SR'', -(alkylene or substituted alkylene)-S--S-(aryl
or substituted aryl), --C(O)R'', --C(O).sub.2R'', or
--C(O)N(R'').sub.2, wherein each R'' is independently hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy,
substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl,
substituted alkaryl, aralkyl, or substituted aralkyl; [0958] each
Z.sub.1 is a bond, CR.sub.17R.sub.17, O, S, NR',
CR.sub.17R.sub.17--CR.sub.17R.sub.17, CR.sub.17R.sub.17--O,
O--CR.sub.17R.sub.17, CR.sub.17R.sub.17--S, S--CR.sub.17R.sub.17,
CR.sub.17R.sub.17--NR', or NR'--CR.sub.17R.sub.17; [0959] each R'
is H, alkyl, or substituted alkyl; [0960] each Z.sub.2 is selected
from the group consisting of a bond, --C(O)--, --C(S)--, optionally
substituted C.sub.1-C.sub.3 alkylene, optionally substituted
C.sub.1-C.sub.3 alkenylene, and optionally substituted heteroalkyl;
[0961] each Z.sub.3 are independently selected from the group
consisting of a bond, optionally substituted C.sub.1-C.sub.4
alkylene, optionally substituted C.sub.1-C.sub.4 alkenylene,
optionally substituted heteroalkyl, --O--, --S--, --C(O)--,
--C(S)--, and --N(R')--; [0962] each T.sub.3 is a bond,
C(R'')(R''), O, or S; with the proviso that when T.sub.3 is O or S,
R'' cannot be halogen; [0963] each R'' is H, halogen, alkyl,
substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0964] m
and p are 0, 1, 2, or 3, provided that at least one of m or p is
not 0; [0965] M.sub.2 is
[0965] ##STR00211## where (a) indicates bonding to the B group and
(b) indicates bonding to respective positions within the
heterocycle group; [0966] M.sub.3 is
[0966] ##STR00212## where (a) indicates bonding to the B group and
(b) indicates bonding to respective positions within the
heterocycle group; [0967] M.sub.4 is
[0967] ##STR00213## where (a) indicates bonding to the B group and
(b) indicates bonding to respective positions within the
heterocycle group; [0968] each R.sub.19 is independently selected
from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkoxy, ester, ether, thioether, aminoalkyl, halogen, alkyl ester,
aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl
sulfonic acid, alkyl nitro, thioester, sulfonyl ester,
halosulfonyl, nitrile, alkyl nitrile, and nitro; [0969] q is 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; and [0970] each R.sub.16 is
independently selected from the group consisting of hydrogen,
halogen, alkyl, NO.sub.2, CN, and substituted alkyl. In other
embodiments of the present invention, R.sub.1 is a polypeptide. In
other embodiments, R.sub.1 is an antibody. In still other
embodiments, R.sub.1 is an .alpha.PSMA antibody. In other
embodiments of the present invention, R.sub.2 is a polypeptide. In
other embodiments, R.sub.2 is an antibody. In still other
embodiments, R.sub.2 is an .alpha.PSMA antibody.
[0971] Some embodiments of the present invention comprise Formula
(XXXI-A):
##STR00214##
[0972] Each of these embodiments are provided as non-limiting
examples of the present invention.
EXAMPLES
Example 1: Synthesis of Compound 1
##STR00215##
[0974] Compound 1-3: Tetra (ethylene glycol) 1-1 (10 g, 51.5 mmol),
N-hydroxyphthalimide 1-2 (8.4 g, 51.15 mmol) and triphenylphosphine
(17.6 g, 67 mmol) were dissolved in 300 mL of tetrahydrofuran
followed by addition of DIAD (12.8 mL, 61.78 mmol) at 0.degree. C.
The resulting solution was stirred at room temperature overnight,
and then concentrated to dryness. The residue was purified by flash
column chromatography to give 5.47 g (31%) of compound 1-3.
[0975] Compound 1-4: To a solution of compound 1-3 (200 mg, 0.59
mmol) in 15 mL dichloromethane was added Dess-Martin Periodinane
(300 mg, 0.71 mmol). The reaction mixture was stirred at ambient
temperature overnight. The reaction was quenched with the solution
of sodium bisulfite in 15 mL of saturated sodium bicarbonate. The
mixture was separated. The organic layer was washed with saturated
sodium bicarbonate, brine, dried over sodium sulfate, filtered and
concentrated in vacuo. The residue was purified by flash column
chromatography to give 150 mg (75%) of compound 1-4.
[0976] Compound 1-6: To a solution of monomethyldolastatin
hydrochloride salt 1-5 (50 mg, 0.062 mmol) in 1 mL of DMF was added
compound 1-4 (63 mg, 0.186 mmol) and 70 .mu.L of acetic acid,
followed by addition of 8 mg of sodium cyanoborohydride. The
resulting mixture was stirred at ambient temperature for 2 hours.
The reaction mixture was diluted with water and purified by HPLC to
give 60 mg (80%) of compound 1-6. MS (ESI) m/z 547 [M+2H], 1092
[M+H].
1d.
[0977] Compound 1: Compound 1-6 (60 mg, 0.05 mmol) was dissolved in
1 mL of DMF. 32 .mu.L of hydrazine was added. The resulting
solution was stirred at ambient temperature for 1 hour. The
reaction was quenched with 1N hydrochloride solution. The reaction
mixture was purified by UPLC to give 33 mg (55%) of compound 1. MS
(ESI) m/z 482 [M+2H], 962 [M+H].
Example 2: Synthesis of Compound 2
##STR00216##
[0979] Compound 2 was synthesized via a similar synthetic route as
described in Example 1. MS 1N (ESI) m/z 460 [M+2H], 918 [M+H].
Example 3: Synthesis of Compound 3
##STR00217##
[0981] Compound 3 was synthesized via similar synthetic route to
Example 1. MS (ESI) m/z 438 [M+2H], 974 [M+H].
Example 4: Synthesis of Compound 4
[0982] Compound 4-2: To a solution of Val (OtBu)-OH.HCl 4-1 (1 g,
4.77 mmol) and bromoethanol (304.7 .mu.L, 4.3 mmol) in 10 mL of DMF
was added 1.68 ml of DIEA. The reaction mixture was stirred at room
temperature for 2 days. 4.8 mmol of Boc.sub.2O was added to the
reaction mixture, followed by 0.84 mL of DIEA. The reaction mixture
was stirred at room temperature for 2 days. The reaction mixture
was concentrated in vacuo and extracted with ethyl acetate, and
washed with water, brine, dried over sodium sulfate and
concentrated in vacuo. The residue was purified by flash column
chromatography to give 0.66 g of compound 4-2.
##STR00218##
[0983] Compound 4-3: To a solution of compound 4-2 (500 mg, 1.58
mmol), N-hydroxyphthalimide (261 mg, 1.6 mmol) and
triphenylphosphine (538 mg, 2.05 mmol) in 15 mL THF was added DIAD
(394 .mu.L, 1.9 mmol) at at 0.degree. C. The resulting solution was
stirred at room temperature overnight, and then concentrated in
vacuo. The residue was purified by flash column chromatography to
give 0.68 g of compound 4-3.
[0984] Compound 4-4: Compound 4-3 was dissolved in 15 mL 4N
HCl/Dioxane. The reaction mixture was stirred at room temperature
for 2 days and concentrated in vacuo. The residue was dissolved in
DMF and treated with Boc.sub.2O (230 .mu.L, 1 mmol) and DIEA (352
.mu.L, 2 mmol). The reaction mixture was stirred at room
temperature for 2 days. The reaction mixture was purified by HPLC
to give 100 mg of compound 4-4.
[0985] Compound 4-5: To a solution of compound
Boc-Val-Dil-methylDap-OH in DMF is added phe(OtBu)-OH.HCl, HATU and
N-methylmorpholine. The reaction mixture is stirred at room
temperature for 4 hours. The reaction mixture is concentrated in
vacuo and extracted with ethyl acetate (100 mL.times.1, 50
mL.times.2). The organic layer is combined and washed with brine,
dried over sodium sulfate and concentrated in vacuo. The residue is
purified by flash chromatography. The resulting compound is treated
with HCl/EtOAC to give compound 4-5.
[0986] Compound 4-6: To a solution of compound 4-5 in DMF is added
compound 4-4, HATU and DIEA. The reaction mixture is stirred at
room temperature for 4 hours. The reaction mixture is concentrated
in vacuo and extracted with ethyl acetate (100 mL.times.1, 50
mL.times.2). The organic layer is combined and washed with brine,
dried over sodium sulfate and concentrated in vacuo. The residue is
purified by flash chromatography to give compound 4-6.
[0987] Compound 4-7: Compound 4-6 is dissolved in 15 mL 4N
HCl/Dioxane. The reaction mixture is stirred at room temperature
for 2 hours and concentrated in vacuo to give compound 4-7.
[0988] Compound 4-8: To a solution of compound 4-7 in 1 mL of DMF
is added formylaldehyde and acetic acid, followed by addition of
sodium cyanoborohydride. The resulting mixture is stirred at
ambient temperature for 2 hours. The reaction mixture is diluted
with water and purified by HPLC to give compound 4-8.
[0989] Compound 4: Compound 4-8 is dissolved in 1 mL of DMF.
Hydrazine is added. The resulting solution is stirred at ambient
temperature for 1 hour. The reaction is quenched with 1N
hydrochloride solution. The reaction mixture is purified by HPLC to
give Compound 4.
Example 5: Synthesis of Compound 5
[0990] Compound 4-7 is dissolved in 1 mL of DMF. Hydrazine is
added. The resulting solution is stirred at ambient temperature for
1 hour. The reaction is quenched with 1N hydrochloride solution.
The reaction mixture is purified by HPLC to give Compound 5.
Example 6: Synthesis of Compound 6
##STR00219##
[0992] Compound 6-2: To a solution of compound 6-1 (500 mg, 0.875
mmol) in 3 mL of DMF was added 283 mg of phenylalanine
hydrochloride, 433 mg of HATU and 581 .mu.L of N-methylmorpholine.
The reaction mixture was stirred at room temperature for 4 hours.
The reaction mixture was concentrated in vacuo and extracted with
ethyl acetate (100 mL.times.1, 50 mL.times.2). The organic layer
was combined and washed with brine, dried over sodium sulfate and
concentrated in vacuo. The residue was purified by flash
chromatography to give 560 mg (76%) of compound 6-2.
[0993] Compound 6-3: Compound 6-2 was dissolved in 15 mL 4N
HCl/Dioxane. The reaction mixture was stirred at room temperature
for 2 hours and concentrated in vacuo to give 511 mg of compound
6-3.
[0994] Compound 6-4: To a solution of compound 6-3 (368 mg, 0.55
mmol) in 3 mL of DMF was added 255 mg of Boc-N-methyl valine, 314
mg of HATU and 303 .mu.L of N-methylmorpholine. The reaction
mixture was stirred at room temperature for 4 hours. The reaction
mixture was concentrated in vacuo and extracted with ethyl acetate
(100 mL.times.1, 50 mL.times.2). The organic layer was combined and
washed with brine, dried over sodium sulfate and concentrated in
vacuo. The residue was purified by flash chromatography to give 370
mg (79%) of compound 6-4.
[0995] Compound 6-5: To a solution of compound 6-4 (170 mg) in 10
mL MeOH was added 5 eq of 1N LiOH. The reaction mixture was stirred
at room temperature for 2 hours. The reaction mixture was acidified
by 1NHCl and extracted with ethyl acetate washed with brine, dried
over sodium sulfate and concentrated in vacuo to give 150 mg (90%)
of compound 6-5.
[0996] Compound 6-6: Compound 6-5 was dissolved in 4N HCl/Dioxane.
The reaction mixture was stirred at room temperature for 2 hours
and concentrated in vacuo and purified by HPLC to give 150 mg of
compound 6-6.
[0997] Compound 6-7: To a solution of compound 6-6 (50 mg, 0.062
mmol) in 1 mL of DMF was added compound 1-4 (63 mg, 0.186 mmol) and
70 .mu.L of acetic acid, followed by addition of 8 mg of sodium
cyanoborohydride. The resulting mixture was stirred at ambient
temperature for 2 hours. The reaction mixture was diluted with
water and purified by HPLC to give 60 mg (80%) of compound 6-7.
[0998] Compound 6: Compound 6-7 (60 mg, 0.05 mmol) was dissolved in
1 mL of DMF. 32 .mu.L of hydrazine was added. The resulting
solution was stirred at ambient temperature for 1 hour. The
reaction was quenched with 1N hydrochloride solution. The reaction
mixture was purified by HPLC to give 33 mg (55%) of Compound 6.
Example 7: Synthesis of Compound 7
##STR00220##
[1000] Compound 7 was synthesized via similar synthetic route to
Compound 1. MS (ESI) m/z 440 [M+2H], 879 [M+H].
Example 8: Synthesis of Compound 8
##STR00221##
[1002] Compound 8 was synthesized via similar synthetic route to
Compound 1. MS (ESI) m/z 418 [M+2H], 835 [M+H].
Example 9: Synthesis of Compound 9
##STR00222##
[1004] Compound 9-1: To a solution of compound
Boc-Val-Dil-methylDap-OH in DMF is added 4-(2-Aminoethyl) pyridine,
HATU and N-methylmorpholine. The reaction mixture is stirred at
room temperature for 4 hours. The reaction mixture is concentrated
in vacuo and extracted with ethyl acetate (100 mL.times.1, 50
mL.times.2). The organic layer is combined and washed with brine,
dried over sodium sulfate and concentrated in vacuo. The residue is
purified by flash chromatography. The resulting compound is treated
with HCl/EtOAC to give compound 9-1.
[1005] Compound 9-2: To a solution of compound 9-1 in DMF is added
compound 4-4, HATU and DIEA. The reaction mixture is stirred at
room temperature for 4 hours. The reaction mixture is concentrated
in vacuo and extracted with ethyl acetate (100 mL.times.1, 50
mL.times.2). The organic layer is combined and washed with brine,
dried over sodium sulfate and concentrated in vacuo. The residue is
purified by flash chromatography to give compound 9-2.
[1006] Compound 9-3: Compound 9-2 is dissolved in 15 mL 4N
HCl/Dioxane. The reaction mixture is stirred at room temperature
for 2 hours and concentrated in vacuo to give compound 9-3.
[1007] Compound 9-4: To a solution of compound 9-3 in 1 mL of DMF
is added formylaldehyde and acetic acid, followed by addition of
sodium cyanoborohydride. The resulting mixture is stirred at
ambient temperature for 2 hours. The reaction mixture is diluted
with water and purified by HPLC to give compound 9-4.
[1008] Compound 9: Compound 9-4 is dissolved in 1 mL of DMF.
Hydrazine is added. The resulting solution is stirred at ambient
temperature for 1 hour. The reaction is quenched with 1N
hydrochloride solution. The reaction mixture is purified by HPLC to
give compound 9.
Example 10: Synthesis of Compound 10
##STR00223##
[1010] Compound 10: Compound 9-3 is dissolved in 1 mL of DMF.
Hydrazine is added. The resulting solution is stirred at ambient
temperature for 1 hour. The reaction is quenched with 1N
hydrochloride solution. The reaction mixture is purified by HPLC to
give Example 10.
Example 11: Synthesis of Compound 11
##STR00224##
[1012] Compound 11-3: To a solution of tetra (ethylene glycol) 11-1
(40.6 mL, 235 mmol) in 100 mL of tetrahedrofuran was added 47 mg of
sodium. 12 mL of tert-butylacrylate was added after sodium was
dissolved. The reaction mixture was stirred at room temperature for
24 hours. The reaction mixture was concentrated in vacuo and
quenched with 2 mL of 1 N HC1. The residue was suspended in brine
and extracted with ethyl acetate (100 mL.times.1, 50 mL.times.2).
The organic layer was combined and washed with brine, dried over
sodium sulfate and concentrated in vacuo to give 6.4 g (23%) of
compound 11-3.
[1013] Compound 11-5: Compound 11-3 (1.0 g, 3.12 mmol),
N-hydroxyphthalimide 11-4 (611 mg, 3.744 mmol) and
triphenylphosphine (1.23 g, 4.68 mmol) were dissolved in 20 mL of
tetrahydrofuran followed by addition of DIAD (0.84 mL, 4.06 mmol)
at 0.degree. C. The resulting solution was stirred at room
temperature overnight, and then concentrated to dryness. The
residue was purified by flash column chromatography using SiliaSep
Cartridges (80 g), eluting with 0-100% ethyl acetate/hexanes, to
give 1.0 g (100%) of compound 11-5.
[1014] Compound 11-6: Compound 11-5 was dissolved in 15 mL 4N
HCl/Dioxane. The reaction mixture was stirred at room temperature
for 2 hours and concentrated in vacuo to give 1.0 g of compound
11-6.
[1015] Compound 11-8: To a solution of 30 mg (0.0372 mmol) of
monomethyldolastatin hydrochloride, 31 mg (0.0744 mmol) of compound
11-6 and 38.2 mg (0.082 mmol) of PyBroP in 1 mL of DMF was added 33
.mu.L (0.186 mmol) of diisopropylethylamine. The reaction mixture
was stirred at room temperature for 5 hours. The reaction mixture
was purified by HPLC to give 28 mg (65%) of compound 11-8. MS (ESI)
m/z 785 [M+2H], 1164[M+H].
[1016] Compound 11: Compound 11-8 (28 mg, 0.024 mmol) was dissolved
in 1 mL of DMF. 23 .mu.L (0.72 mmol) of anhydrous hydrazine was
added. The resulting solution was stirred at room temperature for 1
hour. The reaction was quenched with 1N hydrochloride solution. The
reaction mixture was purified by preparative HPLC, eluting with
20-70% CH.sub.3CN/H.sub.2O in 20 min at 254 nm, to give 20 mg (66%)
of Compound 11. MS (ESI) m/z 518 [M+2H], 1034[M+H].
Example 12: Synthesis of Compound 12
##STR00225## ##STR00226##
[1018] Compound 12-2: To a solution of compound 12-1 (500 mg, 0.875
mmol) in 3 mL of DMF was added 283 mg of phenylalanine
hydrochloride, 433 mg of HATU and 581 .mu.L of N-methylmorpholine.
The reaction mixture was stirred at room temperature for 4 hours.
The reaction mixture was concentrated in vacuo and extracted with
ethyl acetate (100 mL.times.1, 50 mL.times.2). The organic layer
was combined and washed with brine, dried over sodium sulfate and
concentrated in vacuo. The residue was purified by flash
chromatography to give 560 mg (76%) of compound 12-2.
[1019] Compound 12-3: Compound 12-2 was dissolved in 15 mL 4N
HCl/Dioxane. The reaction mixture was stirred at room temperature
for 2 hours and concentrated in vacuo to give 511 mg of compound
12-3.
[1020] Compound 12-4: To a solution of compound 12-3 (368 mg, 0.55
mmol) in 3 mL of DMF was added 255 mg of Boc-N-methyl valine, 314
mg of HATU and 303 .mu.L of N-methylmorpholine. The reaction
mixture was stirred at room temperature for 4 hours. The reaction
mixture was concentrated in vacuo and extracted with ethyl acetate
(100 mL.times.1, 50 mL.times.2). The organic layer was combined and
washed with brine, dried over sodium sulfate and concentrated in
vacuo. The residue was purified by flash chromatography to give 370
mg (79%) of compound 12-4.
[1021] Compound 12-5: To a solution of compound 12-4 (170 mg) in 10
mL MeOH was added 5 eq of 1N LiOH. The reaction mixture was stirred
at room temperature for 2 hours. The reaction mixture was acidified
by 1NHCl and extracted with ethyl acetate washed with brine, dried
over sodium sulfate and concentrated in vacuo to give 150 mg (90%)
of compound 12-5.
[1022] Compound 12-6: Compound 12-5 was dissolved in 4N
HCl/Dioxane. The reaction mixture was stirred at room temperature
for 2 hours and concentrated in vacuo and purified by HPLC to give
150 mg of compound 12-6.
[1023] Compound 12-7: To a solution of compound 12-6 in DMF was
added formylaldehyde (3 eq) and 20 eq of acetic acid, followed by
addition of 2 eq of sodium cyanoborohydride. The resulting mixture
was stirred at ambient temperature for 2 hours. The reaction
mixture was diluted with water and purified by HPLC to give
compound 12-7.
[1024] Compound 12-10: tert-Butyl 2-(2-hydroxyethoxy)ethylcarbamate
(2.05 g, 10 mmol), N-hydroxyphthalimide (1.8 g, 11 mmol) and
triphenylphosphine (3.67 g, 14 mmol) were dissolved in 100 mL of
tetrahydrofuran followed by addition of DIAD (2.48 mL, 12 mmol) at
0.degree. C. The resulting solution was stirred at room temperature
overnight, and then concentrated to dryness. The residue was
treated with 50 mL of 4N HCl/dioxane. The mixture was stirred at
room temperature for 2 hours. The solvent was removed in vacuo. The
residue was treated with ether, filtered, washed with ether and
dried in vacuo to get 2.6 g (91%) of compound 12-10. MS (ESI) m/z
251 [M+H].
[1025] Compound 12-11: To a solution of compound 12-10 (20 mg,
0.026 mmol) in 1 mL of DMF was added 11.2 mg of compound 12-10, 15
mg of HATU and 23 .mu.L of DIEA. The reaction mixture was stirred
at room temperature for 2 hours. The reaction mixture was purified
by HPLC to give 20 mg (70%) of compound 12-4. MS (ESI) m/z 490
[M+2H], 978[M+H].
[1026] Compound 12: Compound 12-11 (20 mg, 0.0183 mmol) was
dissolved in 1 mL of DMF. 18 .mu.L (0.56 mmol) of anhydrous
hydrazine was added. The resulting solution was stirred at room
temperature for 1 hour. The reaction was quenched with 1N
hydrochloride solution. The reaction mixture was purified by
preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254
nm, to give 14 mg (72%) of Compound 12. MS (ESI) m/z 425 [M+2H],
848[M+H].
Example 13: Synthesis of Compound 13
##STR00227## ##STR00228##
[1028] Compound 13-2: Tert-butyl 6-hydroxyhexanoate 13-1 (1.5 g,
1.97 mmol), N-hydroxyplithalimide (1.42 g, 8.76 mmol) and
triphenylphosphine (2.82 g, 10.76 mmol) were dissolved in 50 mL of
tetrahydrofuran followed by addition of DIAD (2 mL, 9.564 mmol) at
0.degree. C. The resulting solution was stirred at room temperature
overnight, and then concentrated to dryness. The residue was
purified by flash column chromatography to give 2.5 g (95%) of
compound 13-2.
[1029] Compound 13-3: The compound 13-2 was treated with 15 mL 4N
HCl in dioxane. The reaction mixture was stirred at ambient
temperature for 12 hours and concentrated to dryness in vacuo to
give 900 mg (100%) of compound 13-3.
[1030] Compound 13-4: To a solution of compound 13-3 (900 mg, 3.0
mmol) in 10 mL of THF was added 397 mg of N-hydroxysuccinimide,
followed by adding 669 mg of DCC. The reaction mixture was stirred
at ambient temperature overnight and filtered. The filtration was
concentrated and treated with 10 mL of DCM. The DCM solution was
stayed at ambient temperature for 1 hour and filtered. The
filtration was concentrated and purified by flash column
chromatography to give 800 mg (71%) of compound 13-4.
[1031] Compound 13-6: The mixture of compound 13-4 (435 mg, 1.16
mmol) and Val-Cit-PABOH 13-5.sup.1 (400 mg, 1.054 mmol) in 12 mL of
DMF was stirred at ambient temperature for 24 hours. The solvent
was removed in vacuo. The residue was treated with ether, filtered
and washed with ether. The solid was dried in vacuo to give 660 mg
(98%) of compound 13-6.
[1032] Compound 13-7: To the solution of compound 13-6 (200 mg,
0.313 mmol) in 6 mL of DMF was added bis (p-nitrophenyl) carbonate
(286 mg, 0.94 mmol), followed by addition of 110.2 .mu.L of DIEA.
The reaction mixture was stirred at ambient temperature for 5 hours
and concentrated. The residue was treated with ether and filtered.
The collected solid was washed with ether, 5% citric acid, water,
ether and dried in vacuo to give 210 mg (83%) compound 13-7.
[1033] Compound 13-9: To a solution of monomethylauristatin
hydrochloride salt 13-8 (100 mg, 0.1325 mmol) in 2 mL of DMF was
added compound 13-7 (159 mg, 0.2 mmol) and 10 mg of HOBt, followed
by addition of 35.2 .mu.L of DIEA. The resulting mixture was
stirred at ambient temperature for 2 days. The reaction mixture was
diluted with water and purified by HPLC to give 93 mg (51%) of
compound 13-9. MS (ESI) m/z 692 [M+2H], 1382 [M+H].
[1034] Compound 13: The compound 13-9 (50 mg, 0.036 mmol) was
dissolved in 1 mL of DMF. 23 .mu.L of hydrazine was added. The
resulting solution was stirred at ambient temperature for 3 hours.
The reaction was quenched with 1N hydrochloride solution. The
reaction mixture was purified by HPLC to give 32 mg (65%) of
Compound 13. MS (ESI) m/z 638.5 [M+Na+2H], 1253.3 [M+H], 1275.8
[M+Na].
Example 14: Synthesis of Compound 14
##STR00229## ##STR00230## ##STR00231##
[1036] Compound 14-3: To a solution of tetra (ethylene glycol) 14-1
(40.6 mL, 235 mmol) in 100 mL of tetrahedrofuran was added 47 mg of
sodium. 12 mL of tert-butylacrylate was added after sodium was
dissolved. The reaction mixture was stirred at room temperature for
24 hours. The reaction mixture was concentrated in vacuo and
quenched with 2 mL of 1 N HCl. The residue was suspended in brine
and extracted with ethyl acetate (100 mL.times.1, 50 mL.times.2).
The organic layer was combined and washed with brine, dried over
sodium sulfate and concentrated in vacuop to give 6.4 g (23%) of
compound 14-3.
[1037] Compound 14-5: Compound 14-3 (1.0 g, 3.12 mmol),
N-hydroxyphthalimide 14-4 (611 mg, 3.744 mmol) and
triphenylphosphine (1.23 g, 4.68 mmol) were dissolved in 20 mL of
tetrahydrofuran followed by addition of DIAD (0.84 mL, 4.06 mmol)
at 0.degree. C. The resulting solution was stirred at room
temperature overnight, and then concentrated to dryness. The
residue was purified by flash column chromatography using SiliaSep
Cartridges (80 g), eluting with 0-100% ethyl acetate/hexanes, to
give 1.0 g (100%) of compound 14-5.
[1038] Compound 14-6: Compound 14-5 was dissolved in 15 mL 4N
HCl/Dioxane. The reaction mixture was stirred at room temperature
for 2 hours and concentrated in vacuo to give 1.0 g of compound
14-6.
[1039] Compound 14-7: To a solution of compound 6 (1.93 g, 4.68
mmol) and N-hydroxysuccinimide (646 mg, 5.616 mmol) in 20 mL of
tetrahedrofuran was added 1.062 g (5.148 mmol) of DCC. The reaction
mixture was stirred at room temperature overnight and filtered. The
filtration was concentrated and purified by flash column
chromatography using SiliaSep Cartridges (80 g), eluting with
0-100% ethyl acetate/hexanes to give 2.37 g (100%) of compound
14-7.
[1040] Compound 14-8: Compound 14-8 was made according to the
literature (Bioconjugat Chem. 2002, 13 (4), 855-869.)
[1041] Compound 14-9: To a solution of compound 14-8 (200 mg, 0.527
mmol) in 2 mL of DMF was added 295 mg (0.58 mmol) of compound 14-7.
The reaction mixture was stirred at room temperature overnight and
concentrated in vacuo. The residue was treated with ether,
filtered, washed with ether and dried in vacuo to give 402 mg (98%)
of compound 14-9.
[1042] Compound 14-10: To a solution of compound 14-9 (406 mg,
0.527 mmol) and bis(p-nitrophenol) carbonate (481 mg, 1.58 mmol) in
10 mL of DMF was added 0.186 mL (1.054 mmol) of
diisopropylethylamine. The reaction mixture was stirred at room
temperature for 5 hours. The solvent was removed in vacuo. The
residue was treated with ether, filtered, washed with ether, 5%
citic acid, water, ether and dried in vacuo to give 350 mg (72%) of
compound 14-10.
[1043] Compound 14-11: To a solution of 50 mg (0.062 mmol) of
monomethyldolastatin hydrochloride, 87.2 mg (0.093 mmol) of
compound 14-10 and 4.7 mg (0.031 mmol) of HOBt in 1 mL of DMF was
added 22 .mu.L (0.124 mmol) of diisopropylethylamine. The reaction
mixture was stirred at room temperature for 16 hours. The reaction
mixture was purified by HPLC to give 41 mg (42%) of compound 14-11.
MS (ESI) m/z 785 [M+2H].
[1044] Compound 14: Compound 14-11 (41 mg, 0.026 mmol) was
dissolved in 1 mL of DMF. 17 .mu.L (0.52 mmol) of anhydrous
hydrazine was added. The resulting solution was stirred at room
temperature for 1 hour. The reaction was quenched with 1N
hydrochloride solution. The reaction mixture was purified by
preparative HPLC, eluting with 20-70% CH.sub.3CN/H2O in 20 min at
254 nm, to give 22 mg (58%) of compound 14. MS (ESI) m/z 720
[M+2H].
Example 15: Synthesis of Compound 15
##STR00232##
[1046] Compound 15-2: To a solution of 50 mg (0.062 mmol) of
monomethyldolastatin hydrochloride, 75 mg (0.093 mmol) of compound
13-7 and 4.7 mg (0.031 mmol) of HOBt in 1 mL of DMF was added 22
.mu.L (0.124 mmol) of diisopropylethylamine. The reaction mixture
was stirred at room temperature for 16 hours. The reaction mixture
was purified by H-PLC to give 41 mg (42%) of compound 15-2. MS
(ESI) m/z 718 [M+2H], 1435 [M+H].
[1047] Compound 15-2: Compound 15-2 (41 mg, 0.026 mmol) was
dissolved in 1 mL of DMF. 17 .mu.L (0.52 mmol) of anhydrous
hydrazine was added. The resulting solution was stirred at room
temperature for 1 hour. The reaction was quenched with 1N
hydrochloride solution. The reaction mixture was purified by
preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254
nm, to give 22 mg (58%) of example 15. MS (ESI) m/z 653 [M+2H],
1305 [M+H].
Example 16: Synthesis of Compound 16
##STR00233## ##STR00234##
[1049] Compound 16-3: To a solution of ethylene glycol 16-1 (13.1
mL, 235 mmol) in 100 mL of tetrahedrofuran was added 47 mg of
sodium. 12 mL of tert-butylacrylate was added after sodium was
dissolved. The reaction mixture was stirred at room temperature for
24 hours. The reaction mixture was concentrated in vacuo and
quenched with 2 mL of 1 N HCl. The residue was suspended in brine
and extracted with ethyl acetate (100 mL.times.1, 50 mL.times.2).
The organic layer was combined and washed with brine, dried over
sodium sulfate and concentrated in vacuo. The residue was purified
by flash column chromatography to give 5.2 g (24%) of compound
16-3.
[1050] Compound 16-5: Compound 16-3 (2.0 g, 10.5 mmol),
N-hydroxyphthalimide (2.05 g, 12.6 mmol) and triphenylphosphine
(3.58 g, 13.65 mmol) were dissolved in 50 mL of tetrahydrofuran
followed by addition of DIAD (3.26 mL, 15.75 mmol) at 0.degree. C.
The resulting solution was stirred at room temperature overnight,
and then concentrated to dryness. The residue was purified by flash
column chromatography to give compound 16-5.
[1051] Compound 16-6: Compound 16-5 was dissolved in 15 mL 4N
HCl/Dioxane. The reaction mixture was stirred at room temperature
for 2 hours and concentrated in vacuo to give compound 16-6.
[1052] Compound 16-7: To a solution of compound 16-6 (5.16 mmol)
and N-hydroxysuccinimide (722 mg, 6.7 mmol) in 20 mL of
tetrahedrofuran was added 1.28 g (6.2 mmol) of DCC. The reaction
mixture was stirred at room temperature overnight and filtered. The
filtration was concentrated and purified by flash column
chromatography to give 500 mg of compound 16-7.
[1053] Compound 16-8: Compound 16-8 was made according to the
literature (Bioconjugat Chem. 2002, 13 (4), 855-869.)
[1054] Compound 16-9: To a solution of compound 16-8 (5.0 g, 8.3
mmol) and bis(p-nitrophenol) carbonate (7.6 g, 25 mmol) in 100 mL
of DMF was added 2.92 mL (16.6 mmol) of diisopropylethylamine. The
reaction mixture was stirred at room temperature for 16 hours. The
solvent was removed in vacuo. The residue was treated with ether,
filtered, washed with ether, 5% citic acid, water, ether and dried
in vacuo to give 5.0 g (81%) of compound 16-9.
[1055] Compound 16-10: To a solution of 1.0 g (1.24 mmol) of
monomethyldolastatin hydrochloride, 1.42 g (1.8575 mmol) of
compound 16-9 and 95 mg (0.62 mmol) of HOBt in 10 mL of DMF was
added 437 .mu.L (2.48 mmol) of diisopropylethylamine. The reaction
mixture was stirred at room temperature for 16 hours. The reaction
mixture was purified by HPLC to give 1.0 g (58%) of compound 16-10.
MS (ESI) m/z 700 [M+2H], 1398 [M+H].
[1056] Compound 16-11: To a solution of compound 16-10 (1.0 g,
0.715 mmol) in 15 mL of tetrahedrofuran was added 5 mL (48 mmol) of
diethylamine. The reaction mixture was stirred at room temperature
for 1.5 hours and concentrated in vacuo. The residue was dissolved
in 20 mL of DCM, treated with 200 mL of ether and filtered, washed
with ether and dried in vacuo to give 860 mg of compound 16-11. MS
(ESI) m/z 589 [M+2H], 1176 [M+H].
[1057] Compound 16: To a solution of 50 mg (0.0425 mmol) of
compound 16-11 in 1 mL of DMF was added 32 mg (0.085 mmol) of
compound 16-7. The reaction mixture was stirred at room temperature
for 16 hours. The HPLC and MS showed reaction done. 27.2 .mu.L
(0.85 mmol) of anhydrous hydrazine was added to the reaction
mixture. The reaction was done in 2 hours. The reaction mixture was
acidified with 1N HCl and purified by HPLC to give 40 mg (66%) of
compound 16. MS (ESI) m/z 654 [M+2H], 1307[M+H].
Example 17: Synthesis of Compound 17
##STR00235##
[1059] Compound 17-2: To a solution of compound 17-1 (1.0 g, 4.52
mmol) and N-hydroxysuccinimide (572 mg, 4.97 mmol) in 20 mL of
tetrahedrofuran was added 1.12 g (5.424 mmol) of DCC. The reaction
mixture was stirred at room temperature overnight and filtered. The
filtration was concentrated to give compound 17-2.
[1060] Compound 17: To a solution of 50 mg (0.0425 mmol) of
compound 16-11 in 1 mL of DMF was added 41 mg (0.1275 mmol) of
compound 17-2. The reaction mixture was stirred at room temperature
for 16 hours. The HPLC and MS showed reaction done. 20 .mu.L (0.625
mmol) of anhydrous hydrazine was added to the reaction mixture. The
reaction was done in 2 hours. The reaction mixture was acidified
with 1N HCl and purified by HPLC to give 35 mg (60%) of compound
17. MS (ESI) m/z 625 [M+2H], 1249[M+H].
Example 18: Synthesis of Compound 18
##STR00236##
[1062] Compound 18-1: To a solution of compound 6-6, mg (0.062
mmol) of compound 14-10 and HOBt in 1 mL of DMF was added
diisopropylethylamine. The reaction mixture was stirred at room
temperature for 16 hours. The reaction mixture was purified by HPLC
to give compound 18-1.
[1063] Compound 18: Compound 18-1 was dissolved in 1 mL of DMF.
Anhydrous hydrazine was added. The resulting solution was stirred
at room temperature for 1 hour. The reaction was quenched with 1N
hydrochloride solution. The reaction mixture was purified by
preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 mi at 254 nm,
to give compound 18.
Example 19: Synthesis of Compound 19
##STR00237## ##STR00238##
[1065] Compound 19-2: tert-Butyl 2-(2-hydroxyethoxy)ethylcarbamate
13 (2.05 g, 10 mmol), N-hydroxyphthalimide (1.8 g, 11 mmol) and
triphenylphosphine (3.67 g, 14 mmol) were dissolved in 100 mL of
tetrahydrofuran followed by addition of DIAD (2.48 mL, 12 mmol) at
0.degree. C. The resulting solution was stirred at room temperature
overnight, and then concentrated to dryness. The residue was
treated with 50 mL of 4N HCl/dioxane. The mixture was stirred at
room temperature for 2 hours. The solvent was removed in vacuo. The
residue was treated with ether, filtered, washed with ether and
dried in vacuo to get 2.6 g (91%) of compound 19-2. MS (ESI) m/z
251 [M+H].
[1066] Compound 19-3: To the mixture of compound 19-2 (315 mg, 1.1
mmol), Boc-Lys(Boc)-OH (365 mg, 1 mmol), EDC (382 mg, 2 mmol) and
HOBt (306 mg, 2 mmol) in 10 mL of DCM was added 1.056 mL (6 mmol)
of diisopropylethylamine. The reaction mixture was stirred at room
temperature for 3 hours and extracted with ethyl acetate, washed
with 5% citric acid, saturate sodium bicarbonate, brine, dried over
sodium sulfate, filtered and concentrated in vacuo. The residue was
purified flash column chromatography using SiliaSep Cartridges (40
g), eluting with 0-100% ethyl acetate/hexanes, to give 405 mg (70%)
of compound 19-3.
[1067] Compound 19-4: Compound 19-3 was dissolved in 15 mL 4N
HCl/Dioxane. The reaction mixture was stirred at room temperature
for 2 hours and concentrated in vacuo to give 315 mg (98%) of
compound 19-4. MS (ESI) m/z 379 [M+H].
[1068] Compound 19-5: To a solution of compound 14-3 (322 mg, 1
mmol) in 20 mL dichloromethane was added Dess-Martin Periodinane
(636 mg, 1.5 mmol). The reaction mixture was stirred at room
temperature for 3 hours. The reaction was quenched with a solution
of sodium thiosulfate (1.4 g, 8.85 mmol) in 15 mL of saturated
sodium bicarbonate. The mixture was separated. The organic layer
was washed with saturated sodium bicarbonate, brine, dried over
sodium sulfate, filtered and concentrated in vacuo. The residue was
purified by flash column chromatography using SiliaSep Cartridges
(40 g), eluting with 0-100% ethyl acetate/hexanes to give 170 mg
(53%) of compound 19-5.
[1069] Compound 19-6: To a solution of monomethyldolastatin
hydrochloride 1.0 g (1.24 mmol) in 20 mL of DMF was added 1.19 g
(3.72 mmol) of compound 17 followed by 1.4 mL (24.8 mmol) of acetic
acid and 156 mg (2.48 mmol) of sodium cyanoborohydride. The
resulting mixture was stirred at room temperature for 2 hours. The
solvent was removed in vacuo. The residue was adjusted to pH 8 by
sodium bicarbonate and extracted with DCM, washed with brine, dried
over sodium sulfate, filtered and concentrated in vacuo. The
residue was purified by flash column chromatography using SiliaSep
Cartridges (40 g), eluting with 0-5% methanol/DCM to give 680 mg
(51%) of compound 19-6. MS (ESI) m/z 538 [M+2H], 1075 [M+II].
[1070] Compound 19-7: To a solution of compound 19-6 (680 mg, 0.632
mmol) in 5 mL of DCM was added 20 mL of 4N HCl/dioxane. The
reaction mixture was stirred at room temperature for 2 hours and
concentrated in vacuo. The residue was treated with ether,
filtered, washed with ether and dried in vacuo to give 660 mg (98%)
of compound 19-7. MS (ESI) m/z 510 [M+2H], 1019 [M+H].
[1071] Compound 19-8: To a solution of compound 19-7 (280 mg, 0.257
mmol), compound 19-4 (38 mg, 0.0857 mmol) and N-methylmorpholine
(0.283 mL, 2.57 mmol) in 5 mL of N-methylmpyrrolidinone was added
98 mg (0.257 mmol) of HATU. The reaction mixture was stirred at
room temperature for 1 hour. The reaction mixture was purified by
HPLC to give 160 mg (71%) of compound 19-8. MS (ESI) m/z 596
[M+4H], 794[M+3H], 1191 [M+2H].
[1072] Compound 19: Compound 19-8 (160 mg, 0.0613 mmol) was
dissolved in 1.5 mL of DMF. 20 .mu.L (0.613 mmol) of anhydrous
hydrazine was added. The resulting solution was stirred at room
temperature for 1 hour. The reaction was quenched with 1N
hydrochloride solution. The reaction mixture was purified by
preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254
nm, to give 120 mg (75%) of compound 19. MS (ESI) m/z 451[M+5H],
563[M+4H], 751 [M+3H], 1126 [M+2H].
Example 20: Synthesis of Compound 20
##STR00239## ##STR00240##
[1074] Compound 20-2: To a solution of tetra (ethylene glycol) 20-1
(8.0 g, 41.2 mmol) in 100 mL of tetrahedrofuran was added 1.65 g of
sodium hydride at 0.degree. C. The reaction mixture was stirred at
room temperature for 30 min, 6.21 g of TBS-C.sub.1 was added to
this solution. The reaction mixture was stirred at room temperature
overnight. The reaction mixture was concentrated in vacuo and
quenched with 2 mL of 1 N HCl. The residue was suspended in brine
and extracted with ethyl acetate (100 mL.times.1, 50 mL.times.2).
The organic layer was combined and washed with brine, dried over
sodium sulfate and concentrated in vacuo. The residue was purified
by flash column chromatography to give 5.7 g of compound 20-2.
[1075] Compound 20-3: To a solution of compound 20-2 (500 mg, 1.62
mmol) in 30 mL dichloromethane was added Dess-Martin Periodinane
(1.03 g, 2.43 mmol). The reaction mixture was stirred at room
temperature for 3 hours. The reaction was quenched with a solution
of sodium thiosulfate (1.4 g, 8.85 mmol) in 15 mL of saturated
sodium bicarbonate. The mixture was separated. The organic layer
was washed with saturated sodium bicarbonate, brine, dried over
sodium sulfate, filtered and concentrated in vacuo. The residue was
purified by flash column chromatography to give 400 mg of compound
20-3.
[1076] Compound 20-4: To a solution of monomethyldolastatin
hydrochloride 213 mg (0.263 mmol) in 4 mL of DMF was added 245 mg
(0.75 mmol) of compound 20-3 followed by 0.303 mL (5 mmol) of
acetic acid and 34 mg (0.5 mmol) of sodium cyanoborohydride. The
resulting mixture was stirred at room temperature for 2 hours. The
solvent was removed in vacuo. 3 mL of 60% acetonitrile was added,
followed by 0.2 mL of HF.Pyridine at 0.degree. C. The resulting
solution was stirred at room temperature for 2 hours. The organic
solvent was removed in vacuo. The residue was adjusted to pH 8 by
sodium bicarbonate and extracted with DCM, washed with brine, dried
over sodium sulfate, filtered and concentrated in vacuo. The
residue was purified by flash column chromatography to give 160 mg
of compound 20-4. MS (ESI) m/z 474 [M+2H], 947[M+H].
[1077] Compound 20-5: To a solution of compound 20-4 (50 mg, 0.062
mmol) in 4 mL of DCM was added 0.3 mL of phosgene/toluene at
0.degree. C. The reaction mixture was stirred at 0.degree. C. for 3
hours and concentrated in vacuo for next step without
purification.
[1078] Compound 20-6: To a solution of compound 19-4 (7.6 mg, 0.017
mmol) and compound 20-5 (0.062 mmol) was added 25 .mu.L of
diisopropylethylamine. The reaction mixture was stirred at room
temperature for 1 hour. The reaction mixture was purified by HPLC,
eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 33 mg of
compound 20-6. MS (ESI) m/z 582[M+4H], 775[M+3H], 1163[M+2H].
[1079] Compound 20: Compound 20-6 (33 mg, 0.014 mmol) was dissolved
in 1 mL of DMF. 14 .mu.L (0.43 mmol) of anhydrous hydrazine was
added. The resulting solution was stirred at room temperature for 1
hour. The reaction was quenched with 1N hydrochloride solution. The
reaction mixture was purified by preparative HPLC, eluting with
20-70% CH3CN/H2O in 20 min at 254 nm, to give 10 mg of compound 20.
MS (ESI) m/z 549[M+41H], 732[M+3H], 1098[M+2H].
Example 21: Synthesis of Compound 21
##STR00241## ##STR00242##
[1081] Compound 21-2: The mixture of N, N'-Dimethylene diamine 21-1
(5 mL, 46.5 mmol) and tert-butyl acrylate 13 mL (116 mmol) was
heated at 85.degree. C. for 1 hour. Another 13 mL (116 mmol) of
tert-butyl acrylate was added. The reaction mixture was continually
heated at 85.degree. C. for 1 hour and stirred at room temperature
overnight. The reaction mixture was concentrated in vacuo. The
residue was diluted with hexanes and purified by flash column
chromatography using SiliaSep Cartridges (120 g), eluting with 0-5%
methanol/DCM to give 10.1 g (62%) of compound 21-2. MS (ESI) m/z
345 [M+H].
[1082] Compound 21-3: To a solution of compound 21-2 (5.0 g, 14.5
mmol) in 50 mL of DCM was added 40 mL of 4N HCl/dioxane. The
reaction mixture was stirred at room temperature for 2 days and
concentrated in vacuo. The residue was treated with ether,
filtered, washed with ether and dried in vacuo to give 4.3 g (97%)
of compound 21-3.
[1083] Compound 21-4: To a solution of 166 mg (0.544 mmol) of
compound 21-3 and 0.15 mL of N-methylmorpholine in 10 mL of
N-methylpyrrolidinone was added 160 mg of compound 16-11, followed
by 0.068 mL (0.408 mmol) of DECP. The reaction mixture was stirred
at room temperature for 1 hour. The reaction mixture was purified
by preparative HPLC, eluting with 35-70% CH3CN/H2O in 20 min at 254
nm, to give 100 mg (50%) of compound 21-4. MS (ESI) m/z 464[M+3H],
696 [M+2H], 1391 [M+H].
[1084] Compound 21-5: To a solution of compound 19-4 (11 mg, 0.025
mmol), compound 21-4 (115 mg, 0.077 mmol) and N-methylmorpholine
(0.028 mL, 0.25 mmol) in 1.5 mL of N-methylmpyrrolidinone was added
29.3 mg (0.077 mmol) of HATU. The reaction mixture was stirred at
room temperature for 1 hour. The reaction mixture was purified by
HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 60
mg (67%) of compound 21-5. MS (ESI) m/z 625 [M+5H], 781 [M+4H],
1041[M+3H].
[1085] Compound 21: Compound 21-5 (60 mg, 0,014 mmol) was dissolved
in 1 mL of DMF. 7 .mu.L (0.21 mmol) of anhydrous hydrazine was
added. The resulting solution was stirred at room temperature for 1
hour. The reaction was quenched with 1N hydrochloride solution. The
reaction mixture was purified by preparative HPLC, eluting with
20-70% CH3CN/H2O in 20 min at 254 nm, to give 29 mg (58%) of
compound 21. MS (ESI) m/z 599[M+51-H], 749[M+4H], 998 [M+3H].
Example 22: Synthesis of Compound 22
##STR00243##
[1087] Compound 22-1: To a solution of compound 19-4 (7.6 mg, 0.017
mmol), compound 12-7 (40 mg, 0.051 mmol) and DIEA (0.030 mL, 0.17
mmol) in 2 mL of DMF was added 32 mg (0.085 mmol) of HATU. The
reaction mixture was stirred at room temperature for 2 hour. The
reaction mixture was purified by HPLC, eluting with 20-70%
CH3CN/H2O in 20 min at 254 nm, to give 24 mg (68%) of compound
22-1. MS (ESI) m/z 612 [M+3H], 917 [M+2H], 1834[M+H].
[1088] Compound 22: Compound 22-1 (24 mg, 0.012 mmol) was dissolved
in 1 mL of DMF. 12 .mu.L (0.36 mmol) of anhydrous hydrazine was
added. The resulting solution was stirred at room temperature for 1
hour. The reaction was quenched with 1N hydrochloride solution. The
reaction mixture was purified by preparative HPLC, eluting with
20-70% CH3CN/H2O in 20 min at 254 nm, to give 15 mg (58%) of
Example 21. MS (ESI) m/z 569 [M+3H], 852[M+2H], 1726[M+2H].
Example 23: Synthesis of Compound 23
##STR00244## ##STR00245##
[1090] Compound 23-1: To a solution of compound 14-3 (4.0 g, 12.4
mmol) and 6.6 mL (37.2 mmol) of DIEA in 50 mL of DCM was added 3.31
g of tolunenesulfonyl chloride at 0.degree. C. The reaction mixture
was stirred at room temperature for 2 days. The reaction mixture
was extracted with ethyl acetate. The organic layer was combined
and washed with 5% citric acid, water, brine, dried over sodium
sulfate and concentrated in vacuo. The residue was purified by
flash column chromatography to give 3.5 g of compound 23-1.
[1091] Compound 23-2: To a solution of compound 23-1 (3.5 g, 7.34
mmol) in 20 mL DMF was added solium azide (1.44 g, 22.02 mmol). The
reaction mixture was stirred at 50.degree. C. for 2 days. The
reaction mixture was extracted with ethyl acetate. The organic
layer was washed with water, brine, dried over sodium sulfate,
filtered and concentrated in vacuo. The residue was purified by
flash column chromatography to give 2.1 g of compound 23-2.
[1092] Compound 23-3: To a solution of compound 23-2 (2.1 g, 6.05
mmol) in 50 mL MeOH was added 400 mg (10%) of Pd--C. The resulting
mixture was stirred at room temperature under 1 atm H.sub.2 for 24
hours. The reaction mixture was filtered and concentrated in vacuo
to give 2.1 g of compound 23-3. MS (ESI) m/z 322[M+H].
[1093] Compound 23-4: To a solution of compound 23-3 (33 mg, 0.102
mmol), compound 12-7 (40 mg, 0.051 mmol) and 54 .mu.L of
diisopropylethylamine in 1 mL of DMF was added 38 mg of HATU. The
reaction mixture was stirred at room temperature for 4 hours. The
reaction mixture was purified by HPLC to give 52 mg of compound
23-4. MS (ESI) m/z 525[M+2H], 1049[M+H].
[1094] Compound 23-5: Compound 23-4 (52 mg, 0.045 mmol) was
dissolved in 5 mL 4N HCl/Dioxane. The reaction mixture was stirred
at room temperature for 2 hours and concentrated in vacuo to give
52 mg (100%) of compound 23-5. MS (ESI) m/z 497[M+2H],
993[M+1].
[1095] Compound 23-6: To a solution of compound 19-4 (7.6 mg, 0.017
mmol), compound 23-6 (52 mg, 0.051 mmol) and DIEA (0.030 mL, 0.17
mmol) in 2 mL of DMF was added 32 mg (0.085 mmol) of HATU. The
reaction mixture was stirred at room temperature for 2 hour. The
reaction mixture was purified by HPLC, eluting with 20-70%
CH3CN/H2O in 20 min at 254 nm, to give 26 mg (61%) of compound
23-6. MS (ESI) m/z 583[M+4H], 777[M+3H], 1165[M+2H].
[1096] Compound 23: Compound 23-6 (26 mg, 0.01 mmol) was dissolved
in 1 mL of DMF. 10 .mu.L (0.31 mmol) of anhydrous hydrazine was
added. The resulting solution was stirred at room temperature for 1
hour. The reaction was quenched with 1N hydrochloride solution. The
reaction mixture was purified by preparative HPLC, eluting with
20-70% CH3CN/H2O in 20 min at 254 nm, to give 10 mg (40%) of
Example 23. MS (ESI) m/z 550[M+4H], 733[M+3H], 1100[M+2H].
TABLE-US-00003 TABLE 1 Structures of Compounds 1-23 Example
Structure 1 ##STR00246## 2 ##STR00247## 3 ##STR00248## 4
##STR00249## 5 ##STR00250## 6 ##STR00251## 7 ##STR00252## 8
##STR00253## 9 ##STR00254## 10 ##STR00255## 11 ##STR00256## 12
##STR00257## 13 ##STR00258## 14 ##STR00259## 15 ##STR00260## 16
##STR00261## 17 ##STR00262## 18 ##STR00263## 19 ##STR00264## 20
##STR00265## 21 ##STR00266## 22 ##STR00267## 23 ##STR00268##
Example 24: Transient Transfection
[1097] CHO--S culture is seeded at 0.75.times.10{circumflex over (
)}6/mL approximately 16 hours pre-transfection in FreeStyle Cho
medium. Cells are ready to transfect the next day when the cell
count has reached 1.4-1.6.times.10{circumflex over ( )}6/mL. When
cells reach target count, 400 mM pAF stock is added to a 1.4 mM
final culture concentration. PEI/DNA complex is prepared as
described: DNA (1.42 ug/1.times.10{circumflex over ( )}6 cells) is
dissolved in RPMI (5% (v/v) of total culture volume), DNA/RPMI
mixture is incubated at room temperature for 2 minutes, PEI stock
(1 mg/mL) is added to DNA solution at a 3:1 ratio (mL PEI/ug DNA),
and the mixture is incubated at room temperature for 5 min. The
culture is gently added to the mixture and swirled. The flasks are
transferred to a 32.degree. C. incubator. At day 6
post-transfection, a western blot analysis is performed. At day 7
post-transfection, the supernatant is harvested.
Example 25: PSMA-ADC In Vivo Efficacy Study
[1098] Anti-tumor efficacy of ARX-.alpha.PSMA conjugated to NCA1
was tested in an LNCaP mouse model. LNCaP prostate cancer cells
expressing high levels of PSMA were obtained from ATCC and expanded
in vitro following ATCC instruction. LNCaP human prostate carcinoma
xenografts were grown in male nude mice. Nude mice were implanted
subcutaneously. Mice were weighed and measured for tumor three
dimensionally using an electronic caliper. Individual tumor volume
was calculated as height.times.width.times.length. Mice with
vascularized tumors (determined by appearance of the tumors) of
appropriate sizes were randomized into treatment groups and were
dosed per individual body weight on Day 33 (three (3) weeks
following injection with cells). Results from this experiment are
shown in FIGS. 1 and 2.
TABLE-US-00004 TABLE 4 Test Groups for PSMA In Vivo Efficacy Study
Grp Treatment Dose Concentration Dose Volume Route Schedule 1
Vehicle N/A .sup. N/A 5 mL/kg IV Once, day 1 2
ARX.alpha.PSMA(A116)-NCA1 1 mg/kg 0.2 mg/mL 5 mL/kg IV Once, day 1
3 ARX.alpha.PSMA(A116)-NCA1 3 mg/kg 0.6 mg/mL 5 mL/kg IV Once, day
1 4 ARX.alpha.PSMA(A116)-NCA1 10 mg/kg .sup. 2 mg/mL 5 mL/kg IV
Once, day 1 5 ARX-.alpha.PSMA (A116) 10 mg/kg .sup. 2 mg/mL 5 mL/kg
IV Once, day 1 *Ten (10) nude mice per group
Example 26: PSMA-ADC (DNA alkylator) In Vivo Efficacy Study
[1099] Anti-tumor efficacy of ARX-.alpha.PSMA conjugated to
ARX-duocarmycin is tested in an LNCaP mouse model. LNCaP prostate
cancer cells expressing high levels of PSMA are obtained from ATCC
and expanded in vitro following ATCC instruction. LNCaP human
prostate carcinoma xenografts are grown in male nude mice. Nude
mice are implanted subcutaneously. Mice are weighed and measured
for tumor three dimensionally using an electronic caliper.
Individual tumor volume is calculated as
height.times.width.times.length. Mice with vascularized tumors
(determined by appearance of the tumors) of appropriate sizes are
randomized into treatment groups and are dosed per individual body
weight on Day 33 (three (3) weeks following injection with
cells).
TABLE-US-00005 TABLE 5 Test Groups for PSMA In Vivo Efficacy Study
Grp Treatment Dose Concentration Dose Vol Route Schedule 1 Vehicle
N/A .sup. N/A 5 mL/kg IV Once, day 1 2 ARX.alpha.PSMA-ARX- 1 mg/kg
0.2 mg/mL 5 mL/kg IV Once, day 1 duocarmycin 3 ARX.alpha.PSMA-ARX-
3 mg/kg 0.6 mg/mL 5 mL/kg IV Once, day 1 duocarmycin 4
ARX.alpha.PSMA-ARX- 10 mg/kg .sup. 2 mg/mL 5 mL/kg IV Once, day 1
duocarmycin 5 ARX-.alpha.PSMA (A116) 10 mg/kg .sup. 2 mg/mL 5 mL/kg
IV Once, day 1 *Ten (10) nude mice per group
Example 27: ARX.alpha.PSMA(A116)-NCA1 72 h Prostate Cancer Cell
Line Viability Study
[1100] Anti-tumor efficacy of ARX-.alpha.PSMA conjugated to tubulin
inhibitor NCA1, an ARX proprietary dolastatin derivative, and
"free", unbound dolastatin were tested on prostate cancer cell
lines LNCaP and MDA-PCa-2b. The two prostate cancer cell lines and
PC-3 cells, used as negative control, were cultured and then
treated either with unconjugated dolastatin, unconjugated
duocarmycin, or ADC ARX-.alpha.PSMA-duocarmycin and 72 hours
following treatment cell viability was measured using the Dojindo
cell counting kit-8 (WST-8 based). Results from this experiment are
shown in FIGS. 3A, 3B, and 4.
Example 28: PSMA-duocarmycin 72 h Prostate Cancer Cell Line
Viability Study
[1101] Anti-tumor efficacy of ARX-.alpha.PSMA conjugated to DNA
alykylator duocarmycin was tested on prostate cancer cell lines
LNCaP and MDA-PCa-2b. The two prostate cancer cell lines and PC-3
cells, used as negative control, were cultured and then treated
either with unconjugated dolastatin, unconjugated duocarmycin, or
ADC ARX-.alpha.PSMA-duocarmycin and 72 hours following treatment
cell viability was measured using the Dojindo cell counting kit-8
(WST-8 based). Results from this experiment are shown in FIGS. 5A,
58, and 6.
Example 29: Human Clinical Trial of ARX.alpha.PSMA(A116)-NCA1
[1102] Human Clinical Trial of the Safety and/or Efficacy of
ARX.alpha.PSMA(A116)-NCA1 for Prostate Cancer Therapy
[1103] Objective: To compare the safety and pharmacokinetics of
administered composition comprising ARX-.alpha.PSMA-linked
dolastatin derivative.
[1104] Study Design: This study will be a Phase I, single-center,
open-label, randomized dose escalation study followed by a Phase II
study in prostate cancer patients. Patients should not have had
exposure to .alpha.PSMA -linked dolastatin derivative prior to the
study entry. Patients must not have received treatment for their
cancer within 2 weeks of beginning the trial. Treatments include
the use of chemotherapy, hematopoietic growth factors, and biologic
therapy such as monoclonal antibodies. Patients must have recovered
from all toxicities (to grade 0 or 1) associated with previous
treatment. All subjects are evaluated for safety and all blood
collections for pharmacokinetic analysis are collected as
scheduled. All studies are performed with institutional ethics
committee approval and patient consent.
[1105] Phase I: Patients receive i.v. ARX.alpha.PSMA(A116)-NCA1 on
days 1, 8, and 15 of each 28-day cycle. Doses of
ARX.alpha.PSMA(A116)-NCA1 may be held or modified for toxicity
based on assessments as outlined below. Treatment repeats every 28
days in the absence of unacceptable toxicity. Cohorts of 3-6
patients receive escalating doses of ARX.alpha.PSMA(A116)-NCA1
until the maximum tolerated dose (MTD) for
ARX.alpha.PSMA(A116)-NCA1 is determined. The MTD is defined as the
dose preceding that at which 2 of 3 or 2 of 6 patients experience
dose-limiting toxicity. Dose limiting toxicities are determined
according to the definitions and standards set by the National
Cancer Institute (NCI) Common Terminology for Adverse Events
(CTCAE) Version 3.0 (Aug. 9, 2006).
[1106] Phase II: Patients receive ARX.alpha.PSMA(A116)-NCA1 as in
phase I at the MTD determined in phase I. Treatment repeats every 4
weeks for 2-6 courses in the absence of disease progression or
unacceptable toxicity. After completion of 2 courses of study
therapy, patients who achieve a complete or partial response may
receive an additional 4 courses. Patients who maintain stable
disease for more than 2 months after completion of 6 courses of
study therapy may receive an additional 6 courses at the time of
disease progression, provided they meet original eligibility
criteria.
[1107] Blood Sampling: serial blood is drawn by direct vein
puncture before and after administration of
ARX.alpha.PSMA(A116)-NCA1. Venous blood samples (5 mL) for
determination of serum concentrations are obtained at about 10
minutes prior to dosing and at approximately the following times
after dosing: days 1, 8, and 15. Each serum sample is divided into
two aliquots. All serum samples are stored at -20.degree. C. Serum
samples are shipped on dry ice.
[1108] Pharmacokinetics: Patients undergo plasma/serum sample
collection for pharmacokinetic evaluation before beginning
treatment and at days 1, 8, and 15. Pharmacokinetic parameters are
calculated by model independent methods on a Digital Equipment
Corporation VAX 8600 computer system using the latest version of
the BIOAVL software. The following pharmacokinetics parameters are
determined: peak serum concentration (C.sub.max); time to peak
serum concentration (t.sub.max); area under the concentration-time
curve (AUC) from time zero to the last blood sampling time
(AUC.sub.0-72) calculated with the use of the linear trapezoidal
rule; and terminal elimination half-life (t.sub.1/2), computed from
the elimination rate constant. The elimination rate constant is
estimated by linear regression of consecutive data points in the
terminal linear region of the log-linear concentration-time plot.
The mean, standard deviation (SD), and coefficient of variation
(CV) of the pharmacokinetic parameters are calculated for each
treatment. The ratio of the parameter means (preserved
formulation/non-preserved formulation) is calculated.
[1109] Patient Response to combination therapy: Patient response is
assessed via imaging with X-ray, CT scans, and MRI, and imaging is
performed prior to beginning the study and at the end of the first
cycle, with additional imaging performed every four weeks or at the
end of subsequent cycles. Imaging modalities are chosen based upon
the cancer type and feasibility/availability, and the same imaging
modality is utilized for similar cancer types as well as throughout
each patient's study course. Response rates are determined using
the RECIST criteria. (Therasse et al, J. Natl. Cancer Inst. 2000
Feb. 2; 92(3):205-16;
http://ctep.cancer.gov/forms/TherasseRECISTJNCI.pdf). Patients also
undergo cancer/tumor biopsy to assess changes in progenitor cancer
cell phenotype and clonogenic growth by flow cytometry, Western
blotting, and IHC, and for changes in cytogenetics by FISH. After
completion of study treatment, patients are followed periodically
for 4 weeks.
Example 30: Treatment for Prostate Cancer
[1110] Human Clinical Trial of the Safety and Efficacy of
ARX.alpha.PSMA(A116)-NCA1 for Prostate Cancer Therapy
[1111] Objective: Compare the efficacy and toxicity of
ARX.alpha.PSMA(A116)-NCA1 alone followed at disease progression by
combination trastuzumab and paclitaxel vs first-line combination
trastuzumab and paclitaxel in women with HER2-overexpressing
metastatic breast cancer.
[1112] Study Design: This study is a randomized, multicenter study.
Patients are stratified according to degree of
HER2/neu-overexpression (2+ vs 3+), prior anthracycline-containing
adjuvant treatment (no prior treatment vs prior treatment without
radiotherapy to left chest wall vs prior treatment with
radiotherapy to left chest wall), estrogen-receptor status
(positive vs negative vs unknown), prior therapy (first-line vs
second/third-line), and center. Patients are randomized to one of
two treatment arms. Arm I: Patients receive trastuzumab-linked
dolastatin derivative IV over 30-90 minutes weekly. At time of
disease progression, patients receive combination
trastuzumab-linked dolastatin derivative IV and paclitaxel IV as in
arm II. Arm II: Patients receive trastuzumab-linked dolastatin
derivative IV over 30-90 minutes weekly. Paclitaxel is administered
IV over 1 hour weekly for 3 weeks followed by 1 week of rest.
[1113] Treatment continues in both arms in the absence of disease
progression or unacceptable toxicity. Quality of life is assessed
at baseline and day 1 of courses 2, 3, 4, 5, 6, 8, 10, and 12.
Patients are followed at 1, 3, and 6 months and then every 6 months
thereafter.
Sequence CWU 1
1
141214PRTHomo sapiens 1Asp Ile Val Met Thr Gln Ser His Lys Phe Met
Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser Ile Ile Cys Lys Ala Ser
Gln Asp Val Gly Thr Ala 20 25 30Val Asp Trp Tyr Gln Gln Lys Pro Gly
Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr
Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Thr Asn Val Gln Ser65 70 75 80Glu Asp Leu Ala Asp
Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Ala
Gly Thr Met Leu Asp Leu Lys Arg Thr Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 2102444PRTHomo sapiens 2Glu Val Gln Leu Gln Gln Ser Gly Pro Glu
Leu Val Lys Pro Gly Thr1 5 10 15Ser Val Arg Ile Ser Cys Lys Thr Ser
Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Thr Ile His Trp Val Lys Gln Ser
His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Asn Ile Asn Pro Asn Asn
Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55 60Glu Asp Lys Ala Thr Leu
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Ala Gly
Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120
125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala145 150 155 160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu225 230 235
240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360
365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln 405 410 415Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 4403214PRTHomo sapiensMISC_FEATURE(7)..(7)X
is non-natural amino acid 3Asp Ile Val Met Thr Gln Xaa His Lys Phe
Met Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser Ile Ile Cys Lys Ala
Ser Gln Asp Val Gly Thr Ala 20 25 30Val Asp Trp Tyr Gln Gln Lys Pro
Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His
Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Thr Asn Val Gln Ser65 70 75 80Glu Asp Leu Ala
Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly
Ala Gly Thr Met Leu Asp Leu Lys Arg Thr Val Ala Ala 100 105 110Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 2104214PRTHomo sapiensMISC_FEATURE(18)..(18)X is non-natural
amino acid 4Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser
Val Gly1 5 10 15Asp Xaa Val Ser Ile Ile Cys Lys Ala Ser Gln Asp Val
Gly Thr Ala 20 25 30Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro
Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Thr Asn Val Gln Ser65 70 75 80Glu Asp Leu Ala Asp Tyr Phe Cys
Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Met
Leu Asp Leu Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150
155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
2105214PRTHomo sapiensMISC_FEATURE(79)..(79)X is non-natural amino
acid 5Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val
Gly1 5 10 15Asp Arg Val Ser Ile Ile Cys Lys Ala Ser Gln Asp Val Gly
Thr Ala 20 25 30Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys
Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp
Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Thr Asn Val Xaa Ser65 70 75 80Glu Asp Leu Ala Asp Tyr Phe Cys Gln
Gln Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Met Leu
Asp Leu Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 2106214PRTHomo
sapiensMISC_FEATURE(109)..(109)X is non-natural amino acid 6Asp Ile
Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly1 5 10 15Asp
Arg Val Ser Ile Ile Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25
30Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val
Gln Ser65 70 75 80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn
Ser Tyr Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Met Leu Asp Leu Lys
Arg Xaa Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 2107214PRTHomo
sapiensMISC_FEATURE(155)..(155)X is non-natural amino acid 7Asp Ile
Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly1 5 10 15Asp
Arg Val Ser Ile Ile Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25
30Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val
Gln Ser65 70 75 80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn
Ser Tyr Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Met Leu Asp Leu Lys
Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Xaa Ser Gly Asn Ser Gln145 150 155 160Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 2108214PRTHomo
sapiensMISC_FEATURE(202)..(202)X is non-natural amino acid 8Asp Ile
Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly1 5 10 15Asp
Arg Val Ser Ile Ile Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25
30Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val
Gln Ser65 70 75 80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn
Ser Tyr Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Met Leu Asp Leu Lys
Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Xaa Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 2109444PRTHomo
sapiensMISC_FEATURE(116)..(116)X is non-natural amino acid 9Glu Val
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr1 5 10 15Ser
Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25
30Thr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys
Phe 50 55 60Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln
Gly Thr Thr Leu Thr 100 105 110Val Ser Ser Xaa Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro 115 120 125Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala145 150 155 160Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170
175Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys 195 200 205Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295
300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys305 310 315 320Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser 340 345 350Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410
415Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435
44010444PRTHomo sapiensMISC_FEATURE(280)..(280)X is non-natural
amino acid 10Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
Pro Gly Thr1 5 10 15Ser Val Arg Ile Ser Cys Lys Thr Ser
Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Thr Ile His Trp Val Lys Gln Ser
His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Asn Ile Asn Pro Asn Asn
Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55 60Glu Asp Lys Ala Thr Leu
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Ala Gly
Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120
125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala145 150 155 160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu225 230 235
240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys 260 265 270Phe Asn Trp Tyr Val Asp Gly Xaa Glu Val His Asn
Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360
365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln 405 410 415Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 44011444PRTHomo
sapiensMISC_FEATURE(294)..(294)X is non-natural amino acid 11Glu
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr1 5 10
15Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30Thr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp
Ile 35 40 45Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln
Lys Phe 50 55 60Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly
Gln Gly Thr Thr Leu Thr 100 105 110Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro 115 120 125Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala145 150 155 160Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170
175Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys 195 200 205Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro
Arg Glu Glu Gln Xaa Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295
300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys305 310 315 320Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser 340 345 350Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410
415Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435
44012444PRTHomo sapiensMISC_FEATURE(340)..(340)X is non-natural
amino acid 12Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
Pro Gly Thr1 5 10 15Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr
Phe Thr Glu Tyr 20 25 30Thr Ile His Trp Val Lys Gln Ser His Gly Lys
Ser Leu Glu Trp Ile 35 40 45Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr
Thr Tyr Asn Gln Lys Phe 50 55 60Glu Asp Lys Ala Thr Leu Thr Val Asp
Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Ala Gly Trp Asn Phe
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135
140Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala145 150 155 160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys 195 200 205Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250
255Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys 275 280 285Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Xaa Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375
380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln 405 410 415Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 435 44013444PRTHomo
sapiensMISC_FEATURE(359)..(359)X is non-natural amino acid 13Glu
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr1 5 10
15Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30Thr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp
Ile 35 40 45Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln
Lys Phe 50 55 60Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly
Gln Gly Thr Thr Leu Thr 100 105 110Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro 115 120 125Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala145 150 155 160Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170
175Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys 195 200 205Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295
300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys305 310 315 320Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser 340 345 350Arg Asp Glu Leu Thr Lys Xaa Gln
Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410
415Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435
44014444PRTHomo sapiensMISC_FEATURE(417)..(417)X is non-natural
amino acid 14Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
Pro Gly Thr1 5 10 15Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr
Phe Thr Glu Tyr 20 25 30Thr Ile His Trp Val Lys Gln Ser His Gly Lys
Ser Leu Glu Trp Ile 35 40 45Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr
Thr Tyr Asn Gln Lys Phe 50 55 60Glu Asp Lys Ala Thr Leu Thr Val Asp
Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Ala Gly Trp Asn Phe
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135
140Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala145 150 155 160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys 195 200 205Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250
255Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys 275 280 285Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375
380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln 405 410 415Xaa Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 435 440
* * * * *
References