U.S. patent application number 16/078080 was filed with the patent office on 2020-03-12 for derivatives of amanita toxins and their conjugation to a cell binding molecule.
This patent application is currently assigned to Hangzhou DAC Biotech Co., Ltd.. The applicant listed for this patent is Hangzhou DAC Biotech Co., Ltd.. Invention is credited to Minjun CAO, Shun GAI, Huihui GUO, Zixiang GUO, Yuanyuan HUANG, Junxiang JIA, Wenjun LI, Chen LIN, Qianqian TONG, Hongsheng XIE, Yifang XU, Chengyu YANG, Qingliang YANG, Hangbo YE, Linyao ZHAO, Robert Yongxin ZHAO, Xiaomai ZHOU, Xiaotao ZUO.
Application Number | 20200079820 16/078080 |
Document ID | / |
Family ID | 58288166 |
Filed Date | 2020-03-12 |
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United States Patent
Application |
20200079820 |
Kind Code |
A1 |
ZHAO; Robert Yongxin ; et
al. |
March 12, 2020 |
DERIVATIVES OF AMANITA TOXINS AND THEIR CONJUGATION TO A CELL
BINDING MOLECULE
Abstract
The present invention is related to novel cytotoxic agents,
derivatives of Amanita toxins of Formula (I), wherein , - - - ,
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7,
R.sub.8, R.sub.9, R.sub.10, X, L, m, n and Q are defined herein,
the preparation and the therapeutic uses in the targeted treatment
of cancers, autoimmune disorders, and infectious diseases.
##STR00001##
Inventors: |
ZHAO; Robert Yongxin;
(Hangzhou, CN) ; YANG; Qingliang; (Hangzhou,
CN) ; HUANG; Yuanyuan; (Hangzhou, CN) ; GAI;
Shun; (Hangzhou, CN) ; YE; Hangbo; (Hangzhou,
CN) ; YANG; Chengyu; (Hangzhou, CN) ; GUO;
Huihui; (Hangzhou, CN) ; ZHOU; Xiaomai;
(Hangzhou, CN) ; XIE; Hongsheng; (Hangzhou,
CN) ; TONG; Qianqian; (Hangzhou, CN) ; CAO;
Minjun; (Hangzhou, CN) ; ZHAO; Linyao;
(Hangzhou, CN) ; JIA; Junxiang; (Hangzhou, CN)
; LI; Wenjun; (Hangzhou, CN) ; ZUO; Xiaotao;
(Hangzhou, CN) ; LIN; Chen; (Hangzhou, CN)
; XU; Yifang; (Hangzhou, CN) ; GUO; Zixiang;
(Hangzhou, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hangzhou DAC Biotech Co., Ltd. |
Hangzhou, Zhejiang |
|
CN |
|
|
Assignee: |
Hangzhou DAC Biotech Co.,
Ltd.
Hangzhou, Zhejiang
CN
|
Family ID: |
58288166 |
Appl. No.: |
16/078080 |
Filed: |
April 20, 2016 |
PCT Filed: |
April 20, 2016 |
PCT NO: |
PCT/IB2016/052246 |
371 Date: |
August 21, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/04 20180101;
A61P 25/28 20180101; A61K 38/00 20130101; C07K 16/32 20130101; A61P
37/06 20180101; A61P 35/00 20180101; A61P 13/12 20180101; C07K 7/64
20130101; A61P 35/02 20180101; A61P 29/00 20180101; A61P 25/00
20180101; A61P 31/12 20180101; Y02A 50/473 20180101; A61P 7/00
20180101; A61P 1/18 20180101; A61P 19/08 20180101 |
International
Class: |
C07K 7/64 20060101
C07K007/64; C07K 16/32 20060101 C07K016/32 |
Claims
1. A compound of Formula (I) ##STR00170## or a pharmaceutically
acceptable salt, hydrate, or hydrated salt thereof; or a
polymorphic crystalline structures thereof; or an optical isomer,
racemate, diastereomer or enantiomer thereof, wherein - - -
represents a single bond that links any carbon position of the
aromatic (indole) ring; represents an optional single bond or an
absent bond; R.sub.1 and R.sub.2 are independently selected from
the group consisting of H, OH, CH.sub.2OH, CH(OH)CH.sub.2OH,
CH(CH.sub.3)CH.sub.2OH, CH(OH)CH.sub.3, C.sub.1-C.sub.8 alkyl,
--OR.sub.12 (ether), C.sub.2-C.sub.8 alkenyl, alkynyl, heteroalkyl,
--OCOR.sub.12 (ester), --OC(.dbd.O)OR.sub.12-(carbonate),
--OC(.dbd.O)NHR.sub.12(carbamate); C.sub.3-C.sub.8 aryl,
heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl,
heteroaralkyl, and alkylcarbonyl; R.sub.3 and R.sub.4 are
independently selected from the group consisting of H, OH,
--OR.sub.12 (ether), --OCOR.sub.12 (ester), --OCOCH.sub.3
(acetate), --OCOOR.sub.12 (carbonate), --OC(.dbd.O)NHR.sub.12
(carbamate), --OP(O)(OR.sub.12)(OR.sub.12') (phosphate),
OP(O)(NHR.sub.12)(NHR.sub.12') (phosphamide), O--SO.sub.3.sup.-,
and O-glycoside; R.sub.5 is selected from the group consisting of
H, OH, NH.sub.2, NHOH, NHNH.sub.2, --OR.sub.12, --NHR.sub.12,
NHNHR.sub.12, --NR.sub.12R.sub.12', and
N(H)(R.sub.12)R.sub.13CO(Aa).sub.p, wherein Aa is an amino acid or
a polypeptide, p represents 0-6; R.sub.6 is selected from the group
consisting of H, OH, CH.sub.2OH, CH(OH)CH.sub.2OH,
CH(CH.sub.2OH).sub.2, CH(CH.sub.3)OH, CH.sub.2CH.sub.2OH, PrOH,
BuOH, C.sub.1.about.C.sub.8 alkyl, --OR.sub.12 (ether),
C.sub.2.about.C.sub.8 alkenyl, alkynyl, heteroalkyl, or
--OCOR.sub.12 (ester); and C.sub.3.about.C.sub.8 aryl,
heterocyclic, or carbocyclic. R.sub.7, R.sub.8 and R.sub.9 are
independently selected from the group consisting of H, OH,
CH.sub.3, CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2CH.sub.3,
CH.sub.2OH, CH(OH)CH.sub.2OH, CH.sub.2CH(OH)CH.sub.2OH,
CH(CH.sub.2OH).sub.2, CH.sub.2C(OH)(CH.sub.2OH).sub.2,
CH.sub.2C(OH)(CH.sub.3)(CH.sub.2OH),
CH.sub.2C(OH)(CH(CH.sub.3)2)(CH.sub.2OH), CH.sub.2CH.sub.2OH, PrOH,
BuOH, CH.sub.2COOH, CH.sub.2CH.sub.2COOH, CH(OH)COOH,
CH.sub.2CONH.sub.2, CH.sub.2CH.sub.2CONH.sub.2,
CH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2,
CH.sub.2CH.sub.2CH.sub.2NHC(.dbd.NH)NH.sub.2, C.sub.1.about.C.sub.8
alkyl, CH.sub.2Ar, CH.sub.2SH, CH.sub.2SR.sub.12,
CH.sub.2SSR.sub.12, CH.sub.2SSAr, CH.sub.2CH.sub.2SCH.sub.3,
--OR.sub.12 (ether), C.sub.2.about.C.sub.8 alkenyl, alkynyl,
heteroalkyl, or --OCOR.sub.12 (ester); and C.sub.3.about.C.sub.8
aryl, heterocyclic, or carbocyclic; R.sub.10 is selected from the
group consisting of H, NH.sub.2, OH, SH, NO.sub.2, halogen, --NHOH,
--N.sub.3 (azido); --CN (cyano); C.sub.1.about.C.sub.8 alkyl,
C.sub.2.about.C.sub.8 alkenyl, alkynyl, heteroalkyl;
C.sub.3.about.C.sub.8 aryl, heterocyclic, or carbocyclic;
--OR.sub.12 (ether), --OCOR.sub.12 (ester), --OCOCH.sub.3
(acetate), --OC(O)OR.sub.12 (carbonate), --OC(O)CH(R.sub.12)NHAa
(Aa is an amino acid group), --NR.sub.12R.sub.12' (amine),
--NR.sub.12COR.sub.12' (amine), --NR.sub.12NR.sub.12'NR.sub.12''
(amine); --OCONR.sub.12R.sub.12'(carbamate);
--NR.sub.12(C.dbd.NH)NR.sub.12'R.sub.12'' (guanidinum);
--NR.sub.12CO(Aa).sub.p, (an amino acid or peptide, wherein Aa is
an amino acid or a polypeptide, p represents 0-6);
--N(R.sub.12)CONR.sub.12'R.sub.12'' (urea); --OCSNHR.sub.12
(thiocarbamate); --SH (thiol); --SR.sub.12 (sulfide);
--S(O)R.sub.12 (sulfoxide); --S(O.sub.2)R.sub.12 (sulfone);
--SO.sub.3, HSO.sub.3, HSO.sub.2, or a salt of HSO.sub.3.sup.-,
SO.sub.3.sup.2- or --HSO.sub.2.sup.- (sulphite); --OSO.sub.3.sup.-;
--N(R.sub.12)SOOR.sub.12' (sulfonamide); H.sub.2S.sub.2O.sub.5 or a
salt of S.sub.2O.sub.5.sup.2-(metabisulfite); PO.sub.3SH.sub.3,
PO.sub.2S.sub.2H.sub.2, POS.sub.3H.sub.2, PS.sub.4H.sub.2 or a salt
of PO.sub.3S.sup.3-, PO.sub.2S.sub.2.sup.3-, POS.sub.3.sup.3-,
PS.sub.4.sup.3- (mono-, di-, tri-, and tetra-thiophosphate);
(R.sub.12O).sub.2POSR.sub.12' (thiophosphate ester);
HS.sub.2O.sub.3 or a salt of S.sub.2O.sub.3.sup.2- (thiosulfate);
HS.sub.2O.sub.4 or a salt of S.sub.2O.sub.4.sup.2- (dithionite);
(P(.dbd.S)(OR.sub.12)(S)(OH) or a salt formed with a cation
(phosphorodithioate); --N(R.sub.12)OR.sub.12' (hydroxylamine
derivative); R.sub.12C(.dbd.O)NOH or a salt formed with a cation
(hydroxamic acid); (HOCH.sub.2SO.sub.2.sup.-, or its salt
(formaldehyde sulfoxylate); --N(R.sub.12)COR.sub.12' (amide);
R.sub.12R.sub.12'R.sub.12''NPO.sub.3H (trialkylphosphor-amidate or
phosphoramidic acid); ArAr'Ar''NPO.sub.3H (triarylphosphonium);
OP(O)(OM.sub.1)(OM.sub.2), OCH.sub.2OP(O)(OM.sub.1)(OM.sub.2),
OSO.sub.3M.sub.1; O-glycoside (glucoside, galactoside, mannoside,
glucuronoside, alloside, fructoside), NH-glycoside, S-glycoside and
CH.sub.2-glycoside; M.sub.1 and M.sub.2 are independently H, Na, K,
Ca, Mg, NH.sub.4, or NR.sub.1'R.sub.2'R.sub.3'; R.sub.1', R.sub.2'
and R.sub.3' are independently H, or C.sub.1.about.C.sub.8 alkyl;
and Ar, Ar', and Ar'' are C.sub.3-C.sub.8 aryl or heteroaromatic
group; R.sub.11 is H, C.sub.1.about.C.sub.8 alkyl;
C.sub.2.about.C.sub.8 alkenyl, alkynyl, or heteroalkyl; or
C.sub.3.about.C.sub.8 aryl or heteroaryl; R.sub.12, R.sub.12', and
R.sub.12'' are independently selected from the group consisting of
H, C.sub.1.about.C.sub.8 alkyl; C.sub.2.about.C.sub.8 alkenyl,
alkynyl, heteroalkyl; C.sub.3.about.C.sub.8 aryl, heteroaryl,
heterocyclic, and carbocyclic; X is S, O, NH, SO, SO.sub.2, or
CH.sub.2; m is 0 or 1; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20; L is a releasable linker having
the formula of: -Ww-(Aa)r-Tt-; or -Ww-(Aa)r-Tt-Q; or
Q-Ww-(Aa)r-Tt-; wherein W is a Stretcher unit; w is 0 or 1; Aa is
an amino acid unit comprising independent amino acids; r is an
integer ranging from 0 to 100, the Stretcher unit W comprises a
self-immolative or a non-self-immolative component, peptidyl unit,
a hydrazone bond, a disulfide, an ester, an oxime, an amide, or a
thioether bond, the self-immolative component comprises an aromatic
compound that is electronically similar to
para-aminobenzylcarbamoyl (PAB) group, 2-aminoimidazol-5-methanol
derivative, heterocyclic PAB analog, beta-glucuronide, and ortho or
para-aminobenzylacetal; the non-self-immolative component is one of
the following structures: ##STR00171## ##STR00172## ##STR00173##
##STR00174## wherein the (*) atom is a point of attachment of
additional spacer or releasable linker, a cytotoxic agent, or a
binding molecule; X.sup.1, Y.sup.1, Q.sup.1, R.sub.12, R.sub.12'
are defined as above; r is 0.about.100; p and q is independently 0,
1, 2, 3, 4, 5 or 6; Spacer (T) is a linear, branched or cyclic
alkyl, alkenyl, alkynyl or aryl having from 1 to 10 carbon atoms,
or a polyethylene glycol (--CH.sub.2CH.sub.2O--) spacer; t is 0, or
1.about.100, or T undergoes cyclization upon amide bond hydrolysis,
of a substituted and unsubstituted 4-aminobutyric acid amide,
substituted bicycle [2.2.1] and bicycle [2.2.2] ring systems or
2-aminophenylpropionic acid amide; Q is a cell-binding agent or a
functional group that enables the compound of Formula (I) to link
with a cell-binding agent, or a functional group that enables the
compound of Formula (I) to link with a linker attached on a
cell-binding agent, the function group being selected from the
group consisting of a thiol, an amine, a hydrazine, an
alkoxylamino, a disulfide substituent, a maleimido, a haloacetyl
group, a carboxy acid, an N-hydroxy succinimide ester, a ketone, an
ester, an aldehyde, an alkynyl, an alkenyl, and a protected thiol
or disulfide group: SAc, SSR.sub.1 or SSAr, wherein Ar is an
aromatic group or hetero aromatic group.
2. The compound according to claim 1 having the following Formula
(Ia) (Ib), or (Ic): ##STR00175## or a pharmaceutically acceptable
salt, hydrate, hydrated salt, or polymorphic crystalline structure
thereof, or an optical isomer, racemate, diastereomer or enantiomer
thereof; wherein R.sub.1, R.sub.2, R.sub.4, R.sub.5, R.sub.7,
R.sub.8, R.sub.9, R.sub.10, L and Q are defined the same as in
claim 1.
3. The compound according to claim 1 having the following Formula
(Id): ##STR00176## or a pharmaceutically acceptable salt, hydrate,
hydrated salt, or polymorphic crystalline structure thereof, or an
optical isomer, racemate, diastereomer or enantiomer thereof;
wherein R.sub.1, R.sub.2, R.sub.4, R.sub.5, R.sub.10, L and Q are
defined the same as in claim 1.
4. The compound according to claim 1 having the following Formula
(Ia-1), (Ia-2), (Ia-3), (Ia-4), (Ia-5), (Ia-6), (Ia-7), (Ia-8),
(Ia-9), (Ia-10), (Ia-11), (Ia-12), (Ia-13), (Ia-14), (Ia-15),
(Ia-16), (Ia-17), (Ia-18), (Ia-19), (Ia-20), (Ia-21), (Ia-22),
(Ia-23), (Ia-24), (Ib-1), (Ib-2), (Ib-3), (Ib-4), (Ib-5), (Ib-6),
(Ib-7), (Ic-1), (Ic-2), (Ic-3), (Ic-4), (Ic-5), or (Ic-6):
##STR00177## ##STR00178## ##STR00179## ##STR00180## ##STR00181##
##STR00182## ##STR00183## ##STR00184## ##STR00185## ##STR00186## or
a pharmaceutically acceptable salt, hydrate, hydrated salt, or
polymorphic crystalline structure thereof, or an optical isomer,
racemate, diastereomer or enantiomers thereof; wherein R.sub.10, L
and Q are defined the same as in claim 1.
5. The compound according to claim 1 having the following Formula
(Id-1), (Id-2), (Id-3), (Id-4), (Id-5), (Id-6), (Id-7), (Id-8),
(Id-9), (Id-10), (Id-11), (Id-12), (Id-13), (Id-14), (Id-15),
(Id-16), (Id-17), (Id-18), (Id-19), (Id-20), or (Id-21):
##STR00187## ##STR00188## ##STR00189## ##STR00190## ##STR00191##
##STR00192## wherein L and Q are defined the same as in claim 1;
R.sub.14 is H, PO.sub.3.sup.2-, SO.sub.3.sup.-, R.sub.12,
--COR.sub.12, --COCH.sub.3, --COOR.sub.12, --CONR.sub.12R.sub.12',
--C(.dbd.O)R.sub.12NH(Aa).sub.t, wherein Aa is an amino acid or a
polypeptide, t represents 0-100; --CSNHR.sub.12 (thiocarbamate);
--SOR.sub.12 (sulfoxide); --SO.sub.2R.sub.12 (sulfone);
--SO.sub.3.sup.-, HSO.sub.3, HSO.sub.2, or a salt of
HSO.sub.3.sup.-, SO.sub.3.sup.2- or --HSO.sub.2.sup.- (sulphite);
P(O)(OM.sub.1)(OM.sub.2), CH.sub.2OP(O)(OM.sub.1)(OM.sub.2),
SO.sub.3M.sub.1; or glycoside (glucoside, galactoside, mannoside,
glucuronoside, alloside, fructoside; M.sub.1 and M.sub.2 are
independently H, Na, K, Ca, Mg, NH.sub.4, or
NR.sub.1'R.sub.2'R.sub.3'; R.sub.1', R.sub.2' and R.sub.3' are
independently H, or C.sub.1.about.C.sub.8 alkyl.
6. The compound according to claim 1 having the following Formula
(I-1), (I-2), (I-3), (I-4), (I-5), (I-6), (I-7), (I-8), (I-9),
(I-10), (I-11), (I-12), (I-13), (I-14), (I-15), (I-16), (I-17),
(I-18), (I-19), (I-20), (I-21), (I-22), (I-23), (I-24), (I-25),
(I-26), (I-27), (I-28), (I-29), (I-30), (I-31), (I-32), (I-33),
(I-34), (I-35), (I-36), (I-37), (I-38), (I-39), (I-40), (I-41),
(I-42), (I-43), (I-44), (I-45), (I-46), (I-47), (I-48), (I-49),
(I-50), (I-51), (I-52), (I-53), (I-54), (I-55), (I-56), (I-57),
(I-58), (I-69), (I-60), (I-61), (I-62), (I-63), (I-64), (I-65),
(I-66), (I-67), (I-68), (I-69), (I-70), (I-71), (I-72), (I-73),
(I-74), (I-75), (I-76), (I-77), (I-78), (I-79), (I-80), (I-81),
(I-82), (I-83), (I-84), (I-85), (I-86), (I-87), (I-88), (I-89),
(I-90), (I-91), (I-92), (I-93), (I-94), (I-95), (I-95) or (I-95):
##STR00193## ##STR00194## ##STR00195## ##STR00196## ##STR00197##
##STR00198## ##STR00199## ##STR00200## ##STR00201## ##STR00202##
##STR00203## ##STR00204## ##STR00205## ##STR00206## ##STR00207##
##STR00208## ##STR00209## ##STR00210## ##STR00211## ##STR00212##
##STR00213## ##STR00214## ##STR00215## ##STR00216## ##STR00217## or
a pharmaceutically acceptable salt, hydrate, hydrated salt, or
polymorphic crystalline structure thereof, or an optical isomer,
racemate, diastereomer or enantiomer thereof, wherein Aa, r, n, p,
q and Q are defined the same as in the claim 1; PEG is polyethylene
glycol with the formula of --(OCH.sub.2CH.sub.2).sub.r.
7. A method for preparing the compound according to claim 1,
comprising sequentially (i) aromatic nitration of an indole unit,
(ii) reduction of a nitro group on a benzene ring of the indole
unit to an amine, and (iii) condensation of the produced amine
compound with a linker having a reactive or a reactable carboxylic
group to form an amide linkage as illustrated below: ##STR00218##
wherein R.sub.10, L and Q are defined the same as in claim 1,
wherein Lv is leaving group selected from the group consisting of
OH, halogen, NHS (N-hydroxyl succinimide), nitrophenol,
pentafluorophenol, and an intermediate generated from peptide
coupling and from Mitsunobu reaction.
8. The compound according to claim 1, wherein the linker L is
selected from the group consisting of: R.sub.12, OR.sub.12,
OR.sub.12O, NHR.sub.12, NHR.sub.12NH, NR.sub.11R.sub.12,
SR.sub.12S, OR.sub.12NH, OR.sub.12Ar, NHR.sub.12Ar,
NR.sub.11R.sub.12NR.sub.12'R.sub.12'',
--(CR.sub.11R.sub.12).sub.p(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q(OCH.sub-
.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(CR.sub.12'R.sub.12'').sub.q(Aa).sub.r(OCH.sub-
.2CH.sub.2).sub.t--,
-(Aa).sub.r(CR.sub.11R.sub.12).sub.p(CR.sub.12'R.sub.12'').sub.q--(OCH.su-
b.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(CR.sub.12'R.sub.12'').sub.n(OCH.sub.2CH.sub.2-
).sub.t(Aa).sub.r-, --(CR.sub.11R.sub.12).sub.p(CH.dbd.CH)
(CR.sub.12'R.sub.12'').sub.q(Aa).sub.r(OCH.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(NR.sub.12'CO)(Aa).sub.r(CR.sub.12'R.sub.12'')-
.sub.q--(OCH.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(Aa).sub.t(NHCO)(CR.sub.12'R.sub.12'').sub.q---
(OCH.sub.2CH.sub.2).sub.r--,
(CR.sub.11R.sub.12).sub.p(OCO)(Aa).sub.r-(CR.sub.12'R.sub.12'').sub.q--(O-
CH.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(OCNR.sub.7)(Aa).sub.r(CR.sub.12'R.sub.12'').s-
ub.q(OCH.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(CO)-(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q(OC-
H.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(NR.sub.11CO)(Aa).sub.r(CR.sub.12'R.sub.12'').-
sub.q(OCH.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p--(OCO)(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q--
-(OCH.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(OCNR.sub.7)(Aa).sub.r(CR.sub.12'R.sub.12'').s-
ub.q(OCH.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(CO)(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q(OCH-
.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p-phenyl-CO(Aa).sub.r(CR.sub.12'R.sub.12'').sub-
.q,
--(CR.sub.11R.sub.12).sub.p-furyl-CO-(Aa).sub.t(CR.sub.12'R.sub.12'').-
sub.q, --(CR.sub.11R.sub.12).sub.p-
oxazolyl-CO(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q,
--(CR.sub.11R.sub.12).sub.p-thiazolyl-CO(Aa).sub.r(CR.sub.12'R.sub.12'').-
sub.q,
--(CR.sub.11R.sub.12).sub.p-thienyl-CO(CR.sub.12'R.sub.12'').sub.q,
--(CR.sub.11R.sub.12).sub.p-imidazolyl-CO--(CR.sub.12'R.sub.12'').sub.q---
,
--(CR.sub.11R.sub.12).sub.p-morpholino-CO(Aa).sub.r(CR.sub.12'R.sub.12''-
).sub.q--,
--(CR.sub.11R.sub.12).sub.p-piperazino-CO(Aa).sub.r-(CR.sub.12'-
R.sub.12'').sub.q--,
--(CR.sub.11R.sub.12).sub.p--N-methylpiperazin-CO(Aa).sub.r(CR.sub.12'R.s-
ub.12'').sub.q--, --(CR.sub.11R.sub.12).sub.p(Aa).sub.r-phenyl-,
--(CR.sub.11R.sub.12).sub.p-(Aa).sub.r-furyl-,
--(CR.sub.11R.sub.12).sub.p-oxazolyl(Aa).sub.r-,
--(CR.sub.11R.sub.12).sub.p-thiazolyl-(Aa).sub.r-,
--(CR.sub.11R.sub.12).sub.p-thienyl-(Aa).sub.t-,
--(CR.sub.11R.sub.12).sub.p-imidazolyl(Aa).sub.r-,
--(CR.sub.11R.sub.12).sub.p-morpholino-(Aa).sub.r-,
--(CR.sub.11R.sub.12).sub.p-piperazino-(Aa).sub.r-,
--(CR.sub.11R.sub.12).sub.p--N-methylpiperazino-(Aa).sub.r-,
--K(CR.sub.11R.sub.12).sub.p-(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q(OCH.s-
ub.2CH.sub.2).sub.t,
--K(CR.sub.11R.sub.12).sub.p(CR.sub.12'R.sub.12'').sub.q(Aa).sub.r(OCH.su-
b.2CH.sub.2).sub.t--,
--K(Aa).sub.r(CR.sub.11R.sub.12).sub.p(CR.sub.12'R.sub.12'').sub.q--(OCH.-
sub.2CH.sub.2).sub.t,
--K(CR.sub.11R.sub.12).sub.p(CR.sub.12'R.sub.12'').sub.q(OCH.sub.2CH.sub.-
2).sub.r(Aa).sub.t,
--K(CR.sub.11R.sub.12).sub.p(CR.sub.7.dbd.CR.sub.8)(CR.sub.12'R.sub.12'')-
.sub.q-(Aa).sub.r(OCH.sub.2CH.sub.2).sub.t--,
--K(CR.sub.11R.sub.12).sub.p(NR.sub.7CO)(Aa).sub.r(CR.sub.12'R.sub.12'').-
sub.q(OCH.sub.2CH.sub.2).sub.t,
--K(CR.sub.11R.sub.12).sub.p(Aa).sub.t(NR.sub.7--CO)(CR.sub.12'R.sub.12''-
).sub.q(OCH.sub.2CH.sub.2).sub.t,
--K(CR.sub.11R.sub.12).sub.p(OCO)(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q(O-
CH.sub.2CH.sub.2).sub.t,
--K(CR.sub.11R.sub.12).sub.p(OCNR.sub.7)(Aa).sub.r(CR.sub.12'R.sub.12'').-
sub.q(OCH.sub.2CH.sub.2).sub.t--,
--K(CR.sub.11R.sub.12).sub.p(CO)(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q(OC-
H.sub.2CH.sub.2).sub.t,
--K(--CR.sub.11R.sub.12).sub.p(NR.sub.11CO)(Aa).sub.r(CR.sub.12'R.sub.12'-
').sub.q(OCH.sub.2CH.sub.2).sub.t,
--K(CR.sub.11R.sub.12).sub.p(OCO)(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q(O-
CH.sub.2CH.sub.2).sub.t,
--K(CR.sub.11R.sub.12).sub.p(OCNR.sub.7)(Aa).sub.r(CR.sub.12'R.sub.12'').-
sub.q(OCH.sub.2CH.sub.2).sub.t,
--K(CR.sub.11R.sub.12).sub.p(CO)(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q(OC-
H.sub.2CH.sub.2).sub.rQ,
--K(CR.sub.11R.sub.12).sub.p-phenyl-CO-(Aa).sub.r(CR.sub.12'R.sub.12'').s-
ub.q--,
--K(CR.sub.11R.sub.12).sub.p-furyl-CO(Aa).sub.t-(CR.sub.12'R.sub.1-
2'').sub.q,
--K(CR.sub.11R.sub.12).sub.p-oxazolyl-CO(Aa).sub.r(CR.sub.12R.sub.12'').s-
ub.q--,
--K(CR.sub.11R.sub.12).sub.p-thiazolyl-CO(Aa).sub.r-(CR.sub.12'R.s-
ub.12'').sub.q,
--K(CR.sub.11R.sub.12).sub.p-thienyl-CO(CR.sub.12'R.sub.12'').sub.q--,
--K(CR.sub.11R.sub.12).sub.p-imidazolyl-CO--(CR.sub.12'R.sub.12'').sub.q,
--K(CR.sub.11R.sub.12).sub.p-morpholino-CO(Aa).sub.t(CR.sub.12'R.sub.12''-
).sub.q--,
--K(CR.sub.11R.sub.12).sub.p-piperazino-CO-(Aa).sub.r(CR.sub.12-
'R.sub.12'').sub.q--,
--K(CR.sub.11R.sub.12).sub.p--N-methylpiperazin-CO(Aa).sub.r-(CR.sub.12'R-
.sub.12'').sub.q, --K(CR.sub.11R.sub.12).sub.p-(Aa).sub.r-phenyl-,
--K(CR.sub.11R.sub.12).sub.m-(Aa).sub.r-furyl-,
--K(CR.sub.11R.sub.12).sub.p-oxazolyl(Aa).sub.r-,
--K(CR.sub.11R.sub.12).sub.m-thiazolyl-(Aa).sub.r-,
--K(CR.sub.11R.sub.12).sub.p-thienyl- (Aa).sub.r,
--K(CR.sub.11R.sub.12).sub.p-imidazolyl(Aa).sub.r-,
--K((CR.sub.11R.sub.12).sub.m-morpholino-(Aa).sub.r,
--K(CR.sub.11R.sub.12).sub.p-piperazino-(Aa).sub.t-, and
--K(CR.sub.11R.sub.12).sub.mN-methylpiperazino-(Aa).sub.r, wherein
K is NR.sub.12, O, S, Se, B, or C.sub.3.about.C.sub.10 of Ar or
heterocyclic; wherein Aa, r, n, p, q, t, R.sub.7, R.sub.11,
R.sub.12, R.sub.12', R.sub.12'' are as defined in claim 1.
9. The compound according to claim 1 having the following formula
(II-1)-(II-91): ##STR00219## ##STR00220## ##STR00221## ##STR00222##
##STR00223## ##STR00224## ##STR00225## ##STR00226## ##STR00227##
##STR00228## ##STR00229## ##STR00230## ##STR00231## ##STR00232##
##STR00233## ##STR00234## ##STR00235## ##STR00236## ##STR00237##
##STR00238## ##STR00239## ##STR00240## ##STR00241## ##STR00242##
##STR00243## ##STR00244## ##STR00245## ##STR00246## ##STR00247##
wherein Aa, L, m, n, p, Q, r, R.sub.1, and R.sub.2, are described
the same as in claim 1; CBA is a cell-binding agent.
10. The compound of claim 1, wherein the cell binding agent is a
full-length antibody (polyclonal and monoclonal antibody); a single
chain antibody; a diabody, a triabody, a fragments of antibody
(Fab, Fab', F(ab').sub.2, F.sub.v, a fragment produced by a Fab
expression library, an anti-idiotypic (anti-Id) antibody, CDR's,
and an epitope-binding fragment of any of the above which
immuno-specifically bind to cancer cell antigens, viral antigens or
microbial antigens; interferon (type I, II, III); a peptide; a
lymphokine IL-2, IL-3, IL-4, IL-6, GM-CSF, interferon-gamma
(IFN-.gamma.); a hormone, insulin, TRH (thyrotropin releasing
hormone), MSH (melanocyte-stimulating hormone), a steroid hormone,
androgens, estrogens, melanocyte-stimulating hormone (MSH); a
growth factor and a colony-stimulating factor, an epidermal growth
factors (EGF), a granulocyte-macrophage colony-stimulating factor
(GM-CSF), a transforming growth factors (TGF), TGF.alpha.,
TGF.beta.; a insulin and insulin like growth factor (IGF-I, IGF-II)
G-CSF, M-CSF and GM-CSF; a vaccinia growth factor (VGF); a
fibroblast growth factor (FGFs); a smaller molecular weight
protein, poly-peptide, peptides and peptide hormones, bombesin,
gastrin, gastrin-releasing peptide; a platelet-derived growth
factor; an interleukin and a cytokine, interleukin-2 (IL-2),
interleukin-6 (IL-6), a leukemia inhibitory factor, a
granulocyte-macrophage colony-stimulating factor (GM-CSF); a
vitamin, folate; an apoprotein and a lycoproteis; transferrin; a
sugar-binding protein or a lipoproteins, a lectin; a cell
nutrient-transport molecule (transferrin); and a small molecular
inhibitor, prostate-specific membrane antigen (PSMA) inhibitor and
small molecular tyrosine kinase inhibitors (TKI); peptides, or
peptide analogs, proteins including conjugated proteins that are
able to bind targeted cells; a non-peptides or any other cell
binding molecule or substance in a form of bioactive polymer,
bioactive dendrimer, nanoparticle, liposome, or viral capside.
11. The compound according to claim 1, wherein L is one or more
linker components of 6-maleimidocaproyl ("MC"), maleimidopropanoyl
("MP"), valine-citrulline ("val-cit" or "vc"),
alanine-phenylalanine ("ala-phe" or "af"), glycine-glycine, a
nature peptides containing up to 6 the same or different natural
amino acids (dipeptide, tripeptide, tetrapeptide, pentapeptide,
hexapeptide), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl
4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl
4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("SMCC"),
N-Succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB"), ethyleneoxy
(--CH.sub.2CH.sub.2O--) as one or up to 100 repeating units ("EO"
or "PEO"), or one or more components that are illustrated below:
##STR00248## ##STR00249## ##STR00250## wherein R.sub.10 is defined
the same as in claim 1; R.sub.15, R.sub.16 and R.sub.17 are
independently selected from the group consisting of
--C.sub.1.about.C.sub.8 alkyl or alkylene-, --C.sub.1.about.C.sub.7
carbocyclo-, --O--(C.sub.1.about.C.sub.8 alkyl)-,
--NH--(C.sub.1.about.C.sub.8 alkyl)-, -arylene-,
--C.sub.1.about.C.sub.8 alkylene-arylene-, -arylene,
--C.sub.1.about.C.sub.8 alkylene-, --C.sub.1.about.C.sub.8
alkylene-(C.sub.1.about.C.sub.8 carbocyclo)-,
--(C.sub.3.about.C.sub.7 carbocyclo)-C.sub.1.about.C.sub.8
alkylene-, --C.sub.3.about.C.sub.8 heterocyclo-,
--C.sub.1.about.C.sub.8 alkylene-(C.sub.3.about.C.sub.8
heterocyclo)-, --(C.sub.3.about.C.sub.8
heterocyclo)-C.sub.1.about.C.sub.9 alkylene-,
--(CH.sub.2CH.sub.2O).sub.k--, --(CH(CH.sub.3)CH.sub.2O).sub.k--,
and --(CH.sub.2CH.sub.2O).sub.k--CH.sub.2--; k is an integer
ranging from 1-50; X''', Y''' and Z''' are independently NH, O or
S.
12. The compound according to claim 1, having any one of the
following specific conjugation structures of (III-1), (III-2),
(III-3), (III-4), (III-5), (III-6), (III-7), (III-8), (III-9),
(III-10), (III-11), or (III-12): wherein the bold structure is an
IgG antibody; "" represents either a single bond or a double bond;
L.sub.1 and L.sub.2 are, the same or different, independently
defined the same as L in claim 1; X.sub.1 and X.sub.2, are, the
same or different, independently NH, N(R.sub.1), O, S, CH.sub.2, or
Ar, wherein R.sub.1 is C.sub.1-C.sub.6 alkyl and Ar is an aromatic
or heteroaromatic ring; wherein Drug.sub.1 and Drug.sub.2 are the
same or different, independently a moiety linked to L other than Q
in Formula (I) of claim 1, or one of Drug.sub.1 and Drug.sub.2 is
the moiety linked to L other than Q in Formula (I) of claim 1, the
other of Drug.sub.1 and Drug.sub.2 is absent,
(OCH.sub.2CH.sub.2).sub.rOR.sub.10,
(OCH.sub.2CH(CH.sub.3)).sub.pOR.sub.10,
NH(CH.sub.2CH.sub.2O).sub.pR.sub.10,
NH(CH.sub.2CH(CH.sub.3)O).sub.pR.sub.10,
N[(CH.sub.2CH.sub.2O).sub.pR.sub.10][(CH.sub.2CH.sub.2O).sub.rR.sub.5],
(OCH.sub.2CH.sub.2).sub.pCOOR.sub.10, or
CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.pCOOR.sub.10, wherein p, r,
R.sub.10 are described the same as in claim 1, when both Drug.sub.2
and L.sub.2 are absent, X.sub.2 is NH.sub.2 or OH.
13. The compound according to claim 1, comprising an IgG antibody
linked to one of the following linkage structures of (IV-1),
(IV-2), (IV-3), (IV-4), (IV-5), and (IV-6): ##STR00251## wherein ""
represents either a single bond or a double bond; Drug is a moiety
linked to L other than Q in Formula (I) of claim 1; ##STR00252##
represents a site on an antibody; L, X, n and R.sub.12 are defined
the same as in claim 1.
14. A process for forming the compound according to claim 12
comprising a step of reducing a dithiol bond in an antibody with
one or more reducing agent selected from the group consisting of
dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH),
tris (2-carboxyethyl) phosphine (TCEP), 2-mercaptoethylamine
(.beta.-MEA), and beta mercaptoethanol (.beta.-ME, 2-ME), the
reducing agent being optionally loaded or covalently bonded to a
solid polymer or a solid particle, wherein the polymer or the
particle is selected from the group consisting of polyethene,
polyacrylate, silica, crossed-linked silica
(2-mercaptoethyl)silica, (aminoethyl)silica, (aminopropyl)silica),
polyethylene terephthalate, polyethylene glycol, polystyrene,
poly(isopropyl acrylate), dextrans (Sephadex, cross-linked
dextran), isopropylacrylamide butyl methacrylate copolymer, and
polysaccharide polymer (agarose, agar, agaropectin, Sepharose).
15. The compound according to claim 12, when one of Drug.sub.1 and
Drug.sub.2 is a moiety linked to L other than Q in Formula (I), the
other one of Drug.sub.1 and Drug.sub.2 is selected from the group
consisting of a protein, an antibody (a monoclonal or polyclonal
antibody, antibody dimers, antibody multimers, a bispecific or
trispecific antibody, a single chain antibody, an antibody fragment
that binds to the target cell), a chromophore molecule, a tubulysin
derivative, a maytansinoid, a taxanoid (taxane), a CC-1065 analog,
a daunorubicin or doxorubicin compound, a benzodiazepine dimer
(dimers of pyrrolobenzodiazepine (PBD), tomaymycin, anthramycin,
indolinobenzodiazepines, imidazobenzothiadiazepines, or
oxazolidinobenzodiazepines), a calicheamicin, a dolastatin or
auristatin derivative (monomethyl auristatin E, MMAE, MMAF,
auristatin PYE, auristatin TP, Auristatins 2-AQ, 6-AQ, EB (AEB),
EFP (AEFP)), a duocarmycin, a siRNA, and an enzyme.
16. A pharmaceutical composition comprising a therapeutically
effective amount of the compound of claim 1, and a pharmaceutically
acceptable excipient selected from the group consisting of one or
more components of 0.002%-1% of polysorbate (polysorbate 20,
polysorbate 40, polysorbate 60, or polysorbate 80), sodium lauryl
sulfate, triton X-100; 0.01%.about.10% of a binder (disaccharides:
sucrose, lactose, trehalose or maltose; or sugar alcohols: xylitol,
sorbitol or maltitol, or polyethylene glycol), and 0.01%.about.10%
of a pharmaceutical buffering agent (citrate, succinate acetate,
phosphate, or borate) at a pH of 4.5-9.5.
17. A method for inhibiting abnormal cell growth or treating a
proliferative disorder including cancers, benign or malignant
tumors; leukemia and lymphoid malignancies; neuronal, glial,
astrocytal, hypothalamic, glandular, macrophagal, epithelial,
stromal, blastocoelic, angiogenic and immunologic disorders;
inflammatory; autoimmune disorders; destructive disorders; bone
disorder; infectious disease; viral disease; fibrotic disease;
neurodegenerative disorder; pancreatitis or kidney disease; in a
mammal, comprising administering to the mammal a pharmaceutically
effective amount of the compound according to claim 1.
18. The pharmaceutical composition according to claim 16 further
comprising one or more synergistic drugs of a chemotherapeutic
agent, radiation therapy, immunotherapy agent, autoimmune disorder
agent, or anti-infectious agent.
19. The compound according to claim 1, wherein the cell binding
agent is an antibody, an antibody fragment, a diabody, a
tri(a)body, an epidermal growth factor (EGF), a prostate specific
membrane antigen (PSMA) inhibitor, a melanocyte stimulating hormone
(MSH), a thyroid stimulating hormone (TSH), a polyclonal antibody,
a somatostatin, a folate, a matriptase inhibitor, an estrogen, an
estrogen analogue, a designed ankyrin repeat proteins (DARPins), an
androgen, or an androgen analogue.
20. The compound according to claim 1, wherein Q targets cells
selected from the group consisting of tumor cells; virus infected
cells; microorganism infected cells; parasite infected cells;
autoimmune cells; activated cells; myeloid cells; activated
T-cells, B cells, or melanocytes; cells expressing antigen of CD3,
CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w,
CD14, CD15, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24,
CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35,
CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46,
CD47, CD48, CD49b, CD49c, CD51, CD52, CD53, CD54, CD55, CD56, CD58,
CD59, CD61, CD62E, CD62L, CD62P, CD63, CD66, CD68, CD69, CD70,
CD72, CD74, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD86, CD87,
CD88, CD89, CD90, CD91, CD95, CD96, CD98, CD100, CD103, CD105,
CD106, CD109, CD117, CD120, CD125, CD126, CD127, CD133, CD134,
CD135, CD137, CD138, CD141, CD142, CD143, CD144, CD147, CD151,
CD147, CD152, CD154, CD156, CD158, CD163, CD166, CD168, CD174,
CD180, CD184, CDw186, CD194, CD195, CD200, CD200a, CD200b, CD209,
CD221, CD227, CD235a, CD240, CD262, CD271, CD274, CD276 (B7-H3),
CD303, CD304, CD309, CD326, 4-1BB, SAC, 5T4 (Trophoblast
glycoprotein, TPBG, 5T4, Wnt-Activated Inhibitory Factor 1 or
WAIF1), Adenocarcinoma antigen, AGS-5, AGS-22M6, Activin
receptor-like kinase 1, AFP, AKAP-4, ALK, Alpha intergrin, Alpha v
beta6, Amino-peptidase N, Amyloid beta, Androgen receptor,
Angiopoietin 2, Angiopoietin 3, Annexin A1, Anthrax toxin
protective antigen, Anti-transferrin receptor, AOC3 (VAP-1), B7-H3,
Bacillus anthracis anthrax, BAFF (B-cell activating factor), BCMA,
B-lymphoma cell, bcr-abl, Bombesin, BORIS, C5, C242 antigen, CA125
(carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic
anhydrase 9), CALLA, CanAg, Canis lupus familiaris IL31, Carbonic
anhydrase IX, Cardiac myosin, CCL11 (C--C motif chemokine 11), CCR4
(C--C chemokine receptor type 4, CD194), CCR5, CD3E (epsilon), CEA
(Carcinoembryonic antigen), CEACAM3, CEACAM5 (carcino-embryonic
antigen), CFD (Factor D), Ch4D5, Cholecystokinin 2 (CCK2R), CLDN18
(Claudin-18), Clumping factor A, cMet, CRIPTO, FCSF1R (Colony
stimulating factor 1 receptor, CD115), CSF2 (colony stimulating
factor 2, Granulocyte-macrophage colony-stimulating factor
(GM-CSF)), CSP4, CTLA4 (cytotoxic T-lymphocyte-associated protein
4), CTAA16.88 tumor antigen, CXCR4 (CD184), C-X-C chemokine
receptor type 4, cyclic ADP ribose hydrolase, Cyclin B1, CYP1B1,
Cytomegalovirus, Cytomegalovirus glycoprotein B, Dabigatran, DLL4
(delta-like-ligand 4), DPP4 (Dipeptidyl-peptidase 4), DR5 (Death
receptor 5), E. coli shiga toxin type-1, E. coli shiga toxin
type-2, ED-B, EGFL7 (EGF-like domain-containing protein 7), EGFR,
EGFRII, EGFRvIII, Endoglin (CD105), Endothelin B receptor,
Endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2,
Episialin, ERBB2 (Epidermal Growth Factor Receptor 2), ERBB3, ERG
(TMPRSS2 ETS fusion gene), Escherichia coli, ETV6-AML, FAP
(Fibroblast activation protein alpha), FCGR1, alpha-Fetoprotein,
Fibrin II, beta chain, Fibronectin extra domain-B, FOLR (folate
receptor), Folate receptor alpha, Folate hydrolase, Fos-related
antigen 1F protein of respiratory syncytial virus, Frizzled
receptor, Fucosyl GM1, GD2 ganglioside, G-28 (a cell surface
antigen glyvolipid), GD3 idiotype, GloboH, Glypican 3,
N-glycolylneuraminic acid, GM3, GMCSF receptor .alpha.-chain,
Growth differentiation factor 8, GP100, GPNMB (Trans-membrane
glycoprotein NMB), GUCY2C (Guanylate cyclase 2C, guanylyl cyclase C
(GC-C), intestinal Guanylate cyclase, Guanylate cyclase-C receptor,
Heat-stable enterotoxin receptor (hSTAR)), Heat shock proteins,
Hemagglutinin, Hepatitis B surface antigen, Hepatitis B virus, HER1
(human epidermal growth factor receptor 1), HER2, HER2/neu, HER3
(ERBB-3), IgG4, HGF/SF (Hepatocyte growth factor/scatter factor),
HHGFR, HIV-1, Histone complex, HLA-DR (human leukocyte antigen),
HLA-DR10, HLA-DRB, HMWMAA, Human chorionic gonadotropin, HNGF,
Human scatter factor receptor kinase, HPV E6/E7, Hsp90, hTERT,
ICAM-1 (Intercellular Adhesion Molecule 1), Idiotype, IGF1R (IGF-1,
insulin-like growth factor 1 receptor), IGHE, IFN-.gamma.,
Influenza hemagglutinin, IgE, IgE Fc region, IGHE, IL-1, IL-2
receptor (interleukin 2 receptor), IL-4, IL-5, IL-6, IL-6R
(interleukin 6 receptor), IL-9, IL-10, IL-12, IL-13, IL-17, IL-17A,
IL-20, IL-22, IL-23, IL31RA, ILGF2 (Insulin-like growth factor 2),
Integrins (.alpha.4, .alpha..sub.IIb.beta..sub.3, .alpha.v.beta.3,
.alpha..sub.4.beta..sub.7, .alpha.5.beta.1, .alpha.6.beta.4,
.alpha.7.beta.7, .alpha.II.beta.3, .alpha.5.beta.5,
.alpha.v.beta.5), Interferon gamma-induced protein, ITGA2, ITGB2,
KIR2D, Kappa Ig, LCK, Le, Legumain, Lewis-Y antigen, LFA-1
(Lymphocyte function-associated antigen 1, CD11a), LHRH, LINGO-1,
Lipoteichoic acid, LIVIA, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1,
MAGE-2, MAGE-3, MAGE A1, MAGE A3, MAGE 4, MART1, MCP-1, MIF
(Macrophage migration inhibitory factor, or
glycosylation-inhibiting factor (GIF)), MS4A1 (membrane-spanning
4-domains subfamily A member 1), MSLN (mesothelin), MUC1 (Mucin 1,
cell surface associated (MUC1) or polymorphic epithelial mucin
(PEM)), MUC1-KLH, MUC16 (CA125), MCP1 (monocyte chemotactic protein
1), MelanA/MART1, ML-IAP, MPG, MS4A1 (membrane-spanning 4-domains
subfamily A), MYCN, Myelin-associated glycoprotein, Myostatin,
NA17, NARP-1, NCA-90 (granulocyte antigen), Nectin-4 (ASG-22ME),
NGF, Neural apoptosis-regulated proteinase 1, NOGO-A, Notch
receptor, Nucleolin, Neu oncogene product, NY-BR-1, NY-ESO-1,
OX-40, OxLDL (Oxidized low-density lipoprotein), OY-TES1, P21, p53
nonmutant, P97, Page4, PAP, Paratope of anti-(N-glycolylneuraminic
acid), PAX3, PAX5, PCSK9, PDCD1 (PD-1, Programmed cell death
protein 1,CD279), PDGF-R.alpha. (Alpha-type platelet-derived growth
factor receptor), PDGFR-.beta., PDL-1, PLAC1, PLAP-like testicular
alkaline phosphatase, Platelet-derived growth factor receptor beta,
Phosphate-sodium co-transporter, PMEL 17, Polysialic acid,
Proteinase3 (PR1), Prostatic carcinoma, PS (Phosphatidylserine),
Prostatic carcinoma cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA,
Rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD240),
Rhesus factor, RANKL, RhoC, Ras mutant, RGSS, ROBO4, Respiratory
syncytial virus, RON, ROR1, Sarcoma translocation breakpoints,
SART3, Sclerostin, SLAMF7 (SLAM family member 7), Selectin P, SDC1
(Syndecan 1), sLe(a), Somatomedin C, SIP (Sphingosine-1-phosphate),
Somatostatin, Sperm protein 17, SSX2, STEAP1 (six-transmembrane
epithelial antigen of the prostate 1), STEAP2, STn, TAG-72 (tumor
associated glycoprotein 72), Survivin, T-cell receptor, T cell
transmembrane protein, TEM1 (Tumor endothelial marker 1), TENB2,
Tenascin C (TN-C), TGF-.alpha., TGF-.beta. (Transforming growth
factor beta), TGF-.beta.1, TGF-.beta.2 (Transforming growth
factor-beta 2), Tie (CD202b), Tie2, TIM-1 (CDX-014), Tn, TNF,
TNF-.alpha., TNFRSF8, TNFRSF10B (tumor necrosis factor receptor
superfamily member 10B), TNFRSF-13B (tumor necrosis factor receptor
superfamily member 13B), TPBG (trophoblast glycoprotein), TRAIL-R1
(Tumor necrosis apoprosis Inducing ligand Receptor 1), TRAILR2
(Death receptor 5 (DR5)), tumor-associated calcium signal
transducer 2, tumor specific glycosylation of MUC1, TWEAK receptor,
TYRP1 (glycoprotein 75), TRP-2, Tyrosinase, VCAM-1 (CD106), VEGF,
VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, vimentin, WT1, XAGE 1,
cells expressing any insulin growth factor receptors, and epidermal
growth factor receptors.
21. A method for treatment of a cancer, autoimmune disorder,
infectious disease or viral disease in vitro, in vivo, or ex vivo,
comprising utilizing the pharmaceutical composition according to
claim 16.
22. The compound according to claim 1, wherein the self-immolative
linker component has one of the following structures: ##STR00253##
wherein the (*) atom is a point of attachment of additional spacer
or releasable linker unit, or a cytotoxic agent, or the cell
binding agent (CBA); X.sup.1, Y.sup.1, Z.sup.2 and Z.sup.3 are
independently NH, O, or S; Z.sup.1 is independently H, NH, O or S;
v is 0 or 1; Q.sup.1 is independently H, OH, C.sub.1.about.C.sub.6
alkyl, (OCH.sub.2CH.sub.2).sub.nF, Cl, Br, I, OR.sub.12, SR.sub.12,
NR.sub.12R.sub.12', N.dbd.NR.sub.12, N.dbd.R.sub.12,
NR.sub.12R.sub.12', NO.sub.2, SOR.sub.12R.sub.12',
SO.sub.2R.sub.12, SO.sub.3R.sub.12, OSO.sub.3R.sub.12,
PR.sub.12R.sub.12', POR.sub.12R.sub.12', PO.sub.2R.sub.12R.sub.12',
OPO(OR.sub.12)(OR.sub.12'), or OCH.sub.2PO(OR.sub.12(OR.sub.12'),
wherein R.sub.12 and R.sub.12' are as defined the same as in claim
1.
23. The compound according to claim 22, wherein R.sub.12 and
R.sub.12' are independently H, C.sub.1.about.C.sub.8 alkyl;
C.sub.2.about.C.sub.8 alkenyl, alkynyl, heteroalkyl;
C.sub.3.about.C.sub.8 aryl, heterocyclic, carbocyclic, cycloalkyl,
heterocycloalkyl, heteroaralkyl, alkylcarbonyl; or a pharmaceutical
cation salt thereof.
24. The compound according to claim 1, wherein Q is one of the
following formulas: ##STR00254## ##STR00255## ##STR00256##
##STR00257## wherein D is H, --NO.sub.2, SO.sub.3.sup.-, CN, or F;
Aa, r, p, q, m, and n are described the same as in claim 1; w and
w' are 0 or 1 independently; R.sub.1', R.sub.2', R.sub.3', and
R.sub.4' are independently H, CH.sub.3, C.sub.2H.sub.5,
C.sub.3H.sub.7, CH.sub.2OH, or CH.sub.2CH.sub.2OH.
25. The compound according to claim 6, wherein Q is H,
C.sub.1.about.C.sub.8 of alkyl, alkenyl, alkynyl, aryl, cyclic,
cyclohetero, haloalkyl, alkoxy, haloalkoxy alkylamino; halogen;
--NO.sub.2; --CN; --SH; --SSCH.sub.3; --SSAc; --SSAr;
--SS-Pyridine; --SS--Ar(--NO.sub.2); --S-cell binding agent; or a
function group of NHS ester, pentafluorophenyl ester;
alkyloxyamine; aldehyde; ketone; carboxyl acid; hydrazine; amine;
or thiolactone; or is linked the cell binding agent via Stretcher
units (Ww) or via Spacer units (Tt), wherein W, w, T, and t are
defined the same as in claim 6.
26. The compound according to claim 12, wherein the IgG antibody is
IgG1, IgG2, IgG3 or IgG4 antibody.
27. The compound according to claim 15, wherein the other one of
Drug.sub.1 and Drug.sub.2 is one of structures illustrated below:
##STR00258## ##STR00259## ##STR00260## ##STR00261## ##STR00262##
##STR00263## wherein R.sub.10 are described the same as in claim
15, and ##STR00264## is a site of link to either linker L.sub.1 or
linker L.sub.2.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel cytotoxic agents,
derivatives of Amanita toxins and their uses to specifically target
cell population by chemically linking these derivatives to a cell
binding agent.
BACKGROUND OF THE INVENTION
[0002] Antibody-drug conjugates (ADCs), which utilize the targeting
ability of monoclonal antibodies (mAbs) to deliver highly toxic
drugs to targeted tumor cells, have been proof that the products
are technically feasible, therapeutically effective, and able to
get regulatory approval after Seattle Genetics/Takeda's Brentuximab
vedotin (Adcetris) and ImmunoGen/Roche's Trastuzumab emtansine
(Kadcyla) are on the market (Sassoon, I. and Blanc, V. Laurent
Ducry (ed.), Antibody-Drug Conjugates, Methods in Molecular
Biology, vol. 1045, p 1-27). Currently there are more than 50 ADC
drugs in the clinic trials according to www.clinictrails.gov and
many big pharmas and biotech firms, as well as plenty of small
start-ups have a lot of incentives to invest in further development
of this technology. Indeed, the complexity of antibody-drug
conjugates with its four moving repertoires of cytotoxic agents,
linker technologies, antibody properties, and conjugation methods
makes this field incredibly challenging yet full of potential for
innovation (Perez, H. L., et al. Drug Discovery Today, 2014, 19(7),
869-81). The development from novel targeting ligands to new site
specific conjugation methods, from multiple payloads to changing
the drug-antibody ratio, from functional linkers to homogeneous
stoichiometry, are challenging the convention for the design and
development of next-generation ADCs (Zhao, R. et al,
PCT/IB2015/055051, PCT/IB2015/055264, introduction part). However,
the important key factor in generating powerful ADCs is the
cytotoxic agents (Bourchard, H., et al, Bioorg. Med. Chem. Lett.
2014, 24, 5357-63). One class of the highly potent cytotoxic agents
that can be used for ADC is the Amanitins (Moldenhauer, G. et al, J
Natl Cancer Inst 2012, 104 (8): 622-34; Flygare, J. A. et al, Chem.
Biol. Drug. Des. 2013, 81, 113-121; WO2010/115629, WO2010/115630,
WO2012041504, WO2012119787, WO2014/009025, WO2014135282, US pat
appl 20120213805, 20120100161, 20130259880, 20140294865,
20150218220, EP2416805, EP1661584, EP1859811, EP2497499,
EP2774624), which are highly toxic components of Amanita toxins
(Vetter, J., Toxicon 1998, 36 (1): 13-24).
[0003] Amanita toxins are mainly cyclic octa- and heptapeptide
toxins of Amanita mushrooms, which contain an unusual Trp-Cys
crossbridge known as tryptathionine (Walton, J. D. et al.
Biopolymers 2010; 94(5): 659-64). The major components of amanita
toxins are amatoxins, phallotoxins, and virotoxins (Wieland, T.,
Faulstich, H., CRC Crit. Rev. Biochem. 1978, 5(3):185-260; Vetter,
J., Toxicon 1998, 36 (1): 13-24; Weiland, T., and Faulstich, H.
1983. Peptide Toxins from Amanita. p. 585-635. In: Handbook of
Natural Toxins, Volume I: Plant and Fungal Toxins. R. F. Keeler and
A. T. Tu, Ed. Marcel Dekker, Inc. New York, N.Y., Wieland, T., Int
J. Pept. Protein Res., 1983, 22, 257-76) and their collectively
known structures are listed in Tables 1, 2 and 3 respectively
below.
TABLE-US-00001 TABLE 1 Ten currently known structures of amatoxins
##STR00002## Name R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5
.alpha.-Amanitin OH OH NH.sub.2 OH OH .beta.-Amanitin OH OH OH OH
OH .gamma.-Amanitin H OH NH.sub.2 OH OH -Amanitin H OH OH OH OH
Amanullin H H NH.sub.2 OH OH Amanullinic acid H H OH OH OH
Amaninamide OH OH NH.sub.2 H OH Amanin OH OH OH H OH Proamanullin H
H H NH.sub.2 OH H
(The .delta.-Amanitin has been reported, but its chemical structure
has not been determined)
TABLE-US-00002 TABLE 2 Structures of the Phallotoxins ##STR00003##
Name R.sub.1' R.sub.2' R.sub.3' R.sub.4' R.sub.5' phalloidin OH H
CH.sub.3 CH.sub.3 OH phalloin H H CH.sub.3 CH.sub.3 OH prophallin H
H CH.sub.3 CH.sub.3 H phallisin OH OH CH.sub.3 CH.sub.3 OH
phallacin H H CH(CH.sub.3).sub.2 COOH OH phallacidin OH H
CH(CH.sub.3).sub.2 COOH OH phallisacin OH OH CH(CH.sub.3).sub.2
COOH OH
TABLE-US-00003 TABLE 3 Structures of the Virotoxins ##STR00004##
Name R.sub.l'' R.sub.2'' X Viroidin CH.sub.3 CH(CH.sub.3).sub.2
S(O.sub.2) Desoxiviroidin CH.sub.3 CH(CH.sub.3).sub.2 S(O)
(Ala)viroidin CH.sub.3 CH.sub.3 S(O.sub.2) (Ala)desoxiviroidin
CH.sub.3 CH.sub.3 S(O) (Ala)viroisin CH.sub.2OH CH(CH.sub.3).sub.2
S(O.sub.2) Desoxiviroisin CH.sub.2OH CH(CH.sub.3).sub.2 S(O)
[0004] Amatoxins which are a subgroup of at least ten toxic
compounds originally found in several genera of poisonous
mushrooms, most notably Amanita phalloides and several other
mushroom species, are bicyclic octapeptides containing
intra-annular tryptathionine crossbridge that is oxidatively formed
from tryptophan and cysteine (Kaya, E., et al, Toxicon 2013, 76,
225-33).
[0005] Amatoxins are potent and selective inhibitors of RNA
polymerase II (Pol II), a vital enzyme in the synthesis of
messenger RNA (mRNA), microRNA, and small nuclear RNA (snRNA)
(Karlson-Stiber C, Persson H. 2003 "Cytotoxic fungi--an overview",
Toxicon 42 (4): 339-49). Thus Amatoxins kill cells by shutting down
gene transcription and protein biosynthesis (Brodner, O. G. and
Wieland, T. 1976 Biochem.,15(16): 3480-4; Fiume, L., Curr Probl
Clin Biochem, 1977, 7: 23-8; Karlson-Stiber C, Persson H. 2003,
Toxicon 42(4): 339-49; Chafin, D. R., Guo, H. & Price, D. H.
1995 J. Biol. Chem. 270 (32): 19114-19; Wieland (1983) Int. J.
Pept. Protein Res. 22(3): 257-76). So far ten known amatoxins,
named .alpha.-Amanitin, .beta.-Amanitin, .gamma.-Amanitin,
.epsilon.-Amanitin, Amanullin, Amanullinic acid, Amaninamide,
Amanin, and Proamanullin are synthesized as 35-amino-acid
proproteins, from which the final eight amino acids are cleaved by
a prolyl oligopeptidase (Litten, W. 1975 Scientific American 232
(3): 90-101; H. E. Hallen, et al 2007 Proc. Nat. Aca. Sci. USA 104,
19097-101; K. Baumann, et al, 1993 Biochemistry 32 (15): 4043-50;
Karlson-Stiber C, Persson H. 2003, Toxicon 42 (4): 339-49; Horgen,
P. A. et al. 1978 Arch. Microbio. 118 (3): 317-9). Amatoxins can be
produced from collected Amanita phalloides mushrooms (Yocum, R. R.
1978 Biochemistry 17(18): 3786-9; Zhang, P. et al, 2005, FEMS
Microbiol. Lett. 252(2), 223-8), or from fermentation using a
basidiomycete (Muraoka, S. and Shinozawa T., 2000 J. Biosci.
Bioeng. 89(1): 73-6, US pat. Appl 20100267019) or from fermentation
using A. fissa (Guo, X. W., et al, 2006 Wei Sheng Wu Xue Bao 46(3):
373-8), or from culturing Galerina fasciculata or Galerina
helvoliceps (WO/1990/009799, JP11137291). However the yields from
these isolation and fermentation were quite low (less than 5 mg/L
culture). Several semi-chemical or synthetic preparations of
amatoxins and their analogs have been reported in the past three
decades (W. E. Savige, A. Fontana, Chem. Commun. 1976, 600-1;
Zanotti, G., et al, Int J Pept Protein Res, 1981. 18(2): 162-8;
Wieland, T., et al, Eur. J. Biochem. 1981, 117, 161-4; P. A.
Bartlett, et al, Tetrahedron Lett. 1982, 23, 619-22; Zanotti, G.,
et al., Biochim Biophys Acta, 1986. 870 (3): 454-62; Zanotti, G.,
et al., Int. J. Peptide Protein Res. 1987, 30, 323-9; Zanotti, G.,
et al., Int. J. Peptide Protein Res. 1987, 30, 450-9; Zanotti, G.,
et al., Int J Pept Protein Res, 1988. 32(1): 9-20; G. Zanotti, T.
et al, Int. J. Peptide Protein Res. 1989, 34, 222-8; Zanotti, G.,
et al., Int J Pept Protein Res, 1990. 35(3): 263-70; Mullersman, J.
E. and J. F. Preston, 3rd, Int J Pept Protein Res, 1991. 37(6):
544-51; Mullersman, J. E., et al, Int J Pept Protein Res, 1991.
38(5): 409-16; Zanotti, G., et al, Int J Pept Protein Res, 1992.
40(6): 551-8; Schmitt, W. et al, J. Am. Chem. Soc. 1996, 118,
4380-7; Anderson, M. O., et al, J. Org. Chem., 2005, 70(12):
4578-84; J. P. May, et al, J. Org. Chem. 2005, 70, 8424-30; F.
Brueckner, P. Cramer, Nat. Struct. Mol. Biol. 2008, 15, 811-8; J.
P. May, D. M. Perrin, Chem. Eur. J. 2008, 14, 3404-9; J. P. May, et
al, Chem. Eur. J. 2008, 14, 3410-17; Q. Wang, et al, Eur. J. Org.
Chem. 2002, 834-9; May, J. P. and D. M. Perrin, Biopolymers, 2007.
88(5): 714-24; May, J. P., et al., Chemistry, 2008. 14(11): 3410-7;
S. De Lamo Marin, et al, Eur. J. Org. Chem. 2010, 3985-9; Pousse,
G., et al., Org Lett, 2010. 12(16): 3582-5; Luo, H., et al., Chem
Biol, 2014. 21(12): 1610-7; Zhao, L., et al., Chembiochem, 2015.
16(10): 1420-5). Because of their extreme potency and unique
mechanism of cytotoxicity, amatoxins have been used as payloads for
conjugations (Fiume, L., Lancet, 1969. 2 (7625): 853-4;
Barbanti-Brodano, G. and L. Fiume, Nat New Biol, 1973. 243(130):
281-3; Bonetti, E., M. et al, Arch Toxicol, 1976. 35(1): p. 69-73;
Davis, M. T., Preston, J. F. Science 1981, 213, 1385-1388; Preston,
J. F., et al, Arch Biochem Biophys, 1981. 209(1): 63-71; H.
Faulstich, et al, Biochemistry 1981, 20, 6498-504; Barak, L. S., et
al., Proc Natl Acad Sci USA, 1981. 78(5): 3034-8; Faulstich, H. and
L. Fiume, Methods Enzymol, 1985. 112: 225-37; Zhelev, Z., A. et al,
Toxicon, 1987. 25(9): 981-7; Khalacheva, K., et al, Eksp Med
Morfol, 1990. 29(3): 26-30; U. Bermbach, H. Faulstich, Biochemistry
1990, 29, 6839-45; Mullersman, J. E. and J. F. Preston, Int. J.
Peptide Protein Res. 1991, 37, 544-51; Mullersman, J. E. and J. F.
Preston, Biochem Cell Biol, 1991. 69(7): 418-27; J. Anderl, H.
Echner, H. Faulstich, Beilstein J. Org. Chem. 2012, 8, 2072-84;
Moldenhauer, G., et al, J. Natl. Cancer Inst. 2012, 104, 622-34; A.
Moshnikova, et al; Biochemistry 2013, 52, 1171-8; Zhao, L., et al.,
Chembiochem, 2015. 16(10): 1420-5; Zhou, B., et al., Biosens
Bioelectron, 2015. 68: 189-96; WO2014/043403, US20150218220, EP
1661584).
[0006] The phallotoxins which are bicyclic heptapeptides, consist
of at least seven compounds as shown in Table 2, were originally
discovered from the death cap mushroom (Amanita phalloides) in 1937
by Feodor Lynen, Heinrich Wieland's student and son-in-law, and
Ulrich Wieland of the University of Munich (Enjalbert, F., et al,
Toxicon 1993, 31, 803-7). The phallotoxins, after i.p.
administration, inhibit the conversion of actin-F into actin-G and
disturb the dynamic equilibrium of these forms necessary for cell
functions (Enjalbert, F., et al., C. R. Acad. Sci. Paris, Sciences
de la vie/Life Sciences, 1999, 322, 855-62; Wieland, T., 50 Jahre
Phalloidin, Naturwissenchaften 1987, 74, 367-73). There are six
known structures of phallotoxins namely prophalloin, phalloin,
phallisin, phallacidin, phallacin and phallisacin as shown in Table
2 (Yocum R. R., Simons M., Lloydia 1977, 40, 178-90; Enjalbert, F.,
et al., C. R. Acad. Sci. Paris, Sciences de la vie/Life Sciences,
1999, 322, 855-62; Schafer A. J., Faulstich H., Anal. Biochem.
1977, 83, 720-723). Though highly toxic to liver cells,
phallotoxins have since been found to have little contribution to
the death cap's toxicity because they are not absorbed through the
gut. On average, the six structure known phallotoxins are about
5-10 times less toxic than (.alpha.-, .beta.-, .gamma.-, or
.epsilon.-) amanitins (Vetter, J., Toxicon, 1998, 36, 13-24).
Furthermore, phalloidin is also found in the edible Blusher
(Amanita rubescens) (Litten, W., Scientific American 1975, 232,
90-101).
[0007] Virotoxins are monocyclic heptapeptides formed by at least
five different compounds: alaviroidin, viroisin, deoxoviroisin,
viroidin, and deoxoviroidin (Vetter, J., Toxicon, 1998, 36, 13-24)
as shown in Table 3. The structure and biological activity of
virotoxins are similar to those of phallotoxins, thus suggesting
that virotoxins are biosynthetically derived from phallotoxins or
share common precursor pathways (Wieland, T., Int J Pept Protein
Res, 1983, 22, 257-76). However, differing from phallotoxins,
virotoxins are monocyclic peptides and contain D-serine instead of
L-cysteine, as well as have two unnatural amino acids:
2,3-trans-3,4-dihydroxy-L-proline and
2'-(methylsulfonyl)-L-tryptophan (Buku, A. et al, Proc Natl Acad
Sci USA. 1980, 77(5), 2370-1). NMR studies revealed that
D-configuration of serine is the structural element that maintains
the phalloidin-like structure, while the hydroxy group does not
contribute to conformational stability but is likely to be in
contact with the actin surface (Zanotti, G., et al, Biochemistry.
1999, 38(33):10723-9). On the molecular level, the virotoxins
behave similarly to the phallotoxins, e.g. the affinity of viroisin
is very similar to that of phalloidin, with an apparent equilibrium
dissociation constant K.sub.d approximately 2.times.10.sup.-8 M
(Faulstich, H., et al, Biochemistry, 1980, 19, 3334-43). However,
the flexibility of the monocyclic structure and the presence of two
additional hydroxy groups in the virotoxins suggest a different
mode of interaction with actin. While there is proof that the
bicyclic phallotoxins possess a rigid binding site, the virotoxins
may adopt the biologically active conformation by an induced-fit
mechanism upon contacting with actin (Faulstich, H., et al,
Biochemistry, 1980, 19, 3334-43).
[0008] There are several methods disclosed so far in conjugation of
amatoxins. Preston and his collegues used diazotization of
p-aminobenzoylglycylglycine and then coupling the linker to
.alpha.-amanitin at the 7'C position of Trptophan (Preston, J. F.
et al, Arch. Biochem. Biophy. 1981, 209, 63-71; Davis, M-T. B. and
Preston, J. F., Science, 1981, 213, 1385-7; Hencin, R. S. and
Preston, J. F., Mol. Pharm., 1979, 16, 961-9). Heidelberg Pharma
GMBH (WO2010/115629, WO2010115630, WO2012/041504, US20120100161,
US20120213805) disclosed the conjugation of amatoxin via the oxygen
atom as an ether linkage to the 6'C-atom of amino acid 4, or via
the oxygen atom as an ester or a carbomate linkage to the
.delta.C-atom of amino acid 3, or via the nitrogen atom as an amide
linkage to the .gamma.C-atom of amino acid 1. Heidelberg Pharma
GMBH (EP2774624, WO2012119787, WO2014/135282, US20140294865,
US20160002298) also disclosed the conjugation via position of
1'N-atom of indole of amatoxin. Agensys Inc (US20150218220)
disclosed that through activation of the C7'-postion of the indole
group of .alpha.-amanitin with a reagent such as iodine, followed
by coupling with a suitably substituted 2.degree.-amino reagent to
introduce a diamine spacer linkage, or introduce a carbon spacer
(C--C bond of the C7-position of the indole) in front of a
2.degree.-amino linkage in the presence of formaldehyde.
[0009] It is known that amatoxins are relatively non-toxic when
coupled to large biomolecule carriers, such as antibody molecules,
and that they exert their cytotoxic activity only after the
biomolecule carrier is cleaved off In light of the toxicity of
amatoxins, particularly their notorious toxicity for liver cells
(Zhou, P. et al, World J. Gastroenterology, 2012, 18(5), 435-4;
Giannini, A., et al, Clin. Toxicology, 2007. 45(5), 539-42), it is
of outmost importance that amatoxin conjugates for targeted tumour
therapy remain highly stable after administration in plasma, and
that the released the amatoxin would not run away from the target
cells or damage liver cells when occurs after internalization in
the target cells.
[0010] Here we disclose novel amanita toxin derivatives that first
can be conjugated cell-binding molecule through stable amide bonds
after simple nitrition, following by reduction and conjugation at
the indole unit of the amanita toxin derivatives. Second, many of
the amanita toxin derivatives of this invention have prodrug units
on the indole units which can bring down the highly potency in
vitro, but can be slowly transformed back the highly potent
cytotoxicity in vivo after their extra prodrug units are removed by
enzymes. Since amatoxins are notorious for their extremely toxic to
hepatitis with centrolobular necrosis and hepatic steatosis, as
well as acute tubulointerstitial nephropathy, which altogether
induce a severe hepatorenal syndrome (Litten, W. 1975 Scientific
American 232: 90-10; Karlson-Stiber C, Persson H. 2003 Toxicon 42
(4): 339-49). The slow conversion of the safer prodrugs to their
mother toxins would minimize the severe side toxicity when the
toxins are off-targets during the delivery. Thus these improvements
of the conjugatable amanita toxin derivatives may have drastic
consequences of wider therapeutic index windows and much more
safety of the amatoxin conjugates for targeted therapeutic
applications.
SUMMARY OF THE INVENTION
[0011] The first embodiment of this invention is to disclose potent
cytotoxic agents, specifically, derivatives of amatoxins,
phallotoxins or virotoxins which can be effectively used to block
cell proliferation. In particular, this invention is to disclose
novel amanita toxin derivatives, optionally linkable or linked to a
cell binding agent to block cell proliferation. The novel cytotoxic
agents and their conjugates to a cell binding agent of this
invention are illustrated in the following formula (I):
##STR00005##
or their pharmaceutically acceptable salts, hydrates, or hydrated
salts; or the polymorphic crystalline structures of these
compounds; or their optical isomers, racemates, diastereomers or
enantiomers.
[0012] Wherein
[0013] - - - represents a single bond that links any carbon
position of the aromatic (indole) ring;
[0014] represents an optional single bond or an absent bond.
[0015] R.sub.1 and R.sub.2 are independently selected from H, OH,
CH.sub.2OH, CH(OH)CH.sub.2OH, CH(CH.sub.3)CH.sub.2OH,
CH(OH)CH.sub.3, C.sub.1-C.sub.8 alkyl, --OR.sub.12 (ether),
C.sub.2-C.sub.8 alkenyl, alkynyl, heteroalkyl, --OCOR.sub.12
(ester), --OC(.dbd.O)OR.sub.12-(carbonate), --OC(.dbd.O)NHR.sub.12
(carbamate); C.sub.3-C.sub.8 aryl, heterocyclic, carbocyclic,
cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl.
[0016] R.sub.3 and R.sub.4 are independently selected from H, OH,
--OR.sub.12 (ether), --OCOR.sub.12 (ester), --OCOCH.sub.3
(acetate), --OCOOR.sub.12 (carbonate), --OC(.dbd.O)NHR.sub.12
(carbamate), --OP(O)(OR.sub.12)(OR.sub.12') (phosphate),
OP(O)(NHR.sub.12)(NHR.sub.12') (phosphamide), O--SO.sub.3.sup.-, or
O-glycoside;
[0017] R.sub.5 is selected from H, OH, NH.sub.2, NHOH, NHNH.sub.2,
--OR.sub.12, --NHR.sub.12, NHNHR.sub.12, --NR.sub.12R.sub.12',
N(H)(R.sub.12)R.sub.13CO(Aa).sub.p, (an amino acid or peptide,
wherein Aa is an amino acid or a polypeptide, p represents
0-6);
[0018] R.sub.6 is selected from H, OH, CH.sub.2OH,
CH(OH)CH.sub.2OH, CH(CH.sub.2OH).sub.2, CH(CH.sub.3)OH,
CH.sub.2CH.sub.2OH, PrOH, BuOH, C.sub.1.about.C.sub.8 alkyl,
--OR.sub.12 (ether), C.sub.2.about.C.sub.8 alkenyl, alkynyl,
heteroalkyl, --OCOR.sub.12 (ester); C.sub.3.about.C.sub.8 aryl,
heterocyclic, or carbocyclic.
[0019] R.sub.7, R.sub.8 and R.sub.9 are independently selected from
H, OH, CH.sub.3, CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2CH.sub.3,
CH.sub.2OH, CH(OH)CH.sub.2OH, CH.sub.2CH(OH)CH.sub.2OH,
CH(CH.sub.2OH).sub.2, CH.sub.2C(OH)(CH.sub.2OH).sub.2,
CH.sub.2C(OH)(CH.sub.3)(CH.sub.2OH),
CH.sub.2C(OH)(CH(CH.sub.3).sub.2)(CH.sub.2OH), CH.sub.2CH.sub.2OH,
PrOH, BuOH, CH.sub.2COOH, CH.sub.2CH.sub.2COOH, CH(OH)COOH,
CH.sub.2CONH.sub.2, CH.sub.2CH.sub.2CONH.sub.2,
CH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2,
CH.sub.2CH.sub.2CH.sub.2NHC(.dbd.NH)NH.sub.2, C.sub.1.about.C.sub.8
alkyl, CH.sub.2Ar, CH.sub.2SH, CH.sub.2SR.sub.12,
CH.sub.2SSR.sub.12, CH.sub.2SSAr, CH.sub.2CH.sub.2SCH.sub.3,
--OR.sub.12 (ether), C.sub.2.about.C.sub.8 alkenyl, alkynyl,
heteroalkyl, --OCOR.sub.12 (ester); C.sub.3.about.C.sub.8 aryl,
heterocyclic, or carbocyclic.
[0020] R.sub.10 is selected from H, NH.sub.2, OH, SH, NO.sub.2,
halogen, --NHOH, --N.sub.3 (azido); --CN (cyano);
C.sub.1.about.C.sub.8 alkyl, C.sub.2.about.C.sub.8 alkenyl,
alkynyl, heteroalkyl; C.sub.3.about.C.sub.8 aryl, heterocyclic, or
carbocyclic; --OR.sub.12 (ether), --OCOR.sub.12 (ester),
--OCOCH.sub.3 (acetate), --OC(O)OR.sub.12 (carbonate),
--OC(O)CH(R.sub.12)NHAa (Aa is an aminoacid group),
--NR.sub.12R.sub.12'(amine), --NR.sub.12COR.sub.12'(amine),
--NR.sub.12NR.sub.12'NR.sub.12'' (amine);
--OCONR.sub.12R.sub.12'(carbamate);
--NR.sub.12(C.dbd.NH)NR.sub.12'R.sub.12'' (guanidinum);
--NR.sub.12CO(Aa).sub.p, (an amino acid or peptide, wherein Aa is
an amino acid or a polypeptide, p represents 0-6);
--N(R.sub.12)CONR.sub.12'R.sub.12'' (urea); --OCSNHR.sub.12
(thiocarbamate); --SH (thiol); --SR.sub.12 (sulfide);
--S(O)R.sub.12 (sulfoxide); --S(O.sub.2)R.sub.12 (sulfone);
--SO.sub.3, HSO.sub.3, HSO.sub.2, or a salt of HSO.sub.3.sup.-,
SO.sub.3.sup.2- or --HSO.sub.2.sup.- (sulphite); --OSO.sub.3.sup.-;
--N(R.sub.12)SOOR.sub.12' (sulfonamide); H.sub.2S.sub.2O.sub.5 or a
salt of S.sub.2O.sub.5.sup.2- (metabisulfite); PO.sub.3SH.sub.3,
PO.sub.2S.sub.2H.sub.2, POS.sub.3H.sub.2, PS.sub.4H.sub.2 or a salt
of PO.sub.3S.sup.3-, PO.sub.2S.sub.2.sup.3-, POS.sub.3.sup.3-,
PS.sub.4.sup.3- (mono-, di-, tri-, and tetra-thiophosphate);
(R.sub.12O).sub.2POSR.sub.12' (thiophosphate ester);
HS.sub.2O.sub.3 or a salt of S.sub.2O.sub.3.sup.2-(thiosulfate);
HS.sub.2O.sub.4 or a salt of S.sub.2O.sub.4.sup.2- (dithionite);
(P(.dbd.S)(OR.sub.12)(S)(OH) or a salt formed with a cation
(phosphorodithioate); --N(R.sub.12)OR.sub.12' (hydroxylamine
derivative); R.sub.12C(.dbd.O)NOH or a salt formed with a cation
(hydroxamic acid); (HOCH.sub.2SO.sub.2.sup.-, or its salts
(formaldehyde sulfoxylate); --N(R.sub.12)COR.sub.12' (amide);
R.sub.12R.sub.12'R.sub.12''NPO.sub.3H (trialkylphosphor-amidate or
phosphoramidic acid); or ArAr'Ar''NPO.sub.3H (triarylphosphonium);
OP(O)(OM.sub.1)(OM.sub.2), OCH.sub.2OP(O)(OM.sub.1)(OM.sub.2),
OSO.sub.3M.sub.1; O-glycoside (glucoside, galactoside, mannoside,
glucuronoside, alloside, fructoside, etc), NH-glycoside,
S-glycoside or CH.sub.2-glycoside; M.sub.1 and M.sub.2 are
independently H, Na, K, Ca, Mg, NH.sub.4,
NR.sub.1'R.sub.2'R.sub.3'; R.sub.1', R.sub.2' and R.sub.3' are
independently H, C.sub.1.about.C.sub.8 alkyl; Ar, Ar', and Ar'' are
C.sub.3-C.sub.8 aryl or heteroaromatic group.
[0021] R.sub.11 is H, C.sub.1.about.C.sub.8 alkyl;
C.sub.2.about.C.sub.8 alkenyl, alkynyl, heteroalkyl;
C.sub.3.about.C.sub.8 aryl, heteraoaryl.
[0022] R.sub.12, R.sub.12', and R.sub.12'' are independently
selected from H, C.sub.1.about.C.sub.8 alkyl; C.sub.2.about.C.sub.8
alkenyl, alkynyl, heteroalkyl; C.sub.3.about.C.sub.8 aryl,
heteraoaryl, heterocyclic, or carbocyclic.
[0023] X is S, O, NH, SO, SO.sub.2, or CH.sub.2.
[0024] m is 0 or 1; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20.
[0025] L is a linker or a linker-cell-binding molecule (Q)
covalently bound cluster, or a linker which has a functional group
on the linker that enables linkage with a cell-binding agent (CBA).
L is preferably a releasable linker, which has the formula of:
-Ww-(Aa)r-Tt-; or -Ww-(Aa)r-Tt-Q; or Q-Ww-(Aa)r-Tt-; wherein W is a
Stretcher unit; w is 0 or 1; Aa is an Amino Acid unit comprising
independent amino acids; r is an integer ranging from 0 to 100. The
Stretcher unit W may contain a self-immolative or a
non-self-immolative component, peptidyl units, a hydrazone bond, a
disulfide, an ester, an oxime, an amide, or a thioether bond. The
self-immolative unit includes, but is not limited to, aromatic
compounds that are electronically similar to the
para-aminobenzylcarbamoyl (PAB) groups such as
2-aminoimidazol-5-methanol derivatives, heterocyclic PAB analogs,
beta-glucuronide, and ortho or para-aminobenzylacetals. Preferably,
the self-immolative linker component has one of the following
structures:
##STR00006##
wherein the (*) atom is the point of attachment of additional
spacer or releasable linker units, or the cytotoxic agent, and/or
the binding molecule (CBA); X.sup.1, Y.sup.1, Z.sup.2 and Z.sup.3
are independently NH, O, or S; Z.sup.1 is independently H, NH, O or
S; v is 0 or 1; Q.sup.1 is independently H, OH,
C.sub.1.about.C.sub.6 alkyl, (OCH.sub.2CH.sub.2).sub.n, F, Cl, Br,
I, OR.sub.12, SR.sub.12, NR.sub.12R.sub.12', N.dbd.NR.sub.12,
N.dbd.R.sub.12, NR.sub.12R.sub.12', NO.sub.2, SOR.sub.12R.sub.12',
SO.sub.2R.sub.12, SO.sub.3R.sub.12, OSO.sub.3R.sub.12,
PR.sub.12R.sub.12', POR.sub.12R.sub.12', PO.sub.2R.sub.12R.sub.12',
OPO(OR.sub.12)(OR.sub.12'), or OCH.sub.2PO(OR.sub.12(OR.sub.12')
wherein R.sub.12 and R.sub.12' are as defined above; preferably
R.sub.12 and R.sub.12' are independently selected from H,
C.sub.1.about.C.sub.8 alkyl; C.sub.2.about.C.sub.8 alkenyl,
alkynyl, heteroalkyl; C.sub.3.about.C.sub.8 aryl, heterocyclic,
carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl,
alkylcarbonyl; or pharmaceutical cation salts.
[0026] The non-self-immolative linker component is one of the
following structures:
##STR00007## ##STR00008## ##STR00009## ##STR00010##
##STR00011##
Wherein the (*) atom is the point of attachment of additional
spacer or releasable linkers, the cytotoxic agents, and/or the
binding molecules; X.sup.1, Y.sup.1, Q.sup.1, R.sub.12, R.sub.12'
are defined as above; r is 0.about.100; p and q are 0.about.6
independently.
[0027] Spacer (T) is a linear, branched or cyclic alkyl, alkenyl,
alkynyl or aryl having from 1 to 10 carbon atoms, or T can be a
polyethylene glycol (--CH.sub.2CH.sub.2O--) spacer; t is 0, or
1.about.100. T can also undergo cyclization upon amide bond
hydrolysis, such amides include substituted and unsubstituted
4-aminobutyric acid amides, appropriately substituted bicycle
[2.2.1] and bicyclo[2.2.2] ring systems, and 2-aminophenylpropionic
acid amides.
[0028] Q is a cell-binding agent/molecule (CBA), or a functional
group that enables linkage with a cell-binding agent, or a
functional group that enables linkage with a linker attached on a
cell-binding agent. The function group is chosen from a thiol, an
amine, a hydrazine, an alkoxylamino, a disulfide substituent, a
maleimido, a haloacetyl group, a carboxy acid, an N-hydroxy
succinimide ester, a ketone, an ester, an aldehyde, an alkynyl, an
alkenyl, or a protected thiol or disulfide group, such as SAc,
SSR.sub.1 or SSAr. Ar is an aromatic group or hetero aromatic
group.
[0029] In a further aspect, the present invention discloses a
therapeutic composition comprising: (1). an effective amount of one
or more derivatives of amanita toxins optionally linkable or linked
to a cell binding agent, and (2). a pharmaceutically acceptable
carrier, diluent, or excipient, of Formula (I) of the patent
application, to kill target cells or tissues containing target
cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 shows the synthesis of dipeptide Tr-Hpi-Gly-OH.
[0031] FIG. 2 shows the solution phase synthesis of
Ile.sup.3-S-deoxo-amanitin_1.
[0032] FIG. 3 shows the solution phase synthesis of
Ile.sup.3-S-deoxo-amanitin_2.
[0033] FIG. 4 shows the solid phase synthesis of
Ile.sup.3-S-deoxo-amanitin precursor.
[0034] FIG. 5 shows the synthesis of Ile.sup.3-S-deoxo-amanitin
derivatives_1.
[0035] FIG. 6 shows the synthesis of linker_1 for the synthesis of
amanita toxins.
[0036] FIG. 7 shows the synthesis of Ile.sup.3-S-deoxo-amanitin
derivatives and conjugates_2.
[0037] FIG. 8 shows the synthesis of linker_2 for the synthesis of
amanita toxins.
[0038] FIG. 9 shows the synthesis of Ile.sup.3-S-deoxo-amanitin
derivatives and conjugates_3.
[0039] FIG. 10 shows the synthesis of linker_3 for the synthesis of
amanita toxins.
[0040] FIG. 11 shows the synthesis of Ile.sup.3-S-deoxo-amanitin
derivatives and conjugates_4.
[0041] FIG. 12 shows the synthesis of Synthesis of linker_4 of
amanita toxins.
[0042] FIG. 13 shows the synthesis of Ile.sup.3-S-deoxo-amanitin
derivatives and conjugates_5
[0043] FIG. 14 shows the synthesis of Ile.sup.3-S-deoxo-amanitin
derivatives and conjugates_6.
[0044] FIG. 15 shows the synthesis of linker_5 of amanita
toxins.
[0045] FIG. 16 shows the synthesis of Ile.sup.3-S-deoxo-amanitin
derivatives and conjugates_7.
[0046] FIG. 17 shows the synthesis of Ile.sup.3-S-deoxo-amanitin
derivatives and conjugates_8.
[0047] FIG. 18 shows the synthesis of linker_6 for amanita
toxins.
[0048] FIG. 19 shows the synthesis of Ile.sup.3-S-deoxo-amanitin
derivatives and conjugates_9.
[0049] FIG. 20 shows the synthesis of Ile.sup.3-S-deoxo-amanitin
derivatives and conjugates_10.
[0050] FIG. 21 shows the synthesis of conjugates of amanitin
derivatives_1.
[0051] FIG. 22 shows the synthesis of conjugates of amanitin
derivatives_2.
[0052] FIG. 23 shows the synthesis of conjugates of amanitin
derivatives_3.
[0053] FIG. 24 shows the synthesis of conjugates of amanitin
derivatives_4.
[0054] FIG. 25 shows the synthesis of conjugates of phalloidin
derivatives_1.
[0055] FIG. 26 shows the synthesis Solution phase synthesis of
amatoxin and derivatives.
[0056] FIG. 27 shows the solution phase synthesis of phallotoxin
and derivatives.
[0057] FIG. 28 shows the solution phase synthesis of virotoxin and
derivatives.
[0058] FIG. 29 shows the comparison of the anti-tumor effect of
conjugate compounds A1, A2, A3, A5, A6, A7, A9, and A10 with T-DM1
using human gastric tumor N87 cell model at dosing of 6 mg/kg,
i.v., one injection. It shows all the conjugates did not cause the
animal body weight loss
[0059] FIG. 30 shows the comparison of the anti-tumor effect of
conjugate compounds A1, A2, A3, A5, A6, A7, A9, and A10 with T-DM1
using human gastric tumor N87 cell model at dosing of 6 mg/kg,
i.v., one injection. The animals at control group were sacrificed
at day 45. All the 8 conjugate compounds (A1, A2, A3, A5, A6, A7,
A9, and A10) had better in vivo activity than T-DM1.
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
[0060] "Alkyl" means an aliphatic hydrocarbon group which may be
straight or branched having 1 to 8 carbon atoms in the chain or
cyclic. "Branched" means that one or much lower alkyl groups such
as methyl, ethyl or propyl are attached to a linear alkyl chain.
Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl,
n-butyl, t-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl,
cyclopentyl, cyclohexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl,
2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl,
2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl,
2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2
4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl,
isoheptyl, n-octyl, and isooctyl. A C.sub.1-C.sub.8 alkyl group can
be unsubstituted or substituted with one or more groups including,
but not limited to, --C.sub.1.about.C.sub.8 alkyl,
--O--(C.sub.1.about.C.sub.8 alkyl), -aryl, --C(O)R', --OC(O)R',
--C(O)OR', --C(O)NH.sub.2, --C(O)NHR', --C(O)N(R').sub.2--NHC(O)R',
--S(O).sub.2R', --S(O)R', --OH, -halogen (F, Cl, Br or I),
--N.sub.3, --NH.sub.2, --NH(R'), --N(R').sub.2 and --CN; where each
R' is independently selected from --C.sub.1.about.C.sub.8 alkyl and
aryl.
[0061] A "cyclic alkyl", "cycloalkyl" and "C.sub.3.about.C.sub.8
carbocycle" can be used interchangeably. They mean a 3-, 4-, 5-,
6-, 7- or 8-membered saturated or unsaturated non-aromatic
carbocyclic ring. Representative C.sub.3.about.C.sub.8 carbocycles
include, but are not limited to, cyclopropyl, cyclobutyl,
cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl,
1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl,
1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, and
cyclooctadienyl. A C.sub.3.about.C.sub.8 carbocycle group can be
unsubstituted or substituted with one or more groups including, but
not limited to, --C.sub.1.about.C.sub.8 alkyl, --OR.sub.5, -aryl,
--C(O)R.sub.5, --OC(O)R.sub.5, --C(O)OR.sub.5, --C(O)NH.sub.2,
--C(O)NHR.sub.5, --C(O)NR.sub.5R.sub.5'--NHC(O)R.sub.5,
--S(O).sub.2R.sub.5, --S(O)R.sub.5, --OH, -halogen, --N.sub.3,
--NH.sub.2, --NHR.sub.5, --NR.sub.5R.sub.5' and --CN; wherein
R.sub.5 and R.sub.5' are independently H; C.sub.1.about.C.sub.8 of
alkyl, alkenyl, alkynyl, heteroalkyl, aryl, arylalkyl, or
carbonylalkyl; or pharmaceutical salts. R.sub.5 and R.sub.5' can
further be substituted with at least one substituent selected from
--N(R.sub.5)(R.sub.5'), --CO.sub.2H, --SO.sub.3H, --OR.sub.5,
--CO.sub.2R.sub.5, --CONR.sub.5, and --PO.sub.3H.
[0062] A "C.sub.3.about.C.sub.8 carbocyclo" refers to a
C.sub.3.about.C.sub.8 carbocycle group defined above wherein one of
hydrogen atoms on the carbocycle is replaced with a bond.
[0063] Alkenyl refers to an aliphatic hydrocarbon group containing
a carbon-carbon double bond and which may be straight or branched
having 2 to 8 carbon atoms in the chain. The alkenyl double bond
may have "cis" and "trans" orientations, or alternatively, "E" and
"Z" orientations. Exemplary alkenyl groups include, but are not
limited to, ethylenyl or vinyl, propenyl or allyl, n-butenyl,
i-butenyl, 3-methylbut-2-enyl, n-pentenyl, hexylenyl, heptenyl,
octenyl.
[0064] "Alkynyl" or "alkinyl" means an aliphatic hydrocarbon group
containing a carbon-carbon triple bond and which may be straight or
branched having 2 to 8 carbon atoms in the chain. Exemplary alkynyl
groups include ethynyl, propynyl, n-butynyl, 2-butynyl,
3-methylbutynyl, pentynyl, n-pentynyl, hexylynyl, heptynyl, and
octynyl.
[0065] "Heteroalkyl" is C.sub.2.about.C.sub.8 alkyl in which one to
four carbon atoms are independently replaced with a heteroatom from
the group consisting of O, S and N.
[0066] "Heterocycle" refers to an aromatic or non-aromatic
C.sub.3.about.C.sub.14 carbocycle in which one to four of the ring
carbon atoms are independently replaced with a heteroatom from the
group of O, N, P, S and Se. Preferable heteroatoms are oxygen,
nitrogen and sulphur. Suitable heterocyclics are also disclosed in
The Handbook of Chemistry and Physics, 76.sup.th Edition, CRC
Press, Inc., 1995-1996, p 2-25 to 2-26, the disclosure of which is
hereby incorporated by reference. Preferred non aromatic
heterocyclic include, but are not limited to pyrrolidinyl,
pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl,
dioxolanyl, tetrahydro-pyranyl, dioxanyl, dioxolanyl, piperidyl,
piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl,
pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl,
thiomorpholinyl, dihydro-pyranyl, tetrahydropyranyl,
dihydropyranyl, tetrahydro-pyridyl, dihydropyridyl,
tetrahydropyrinidinyl, dihydrothiopyranyl, azepanyl, as well as the
fused systems resulting from the condensation with a phenyl
group.
[0067] "Aryl" or Ar refers to an aromatic or hetero aromatic group,
composed of one or several rings, comprising three to fourteen
carbon atoms, preferentially six to ten carbon atoms. The term of
hetero aromatic group refers one or several carbon on aromatic
group, preferentially one, two, three or four carbon atoms are
replaced by O, N, Si, Se, P or S, preferentially by O, S, N. The
term aryl or Ar also refers to a aromatic group, wherein one or
several H atoms are replaced independently by alkyl, F, Cl, Br, I,
OR.sub.5, or SR.sub.5, NR.sub.5R.sub.5', N.dbd.NR.sub.5,
N.dbd.R.sub.5, NR.sub.5R.sub.5', NO.sub.2, SOR.sub.5R.sub.5,
SO.sub.2R.sub.5, SO.sub.3R.sub.5, OSO.sub.3R.sub.5,
PR.sub.5R.sub.5', POR.sub.5R.sub.5', PO.sub.5R.sub.5',
OPO.sub.3R.sub.5R.sub.5', or PO.sub.3R.sub.5R.sub.5' wherein
R.sub.5 and R.sub.5' are independently H, alkyl, alkenyl, alkinyl,
heteroalkyl, aryl, arylalkyl, carbonyl, or pharmaceutical
salts.
[0068] The term "heteroaryl" or aromatic heterocycles refers to a 5
to 14, preferably 5 to 10 membered aromatic hetero, mono-, bi- or
multi cyclic ring. Examples include pyrrolyl, pyridyl, pyrazolyl,
thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl,
purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl,
benzofuranyl, 1,2,4-thiadiazolyl, isothiazolyl, triazoyl,
tetrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl,
carbazolyl, benzimidazolyl, isoxazolyl, pyridyl-N-oxide, as well as
the fused systems resulting from the condensation with a phenyl
group.
[0069] "Alkyl", "cycloalkyl", "alkenyl", "alkynyl", "aryl",
"heteroaryl", "heterocyclic" and the like refer also to the
corresponding "alkylene", "cycloalkylene", "alkenylene",
"alkynylene", "arylene", "heteroarylene", "heterocyclene" and the
likes which are formed by the removal of two hydrogen atoms.
[0070] "Halogen atom" refers to fluorine, chlorine, bromine or
iodine atom; preferably bromine and chlorine atom.
[0071] "Pharmaceutically" or "pharmaceutically acceptable" refer to
molecular entities and compositions that do not produce an adverse,
allergic or other untoward reaction when administered to an animal,
or a human, as appropriate.
[0072] "Pharmaceutically acceptable excipient" includes any
carriers, diluents, adjuvants, or vehicles, such as preserving or
antioxidant agents, fillers, disintegrating agents, wetting agents,
emulsifying agents, suspending agents, solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents and the like. The use of such media and
agents for pharmaceutical active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with the active ingredient, its use in the therapeutic
compositions is contemplated. Supplementary active ingredients can
also be incorporated into the compositions as suitable therapeutic
combinations.
[0073] As used herein, "pharmaceutical salts" refer to derivatives
of the disclosed compounds wherein the parent compound is modified
by making acid or base salts thereof. The pharmaceutically
acceptable salts include the conventional non-toxic salts or the
quaternary ammonium salts of the parent compound formed, for
example, from non-toxic inorganic or organic acids. For example,
such conventional non-toxic salts include those derived from
inorganic acids such as hydrochloric, hydrobromic, sulfuric,
sulfamic, phosphoric, nitric and the like; and the salts prepared
from organic acids such as acetic, propionic, succinic, tartaric,
citric, methanesulfonic, benzenesulfonic, glucoronic, glutamic,
benzoic, salicylic, toluenesulfonic, oxalic, fumaric, maleic,
lactic and the like. Further addition salts include ammonium salts
such as tromethamine, triethanolamine, meglumine, epolamine, etc.,
metal salts such as sodium, potassium, calcium, zinc or
magnesium.
[0074] The pharmaceutical salts of the present invention can be
synthesized from the parent compound which contains a basic or
acidic moiety by conventional chemical methods. Generally, such
salts can be prepared by reaction of the free acid or base forms of
these compounds with a stoichiometric amount of the appropriate
base or acid in water or in an organic solvent, or in a mixture of
the two. Generally, non-aqueous media like ether, ethyl acetate,
ethanol, isopropanol, or acetonitrile are preferred. Lists of
suitable salts are found in Remington's Pharmaceutical Sciences,
17.sup.th ed., Mack Publishing Company, Easton, Pa., 1985, p 1418,
the disclosure of which is hereby incorporated by reference.
[0075] The term "compound", "cytotoxic agent", "cytotoxic
compound," "cytotoxic dimer" and "cytotoxic dimer compound" are
used interchangeably. They are intended to include compounds for
which a structure or formula or any derivative of thereof has been
disclosed in the present invention or a structure or formula or any
derivative thereof that has been incorporated by reference. The
term also includes, stereoisomers, geometric isomers, tautomers,
solvates, metabolites, salts (e.g., pharmaceutically acceptable
salts) and prodrugs, and prodrug salts of a compound of all the
formulae disclosed in the present invention. The term also includes
any solvates, hydrates, and polymorphs of any of the foregoing. The
specific recitation of "stereoisomers," "geometric isomers,"
"tautomers," "solvates," "metabolites," "salt" "prodrug," "prodrug
salt," "conjugates," "conjugates salt," "solvate," "hydrate," or
"polymorph" in certain aspects of the invention described in this
application shall not be interpreted as an intended omission of
these forms in other aspects of the invention where the term
"compound" is used without recitation of these other forms.
[0076] "Cell binding agents" or "Cell binding molecules" may be of
any kind presently known, or those become known, and include
peptides and non-peptides. Generally, these can be antibodies
(especially monoclonal antibodies) or a fragment of an antibody
that contains at least one binding site, lymphokines, hormones,
growth factors, nutrient-transport molecules (such as transferrin),
or any other cell binding molecule or substance (such as
vitamins).
[0077] More specific examples of cell binding agents that can be
used include:
[0078] monoclonal antibodies (mAb);
[0079] single chain antibodies;
[0080] fragments of antibodies such as Fab, Fab', F(ab').sub.2,
F.sub.v, (Parham, J. Immunol. 131, 2895-2902 (1983); Spring et al,
J. Immunol. 113, 470-478 (1974); Nisonoff et al, Arch. Biochem.
Biophys. 89, 230-244 (1960)), fragments produced by a Fab
expression library, anti-idiotypic (anti-Id) antibodies, CDR's, and
epitope-binding fragments of any of the above which
immunospecifically bind to cancer cell antigens, viral antigens or
microbial antigens.
[0081] interferons;
[0082] peptides; or conjugated proteins or peptides;
[0083] lymphokines such as IL-2, IL-3, IL-4, IL-5, IL-6;
[0084] hormones such as insulin, TRH (thyrotropin releasing
hormones), MSH (melanocyte-stimulating hormone), steroid hormones,
such as androgens and estrogens;
[0085] growth factors and colony-stimulating factors such as EGF,
TGF.alpha., insulin like growth factor (IGF-I, IGF-II) G-CSF, M-CSF
and GM-CSF (Burgess, Immunology Today 5, 155-158 (1984)); vitamins,
such as folate and
[0086] transferrin (O'Keefe et al, J. Biol. Chem. 260, 932-937
(1985)).
[0087] Monoclonal antibodies (mAb), mAb single chain or fragments
can be produced in the well known state of art technology. The
technology permits the production of extremely selective cell
binding agents in the form of specific monoclonal antibodies. The
well known in the art are techniques for creating monoclonal
antibodies produced by immunizing mice, rats, hamsters or any other
mammal with the antigen of interest such as the intact target cell,
antigens isolated from the target cell, whole virus, attenuated
whole virus, and viral proteins such as viral coat proteins.
[0088] Selection of appropriate cell binding agents is a matter of
choice that depends upon the particular cell population that is to
be targeted, but in general monoclonal antibodies are preferred if
an appropriate one is available.
[0089] For example, an anti-CD20 antigen monoclonal antibody, known
as Rituximab is a chimeric (mouse/human) monoclonal antibody and it
was the first therapeutic antibody approved by the United States
Food and Drug Administration for treatment of relapsed or
refractory low-grade or follicular NHL (Leonard, J. P. et al.,
Clin. Canc. Res. 10:5327-5334 (2004)). Another anti-CD20 antibody,
known as Ofatumumab, is a human monoclonal antibody targeting an
epitope different from that of rituximab and most other CD20
directed antibodies. It was approved by US FDA for treating chronic
lymphocytic leukemia and has also shown potential in treating
Follicular non-Hodgkin's lymphoma, Diffuse large B cell lymphoma,
rheumatoid arthritis and relapsing remitting multiple sclerosis
(Coiffier, B. et al Blood 111: 1094-100 (2008); Zhang, B. MAbs 1
(4): 326-31 (2009)). A third-generation, humanized and
glyco-engineered anti-CD20 mAb for the treatment of B-cell lymphoid
malignancies named Afutuzumab (now called obinutuzumab) has been
developed (Robak, T (2009) Current opinion in investigational drugs
(London, England: 2000) 10 (6): 588-96). Obinutuzumab is fully
humanized IgG1 type II anti-CD20 antibody and it selectivity binds
to the extracellular domain of the human CD20 antigen on malignant
human B cells. Similarly, an anti-CD19 antigen monoclonal antibody
B4 is a murine IgG.sub.1, that binds to the CD19 antigen on B cells
(Nadler et al, 131 J. Immunol. 244-250 (1983)) and can be used if
the target cells are B cells or diseased cells that express CD19
antigen such as in non-Hodgkin's lymphoma or chronic lymphoblastic
leukemia. In addition, the anti-CD22 antibodies that include RFB4
(Mansfield, E. et al., Blood 90:2020-2026 (1997)), CMC-544
(DiJoseph, J. F., Blood 103:1807-1814 (2004)) and LL2
(Pawlak-Byczkowska, E. J. et al., Cancer Res. 49:4568-4577 (1989))
can be used as potential therapies for B cell cancers and other B
cell proliferative diseases. The LL2 antibody (formerly called
HPB-2) is an IgG2a mouse monoclonal antibody directed against the
CD22 antigen (Pawlak-Byczkowska, E. J. et al. (1989), supra).
Furthermore, the anti CD33 antigen monoclonal antibody, named.
Gemtuzumab was first monoclonal antibody conjugated with a
cytotoxic drug to treat acute myelogenous leukemia (AML) (P. F.
Bross et al Clin Cancer Res 7 (6): 1490-6). A similar anti CD33
antigen antibody, named My9-6 is a murine IgG.sub.1 antibody that
binds specifically to the CD33 Antigen (J. D. Griffin et al 8
Leukemia Res., 521 (1984)) and can be used to target cells express
CD33 as in the disease of acute myelogenous leukemia (AML).
Additionally, GM-CSF antibody which binds to myeloid cells can be
used as a cell binding agent to diseased cells from acute
myelogenous leukemia. IL-2 antibody, which binds to activated
T-cells, can be used for prevention of transplant graft rejection,
for therapy and prevention of graft-versus-host disease, and for
the treatment of acute T-cell leukemia. MSH antibody, which binds
to melanocytes, can be used for the treatment of melanoma.
Novel Cytotoxic Agents and Their Conjugation of the Invention.
[0090] The novel amanita toxin derivatives according to the present
invention comprises one or more derivatives of amatoxins,
phallotoxins or virotoxins, optionally linkable or linked to a cell
binding agent via a linking group. The linking group is part of a
chemical moiety that is covalently bound to amanita toxin
derivatives through conventional methods.
[0091] The amanita toxin derivatives disclosed in the present
invention have the formula (I) shown below:
##STR00012##
or their pharmaceutically acceptable salts, hydrates, or hydrated
salts; or the polymorphic crystalline structures of these
compounds; or their optical isomers, racemates, diastereomers or
enantiomers.
[0092] Wherein
[0093] - - - represents a single bond that links any carbon
position of the aromatic (indole) ring;
[0094] represents an optional single bond or an absent bond.
[0095] R.sub.1 and R.sub.2 are independently selected from H, OH,
CH.sub.2OH, CH(OH)CH.sub.2OH, CH(CH.sub.3)CH.sub.2OH,
CH(OH)CH.sub.3, C.sub.1-C.sub.8 alkyl, --OR.sub.12 (ether),
C.sub.2-C.sub.8 alkenyl, alkynyl, heteroalkyl, --OCOR.sub.12
(ester), --OC(.dbd.O)OR.sub.12-(carbonate),
--OC(.dbd.O)NHR.sub.12(carbamate); C.sub.3-C.sub.8 aryl,
heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl,
heteroaralkyl, alkylcarbonyl.
[0096] R.sub.3 and R.sub.4 are independently selected from H, OH,
--OR.sub.12 (ether), --OCOR.sub.12 (ester), --OCOCH.sub.3
(acetate), --OCOOR.sub.12 (carbonate),
--OC(.dbd.O)NHR.sub.12(carbamate), --OP(O)(OR.sub.12)(OR.sub.12')
(phosphate), OP(O)(NHR.sub.12)(NHR.sub.12') (phosphamide),
O--SO.sub.3.sup.-, or O-glycoside.
[0097] R.sub.5 is selected from H, OH, NH.sub.2, --OR.sub.12,
--NHR.sub.12, --NR.sub.12R.sub.12',
N(H)(R.sub.12)R.sub.13CO(Aa).sub.p, (an amino acid or peptide,
wherein Aa is an amino acid or a polypeptide, p represents
0-6).
[0098] R.sub.6 is selected from H, OH, CH.sub.2OH,
CH(OH)CH.sub.2OH, CH(CH.sub.2OH).sub.2, CH(CH.sub.3)OH,
CH.sub.2CH.sub.2OH, PrOH, BuOH, C.sub.1.about.C.sub.8 alkyl,
--OR.sub.12 (ether), C.sub.2.about.C.sub.8 alkenyl, alkynyl,
heteroalkyl, --OCOR.sub.12 (ester); C.sub.3.about.C.sub.8 aryl,
heterocyclic, or carbocyclic.
[0099] R.sub.7, R.sub.8 and R.sub.9 are independently selected from
H, OH, CH.sub.3, CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2CH.sub.3,
CH.sub.2OH, CH(OH)CH.sub.2OH, CH.sub.2CH(OH)CH.sub.2OH,
CH(CH.sub.2OH).sub.2, CH.sub.2C(OH)(CH.sub.2OH).sub.2,
CH.sub.2C(OH)(CH.sub.3)(CH.sub.2OH),
CH.sub.2C(OH)(CH(CH.sub.3)2)(CH.sub.2OH), CH.sub.2CH.sub.2OH, PrOH,
BuOH, CH.sub.2COOH, CH.sub.2CH.sub.2COOH, CH(OH)COOH,
CH.sub.2CONH.sub.2, CH.sub.2CH.sub.2CONH.sub.2,
CH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2,
CH.sub.2CH.sub.2CH.sub.2NHC(.dbd.NH)NH.sub.2, C.sub.1.about.C.sub.8
alkyl, CH.sub.2Ar, CH.sub.2SH, CH.sub.2SR.sub.12,
CH.sub.2SSR.sub.12, CH.sub.2SSAr, CH.sub.2CH.sub.2SCH.sub.3,
--OR.sub.12 (ether), C.sub.2.about.C.sub.8 alkenyl, alkynyl,
heteroalkyl, --OCOR.sub.12 (ester); C.sub.3.about.C.sub.8 aryl,
heterocyclic, or carbocyclic.
[0100] R.sub.10 is selected from H, NH.sub.2, OH, SH, NO.sub.2,
halogen, --NHOH, --N.sub.3 (azido); --CN (cyano);
C.sub.1.about.C.sub.8 alkyl, C.sub.2.about.C.sub.8 alkenyl,
alkynyl, heteroalkyl; C.sub.3.about.C.sub.8 aryl, heterocyclic, or
carbocyclic; --OR.sub.12 (ether), --OCOR.sub.12 (ester),
--OCOCH.sub.3 (acetate), --OC(O)OR.sub.12 (carbonate),
--OC(O)CH(R.sub.12)NHAa (Aa is an aminoacid group),
--NR.sub.12R.sub.12'(amine), --NR.sub.12COR.sub.12'(amine),
--NR.sub.12NR.sub.12'NR.sub.12'' (amine);
--OCONR.sub.12R.sub.12'(carbamate);
--NR.sub.12(C.dbd.NH)NR.sub.12'R.sub.12'' (guanidinum);
--NR.sub.12CO(Aa).sub.p, (an amino acid or peptide, wherein Aa is
an amino acid or a polypeptide, p represents 0-6);
--N(R.sub.12)CONR.sub.12'R.sub.12'' (urea); --OCSNHR.sub.12
(thiocarbamate); --SH (thiol); --SR.sub.12 (sulfide);
--S(O)R.sub.12 (sulfoxide); --S(O.sub.2)R.sub.12 (sulfone);
--SO.sub.3, HSO.sub.3, HSO.sub.2, or a salt of HSO.sub.3.sup.-,
SO.sub.3.sup.2- or --HSO.sub.2.sup.- (sulphite); --OSO.sub.3.sup.-;
--N(R.sub.12)SOOR.sub.12' (sulfonamide); H.sub.2S.sub.2O.sub.5 or a
salt of S.sub.2O.sub.5.sup.2- (metabisulfite); PO.sub.3SH.sub.3,
PO.sub.2S.sub.2H.sub.2, POS.sub.3H.sub.2, PS.sub.4H.sub.2 or a salt
of PO.sub.3S.sup.3-, PO.sub.2S.sub.2.sup.3-, POS.sub.3.sup.3-,
PS.sub.4.sup.3- (mono-, di-, tri-, and tetra-thiophosphate);
(R.sub.12O).sub.2POSR.sub.12' (thiophosphate ester);
HS.sub.2O.sub.3 or a salt of S.sub.2O.sub.3.sup.2-(thiosulfate);
HS.sub.2O.sub.4 or a salt of S.sub.2O.sub.4.sup.2- (dithionite);
(P(.dbd.S)(OR.sub.12)(S)(OH) or a salt formed with a cation
(phosphorodithioate); --N(R.sub.12)OR.sub.12' (hydroxylamine
derivative); R.sub.12C(.dbd.O)NOH or a salt formed with a cation
(hydroxamic acid); (HOCH.sub.2SO.sub.2.sup.-, or its salts
(formaldehyde sulfoxylate); --N(R.sub.12)COR.sub.12' (amide);
R.sub.12R.sub.12'R.sub.12''NPO.sub.3H (trialkylphosphor-amidate or
phosphoramidic acid); or ArAr'Ar''NPO.sub.3H (triarylphosphonium);
OP(O)(OM.sub.1)(OM.sub.2), OCH.sub.2OP(O)(OM.sub.1)(OM.sub.2),
OSO.sub.3M.sub.1; O-glycoside (glucoside, galactoside, mannoside,
glucuronoside, alloside, fructoside, etc), NH-glycoside,
S-glycoside or CH.sub.2-glycoside; M.sub.1 and M.sub.2 are
independently H, Na, K, Ca, Mg, NH.sub.4,
NR.sub.1'R.sub.2'R.sub.3'; R.sub.1', R.sub.2' and R.sub.3' are
independently H, C.sub.1.about.C.sub.8 alkyl; Ar, Ar', and Ar'' are
C.sub.3-C.sub.8 aryl or heteroaromatic group.
[0101] R.sub.11 is H, C.sub.1.about.C.sub.8 alkyl;
C.sub.2.about.C.sub.8 alkenyl, alkynyl, heteroalkyl;
C.sub.3.about.C.sub.8 aryl, heteraoaryl.
[0102] R.sub.12, R.sub.12', and R.sub.12'' are independently
selected from H, C.sub.1.about.C.sub.8 alkyl; C.sub.2.about.C.sub.8
alkenyl, alkynyl, heteroalkyl; C.sub.3.about.C.sub.8 aryl,
heteraoaryl, heterocyclic, or carbocyclic.
[0103] X is S, O, NH, SO, SO.sub.2, or CH.sub.2.
[0104] m is 0 or 1; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20.
[0105] L is a linker or a linker-cell-binding molecule (Q)
covalently bound cluster, or a linker which has a functional group
on the linker that enables linkage with a cell-binding agent (CBA).
L is preferably a releasable linker, which has the formula of:
-Ww-(Aa)r-Tt-; or -Ww-(Aa)r-Tt-Q; or Q-Ww-(Aa)r-Tt-; wherein W is a
Stretcher unit; w is 0 or 1; Aa is an Amino Acid unit comprising
independent amino acids; r is an integer ranging from 0 to 100. The
Stretcher unit W may contain a self-immolative or a
non-self-immolative component, peptidyl units, a hydrazone bond, a
disulfide, an ester, an oxime, an amide, or a thioether bond. The
self-immolative unit includes, but is not limited to, aromatic
compounds that are electronically similar to the
para-aminobenzylcarbamoyl (PAB) groups such as
2-aminoimidazol-5-methanol derivatives, heterocyclic PAB analogs,
beta-glucuronide, and ortho or para-aminobenzylacetals. Preferably,
the self-immolative linker component has one of the following
structures:
##STR00013##
wherein the (*) atom is the point of attachment of additional
spacer or releasable linker units, or the cytotoxic agent, and/or
the binding molecule (CBA); X.sup.1, Y.sup.1, Z.sup.2 and Z.sup.3
are independently NH, O, or S; Z.sup.1 is independently H, NH, O or
S; v is 0 or 1; Q.sup.1 is independently H, OH,
C.sub.1.about.C.sub.6 alkyl, (OCH.sub.2CH.sub.2).sub.n, F, Cl, Br,
I, OR.sub.12, SR.sub.12, NR.sub.12R.sub.12', N.dbd.NR.sub.12,
N.dbd.R.sub.12, NR.sub.12R.sub.12', NO.sub.2, SOR.sub.12R.sub.12',
SO.sub.2R.sub.12, SO.sub.3R.sub.12, OSO.sub.3R.sub.12,
PR.sub.12R.sub.12', POR.sub.12R.sub.12', PO.sub.2R.sub.12R.sub.12',
OPO(OR.sub.12)(OR.sub.12'), or OCH.sub.2PO(OR.sub.12(OR.sub.12')
wherein R.sub.12 and R.sub.12' are as defined above; preferably
R.sub.12 and R.sub.12' are independently selected from H,
C.sub.1.about.C.sub.8 alkyl; C.sub.2.about.C.sub.8 alkenyl,
alkynyl, heteroalkyl; C.sub.3.about.C.sub.8 aryl, heterocyclic,
carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl,
alkylcarbonyl; or pharmaceutical cation salts.
[0106] The non-self-immolative linker component is one of the
following structures:
##STR00014## ##STR00015## ##STR00016## ##STR00017##
Wherein the (*) atom is the point of attachment of additional
spacer or releasable linkers, the cytotoxic agents, and/or the
binding molecules; X.sup.1, Y.sup.1, Q.sup.1, R.sub.12, R.sub.12'
are defined as above; r is 0.about.100; p and q are 0.about.6
independently.
[0107] Spacer (T) is a linear, branched or cyclic alkyl, alkenyl,
alkynyl or aryl having from 1 to 10 carbon atoms, or T can be a
polyethylene glycol (--CH.sub.2CH.sub.2O--) spacer; t is 0, or
1.about.100. T can also undergo cyclization upon amide bond
hydrolysis, such amides include substituted and unsubstituted
4-aminobutyric acid amides, appropriately substituted bicycle
[2.2.1] and bicycle [2.2.2] ring systems, and
2-aminophenylpropionic acid amides.
[0108] The linker L can be also selected from the group consisting
of: R.sub.12, OR.sub.12, OR.sub.12O, NHR.sub.12, NHR.sub.12NH,
NR.sub.11R.sub.12, SR.sub.12S, OR.sub.12NH, OR.sub.12Ar,
NHR.sub.12Ar, NR.sub.11R.sub.12NR.sub.12'R.sub.12'',
--(CR.sub.11R.sub.12).sub.p-(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q(OCH.su-
b.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(CR.sub.12'R.sub.12'').sub.q(Aa).sub.r(OCH.sub-
.2CH.sub.2).sub.t--,
-(Aa).sub.r-(CR.sub.11R.sub.12).sub.p(CR.sub.12'R.sub.12'').sub.q--(OCH.s-
ub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(CR.sub.12'R.sub.12'').sub.n(OCH.sub.2CH.sub.2-
).sub.t(Aa).sub.r-, --(CR.sub.11R.sub.12).sub.p(CH.dbd.CH)
(CR.sub.12'R.sub.12'').sub.q(Aa).sub.r(OCH.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(NR.sub.12'CO)(Aa).sub.r(CR.sub.12'R.sub.12'')-
.sub.q--(OCH.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(Aa).sub.t(NHCO)(CR.sub.12'R.sub.12'').sub.q---
(OCH.sub.2CH.sub.2).sub.r--,
(CR.sub.11R.sub.12).sub.p(OCO)(Aa).sub.r-(CR.sub.12'R.sub.12'').sub.q--(O-
CH.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(OCNR.sub.7)(Aa).sub.r(CR.sub.12'R.sub.12'').s-
ub.q(OCH.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p--(CO)-(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q(-
OCH.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(NR.sub.11CO)(Aa).sub.r(CR.sub.12'R.sub.12'').-
sub.q(OCH.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p--(OCO)(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q--
-(OCH.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(OCNR.sub.7)(Aa).sub.r(CR.sub.12'R.sub.12'').s-
ub.q--(OCH.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p(CO)(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q(OCH-
.sub.2CH.sub.2).sub.t,
--(CR.sub.11R.sub.12).sub.p-phenyl-CO-(Aa).sub.r(CR.sub.12'R.sub.12'').su-
b.q,
--(CR.sub.11R.sub.12).sub.p-furyl-CO-(Aa).sub.t(CR.sub.12'R.sub.12'')-
.sub.q,
--(CR.sub.11R.sub.12).sub.p-oxazolyl-CO-(Aa).sub.r(CR.sub.12'R.sub-
.12'').sub.q,
--(CR.sub.11R.sub.12).sub.p-thiazolyl-CO(Aa).sub.r(CR.sub.12'R.sub.12'').-
sub.q,
--(CR.sub.11R.sub.12).sub.p-thienyl-CO--(CR.sub.12'R.sub.12'').sub.-
q,
--(CR.sub.11R.sub.12).sub.p-imidazolyl-CO--(CR.sub.12'R.sub.12'').sub.q-
--,
--(CR.sub.11R.sub.12).sub.p-morpholino-CO-(Aa).sub.r(CR.sub.12'R.sub.1-
2'').sub.q--,
--(CR.sub.11R.sub.12).sub.p-Piperazino-CO(Aa).sub.r-(CR.sub.12'R.sub.12''-
).sub.q--,
--(CR.sub.11R.sub.12).sub.p--N-methyl-piperazin-CO(Aa).sub.r(CR-
.sub.11'R.sub.12'').sub.q--,
--(CR.sub.11R.sub.12).sub.p(Aa).sub.r-phenyl-,
--(CR.sub.11R.sub.12).sub.p-(Aa).sub.r-furyl-,
--(CR.sub.11R.sub.12).sub.p-oxazolyl(Aa).sub.r-,
--(CR.sub.11R.sub.12).sub.p-thiazolyl-(Aa).sub.r-,
--(CR.sub.11R.sub.12).sub.p-thienyl-(Aa).sub.t-,
--(CR.sub.11R.sub.12).sub.p-imidazolyl(Aa).sub.r-,
--(CR.sub.11R.sub.12).sub.p-morpholino-(Aa).sub.r-,
--(CR.sub.11R.sub.12).sub.p-piperazino-(Aa).sub.r-,
--(CR.sub.11R.sub.12).sub.p--N-methylpiperazino-(Aa).sub.r-,
--K(CR.sub.11R.sub.12).sub.p-(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q(OCH.s-
ub.2CH.sub.2).sub.t--,
--K(CR.sub.11R.sub.12).sub.p(CR.sub.12'R.sub.12'').sub.q(Aa).sub.r(OCH.su-
b.2CH.sub.2).sub.t--,
--K(Aa).sub.r(CR.sub.11R.sub.12).sub.p(CR.sub.12'R.sub.12'').sub.q--(OCH.-
sub.2CH.sub.2).sub.t--,
--K(CR.sub.11R.sub.12).sub.p(CR.sub.12'R.sub.12'').sub.q(OCH.sub.2CH.sub.-
2).sub.r(Aa).sub.t,
--K(CR.sub.11R.sub.12).sub.p(CR.sub.7.dbd.CR.sub.8)(CR.sub.12'R.sub.12'')-
.sub.q-(Aa).sub.r(OCH.sub.2CH.sub.2).sub.t--,
--K(CR.sub.11R.sub.12).sub.p(NR.sub.7CO)(Aa).sub.r(CR.sub.12'R.sub.12'').-
sub.q(OCH.sub.2CH.sub.2).sub.t,
--K(CR.sub.11R.sub.12).sub.p-(Aa).sub.t(NR.sub.7--CO)(CR.sub.12'R.sub.12'-
').sub.q(OCH.sub.2CH.sub.2).sub.t,
--K(CR.sub.11R.sub.12).sub.p(OCO)(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q(O-
CH.sub.2CH.sub.2).sub.t--,
--K(CR.sub.11R.sub.12).sub.p(OCNR.sub.7)(Aa).sub.r(CR.sub.12'R.sub.12'').-
sub.q(OCH.sub.2CH.sub.2).sub.t--,
--K(CR.sub.11R.sub.12).sub.p(CO)(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q--(-
OCH.sub.2CH.sub.2).sub.t,
--K(--CR.sub.11R.sub.12).sub.p(NR.sub.11CO)(Aa).sub.r(CR.sub.12'R.sub.12'-
').sub.q(OCH.sub.2CH.sub.2).sub.t,
--K(CR.sub.11R.sub.12).sub.p(OCO)-(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q(-
OCH.sub.2CH.sub.2).sub.t,
--K(CR.sub.11R.sub.12).sub.p(OCNR.sub.7)(Aa).sub.r(CR.sub.12'R.sub.12'').-
sub.q(OCH.sub.2CH.sub.2).sub.t,
--K(CR.sub.11R.sub.12).sub.p(CO)(Aa).sub.r(CR.sub.12'R.sub.12'').sub.q(OC-
H.sub.2CH.sub.2).sub.rQ,
--K(CR.sub.11R.sub.12).sub.p-phenyl-CO-(Aa).sub.r(CR.sub.12'R.sub.12'').s-
ub.q--,
--K(CR.sub.11R.sub.12).sub.p-furyl-CO(Aa).sub.t-(CR.sub.12'R.sub.1-
2'').sub.q,
--K(CR.sub.11R.sub.12).sub.p-oxazolyl-CO(Aa).sub.r(CR.sub.12'R.sub.12'').-
sub.q--,
--K(CR.sub.11R.sub.12).sub.p-thiazolyl-CO(Aa).sub.r-(CR.sub.12'R.-
sub.12'').sub.q,
--K(CR.sub.11R.sub.12).sub.p-thienyl-CO(CR.sub.12'R.sub.12'').sub.q--,
--K(CR.sub.11R.sub.12).sub.p-imidazolyl-CO--(CR.sub.12'R.sub.12'').sub.q,
--K(CR.sub.11R.sub.12).sub.p-morpholino-CO(Aa).sub.t(CR.sub.12'R.sub.12''-
).sub.q--,
--K(CR.sub.11R.sub.12).sub.p-piperazino-CO-(Aa).sub.r(CR.sub.12-
'R.sub.12'').sub.q--,
--K(CR.sub.11R.sub.12).sub.p--N-methylpiperazin-CO(Aa).sub.r-(CR.sub.12'R-
.sub.12'').sub.q, --K(CR.sub.11R.sub.12).sub.p-(Aa).sub.r-phenyl-,
--K(CR.sub.11R.sub.12).sub.m-(Aa).sub.r-furyl-,
--K(CR.sub.11R.sub.12).sub.p-oxazolyl(Aa).sub.r-,
--K(CR.sub.11R.sub.12).sub.m-thiazolyl-(Aa).sub.r-,
--K(CR.sub.11R.sub.12).sub.p-thienyl-(Aa).sub.r,
--K(CR.sub.11R.sub.12).sub.p-imidazolyl(Aa).sub.r-,
--K((CR.sub.11R.sub.12).sub.m-morpholino-(Aa).sub.r,
--K(CR.sub.11R.sub.12).sub.p-piperazino-(Aa).sub.t-,
--K(CR.sub.11R.sub.12).sub.mN-methylpiperazino-(Aa).sub.r.
[0109] Wherein Aa, r, n, p, q, t, R.sub.7, R.sub.11, R.sub.12,
R.sub.12', R.sub.12'' are as defined above. K is NR.sub.12, O, S,
Se, B, C.sub.3.about.C.sub.10 of Ar or Heterocyclic.
[0110] Q is a cell-binding molecule (CBA), or a functional group
that enables linkage with a cell-binding agent, or a functional
group that enables linkage with a linker attached on a cell-binding
agent. The function group is chosen from a thiol, an amine, a
hydrazine, an alkoxylamino, a disulfide substituent, a maleimido, a
haloacetyl group, a carboxy acid, an N-hydroxy succinimide ester, a
ketone, an ester, an aldehyde, an alkynyl, an alkenyl, or a
protected thiol or disulfide group, such as SAc, SSR.sub.1 or SSAr.
Ar is an aromatic group or hetero aromatic group.
[0111] The compounds of the general formula (I) having geometrical
and stereoisomers are also a part of the invention.
[0112] In certain embodiments, the amanita toxin derivatives are
presented by the following formula (Ia) (Ib), and (Ic), wherein an
amide linker linked to C-5 position of the indole unit.
##STR00018##
[0113] Wherein "- - - ", "", R.sub.1, R.sub.2, R.sub.4, R.sub.5,
R.sub.7, R.sub.8, R.sub.9, R.sub.10, L and Q are defined the same
as in Formula (I).
[0114] In certain embodiments, the amanita toxin derivatives are
presented by the following formula (Id), wherein an amide linker
linked to C-7 position of the indole unit.
##STR00019##
[0115] Wherein R.sub.1, R.sub.2, R.sub.4, R.sub.5, R.sub.10, L and
Q are defined the same as in Formula (I).
[0116] In certain embodiments, the amanita toxin derivatives of
formula (Ia), (Ib), and (Ic) are represented by the following
formulas (Ia-1), (Ia-2), (Ia-3), (Ia-4), (Ia-5), (Ia-6), (Ia-7),
(Ia-8), (Ia-9), (Ia-10), (Ia-11), (Ia-12), (Ia-13), (Ia-14),
(Ia-15), (Ia-16), (Ia-17), (Ia-18), (Ia-19), (Ia-20), (Ia-21),
(Ia-22), (Ia-23), (Ia-24), (Ib-1), (Ib-2), (Ib-3), (Ib-4), (Ib-5),
(Ib-6), (Ib-7), (Ic-1), (Ic-2), (Ic-3), (Ic-4), (Ic-5), and
(Ic-6):
##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024##
##STR00025## ##STR00026## ##STR00027##
[0117] Wherein R.sub.10, L and Q are defined the same as in Formula
(I).
[0118] In certain embodiments, the amanita toxin derivatives of
formula (Id) are represented by the following formulas (Id-1),
(Id-2), (Id-3), (Id-4), (Id-5), (Id-6), (Id-7), (Id-8), (Id-9),
(Id-10), (Id-11), (Id-12), (Id-13), (Id-14), (Id-15), (Id-16),
(Id-17), (Id-18), (Id-19), (Id-20), and (Id-21):
##STR00028## ##STR00029## ##STR00030##
[0119] Wherein R.sub.14 is H, PO.sub.3.sup.2-, SO.sub.3.sup.-,
R.sub.12, --COR.sub.12, --COCH.sub.3, --COOR.sub.12,
--CONR.sub.12R.sub.12', --C(.dbd.O)R.sub.12NH(Aa).sub.t, (an amino
acid or peptide, wherein Aa is an amino acid or a polypeptide, t
represents 0-100); --CSNHR.sub.12 (thiocarbamate); --SOR.sub.12
(sulfoxide); --SO.sub.2R.sub.12 (sulfone); --SO.sub.3.sup.-,
HSO.sub.3, HSO.sub.2, or a salt of HSO.sub.3.sup.-, SO.sub.3.sup.2-
or --HSO.sub.2.sup.- (sulphite); P(O)(OM.sub.1)(OM.sub.2),
CH.sub.2OP(O)(OM.sub.1)(OM.sub.2), SO.sub.3M.sub.1; glycoside
(glucoside, galactoside, mannoside, glucuronoside, alloside,
fructoside, etc), M.sub.1 and M.sub.2 are independently H, Na, K,
Ca, Mg, NH.sub.4, NR.sub.1'R.sub.2'R.sub.3'; R.sub.1', R.sub.2' and
R.sub.3' are independently H, C.sub.1.about.C.sub.8 alkyl.
[0120] In further embodiments, the cytotoxic agent and its
conjugate of this invention are preferred one of the following
structures:
##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035##
##STR00036## ##STR00037## ##STR00038## ##STR00039## ##STR00040##
##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045##
##STR00046## ##STR00047## ##STR00048## ##STR00049## ##STR00050##
##STR00051## ##STR00052## ##STR00053## ##STR00054##
##STR00055##
[0121] or a pharmaceutically acceptable salt, hydrates, or hydrated
salt, or the polymorphic crystalline structure of the compounds
thereof, or the optical isomers, racemates, diastereomers or
enantiomers.
[0122] Wherein Aa, r, n, L and Q are the same in the Formula (I).
PEG is polyethylene glycol with the formula of --OCH.sub.2CH.sub.2.
Preferably, Q is H, C.sub.1.about.C.sub.8 of alkyl, alkenyl,
alkinyl, aryl, cyclic, cyclohetero, haloalkyl, alkoxy, haloalkoxy
alkylamino; or halogen; or --NO.sub.2; or --CN; --SH; --SSCH.sub.3;
--SSAc; --SSAr; --SS-Pyridine; --SS--Ar(--NO.sub.2); --S-cell
binding agent; or a function group of NHS ester, pentafluorophenyl
ester; alkyloxyamine; aldehyde; ketone; carboxyl acid; hydrazine;
amine; or thiolactone; or linked a cell binding agent via.
Stretcher units (Ww) or via. Spacer units (Tt), wherein W, w, T,
and t are as defined in Formula (I); or Q is selected from any one
of the following formulas:
##STR00056## ##STR00057##
[0123] Wherein D is H, --NO.sub.2, SO.sub.3.sup.-, CN, or F;
R.sub.1, R.sub.2, R.sub.3, R.sub.4, r, m, and n are described in
Formula (I); w and w' are 0 or 1 independently.
[0124] Synthesis of the Derivatives of Amanita Toxins as Cytotoxic
Agents.
[0125] The compounds and process of the present invention can be
prepared in a number of ways well known to those skilled in the
art. The compounds can be synthesized, for example, by application
or adaptation of the methods described in the examples, or
variations thereon as appreciated by the skilled artisan. The
appropriate modifications and substitutions will be readily
apparent and well known or readily obtainable from the scientific
literature to those skilled in the art. In particular, such methods
can be found in Richard C. Larock, "Comprehensive Organic
Transformations, A Guide to Functional Group Preparations", Two
Volume Set, 2nd Edition, Wiley Publishers, 2010.
[0126] One preferred aspect of preparation of theses derivatives of
Amanita toxins of the invention is semi-synthesis. Thus, the core
structure compounds of theses derivatives of Amanita toxins are
isolated from theses derivatives-containing mushroom species from
the genera Amanita, Galerina and Lepiota, in particular, A.
bisporigera, A. visosa, A. suballiacea, A. phalloides and allied
species (Hallen, H. E. et al, 2007 Proc. Nat. Acad. Sci. USA, 104,
19097-101), or isolated from fermentation using a basidiomycete, or
using A. fissa (Guo, X. W., et al, 2006 Wei Sheng Wu Xue Bao 46(3):
373-8). The isolated toxin compounds are sequentially undergone
aromatic nitration of the indole units, followed by reduction of
the nitro group to an amine, and then the produced amine compounds
are condensated with a linker having a reactive or a reactable
carboxylic group to form an amide linkage. An illustration of the
semi-synthesis steps are shown below:
##STR00058##
Wherein R.sub.10, L and Q are defined the same in the Formula (I).
Wherein Lv is leaving group selected from OH, halogen, NHS
(N-hydroxyl succinimide), nitrophenol, pentalfluorophenol, etc.
[0127] The nitrition above can be using nitric acid, or mixture of
nitric acid and sulfuric acids, or mixture of nitric acid and
acetic acid optionally containing acetic anhydride, or tert-Butyl
nitrite (TBN), or nitrosonium salts (NO.sup.+BF.sub.4.sup.-,
NO.sup.+ClO.sub.4.sup.-, NO.sup.+PF.sup.-.sub.6,
NO.sup.-AsF.sup.-.sub.6, NO.sup.+SbF.sup.-.sub.6), or nitronium
salts (NO.sup.2+BF4.sup.-, NO.sup.2+ClO4.sup.-,
NO.sup.2+PF.sup.-.sub.6, NO.sup.2+AsF.sup.-.sub.6,
NO.sup.2+SbF.sup.-.sub.6). The reduction of the nitro group to the
amine can be by many different ways, such as catalytic
hydrogenation reduction using palladium-on-carbon, platinum(IV)
oxide, or Raney nickel; or direct reduction by using iron in acidic
media, sodium hydrosulfite; sodium sulfide (or hydrogen sulfide and
base), tin(II) chloride, titanium(III) chloride,
triphenylphosphine, trialkylphosphine, zinc, or samarium. The final
condensation of the amino group to the linker L containing a
carboxylic acid derivative, --C(O)-Lv. Wherein Lv can be selected
from F, Cl, Br, I, nitrophenol; N-hydroxysuccinimide (NHS); phenol;
dinitrophenol; pentafluorophenol; tetrafluorophenol;
difluorophenol; monofluorophenol; pentachlorophenol; triflate;
imidazole; dichlorophenol; tetrachlorophenol;
1-hydroxybenzotriazole; tosylate; mesylate;
2-ethyl-5-phenylisoxazolium-3'-sulfonate, anhydrides formed its
self, or formed with the other anhydride, e.g. acetyl anhydride,
formyl anhydride. When Lv is OH group, then the condensation with
the amino group is through a coupling reagent, such as a peptide
coupling agent, or coupling agent for Mitsunobu reaction. These
coupling reagents are, but not limited:
N-(3-dimethylamino-propyl)-N'-ethylcarbodiimide (EDC),
dicyclohexyl-carbodiimide (DCC), N,N'-diisopropyl-carbodiimide
(DIC), N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide
metho-p-toluenesulfonate (CMC,or CME-CDI), 1,1'-carbonyldiimidazole
(CDI), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TBTU),
N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium
hexafluorophosphate (HBTU),
benzotriazol-1-yloxy)tris(dimethyl-amino)phosphonium
hexafluorophosphate (BOP),
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP), diethyl cyanophosphonate (DEPC),
chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate,
1-[bis(dimethylamino)-methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate (HATU), 1-[(dimethylamino)(morpholino)
methylene]-1H-[1,2,3]triazolo[4,5-b]pyridine-1-ium 3-oxide
hexafluorophosphate (HDMA), 2-chloro-1,3-dimethylimidazolidinium
hexafluorophosphate (CIP), chlorotripyrrolidinophosphonium
hexafluorophosphate (PyCloP),
fluoro-N,N,N',N'-bis(tetramethylene)formamidinium
hexafluorophosphate (BTFFH),
N,N,N',N'-tetramethyl-S-(1-oxido-2-pyridyl)thiuronium
hexafluorophosphate,
O-(2-oxo-1(2H)pyridyl)-N,N,N,'N'-tetramethyluronium
tetrafluoroborate (TPTU),
S-(1-oxido-2-pyridyl)-N,N,N,'N'-tetramethylthiuronium
tetrafluoroborate, O-[(ethoxycarbonyl)
cyano-methylenamino]-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HOTU),
(1-cyano-2-ethoxy-2-oxoethylidenaminooxy)
dimethylamino-morpholino-carbenium hexafluorophosphate (COMU),
O-(benzotriazol-1-yl)-N,N,N',N'-bis(tetramethylene) uronium
hexafluorophosphate (HBPyU), N-benzyl-N'-cyclohexylcarbodiimide
(with, or without polymer-bound),
dipyrrolidino(N-succinimidyloxy)carbenium hexafluoro-phosphate
(HSPyU), chlorodipyrrolidinocarbenium hexafluorophosphate (PyClU),
2-chloro-1,3-dimethylimidazolidinium tetrafluoroborate (CIB),
(benzotriazol-1-yloxy) dipiperidinocarbenium hexafluorophosphate
(HBPipU),
O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TCTU), bromotris(dimethylamino)-phosphonium
hexafluorophosphate (BroP), propylphosphonic anhydride (PPACA,
T3P.RTM.), 2-morpholinoethyl isocyanide (MEI),
N,N,N',N'-tetramethyl-O--(N-succinimidyl)uronium
hexafluorophosphate (HSTU), 2-bromo-1-ethyl-pyridinium
tetrafluoroborate (BEP),
O-[(ethoxycarbonyl)cyanomethylenamino]-N,N,N',N'-tetramethyl-uronium
tetrafluoroborate (TOTU),
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride
(MMTM, DMTMM), N,N,N',N'-tetramethyl-O--(N-succinimidyl)-uronium
tetrafluoroborate (TSTU),
O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N',N'-tetramethyluroniu-
m tetrafluoro-borate (TDBTU), 1,1'-(azodicarbonyl)dipiperidine
(ADD), di-(4-chlorobenzyl) azodicarboxylate (DCAD), di-tert-butyl
azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD),
diethyl azodicarboxylate (DEAD).
[0128] Another aspect of the invention provides a chemical
synthetic preparation of derivatives of amanita toxins of Formula
(I) which are illustrated in FIGS. 1-28. The synthetic preparations
can be in solid phase, solution phase or combination of solid and
solution phases.
[0129] Because the cytotoxic agents of formula (I) of the present
invention contain asymmetrically substituted carbon atoms, and may
be isolated in optically active or racemic forms, all chiral,
diastereomeric, racemic forms and all geometric isomeric forms of a
structure are intended, unless the specific stereochemistry or
isomeric form is specifically indicated. It is well known in the
art how to prepare and isolate such optically active forms. For
example, mixtures of stereoisomers may be separated by standard
techniques including, but not limited to, resolution of racemic
forms, normal, reverse-phase, and chiral chromatography,
preferential salt formation, recrystallization, and the like, or by
chiral synthesis either from chiral starting materials or by
deliberate synthesis of target chiral centers.
[0130] The cytotoxic agents of the present invention may be
prepared by a variety of synthetic routes. The reagents and
starting materials are commercially available, or readily
synthesized by well-known techniques by one of ordinary skill in
the arts. All substituents, unless otherwise indicated, are as
previously defined.
[0131] In the synthetic reactions of the cytotoxic agents of the
present invention, it may be necessary to protect reactive
functional groups, for example hydroxy, amino, imino, thio or
carboxy groups, where these are desired in the final product, to
avoid their unwanted participation in the reactions. Conventional
protecting groups may be used in accordance with standard practice,
for examples see Peter G. M. Wuts, Theodora W. Greene in "Greene's
Protective Groups in Organic Synthesis", 4th Edition, John Wiley
and Sons, 2006; Ian T. Harrison, Shuyen Harrison in "Compendium of
Organic Synthetic Methods", Vol 1, 2 Vols. 1 & 2 By Ian T.
Harrison & Shuyen Harrison , Vols 3-5 by Louis S. Hegedus,
Leroy Wade Vols 6.about.Vol 12 by Michael B. Smith, John Wiley and
Sons,2006.about.2012.
[0132] Normally the synthetic reactions are carried out in suitable
solvents, temperatures and time. A variety of solvents which have
no adverse effect on the reaction or on the reagents involved can
be used in a synthetic reaction of the cytotoxic agent. Examples of
suitable solvents include: hydrocarbons, which may be aromatic,
aliphatic or cycloaliphatic hydrocarbons, such as hexane,
cyclohexane, benzene, toluene and xylene; hydrocarbons containing
halogens, such as chloroform, dichloromethane, dichloroethane;
amides, such as dimethylactamide or dimethylformamide; alcohols
such as propanol, ethanol or methanol, and ethers, such as diethyl
ether, tetrahydrofuran, or dioxane. The reactions can take place
over a wide range of temperatures, from -100.degree.
C..about.300.degree. C., preferably from 0.degree. C. to
100.degree. C. The time required for the synthetic reaction may
also vary widely, depending on many factors, notably the reaction
temperature and the nature of the reagents and can be from 5 second
to 4 weeks, more preferably from 10 min to 20 hours. In addition,
the cytotoxic agents prepared may be isolated or purified from the
reaction mixture by conventional means, such as evaporating or
distilling off the solvent from the reaction mixture, or after
distilling off the solvent from the reaction mixture, pouring the
residue into water followed by extraction with a water-immiscible
organic solvent and then distilling off the solvent from the
extract. It may also involve various well known techniques, such as
re-crystallization, re-precipitation or the various chromatography
techniques, notably column chromatography, preparative thin layer
chromatography, or high performance liquid chromatography.
[0133] Some of the synthetic reactions of the cytotoxic agents and
their conjugates to a cell binding agent are further exampled but
not restricted in the FIGS. 1.about.28 and in the examples
1.about.70 of the description.
The Conjugates of Cell-Binding Agent-Cytotoxic Agent
[0134] The present invention also provides a conjugate molecule
comprising at least one derivative of amanita toxins covalently
linked to a cell binding agent (CBA) through the linking group of
the crosslinker (L). Preferably said conjugate comprises one to
twenty molecules of derivatives of amanita toxins according to the
invention covalently linked to a cell binding agent through the
linking group of the linker of the derivatives of amanita
toxins.
[0135] As stated above, the conjugates of a cell surface binding
molecule-cytotoxic agent are illustrated in the Formula (I):
##STR00059##
or their pharmaceutically acceptable salts, hydrates, or hydrated
salts; or the polymorphic crystalline structures of these
compounds; or their optical isomers, racemates, diastereomers or
enantiomers.
[0136] wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, R.sub.11, L, m, n and
Q are described the same as early in Formula (I). L is preferred a
linker-cell binding molecule covalently bound cluster.
[0137] In certain embodiments, the conjugates of the invention are
illustrated in the following formula:
##STR00060## ##STR00061## ##STR00062## ##STR00063## ##STR00064##
##STR00065## ##STR00066## ##STR00067## ##STR00068## ##STR00069##
##STR00070## ##STR00071## ##STR00072## ##STR00073## ##STR00074##
##STR00075## ##STR00076## ##STR00077## ##STR00078## ##STR00079##
##STR00080## ##STR00081## ##STR00082## ##STR00083## ##STR00084##
##STR00085##
[0138] Wherein Aa, L, m, n, p, Q, r, R.sub.1, and R.sub.2, are
described above in Formula (I). CBA is a Cell binding agent.
[0139] Drug loading (DAR) of the conjugates may range from 1 to 20
drug moieties (D) per cell binding agent and is preferred the
average number of 2.about.8 drug moieties per cell binding agent in
a molecule of Formula (II-1)-(II-91). When CBA is antibody in
preparations of ADC, the preferred drug loading is 3.about.6 drug
per antibody and the average number of drug moieties per antibody
from conjugation reactions may be characterized by conventional
means such as mass spectroscopy (HPLC-MS, UPLC-QTOF, HPLC-MS/MS),
ELISA assay, and HPLC (SEC-HPLC, HIC-HPLC). The quantitative
distribution of the conjugates in terms of the drug loading may
also be determined. In some instances, separation, purification,
and characterization of homogeneous the conjugates where drug
loading is a certain value from the conjugates with the drug
loadings may be achieved by means such as reverse phase HPLC or
electrophoresis.
[0140] The Cell binding agents (CBA) may be of any kind and include
peptides and non-peptides. Generally, the cell binding agents
include, but are not limited to, large molecular weight proteins
such as, for example, full-length antibodies (polyclonal and
monoclonal antibodies); single chain antibodies; fragments of
antibodies such as Fab, Fab', F(ab').sub.2, F.sub.v, [Parham, J.
Immunol. 131, 2895-2902 (1983)], fragments produced by a Fab
expression library, anti-idiotypic (anti-Id) antibodies, CDR's, and
epitope-binding fragments of any of the above which
immuno-specifically bind to cancer cell antigens, viral antigens or
microbial antigens; antibody mimetic, such as an affibody; domain
antibodies (dAb); nanobodies; unibodies; DARPins; anticalins;
versabodies; duocalins; lipocalins; vimers; interferons (such as
type I, II, III); peptides; lymphokines such as IL-2, IL-3, IL-4,
IL-5, IL-6, GM-CSF, interferon-gamma (IFN-.gamma.); hormones such
as insulin, TRH (thyrotropin releasing hormones), MSH
(melanocyte-stimulating hormone), steroid hormones, such as
androgens and estrogens, melanocyte-stimulating hormone (MSH);
growth factors and colony-stimulating factors such as epidermal
growth factors (EGF), granulocyte-macrophage colony-stimulating
factor (GM-CSF), transforming growth factors (TGF), such as
TGF.alpha., TGF.beta., insulin and insulin like growth factors
(IGF-I, IGF-II) G-CSF, M-CSF and GM-CSF [Burgess, Immunology Today,
5, 155-158 (1984)]; vaccinia growth factors (VGF); fibroblast
growth factors (FGFs); smaller molecular weight proteins,
poly-peptide, peptides and peptide hormones, such as bombesin,
gastrin, gastrin-releasing peptide; platelet-derived growth
factors; interleukin and cytokines, such as interleukin-2 (IL-2),
interleukin-6 (IL-6), leukemia inhibitory factors,
granulocyte-macrophage colony-stimulating factor (GM-CSF);
vitamins, such as folate; apoproteins and glycoproteins, such as
transferrin {O'Keefe et al, 260 J. Biol. Chem. 932-937 (1985)};
sugar-binding proteins or lipoproteins, such as lectins; cell
nutrient-transport molecules; and small molecular inhibitors, such
as prostate-specific membrane antigen (PSMA) inhibitors and small
molecular tyrosine kinase inhibitors (TKI), non-peptides or any
other cell binding molecule or substance, such as bioactive
polymers (Dhar, et al, Proc. Natl. Acad. Sci. 2008, 105, 17356-61)
or a polymer having a cell binding agent on its surface; dendrimers
(Lee, et al, Nat. Biotechnol. 2005, 23, 1517-26; Almutairi, et al;
Proc. Natl. Acad. Sci. 2009, 106, 685-90) or a dendrimer containing
a cell binding agent; nanoparticles (Liong, et al, ACS Nano, 2008,
19, 1309-12; Medarova, et al, Nat. Med. 2007, 13, 372-7; Javier, et
al, Bioconjugate Chem. 2008, 19, 1309-12) or a nanoparticles having
a cell binding agent on its surface; liposomes (Medinai, et al,
Curr. Phar. Des. 2004, 10, 2981-9) or a liposome having a cell
binding agent; viral capsides (Flenniken, et al, Viruses
Nanotechnol. 2009, 327, 71-93). In general monoclonal antibodies
are preferred as a cell-surface binding agent if an appropriate one
is available.
[0141] The linker used for the conjugation of this invention
includes, but not limited to, a disulfide linker, a thioether
linker, an amide bonded linker, a peptidase-labile linker, a
photolabile linker, an acid-labile linkers (such as hydrazone
liner), an esterase-labile linker, an oxidatively labile linker, a
metabolically labile linker, a biochemically labile linker.
[0142] Preferably, the linker is linked to the cell binding agent
via a function reactive towards for instance thiol and amino
functions of the cell binding agent coming from reduced disulfide
bonds and lysine residues respectively. More particularly, said
derivative is linked through the --CO-- group to the amino function
of the lysine residue of said cell binding agent, so as to form an
amide bond.
[0143] In addition, the linker may be composed of one or more
linker components. Exemplary linker components include
6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"),
valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine
("ala-phe" or "af"), glycine-glycine, a nature peptides containing
up to 6 the same or different aminoacides (dipeptide, tripeptide,
tetrapeptide, pentapeptide, hexapeptide), p-aminobenzyloxycarbonyl
("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"),
N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxyl ate
("SMCC"), N-Succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB"),
ethyleneoxy (--CH.sub.2CH.sub.2O--) as one or up to 100 repeating
units ("EO" or "PEO"). The linker may be a "cleavable linker,"
facilitating release of a drug in the cell. Additional linker
components are known in the art and some are illustrated below:
##STR00086## ##STR00087## ##STR00088##
Wherein R.sub.10 is defined in Formula (I) above. Wherein R.sub.15,
R.sub.16 and R.sub.17 are independently selected from
--C.sub.1.about.C.sub.8 alkyl or alkylene-, --C.sub.1.about.C.sub.2
carbocyclo-, --O--(C.sub.1.about.C.sub.8 alkyl)-,
--NH--(C.sub.1.about.C.sub.8 alkyl)-, -arylene-,
--C.sub.1.about.C.sub.8 alkylene-arylene-, -arylene,
--C.sub.1.about.C.sub.8 alkylene-, --C.sub.1.about.C.sub.8
alkylene-(C.sub.1.about.C.sub.8 carbocyclo)-,
--(C.sub.3.about.C.sub.7 carbocyclo)-C.sub.1.about.C.sub.8
alkylene-, --C.sub.3.about.C.sub.8 heterocyclo-,
--C.sub.1.about.C.sub.8 alkylene-(C.sub.3.about.C.sub.8
heterocyclo)-, --(C.sub.3.about.C.sub.8
heterocyclo)-C.sub.1.about.C.sub.9 alkylene-,
--(CH.sub.2CH.sub.2O).sub.k--, --(CH(CH.sub.3)CH.sub.2O).sub.k--,
and --(CH.sub.2CH.sub.2O).sub.k--CH.sub.2--; k is an integer
ranging from 1-30; X''', Y''' and Z''' are independently selected
from NH, O or S.
[0144] In a preferred embodiment, conjugates of the invention are
antibody/cytotoxic agent, antibody fragment/cytotoxic agent,
diabody/cytotoxic agent, tri(a)body/cytotoxic agent, epidermal
growth factor (EGF)/cytotoxic agent, prostate specific membrane
antigen (PSMA) inhibitor/cytotoxic agent, melanocyte stimulating
hormone (MSH)/cytotoxic agent, thyroid stimulating hormone
(TSH)/cytotoxic agent, polyclonal antibody/cytotoxic agent,
somatostatin/cytotoxic agent, folate/cytotoxic agent, matriptase
inhibitor/cytotoxic agent, estrogen/cytotoxic agent, estrogen
analogue/cytotoxic agent, designed ankyrin repeat proteins
(DARPins)/cytotoxic agent, androgen/cytotoxic agent, and androgen
analogue/cytotoxic agent.
[0145] In a more preferred embodiment, conjugates of the invention
are monoclonal antibody/cytotoxic agent. Examples of antibodies
used for conjugation of cyotoxic agents in this prevention include,
but are not limited to, 3F8 (anti-GD2), Abagovomab (anti CA-125),
Abciximab (anti CD41 (integrin alpha-IIb), Adalimumab
(anti-TNF-.alpha.), Adecatumumab (anti-EpCAM, CD326), Afelimomab
(anti-TNF-.alpha.); Afutuzumab (anti-CD20), Alacizumab pegol
(anti-VEGFR2), ALD518 (anti-IL-6), Alemtuzumab (Campath,
MabCampath, anti-CD52), Altumomab (anti-CEA), Anatumomab
(anti-TAG-72), Anrukinzumab (IMA-638, anti-IL-13), Apolizumab
(anti-HLA-DR), Arcitumomab (anti-CEA), Aselizumab (anti-L-selectin
(CD62L), Atlizumab (tocilizumab, Actemra, RoActemra, anti-IL-6
receptor), Atorolimumab (anti-Rhesus factor), Bapineuzumab
(anti-beta amyloid), Basiliximab (Simulect, antiCD25 (.alpha. chain
of IL-2 receptor), Bavituximab (anti-phosphatidylserine),
Bectumomab (LymphoScan, anti-CD22), Belimumab (Benlysta,
LymphoStat-B, anti-BAFF), Benralizumab (anti-CD125), Bertilimumab
(anti-CCL11 (eotaxin-1)), Besilesomab (Scintimun, anti-CEA-related
antigen), Bevacizumab (Avastin, anti-VEGF-A), Biciromab
(FibriScint, anti-fibrin II beta chain), Bivatuzumab (anti-CD44
v6), Blinatumomab (BiTE, anti-CD19), Brentuximab (cAC10, anti-CD30
TNFRSF8), Briakinumab (anti-IL-12, IL-23) Canakinumab (Ilaris,
anti-IL-1), Cantuzumab (C242, anti-CanAg), Capromab, Catumaxomab
(Removab, anti-EpCAM, anti-CD3), CC49 (anti-TAG-72), Cedelizumab
(anti-CD4), Certolizumab pegol (Cimzia anti-TNF-.alpha.), Cetuximab
(Erbitux, IMC-C225, anti-EGFR), Citatuzumab bogatox (anti-EpCAM),
Cixutumumab (anti-IGF-1), Clenoliximab (anti-CD4), Clivatuzumab
(anti-MUC1), Conatumumab (anti-TRAIL-R2), CR6261 (anti-Influenza A
hemagglutinin), Dacetuzumab (anti-CD40), Dadizumab (Zenapax,
anti-CD25 (.alpha. chain of IL-2 receptor)), Daratumumab (anti-CD38
(cyclic ADP ribose hydrolase), Denosumab (Prolia, anti-RANKL),
Detumomab (anti-B-lymphoma cell), Dorlimomab, Dorlixizumab,
Ecromeximab (anti-GD3 ganglioside), Eculizumab (Soliris, anti-C5),
Edobacomab (anti-endotoxin), Edrecolomab (Panorex, MAb17-1A,
anti-EpCAM), Efalizumab (Raptiva, anti-LFA-1 (CD11a), Efungumab
(Mycograb, anti-Hsp90), Elotuzumab (anti-SLAMF7), Elsilimomab
(anti-IL-6), Enlimomab pegol (anti-ICAM-1 (CD54)), Epitumomab
(anti-episialin), Epratuzumab (anti-CD22), Erlizumab (anti-ITGB2
(CD18)), Ertumaxomab (Rexomun, anti-HER2/neu, CD3), Etaracizumab
(Abegrin, anti-integrin .alpha..sub.v.beta..sub.3), Exbivirumab
(anti-hepatitis B surface antigen), Fanolesomab (NeutroSpec,
anti-CD15), Faralimomab (anti-interferon receptor), Farletuzumab
(anti-folate receptor 1), Felvizumab (anti-respiratory syncytial
virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF-1
receptor), Fontolizumab (anti-IFN-.gamma.), Foravinimab
(anti-rabies virus glycoprotein), Fresolimumab (anti-TGF-.beta.),
Galiximab (anti-CD80), Gantenerumab (anti-beta amyloid),
Gavilimomab (anti-CD147 (basigin)), Gemtuzumab (anti-CD33),
Girentuximab (anti-carbonic anhydrase 9), Glembatumumab (CR011,
anti-GPNMB), Golimumab (Simponi, anti-TNF-.alpha.), Gomiliximab
(anti-CD23 (IgE receptor)), Ibalizumab (anti-CD4), Ibritumomab
(anti-CD20), Igovomab (Indimacis-125, anti-CA-125), Imciromab
(Myoscint, anti-cardiac myosin), Infliximab (Remicade,
anti-TNF-.alpha.), Intetumumab (anti-CD51), Inolimomab (anti-CD25
(.alpha. chain of IL-2 receptor)), Inotuzumab (anti-CD22),
Ipilimumab (anti-CD152), Iratumumab (anti-CD30 (TNFRSF8)),
Keliximab (anti-CD4), Labetuzumab (CEA-Cide, anti-CEA),
Lebrikizumab (anti-IL-13), Lemalesomab (anti-NCA-90 (granulocyte
antigen)), Lerdelimumab (anti-TGF beta 2), Lexatumumab
(anti-TRAIL-R2), Libivirumab (anti-hepatitis B surface antigen),
Lintuzumab (anti-CD33), Lucatumumab (anti-CD40), Lumiliximab
(anti-CD23 (IgE receptor), Mapatumumab (anti-TRAIL-R1), Maslimomab
(anti-T-cell receptor), Matuzumab (anti-EGFR), Mepolizumab
(Bosatria, anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab
(anti-CD74), Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3
ganglioside), Morolimumab (anti-Rhesus factor), Motavizumab (Numax,
anti-respiratory syncytial virus), Muromonab-CD3 (Orthoclone OKT3,
anti-CD3), Nacolomab (anti-C242), Naptumomab (anti-5T4),
Natalizumab (Tysabri, anti-integrin .alpha..sub.4), Nebacumab
(anti-endotoxin), Necitumumab (anti-EGFR), Nerelimomab
(anti-TNF-.alpha.), Nimotuzumab (Theracim, Theraloc, anti-EGFR),
Nofetumomab, Ocrelizumab (anti-CD20), Odulimomab (Afolimomab,
anti-UFA-1 (CD11a)), Ofatumumab (Arzerra, anti-CD20), Olaratumab
(anti-PDGF-R .alpha.), Omalizumab (Xolair, anti-IgE Fc region),
Oportuzumab (anti-EpCAM), Oregovomab (OvaRex, anti-CA-125),
Otelixizumab (anti-CD3), Pagibaximab (anti-lipoteichoic acid),
Palivizumab (Synagis, Abbosynagis, anti-respiratory syncytial
virus), Panitumumab (Vectibix, ABX-EGF, anti-EGFR), Panobacumab
(anti-Pseudomonas aeruginosa), Pascolizumab (anti-IL-4), Pemtumomab
(Theragyn, anti-MUC1), Pertuzumab (Omnitarg, 2C4, anti-HER2/neu),
Pexelizumab (anti-C5), Pintumomab (anti-adenocarcinoma antigen),
Priliximab (anti-CD4), Pritumumab (anti-vimentin), PRO 140
(anti-CCR5), Racotumomab (1E10, anti-(N-glycolylneuraminic acid
(NeuGc, NGNA)-gangliosides GM3)), Rafivinimab (anti-rabies virus
glycoprotein), Ramucirumab (anti-VEGFR2), Ranibizumab (Lucentis,
anti-VEGF-A), Raxibacumab (anti-anthrax toxin, protective antigen),
Regavirumab (anti-cytomegalovirus glycoprotein B), Reslizumab
(anti-IL-5), Rilotumumab (anti-HGF), Rituximab (MabThera,
Rituxanmab, anti-CD20), Robatumumab (anti-IGF-1 receptor),
Rontalizumab (anti-IFN-.alpha.), Rovelizumab (LeukArrest,
anti-CD11, CD18), Ruplizumab (Antova, anti-CD154 (CD40L)),
Satumomab (anti-TAG-72), Sevirumab (anti-cytomegalovirus),
Sibrotuzumab (anti-TAP), Sifalimumab (anti-IFN-.alpha.), Siltuximab
(anti-IL-6), Siplizumab (anti-CD2), (Smart) MI95 (anti-CD33),
Solanezumab (anti-beta amyloid), Sonepcizumab
(anti-sphingosine-1-phosphate), Sontuzumab (anti-episialin),
Stamulumab (anti-myostatin), Sulesomab (LeukoScan, (anti-NCA-90
(granulocyte antigen), Tacatuzumab (anti-alpha-fetoprotein),
Tadocizumab (anti-integrin .alpha..sub.IIb.beta..sub.3), Talizumab
(anti-IgE), Tanezumab (anti-NGF), Taplitumomab (anti-CD19),
Tefibazumab (Aurexis, (anti-clumping factor A), Telimomab,
Tenatumomab (anti-tenascin C), Teneliximab (anti-CD40), Teplizumab
(anti-CD3), TGN1412 (anti-CD28), Ticilimumab (Tremelimumab,
(anti-CTLA-4), Tigatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13),
Tocilizumab (Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor),
Toralizumab (anti-CD154 (CD40L)), Tositumomab (anti-CD20),
Trastuzumab (Herceptin, (anti-HER2/neu), Tremelimumab
(anti-CTLA-4), Tucotuzumab celmoleukin (anti-EpCAM), Tuvirumab
(anti-hepatitis B virus), Urtoxazumab (anti-Escherichia coli),
Ustekinumab (Stelara, anti-IL-12, IL-23), Vapaliximab (anti-AOC3
(VAP-1)), Vedolizumab, (anti-integrin .alpha..sub.4.beta..sub.7),
Veltuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1), Visilizumab
(Nuvion, anti-CD3), Vitaxin (anti-vascular integrin avb3),
Volociximab (anti-integrin .alpha..sub.5.beta..sub.1), Votumumab
(HumaSPECT, anti-tumor antigen CTAA16.88), Zalutumumab (HuMax-EGFr,
(anti-EGFR), Zanolimumab (HuMax-CD4, anti-CD4), Ziralimumab
(anti-CD147 (basigin)), Zolimomab (anti-CD5), Etanercept
(Enbrel.RTM.), Alefacept (Amevive.RTM.), Abatacept (Orencia.RTM.),
Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (Iron Regulatory Protein
2)], 14G2a (anti-GD2 ganglioside, from Nat. Cancer Inst. for
melanoma and solid tumors), J591 (anti-PSMA, Weill Cornell Medical
School for prostate cancers), 225.28S [anti-HMW-MAA (High molecular
weight-melanoma-associated antigen), Sorin Radiofarmaci S.R.L.
(Milan, Italy) for melanoma], COL-1 (anti-CEACAM3, CGM1, from Nat.
Cancer Inst. USA for colorectal and gastric cancers), CYT-356
(Oncoltad.RTM., for prostate cancers), HNK20 (OraVax Inc. for
respiratory syncytial virus), ImmuRAIT (from Immunomedics for NHL),
Lym-1 (anti-HLA-DR10, Peregrine Pharm. for Cancers), MAK-195F
[anti-TNF (tumor necrosis factor; TNFA, TNF-alpha; TNFSF2), from
Abbott/Knoll for Sepsis toxic shock], MEDI-500 [T10B9, anti-CD3,
TR.alpha..beta. (T cell receptor alpha/beta), complex, from
MedImmune Inc for Graft-versus-host disease], RING SCAN [anti-TAG
72 (tumour associated glycoprotein 72), from Neoprobe Corp. for
Breast, Colon and Rectal cancers], Avicidin (anti-EPCAM (epithelial
cell adhesion molecule), anti-TACSTD1 (Tumor-associated calcium
signal transducer 1), anti-GA733-2 (gastrointestinal
tumor-associated protein 2), anti-EGP-2 (epithelial glycoprotein
2); anti-KSA; KS1/4 antigen; M4S; tumor antigen 17-1A; CD326, from
NeoRx Corp. for Colon, Ovarian, Prostate cancers and NHL];
LymphoCide (Immunomedics, NJ), Smart ID10 (Protein Design Labs),
Oncolym (Techniclone Inc, CA), Allomune (BioTransplant, CA),
anti-VEGF (Genentech, CA); CEAcide (Immunomedics, NJ), IMC-1C11
(ImClone, NJ) and Cetuximab (ImClone, NJ).
[0146] Other antibodies as binding ligands include, but are not
limited to, are antibodies against the following antigens:
Aminopeptidase N (CD13), Annexin A1, B7-H3 (CD276, various
cancers), CA125 (ovarian), CA15-3 (carcinomas), CA19-9
(carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X
(carcinomas), alpha fetoprotein (carcinomas), CA242 (colorectal),
placental alkaline phosphatase (carcinomas), prostate specific
antigen (prostate), prostatic acid phosphatase (prostate),
epidermal growth factor (carcinomas), CD2 (Hodgkin's disease, NHL
lymphoma, multiple myeloma), CD3 epsilon (T cell lymphoma, lung,
breast, gastric, ovarian cancers, autoimmune diseases, malignant
ascites), CD19 (B cell malignancies), CD20 (non-Hodgkin's
lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, SLE), CD30
(Hodgkin's lymphoma), CD33 (leukemia, autoimmune diseases), CD38
(multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia
(CLL)), CD51 (Metastatic melanoma, sarcoma), CD52 (leukemia), CD56
(small cell lung cancers, ovarian cancer, Merkel cell carcinoma,
and the liquid tumor, multiple myeloma), CD66e (cancers), CD70
(metastatic renal cell carcinoma and non-Hodgkin lymphoma), CD74
(multiple myeloma), CD80 (lymphoma), CD98 (cancers), mucin
(carcinomas), CD221 (solid tumors), CD227 (breast, ovarian
cancers), CD262 (NSCLC and other cancers), CD309 (ovarian cancers),
CD326 (solid tumors), CEACAM3 (colorectal, gastric cancers),
CEACAM5 (carcinoembryonic antigen; CEA, CD66e) (breast, colorectal
and lung cancers), DLL4 (.DELTA.-like-4), EGFR (Epidermal Growth
Factor Receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD184,
Heme-oncology, solid tumors), Endoglin (CD105, solid tumors), EPCAM
(epithelial cell adhesion molecule, bladder, head, neck, colon, NHL
prostate, and ovarian cancers), ERBB2 (Epidermal Growth Factor
Receptor 2; lung, breast, prostate cancers), FCGR1 (autoimmune
diseases), FOLR (folate receptor, ovarian cancers), GD2 ganglioside
(cancers), G-28 (a cell surface antigen glyvolipid, melanoma), GD3
idiotype (cancers), Heat shock proteins (cancers), HER1 (lung,
stomach cancers), HER2 (breast, lung and ovarian cancers), HLA-DR10
(NHL), HLA-DRB (NHL, B cell leukemia), human chorionic gonadotropin
(carcinoma), IGF1R (insulin-like growth factor 1 receptor, solid
tumors, blood cancers), IL-2 receptor (interleukin 2 receptor,
T-cell leukemia and lymphomas), IL-6R (interleukin 6 receptor,
multiple myeloma, RA, Castleman's disease, IL6 dependent tumors),
Integrins (.alpha.v.beta.3, .alpha.5.beta.1, .alpha.6.beta.4,
.alpha.11.beta.3, .alpha.5.beta.5, .alpha.v.beta.5, for various
cancers), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3
(carcinomas), MAGE 4 (carcinomas), anti-transferrin receptor
(carcinomas), p97 (melanoma), MS4A1 (membrane-spanning 4-domains
subfamily A member 1, Non-Hodgkin's B cell lymphoma, leukemia),
MUC1 or MUC1-KLH (breast, ovarian, cervix, bronchus and
gastrointestinal cancer), MUC16 (CA125) (Ovarian cancers), CEA
(colorectal), gp100 (melanoma), MART1 (melanoma), MPG (melanoma),
MS4A1 (membrane-spanning 4-domains subfamily A, small cell lung
cancers, NHL), Nucleolin, Neu oncogene product (carcinomas), P21
(carcinomas), Paratope of anti-N-glycolylneuraminic acid (Breast,
Melanoma cancers), PLAP-like testicular alkaline phosphatase
(ovarian, testicular cancers), PSMA (prostate tumors), PSA
(prostate), ROBO4, TAG 72 (tumour associated glycoprotein 72, AML,
gastric, colorectal, ovarian cancers), T cell transmembrane protein
(cancers), Tie (CD202b), TNFRSF10B (tumor necrosis factor receptor
superfamily member 10B, cancers), TNFRSF13B (tumor necrosis factor
receptor superfamily member 13B, multiple myeloma, NHL, other
cancers, RA and SLE), TPBG (trophoblast glycoprotein, Renal cell
carcinoma), TRAIL-R1 (Tumor necrosis apoprosis Inducing ligand
Receptor 1, lymphoma, NHL, colorectal, lung cancers), VCAM-1
(CD106, Melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (various cancers).
Some other tumor associated antigens recognized by antibodies have
been reviewed (Gerber, et al, mAbs 1:3, 247-253 (2009); Novellino
et al, Cancer Immunol Immunother. 54(3), 187-207 (2005). Franke, et
al, Cancer Biother Radiopharm. 2000, 15, 459-76). Examples of these
antigens that antibodies against are: Many other Cluster of
Differentiations (CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b,
CD11c, CD12w, CD14, CD15, CD16, CDw17, CD18, CD20, CD21, CD22,
CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD34,
CD35, CD36, CD37, CD38, CD41, CD42, CD43, CD44, CD45, CD46, CD47,
CD48, CD49b, CD49c, CD53, CD54, CD55, CD56, CD58, CD59, CD61,
CD62E, CD62L, CD62P, CD63, CD68, CD69, CD70, CD71, CD72, CD79,
CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96,
CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD125, CD127,
CD133, CD134, CD135, CD138, CD141, CD142, CD143, CD144, CD147,
CD151, CD152, CD154, CD156, CD158, CD163, CD166, .CD168, CD184,
CDw186, CD195, CD202 (a, b), CD209, CD235a, CD271, CD303, CD304),
Annexin A1, Nucleolin, Endoglin (CD105), ROBO4, Amino-peptidase N,
.DELTA.-like-4 (DLL4), VEGFR-2 (CD309), CXCR4 9CD184), Tie2, B7-H3,
WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, Idiotype, MAGE A3,
p53 nonmutant, NY-ESO-1, GD2, CEA, MelanA/MART1, Ras mutant, gp100,
p53 mutant, Proteinase3 (PR1), bcr-abl, Tyrosinase, Survivin,
hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP,
EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, Androgen
receptor, Cyclin B1, Polysialic acid, MYCN, RhoC, TRP-2, GD3,
Fucosyl GM1, Mesothelin, PSCA, MAGE A1, sLe(a), CYP1B1, PLAC1, GM3,
BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic
anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4,
SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP,
PDGFR-.beta., MAD-CT-2, Fos-related antigen 1.
[0147] Production of antibodies used in the present invention
involves in vivo or in vitro procedures or combinations thereof.
Methods for producing polyclonal anti-receptor peptide antibodies
are well-known in the art, such as in U.S. Pat. No. 4,493,795 (to
Nestor et al). A monoclonal antibody is typically made by fusing
myeloma cells with the spleen cells from a mouse that has been
immunized with the desired antigen (Kohler, G.; Milstein, C.
(1975). Nature 256: 495-497). The detailed procedures are described
in "Antibodies--A Laboratory Manual", Harlow and Lane, eds., Cold
Spring Harbor Laboratory Press, New York (1988), which is
incorporated herein by reference. Particularly monoclonal
antibodies are produced by immunizing mice, rats, hamsters or any
other mammal with the antigen of interest such as the intact target
cell, antigens isolated from the target cell, whole virus,
attenuated whole virus, and viral proteins. Splenocytes are
typically fused with myeloma cells using polyethylene glycol (PEG)
6000. Fused hybrids are selected by their sensitivity to HAT
(hypoxanthine-aminopterin-thymine). Hybridomas producing a
monoclonal antibody useful in practicing this invention are
identified by their ability to immunoreact specified receptors or
inhibit receptor activity on target cells.
[0148] A monoclonal antibody used in the present invention can be
produced by initiating a monoclonal hybridoma culture comprising a
nutrient medium containing a hybridoma that secretes antibody
molecules of the appropriate antigen specificity. The culture is
maintained under conditions and for a time period sufficient for
the hybridoma to secrete the antibody molecules into the medium.
The antibody-containing medium is then collected. The antibody
molecules can then be further isolated by well-known techniques,
such as using protein-A, or protein-G affinity chromatography;
anion, cation, hydrophobic, or size exclusive chromatographies
(particularly by affinity for the specific antigen after Protein A
or G, and sizing column chromatography); centrifugation,
differential solubility, or by any other standard technique for the
purification of proteins.
[0149] Media useful for the preparation of these compositions are
both well-known in the art and commercially available and include
synthetic culture media. An exemplary synthetic medium is
Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol.
8:396 (1959)) supplemented with 4.5 gm/l glucose, 20 mm glutamine,
20% fetal calf serum and with an anti-foaming agent, such as
polyoxyethylene-polyoxypropylene block copolymer.
[0150] In addition, antibody-producing cell lines can also be
created by techniques other than cell fusion, such as direct
transformation of B lymphocytes with oncogenic DNA, or transfection
with an oncovirus, such as Epstein-Barr virus (EBV, also called
human herpesvirus 4 (HHV-4)) or Kaposi's sarcoma-associated
herpesvirus (KSHV). See, U.S. Pat. Nos. 4,341,761; 4,399,121;
4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632;
4,493,890. A monoclonal antibody may also be produced via an
anti-receptor peptide or peptides containing the carboxyl terminal
as described well-known in the art. See Niman et al., Proc. Natl.
Acad. Sci. USA, 80: 4949-4953 (1983); Geysen et al., Proc. Natl.
Acad. Sci. USA, 82: 178-182 (1985); Lei et al. Biochemistry 34(20):
6675-6688, (1995). Typically, the anti-receptor peptide or a
peptide analog is used either alone or conjugated to an immunogenic
carrier, as the immunogen for producing anti-receptor peptide
monoclonal antibodies.
[0151] There are also a number of other well-known techniques for
making monoclonal antibodies as binding molecules in this
invention. Particularly useful are methods of making fully human
antibodies. One method is phage display technology which can be
used to select a range of human antibodies binding specifically to
the antigen using methods of affinity enrichment. Phage display has
been thoroughly described in the literature and the construction
and screening of phage display libraries are well known in the art,
see, e.g., Dente et al, Gene. 148(1):7-13 (1994); Little et al,
Biotechnol Adv. 12(3):539-55 (1994); Clackson et al., Nature
352:264-268 (1991); Huse et al., Science 246:1275-1281 (1989),
Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (2001)
(O'Brien et al., ed., Human Press, Totowa, N.J.), and in certain
embodiments, in Lee et al. J. Mol. Biol. 340:1073-1093 (2004).
[0152] Moncolonal antibodies derived by hybridoma technique from
another species than human, such as mouse, can be humanized to
avoid human anti-mouse antibodies when infused into humans. Among
the more common methods of humanization of antibodies are
complementarity-determining region grafting and resurfacing. These
methods have been extensively described, see e.g. U.S. Pat. Nos.
5,859,205 and 6,797,492; Liu et al, Immunol Rev. 222:9-27 (2008);
Almagro et al, Front Biosci. 1;13:1619-33 (2008); Lazar et al, Mol
Immunol. 44(8):1986-98 (2007); Li et al, Proc. Natl. Acad. Sci.
USA. 103(10):3557-62 (2006) each incorporated herein by reference.
Fully human antibodies can also be prepared by immunizing
transgenic mice, rabbits, monkeys, or other mammals, carrying large
portions of the human immunoglobulin heavy and light chains, with
an immunogen. Examples of such mice are: the Xenomouse
(Abgenix/Amgen.), the HuMAb-Mouse (Medarex/BMS), the VelociMouse
(Regeneron), see also U.S. Pat. Nos. 6,596,541, 6,207,418,
6,150,584, 6,111,166, 6,075,181, 5,922,545, 5,661,016, 5,545,806,
5,436,149 and 5,569,825. In human therapy, murine variable regions
and human constant regions can also be fused to construct called
"chimeric antibodies" that are considerably less immunogenic in man
than murine mAbs (Kipriyanov et al, Mol Biotechnol. 26: 39-60
(2004); Houdebine, Curr Opin Biotechnol. 13: 625-9 (2002) each
incorporated herein by reference). In addition, site-directed
mutagenesis in the variable region of an antibody can result in an
antibody with higher affinity and specificity for its antigen
(Brannigan et al, Nat Rev Mol Cell Biol. 3: 964-70, (2002)); Adams
et al, J Immunol Methods. 231: 249-60 (1999)) and exchanging
constant regions of a mAb can improve its ability to mediate
effector functions of binding and cytotoxicity.
[0153] Antibodies immunospecific for a malignant cell antigen can
also be obtained commercially or produced by any method known to
one of skill in the art such as, e.g., chemical synthesis or
recombinant expression techniques. The nucleotide sequence encoding
antibodies immunospecific for a malignant cell antigen can be
obtained commercially, e.g., from the GenBank database or a
database like it, the literature publications, or by routine
cloning and sequencing.
[0154] DNA encoding hybridoma-derived monoclonal antibodies or
phage display Fv clones of the antibody can be readily isolated and
sequenced using conventional procedures (e.g. by using
oligonucleotide primers designed to specifically amplify the heavy
and light chain coding regions of interest from hybridoma or phage
DNA template). Once isolated, the DNA can be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of the desired monoclonal antibodies in the
recombinant host cells (Skerra et al., Curr. Opinion in Immunol.,
5: 256 (1993) and Pluckthun, Immunol. Revs, 130: 151 (1992)).
Antibodies can also be produced by using an expression system in
which the quantitative ratio of expressed polypeptide components
can be modulated in order to maximize the yield of secreted and
properly assembled antibodies. Such modulation is accomplished at
least in part by simultaneously modulating translational strengths
for the polypeptide components. After the fermentation which is
known in the art, the produced antibody protein is further purified
to obtain preparations that are substantially homogeneous for
further assays and uses. Standard protein purification methods
known in the art can be employed. The exemplary purification
procedures: fractionation on immunoaffinity (such as Protein A
columns) or ion-exchange columns, ethanol precipitation, reverse
phase HPLC, chromatography on silica or on a cation-exchange resin
such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate
precipitation, and gel filtration using, for example, Sephadex
G-75.
[0155] Apart from an antibody, a peptide or protein that
bind/block/target or in some other way interact with the epitopes
or corresponding receptors on a targeted cell can be used as a
binding molecule. These peptides or proteins could be any random
peptide or proteins that have an affinity for the epitopes or
corresponding receptors and they don't necessarily have to be of
the immunoglobulin family. These peptides can be isolated by
similar techniques as for phage display antibodies (Szardenings, J
Recept Signal Transduct Res. 23(4): 307-49, 2003). The use of
peptides from such random peptide libraries can be similar to
antibodies and antibody fragments. The binding molecules of
peptides or proteins may be conjugated on or linked to a large
molecules or materials, such as, but is not limited, an albumin, a
polymer, a liposome, a nano particle, as long as such attachment
permits the peptide or protein to retain its antigen binding
specificity.
[0156] Any one of several different reactive groups on a cell
binding agent, preferably on an antibody, can be a conjugation
site, such as .epsilon.-amino groups in lysine residues, pendant
carbohydrate moieties, carboxylic acid groups, disulfide groups,
and thiol groups. For reviews on antibody reactive groups suitable
for conjugation, see, e.g., Hermanson, G. T. (2008). Bioconjugate
Techniques, Academic Press; Garnett, Adv. Drug Delivery Rev. 53
(2001), 171-216 and Dubowchik and Walker, Pharmacology &
Therapeutics 83 (1999), 67-123, the disclosures of which are
incorporated herein by reference.
[0157] The cytotoxic agents of this invention can be directly
conjugated (linked) to a cell binding agent, or via a bifunctional
linker or a crosslinking agent to a cell binding agent. The
bifunctional linker possess two reactive groups; one of which is
capable of reacting with a cell binding agent while the other one
reacts with one or more molecules of cytotoxic agent of the
invention. The bifunctional crosslinkers are well known in the art
(see, for example, U.S. Pat. No. 5,208,020; Isalm and Dent in
"Bioconjugation" chapter 5, p 218-363, Groves Dictionaries Inc. New
York, 1999). Examples of bifunctional linker are:
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB),
N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP),
N-succinimidyl-3-(2-pyridyldithio)-butyrate (SDPB),
2-iminothiolane, N-succinimidyl-4-(5-nitro-2-pyridyldithio)
butyrate (SNPB), N-succinimidyl
4-(5-nitro-2-pyridyldithio)-pentanoate (SNPP),
N-sulfosuccinimidyl-4-(5-nitro-2-pyridyldithio) butyrate (SSNPB),
N-succinimidyl-4-methyl-4-(5-nitro-2-pyridyldithio)pentanoate
(SMNP), N-sulfosuccinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate
(SSNPP),
4-succinimidyl-oxycarbonyl-.alpha.-methyl-.alpha.-(2-pyridyldithio)-tolue-
ne (SMPT),
N-sulfosuccinimidyl-4-methyl-4-(5-nitro-2-pyridyldithio)pentano-
ate (SSMNP); N-succinimidyl-4-methyl-4-(2-pyridyldithio)pentanoate
(SMPDP),
N-succinimidyl-4-(5-N,N-dimethyl-carboxamido-2-pyridyldithio)
butyrate (SCPB),
N-sulfosuccinimidyl-4-(5-N,N-dimethyl-carboxamido-2-pyridyldithio)
butyrate (SSCPB),
N-succinimidyl-4,4-dimethyl-4-(2-pyridyldithio)pentanoate (SDMPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB),
bis-maleimidopolyethyleneglycol (BMPEG), BM(PEG).sub.1.about.20,
N-(.beta.-maleimidopropyloxy)-succinimide ester (BMPS),
iminothiolane (IT), dimethyl adipimidate HCl or derivatives of
imidoesters, active esters (such as disuccinimidyl suberate),
aldehydes (such as glutaraldehyde), bis-azido compounds (such as
bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such
as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such
as toluene 2,6-diisocyanate), and bis-active fluorine compounds
(such as 1,5-difluoro-2,4-dinitrobenzene), gamma-maleimidobutyric
acid N-succinimidyl ester (GMBS), E-maleimidocaproic acid
N-hydroxysuccinimide ester (EMCS), 5-maleimidovaleric acid NHS,
HBVS,
N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproa-
te) (a "long chain" analog of SMCC (LC-SMCC)),
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),
4-(4-N-maleimidophenyl)-butyric acid hydrazide or HCl salt (MPBH),
N-succinimidyl 3-(bromoacetamido)propionate (SBAP), N-succinimidyl
iodoacetate (SIA), kappa-maleimidoundecanoic acid N-succinimidyl
ester (KMUA), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB),
succinimidyl-6-(beta-maleimidopropionamido)-hexanoate (SMPH),
succinimidyl-(4-vinylsulfonyl)benzoate (SVSB),
dithiobis-maleimidoethane (DTME), 1,4-bis-maleimidobutane (BMB),
1,4 bismaleimidyl-2,3-dihydroxybutane (BMDB), bis-maleimidohexane
(BMH), bis-maleimidoethane (BMOE), sulfosuccinimidyl
4-(N-maleimido-methyl)cyclohexane-1-carboxylate (sulfo-SMCC),
sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate (sulfo-SIAB),
m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS),
N-(gamma-maleimidobutryloxy)sulfo-succinimdeester (sulfo-GMBS),
N-(epsilon-maleimidocaproyloxy)sulfosuccimido ester (sulfo-EMCS),
N-(kappa-maleimidoundecanoyloxy)sulfosuccinimide ester
(sulfo-KMUS), and sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate
(sulfo-SMPB); or the commercially available linkers (such as from
Thermo Scientific's Pierce: Imidoester Crosslinkers: DMA (Dimethyl
adipimidate.2 HCl), DMP (Dimethyl pimelimidate.2 HCl), DMS
(Dimethyl Suberimidate.2 HCl), DTBP (Dimethyl
3,3'-dithiobispropionimidate.2 HCl); NHS-ester Crosslinkers-Amine
Reactive: BS(PEG).sub.5 (Bis(succinimidyl) penta(ethylence glycol),
BS(PEG).sub.9 (Bis(succinimidyl) nona(ethylence glycol), BS.sup.3
(Bis[sulfosuccinimidyl] suberate), BSOCOES
(Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone), DSG
(Disuccinimidyl glutarate), DSP (Dithiobis[succinimidyl
propionate]), DSS (Disuccinimidyl suberate), DST (Disuccinimidyl
tartarate), DTSSP (3,3'-Dithiobis[sulfosuccinimidylpropionate]),
EGS (Ethylene glycol bis[succinimidylsuccinate]), Sulfo-EGS
(Ethylene glycol bis[sulfosuccinimidylsuccinate]), TSAT
(Tris-succinimidyl aminotriacetate), DFDNB
(1,5-Difluoro-2,4-dinitrobenzene); Amine-to-Sulfhydryl
Crosslinkers: Sulfo-SIAB (Sulfosuccinimidyl
(4-iodoacetyl)aminobenzoate), SIAB (Succinimidyl
(4-iodoacetyl)aminobenzoate), SBAP (Succinimidyl
3-(bromoacetamido)propionate), SIA (Succinimidyl iodoacetate),
Sulfo-SMCC
(Sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate),
SM(PEG)n (NHS-PEG-Maleimide Crosslinkers:
Succinimidyl-([N-maleimidopropionamido])-#ethyleneglycol)ester, #=1
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22,
24), LC-SMCC (Succinimidyl 4-(N-maleimidomethyl)
cyclohexane-1-carboxy-(6-amidocaproate)), Sulfo-EMCS
(N-epsilon-Maleimidocaproyl-oxysulfosuccinimide ester), EMCS
(N-epsilon-Malemidocaproyl-oxysuccinimide ester), Sulfo-GMBS
(N-gamma-Maleimidobutyryl-oxysulfosuccinimide ester), GMBS
(N-gamma-Maleimidobutyryl-oxysuccinimide ester), Sulfo-KMUS
(N-kappa-Maleimidoundecanoyl-oxysulfosuccinimide ester), Sulfo-MBS
(m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), MBS
(m-Maleimidobenzoyl-N-hydroxysuccinimide ester), Sulfo-SMPB
((Sulfosuccinimidyl 4-(p-maleiimidophenyl)butyrate), SMPB
(Succinimidyl 4-(p-maleimidophenyl)butyrate), AMAS
N-(.alpha.-Maleimidoacetoxy) succinimide ester), BMPS
(N-beta-Maleimidopropyl-oxysuccinimide ester), SMPH (Succinimidyl
6-[(beta-maleimidopropionamido)hexanoate]), PEG12-SPDP
(2-Pyridyldithiol-tetraoxaoctatriacontane-N-hydroxysuccinimide),
PEG4-SPDP
(2-Pyridyldithiol-tetraoxatetradecane-N-hydroxysuccinimide),
Sulfo-LC-SPDP (Sulfosuccinimidyl
6-[3'-(2-pyridyldithio)propionamido]hexanoate), LC-SPDP
(Succinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate), SMPT
(4-Succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)toluene);
Carboxyl-to-Amine Crosslinkers: DCC (Dicyclohexylcarbodiimide), EDC
(1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide); Photoreactive
Crosslinkers: ANB-NOS (N-5-Azido-2-nitrobenzoyloxysuccinimide),
NHS-Diazirine (SDA) Crosslinkers: SDA (NHS-Diazirine) (Succinimidyl
4,4'-azipentanoate), LC-SDA (NHS-LC-Diazirine) (Succinimidyl
6-(4,4'-azipentanamido)hexanoate), SDAD (NHS-SS-Diazirine)
(Succinimidyl
2-([4,4'-azipentanamido]ethyl)-1,3'-dithiopropionate), Sulfo-SDA
(Sulfo-NHS-Diazirine) (Sulfosuccinimidyl 4,4'-azipentanoate),
Sulfo-LC-SDA (Sulfo-NHS-LC-Diazirine) (Sulfosuccinimidyl
6-(4,4'-azipentanamido)hexanoate), Sulfo-SDAD
(Sulfo-NHS-SS-Diazirine) (Sulfosuccinimidyl
2-([4,4'-azipentanamido]ethyl)-1,3'-dithiopropionate), Sulfo-SANPAH
(Sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)-hexanoate), SPB
(Succinimidyl-[4-(psoralen-8-yloxy)]-butyrate);
Sulfhydryl-to-Carbohydrate Crosslinkers: BMPH
(N-beta-Maleimidopropionic acid hydrazide-TFA), EMCH
(N-epsilon-Maleimidocaproic acid hydrazide-TFA), KMUH
(N-kappa-Maleimidoundecanoic acid hydrazide-TFA), MPBH
(4-(4-N-Maleimidophenyl)butyric acid hydrazide-HCl), PDPH
(3-(2-Pyridyldithio)propionyl hydrazide); Sulfhydryl-to-Hydroxyl
Crosslinkers: PMPI (p-Maleimidophenyl isocyanate);
Sulthydryl-to-Sulfhydryl Crosslinkers: BM(PEG)2
(1,8-Bismaleimido-diethyleneglycol), BM(PEG)3
(1,11-Bismaleimido-triethyleneglycol), BMB
(1,4-Bismaleimidobutane), BMDB
(1,4-Bismaleimidyl-2,3-dihydroxybutane), BMH (Bismaleimidohexane),
BMOE (Bismaleimidoethane), DTME (Dithiobismaleimido-ethane), TMEA
(Tris(2-maleimidoethyl)amine) and SVSB
(succinimidyl-(4-vinylsulfone)benzoate).
[0158] The bis-maleimide or bis-2-pyridyldithiol reagents allow the
attachment of the thiol group of a thiol-containing cell binding
agent (such as antibody) to a thiol-containing drug moiety, label,
or linker intermediate, in a sequential or concurrent fashion.
Other functional groups besides maleimide and pyridyldithiol, which
are reactive with a thiol group of a cell binding agent, drug
moiety, label, or linker intermediate include iodoacetamide,
bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide,
isocyanate, and isothiocyanate.
[0159] In additional embodiments, the linker may be composed of one
or more linker components. The exemplary linker components are:
[0160] 1. The Self-Immolative Linker Components:
##STR00089##
wherein the (*) atom is the point of attachment of additional
spacer or releasable linker units, or the cytotoxic agent, and/or
the binding molecule (CBA); X.sup.1, Y.sup.1, Z.sup.2 and Z.sup.3
are independently NH, or O, or S; Z.sup.1 is H, or NH, or O or S
independently. v is 0 or 1; Q.sup.1 is independently H, OH,
C.sub.1.about.C.sub.6 alkyl, (OCH.sub.2CH.sub.2).sub.nF, Cl, Br, I,
OR.sub.5, or SR.sub.5, NR.sub.5R.sub.5', N.dbd.NR.sub.5,
N.dbd.R.sub.5, NR.sub.5R.sub.5', NO.sub.2, SOR.sub.5R.sub.5',
SO.sub.2R.sub.5, SO.sub.3R.sub.5, OSO.sub.3R.sub.5,
PR.sub.5R.sub.5', POR.sub.5R.sub.5', PO.sub.2R.sub.5R.sub.5',
OPO(OR.sub.5)(OR.sub.5'), or OCH.sub.2PO(OR.sub.5(OR.sub.5')
wherein R.sub.5 and R.sub.5' are described in the Formula (I),
preferably R.sub.5 and R.sub.5' are independently selected from H,
C.sub.1.about.C.sub.8 of alkyl; C.sub.2.about.C.sub.8 of alkenyl,
alkynyl, heteroalkyl; C.sub.3.about.C.sub.8 of aryl, heterocyclic,
carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkyl
carbonyl; or pharmaceutical cation salts
[0161] 2. The Examples of Non-Self-Immolative Linker
Components:
##STR00090## ##STR00091## ##STR00092## ##STR00093##
Wherein the (*) atom is the point of attachment of additional
spacer or releasable linkers, the cytotoxic agents, and/or the
binding molecules; X.sup.1, Y.sup.1, Q.sup.1, R.sub.5, R.sub.5' and
r are as defined in Formula (I); m, n and p are 0.about.6.
[0162] 3. Exemplary linker components may include
6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"),
valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine
("ala-phe" or "af"), p-aminobenzyloxy-carbonyl ("PAB"),
N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl
4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("SMCC"),
N-Succinimidyl (4-iodo-acetyl)amino-benzoate ("SIAB"), ethyleneoxy
(--CH.sub.2CH.sub.2O--) as one or more repeating units ("EO" or
"PEO"). Additional linker components are known in the art and some
are described through this patent application.
[0163] In additional embodiments, the linker may comprise amino
acid residues. Exemplary amino acid linker components include a
dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
Exemplary dipeptides include: valine-citrulline (VC or val-cit),
alanine-phenylalanine (af or ala-phe). Exemplary tripeptides
include: glycine-valine-citrulline (gly-val-cit) and
glycine-glycine-glycine (gly-gly-gly). Amino acid residues which
comprise an amino acid linker component include those occurring
naturally, as well as minor amino acids and non-naturally occurring
amino acid analogs, such as citrulline. Amino acid linker
components can be designed and optimized in their selectivity for
enzymatic cleavage by particular enzymes, for example, a
tumor-associated protease, cathepsin B, C and D, or a plasmin
protease.
[0164] In the cell-binding agent-drug conjugates of the invention,
cell-binding agent (CBA) is conjugated to one or more drug moieties
(Drug, or PBD derivatives), e.g. about 1 to about 20 drug moieties
per cell-binding agent, through a bifunctional linker (L). The
conjugate of Formula (II-1)-(II-91) may be prepared by several
routes, employing organic chemistry reactions, conditions, and
reagents known to those skilled in the art, including: (1) the
first modification of cell-binding agent (CBA) with a crosslinker
(L) in an aqueous buffer pH 3.about.9 having optionally 0.about.30%
organic co-solvents to introduce reactive disulfide, maleimido,
haloacetyl, hydrazide, nitrile, alkynyl, alkyloxyamino or aldehyde
groups on the cell-binding agent, to form a covalent bonded CBA-L.
The CBA-L molecule then reacts with a drug moiety (Drug) of formula
(I) to generate a cell binding agent-drug conjugate; or (2) the
first modification of drug moiety (Drug) of the formula (I) with a
crosslinker (L) in organic media or in an aqueous buffer pH
3.about.9 having optionally 0.about.99% organic co-solvents to
introduce a reactive disulfide, maleimido, haloacetyl, hydrazide,
nitrite, alkynyl, alkyloxyamino, aldehyde, N-hydroxysuccinimide
(NHS) or pentafluorophenyl ester group on the drug moiety (a
covalent bonded Drug-L molecule). The Drug-L molecule then reacts
with a cell binding agent (CBA), or pre-modified CBA to generate a
cell binding agent-drug conjugate; or (3) directly through reaction
of a cell-binding agent with drug moieties of formula (I) bearing
reactive function groups of disulfide, maleimido, haloacetyl,
hydrazide, nitrile, alkynyl, alkyloxyamino, aldehyde,
N-hydroxysuccinimide (NHS) or pentafluorophenyl esters in an
aqueous buffer pH 3.about.9 having optionally 0.about.30% organic
co-solvents.
[0165] The thiol or amine groups on a cell-binding agents, such as
an antibody, are nucleophilic and capable of reacting to form
covalent bonds with electrophilic groups on linker reagents and
drug-linker intermediates including: (i) active esters such as NHS
esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and
benzyl halides, such as haloacetamides; (iii) aldehydes, ketones,
carboxyl, and maleimide groups; and (iv) disulfides, including
pyridyl disulfides, via sulfide exchange. Nucleophilic groups on a
drug moiety include, but are not limited to: amine, thiol,
hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine
carboxylate, and arylhydrazide groups capable of reacting to form
covalent bonds with electrophilic groups on linker moieties and
linker reagents.
[0166] Nucleophilic groups on antibodies or proteins can react to
electrophilic groups on a function linker following by reaction
with a cytotoxic agent, or directly react to a linker-cytotoxic
agent moiety to form covalent bond conjugate of a cell binding
agent-cytotoxic agent. Nucleophilic groups on antibodies or
proteins include, but are not limited to: (i) N-terminal amine
groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain
thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino
groups where the antibody is glycosylated. Amine, thiol, and
hydroxyl groups are nucleophilic and capable of reacting to form
covalent bonds with electrophilic groups on linker moieties and
linker-cytotoxic agent moieties including: (i) active esters such
as NHS esters, HOBt esters, haloformates, and acid halides; (ii)
alkyl and benzyl halides such as haloacetamides; (iii) aldehydes,
ketones, carboxyl, and maleimide groups. Certain antibodies have
reducible interchain disulfides, i.e. cysteine bridges which may be
made reactive by treatment with a reducing agent such as DTT
(dithiothreitol), L-glutathione (GSH), dithioerythritol (DTE),
2-mercaptoethylamine (.beta.-MEA), beta mercaptoethanol (.beta.-ME,
2-ME), or tricarbonylethylphosphine (TCEP) (Getz et al (1999) Anal.
Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.). Each
cysteine bridge will thus form, theoretically, two reactive thiol
nucleophiles. Alternatively, sulfhydryl groups can be introduced
into antibodies through modification of lysine residues, e.g., by
reacting lysine residues with 2-iminothiolane (Traut's reagent),
resulting in conversion of an amine into a thiol. Reactive thiol
groups may be introduced into an antibody by introducing one, two,
three, four, or more cysteine residues (e.g., by preparing variant
antibodies comprising one or more non-native cysteine amino acid
residues). Thus free thiol on the cell binding agents can be
conjugated to the thiol-reactive groups, such as, a maleimide, an
iodoacetamide, a pyridyl disulfide, or other thiol-reactive groups
on the cytotoxic agents, or linker-cytotoxic agent intermediates of
the invention. Some unconjugated free thiols on the antibodies can
be reoxidized to reform interchain and intrachain disulfide
bonds.
[0167] Antibody-drug conjugates of the invention may also be
produced by reaction between an electrophilic group on an antibody,
such as an aldehyde or ketone carbonyl group, with a nucleophilic
group on a linker reagent or drug. Useful nucleophilic groups on a
linker reagent include, but are not limited to, hydrazide, oxime,
amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and
arylhydrazide. In one embodiment, an antibody is modified to
introduce electrophilic moieties that are capable of reacting with
nucleophilic substituents on the linker reagent or drug. In another
embodiment, the sugars of glycosylated antibodies may be oxidized,
e.g. with periodate oxidizing reagents, to form aldehyde or ketone
groups which may react with the amine group of linker reagents or
drug moieties. The resulting imine Schiff base groups may form a
stable linkage, or may be reduced, e.g. by borohydride reagents to
form stable amine linkages. In one embodiment, reaction of the
carbohydrate portion of a glycosylated antibody with either
galactose oxidase or sodium meta-periodate may yield carbonyl
(aldehyde and ketone) groups in the antibody that can react with
appropriate groups on the drug (Hermanson, Bioconjugate
Techniques). In another embodiment, antibodies containing
N-terminal serine or threonine residues can react with sodium
meta-periodate, resulting in production of an aldehyde in place of
the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate
Chem. 3:138146; U.S. Pat. No. 5,362,852). Such an aldehyde can be
reacted with a drug moiety or linker nucleophile.
[0168] Examples of the general conjugations which a linker reacts
to a drug to form a linker-drug intermediate first, following by
conjugation reaction with a cell-binding molecule are depicted
below:
##STR00094## ##STR00095##
wherein E includes, but is not limited to, such as
hydroxysuccinimidyl esters (NHS, Sulfo-NHS, etc), 4-nitrophenyl
esters, pentafluorophenyl esters, tetrafluorophenyl (includes
sulfo-tetrafluorophenyl) esters, anhydrides, acid chlorides,
sulfonyl chlorides, isocyanates and isothiocyanates. R' and R'' are
independently Fl or CH.sub.3, or C.sub.2H.sub.5; J is F, Cl, Br, I,
tosylate (TsO), mesylate (MsO), nitrophenol, dinitrophenol, or
pentafluorophenol; wherein Drug is a compound of the Formula (I),
(Ia), (Ib), (Ic) or (Id) of this invention.
[0169] It is to be understood that where more than one nucleophilic
group on the cell binding agents, such as an antibody, reacts with
a drug-linker intermediate or linker reagent followed by drug
moiety reagent, then the resulting product is a mixture of the cell
binding agent-cytoxic agent conjugates with a distribution of one
or more drug moieties attached to an antibody. The average number
of drugs per antibody may be calculated from the mixture by a dual
ELISA antibody assay, which is specific for antibody and specific
for the drug. Individual conjugate molecules may be identified in
the mixture by mass spectroscopy and separated by HPLC, e.g.
hydrophobic interaction chromatography. In certain embodiments, a
homogeneous conjugate with a single loading value may be isolated
from the conjugation mixture by electrophoresis or
chromatography.
[0170] In yet another embodiment, when an IgG antibody conjugated
with one, or two, or more the same or different of derivatives of
amanita toxins of Formula (I), (Ia), (Ib), (Ic) or (Id), optionally
together with other different function molecules or cytotoxic
agents, through a pair of thiols on the IgG antibody, as exampled
in formula (II-11), (II-12), (II-13), (II-14), (II-17), (II-18),
(II-19), (II-20), (II-24), (II-25), (II-29), (II-30), (II-32),
(II-33), (II-34), (II-35), (II-36), (II-37), (II-38), (II-39),
(II-40), (II-41), (II-42), (II-43), (II-44), (II-45), (II-46),
(II-47), (II-48), (II-49), (II-50), (II-51), (II-52), (II-53), and
(II-64), the ADCs containing an amanita toxin of Formula (I), (Ia),
(Ib), (Ic) or (Id) are preferred to be formed via a bridge linker
specifically at the pair of thiols (through reduction of the
disulfide bonds) between the light chain and heavy chain, or/and
the upper disulfide bonds between the two heavy chains, or/and the
lower disulfide bonds between the two heavy chains. The ADCs
containing one, or two, or more the same or different of
derivatives of amanita toxins of Formula (I), (Ia), (Ib), (Ic) or
(Id) with a bridge linker are preferred having the following
formula of (III-1), (III-2), (III-3), (III-4), (III-5), (III-6),
(III-7), (III-8), (III-9), (III-10), (III-11), or (III-12):
Wherein the bold structure is an IgG antibody, preferably IgG1,
IgG2, IgG3 or IgG4 antibody; "" represents either a single bond or
a double bond; L.sub.1 and L.sub.2 are, the same or deferent,
independently defined the same as L in Formula (I); X.sub.1 and
X.sub.2, are, the same or deferent, independently selected from NH,
N(R.sub.1), O, S, CH.sub.2, or Ar, wherein R.sub.1 is
C.sub.1-C.sub.6 alkyl, Ar is aromatic or heteroaromatic ring;
wherein Drug.sub.1 and Drug.sub.2 are the same or deferent, a
derivative of amanita toxins of Formula (I), (Ia), (Ib), (Ic) or
(Id). In addition, either Drug.sub.1 or Drug.sub.2 can be absent,
but not be absent at the same time. When any single one of
Drug.sub.1 or Drug.sub.2 is a derivative of Amanita toxin of
Formula (I), (Ia), (Ib), (Ic) or (Id), the other one of either
Drug.sub.1 or Drug.sub.2 can be selected from
(OCH.sub.2CH.sub.2).sub.rOR.sub.10, or
(OCH.sub.2CH(CH.sub.3)).sub.pOR.sub.10, or
NH(CH.sub.2CH.sub.2O).sub.pR.sub.10, or
NH(CH.sub.2CH(CH.sub.3)O).sub.pR.sub.10, or
N[(CH.sub.2CH.sub.2O).sub.pR.sub.10][(CH.sub.2CH.sub.2O).sub.rR.sub.5],
or (OCH.sub.2CH.sub.2).sub.pCOOR.sub.10, or
CH.sub.2CH2(OCH.sub.2CH.sub.2).sub.pCOOR.sub.10, wherein p, r,
R.sub.10 are described in many embodiments throughout this patent
application. In further addition, Drug.sub.2 and L.sub.2 can be
absent, thus X.sub.2 is NH.sub.2 or OH.
[0171] In yet another embodiment, when an IgG antibody conjugated
with one derivative of amanita toxins of Formula (I), (Ia), (Ib),
(Ic) or (Id) through a pair of thiols of an IgG antibody, the ADCs
containing an amanita toxin of Formula (I), (Ia), (Ib), (Ic) or
(Id) are also preferred to be formed via a bridge linker
specifically at the pair of thiols of IgG antibody (through
reduction of the disulfide bonds) between the light chain and heavy
chain, the upper disulfide bonds between the two heavy chains, and
the lower disulfide bonds between the two heavy chains. The bridge
linkage is preferred having the following structures of (IV-1),
(IV-2), (IV-3), (IV-4), (IV-5), and (IV-6):
##STR00096##
wherein"" represents either a single bond or a double bond; Drug is
a derivative of Amanita toxin of Formula (I), (Ia), (Ib), (Ic) or
(Id);
##STR00097##
represents the sites on an antibody; L, X, n and R.sub.12 are
defined the same as in Formula (I).
[0172] The pairs of thiols on the antibody are preferred to be
reduced by a reducing agent selected from dithiothreitol (DTT),
dithioerythritol (DTE), L-glutathione (GSH), tris (2-carboxyethyl)
phosphine (TCEP), 2-mercaptoethylamine (.beta.-MEA), or/and beta
mercaptoethanol (.beta.-ME, 2-ME). More preferably, these reducing
agents are loaded or covalently bonded to a solid polymer or a
solid particle. The polymer or the particle can be polyethene,
polyacrylate, silican, crossed-linked silica (e.g.
2-mercaptoethyl)silica, (aminoethyl)silica, (aminopropyl)silica),
polyethylene terephthalate, polyethylene glycol, polystyrene,
poly(isopropyl acrylate), dextrans (e.g. sephadex, cross-linked
dextran), isopropylacrylamide butyl methacrylate copolymer,
polysaccharide polymer (e.g. agarose, agar, agaropectin,
sepharose). Under the reduction of the polymer bonded reducing
agents, the pairs of disulfide bonds can be selectively reduced at
a certain position, for instance, at the hinge region of IgG1,IgG2,
IgG3 or IgG4 antibody, or disulfide bonds between heavy chain and
the light chain of the IgG1,IgG2, IgG3 or IgG4 antibody, or the
certain outer surfaces of a protein or a cell binding molecule.
Thus the loading ratios as well as the positions of derivatives of
amanita toxins conjugated on the cell binding molecules are
controlled.
[0173] In addition, when any single one of either Drug.sub.1 or
Drug.sub.2 of Formula (III-1), (III-2), (III-3), (III-4), (III-5),
(III-6), (III-7), (III-8), (III-9), (III-10), (III-11), or
(III-12), is a derivative of Amanita toxin of Formula (I), the
other one of either Drug.sub.1 or Drug.sub.2 can be selected from a
protein, an antibody, antibody dimers, antibody multimers, a
bispecific or trispecific antibody, a single chain antibody, an
antibody fragment that binds to the target cell, a monoclonal or
polyclonal antibody, a chromophore molecule, a tubulysin
derivative, a maytansinoid, a taxanoid (taxane), a CC-1065 analog,
a daunorubicin or doxorubicin compound, a benzodiazepine dimer
(e.g., dimers of pyrrolobenzodiazepine (PBD), tomaymycin,
anthramycin, indolinobenzodiazepines, imidazobenzothiadiazepines,
or oxazolidinobenzodiazepines), a calicheamicin or the enediyne
antibiotic, an actinomycin, an azaserine, a bleomycin, a
epirubicin, a tamoxifen, a idarubicin, a dolastatins, an auristatin
derivative (e.g. monomethyl auristatin E, MMAE, MMAF, auristatin
PYE, auristatin TP, Auristatins 2-AQ, 6-AQ, EB (AEB), and EFP
(AEFP)), a duocarmycin, a thiotepa, a vincristine, a hemiasterlin,
a nazumamide, a microginin, a radiosumins, an alterobactin, a
microsclerodermin, a theonellamide, an esperamicin, a PNU-159682, a
geldanamycin, a methotrexate, a vindesines, a siRNA, a nucleolytic
enzyme. The examples of the preferred structures of some of these
function molecules or cytotoxic drugs are illustrated below:
##STR00098## ##STR00099## ##STR00100## ##STR00101## ##STR00102##
##STR00103##
wherein R.sub.10 are described in Formula (I),
##STR00104##
is the site to link either linker L.sub.1 or linker L.sub.2.
[0174] In conjugation, the loading (drug/antibody ratio) and/or
position of an ADC with these derivatives of amanita toxins of this
invention can be controlled in other different ways, e.g., by: (i)
limiting the molar excess of drug-linker intermediate or linker
reagent relative to antibody, (ii) limiting the conjugation
reaction time or temperature, (iii) partial or limiting reductive
conditions for cysteine thiol modification, (iv) engineering by
recombinant techniques the amino acid sequence of the antibody such
that the number and position of lysine, carbohydrate, serine,
tyrosine, glutamine, histidine, cysteine or the residues is
modified, or unnatural amino acids are inserted to the antibody
sequences. These engineering methods are, but not limited, thioMab,
thioFab, thiocarbohydrate, selenocysteine, cell-based approach of
unnatural amino acids, open cell-free synthesis (OCFS) of unnatural
amino acids, oxidation or chemo-enzymatic modification of glycans,
Formylglycine generating enzyme (FGE), Sortase or
Transglutaminases, Conjugation via Fc-Binding Domains (FcBD) or via
the Nucleotide Binding Site, or Catalytic antibodies) (Dennler, P.
et al, Antibodies 2015, 4, 197-224)
[0175] Other further exemplary methods for preparing ADC of these
derivatives of amanita toxins are described in FIGS. 1-28 and
examples 1-70 in the description of the patent.
[0176] Treatments of Cell-Binding Agent-Drug Conjugates of These
Derivatives of Amanita Toxins
[0177] It is contemplated that the cell-binding agent-drug
conjugate, preferably antibody-drug conjugates (ADC) of the present
invention may be used to treat various diseases or disorders, e.g.
characterized by the overexpression of a tumor antigen. Exemplary
conditions or hyperproliferative disorders include benign or
malignant tumors; leukemia and lymphoid malignancies. Others
include neuronal, glial, astrocytal, hypothalamic, glandular,
macrophagal, epithelial, stromal, blastocoelic, inflammatory,
angiogenic and immunologic, including autoimmune disorders.
[0178] In specific embodiment, the conjugates of the invention are
used in accordance with the compositions and methods of the
invention for the treatment of cancers. The cancers include, but
are not limited, Adrenocortical Carcinoma, Anal Cancer, Bladder
Cancer, Brain Tumor (Adult, Brain Stem Glioma, Childhood,
Cerebellar Astrocytoma, Cerebral Astrocytoma, Ependymoma,
Medulloblastoma, Supratentorial Primitive Neuroectodermal and
Pineal Tumors, Visual Pathway and Hypothalamic Glioma), Breast
Cancer, Carcinoid Tumor, Gastrointestinal, Carcinoma of Unknown
Primary, Cervical Cancer, Colon Cancer, Endometrial Cancer,
Esophageal Cancer, Extrahepatic Bile Duct Cancer, Ewings Family of
Tumors (PNET), Extracranial Gel in Cell Tumor, Eye Cancer,
Intraocular Melanoma, Gallbladder Cancer, Gastric Cancer (Stomach),
Germ Cell Tumor, Extragonadal, Gestational Trophoblastic Tumor,
Head and Neck Cancer, Hypopharyngeal Cancer, Islet Cell Carcinoma,
Kidney Cancer (renal cell cancer), Laryngeal Cancer, Leukemia
(Acute Lymphoblastic, Acute Myeloid, Chronic Lymphocytic, Chronic
Myelogenous, Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer,
Lung Cancer (Non-Small Cell, Small Cell, Lymphoma (AIDS-Related,
Central Nervous System, Cutaneous T-Cell, Hodgkin's Disease,
Non-Hodgkin's Disease, Malignant Mesothelioma, Melanoma, Merkel
Cell Carcinoma, Metasatic Squamous Neck Cancer with Occult Primary,
Multiple Myeloma, and Other Plasma Cell Neoplasms, Mycosis
Fungoides, Myelodysplastic Syndrome, Myeloproliferative Disorders,
Nasopharyngeal Cancer, Neuroblastoma, Oral Cancer, Oropharyngeal
Cancer, Osteosarcoma, Ovarian Cancer (Epithelial, Germ Cell Tumor,
Low Malignant Potential Tumor), Pancreatic Cancer (Exocrine, Islet
Cell Carcinoma), Paranasal Sinus and Nasal Cavity Cancer,
Parathyroid Cancer, Penile Cancer, Pheochromocytoma Cancer,
Pituitary Cancer, Plasma Cell Neoplasm, Prostate Cancer
Rhabdomyosarcoma, Rectal Cancer, Renal Cell Cancer (kidney cancer),
Renal Pelvis and Ureter (Transitional Cell), Salivary Gland Cancer,
Sezary Syndrome, Skin Cancer, Skin Cancer (Cutaneous T-Cell
Lymphoma, Kaposi's Sarcoma, Melanoma), Small Intestine Cancer, Soft
Tissue Sarcoma, Stomach Cancer, Testicular Cancer, Thymoma
(Malignant), Thyroid Cancer, Urethral Cancer, Uterine Cancer
(Sarcoma), Unusual Cancer of Childhood, Vaginal Cancer, Vulvar
Cancer, Wilms' Tumor
[0179] In another specific embodiment, the compounds and the
conjugates of the invention are used in accordance with the
compositions and methods of the invention for the treatment or
prevention of an autoimmune disease. The autoimmune diseases
include, but are not limited, Achlorhydra Autoimmune Active Chronic
Hepatitis, Acute Disseminated Encephalomyelitis, Acute hemorrhagic
leukoencephalitis, Addison's Disease, Agammaglobulinemia, Alopecia
areata, Amyotrophic Lateral Sclerosis, Ankylosing Spondylitis,
Anti-GBM/TBM Nephritis, Antiphospholipid syndrome, Anti synthetase
syndrome, Arthritis, Atopic allergy, Atopic Dermatitis, Autoimmune
Aplastic Anemia, Autoimmune cardiomyopathy, Autoimmune hemolytic
anemia, Autoimmune hepatitis, Autoimmune inner ear disease,
Autoimmune lymphoproliferative syndrome, Autoimmune peripheral
neuropathy, Autoimmune pancreatitis, Autoimmune polyendocrine
syndrome Types I, II, & III, Autoimmune progesterone
dermatitis, Autoimmune thrombocytopenic purpura, Autoimmune
uveitis, Balo disease/Balo concentric sclerosis, Bechets Syndrome,
Berger's disease, Bickerstaffs encephalitis, Blau syndrome, Bullous
Pemphigoid, Castleman's disease, Chagas disease, Chronic Fatigue
Immune Dysfunction Syndrome, Chronic inflammatory demyelinating
polyneuropathy, Chronic recurrent multifocal ostemyelitis, Chronic
lyme disease, Chronic obstructive pulmonary disease, Churg-Strauss
syndrome, Cicatricial Pemphigoid, Coeliac Disease, Cogan syndrome,
Cold agglutinin disease, Complement component 2 deficiency, Cranial
arteritis, CREST syndrome, Crohns Disease (a type of idiopathic
inflammatory bowel diseases), Cushing's Syndrome, Cutaneous
leukocytoclastic angiitis, Dego's disease, Dercum's disease,
Dermatitis herpetiformis, Dermatomyositis, Diabetes mellitus type
1, Diffuse cutaneous systemic sclerosis, Dressler's syndrome,
Discoid lupus erythematosus, Eczema, Endometriosis,
Enthesitis-related arthritis, Eosinophilic fasciitis, Epidermolysis
bullosa acquisita, Erythema nodosum, Essential mixed
cryoglobulinemia, Evan's syndrome, Fibrodysplasia ossificans
progressiva, Fibromyalgia, Fibromyositis, Fibrosing aveolitis,
Gastritis, Gastrointestinal pemphigoid, Giant cell arteritis,
Glomerulonephritis, Goodpasture's syndrome, Graves' disease,
Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's
thyroiditis, Haemolytic anaemia, Henoch-Schonlein purpura, Herpes
gestationis, Hidradenitis suppurativa, Hughes syndrome (See
Antiphospholipid syndrome), Hypogammaglobulinemia, Idiopathic
Inflammatory Demyelinating Diseases, Idiopathic pulmonary fibrosis,
Idiopathic thrombocytopenic purpura (See Autoimmune
thrombocytopenic purpura), IgA nephropathy (Also Berger's disease),
Inclusion body myositis, Inflammatory demyelinating polyneuopathy,
Interstitial cystitis, Irritable Bowel Syndrome, Juvenile
idiopathic arthritis, Juvenile rheumatoid arthritis, Kawasaki's
Disease, Lambert-Eaton myasthenic syndrome, Leukocytoclastic
vasculitis, Lichen planus, Lichen sclerosus, Linear IgA disease
(LAD), Lou Gehrig's Disease (Also Amyotrophic lateral sclerosis),
Lupoid hepatitis, Lupus erythematosus, Majeed syndrome, Meniere's
disease, Microscopic polyangiitis, Miller-Fisher syndrome, Mixed
Connective Tissue Disease, Morphea, Mucha-Habermann disease,
Muckle-Wells syndrome, Multiple Myeloma, Multiple Sclerosis,
Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica
(Devic's Disease), Neuromyotonia, Occular cicatricial pemphigoid,
Opsoclonus myoclonus syndrome, Ord thyroiditis, Palindromic
rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders
Associated with Streptococcus), Paraneoplastic cerebellar
degeneration, Paroxysmal nocturnal hemoglobinuria, Parry Romberg
syndrome, Parsonnage-Turner syndrome, Pars planitis, Pemphigus,
Pemphigus vulgaris, Pernicious anaemia, Peri venous
encephalomyelitis, POEMS syndrome, Polyarteritis nodosa,
Polymyalgia rheumatica, Polymyositis, Primary biliary cirrhosis,
Primary sclerosing cholangitis, Progressive inflammatory
neuropathy, Psoriasis, Psoriatic Arthritis, Pyoderma gangrenosum,
Pure red cell aplasia, Rasmussen's encephalitis, Raynaud
phenomenon, Relapsing polychondritis, Reiter's syndrome, Restless
leg syndrome, Retroperitoneal fibrosis, Rheumatoid arthritis,
Rheumatoid fever, Sarcoidosis, Schizophrenia, Schmidt syndrome,
Schnitzler syndrome, Scleritis, Scleroderma, Sjogren's syndrome,
Spondyloarthropathy, Sticky blood syndrome, Still's Disease, Stiff
person syndrome, Subacute bacterial endocarditis, Susac's syndrome,
Sweet syndrome, Sydenham Chorea, Sympathetic ophthalmia, Takayasu's
arteritis, Temporal arteritis (giant cell arteritis), Tolosa-Hunt
syndrome, Transverse Myelitis, Ulcerative Colitis (a type of
idiopathic inflammatory bowel diseases), Undifferentiated
connective tissue disease, Undifferentiated spondyloarthropathy,
Vasculitis, Vitiligo, Wegener's granulomatosis, Wilson's syndrome,
Wiskott-Aldrich syndrome
[0180] In another specific embodiment, a binding molecule used for
the conjugate for the treatment or prevention of an autoimmune
disease includes, but are not limited to, anti-elastin antibody;
Abys against epithelial cells antibody; Anti-Basement Membrane
Collagen Type IV Protein antibody; Anti-Nuclear Antibody; Anti ds
DNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG; anti-celiac
antibody; Anti Phospholipid Antibody IgK, IgG; Anti SM Antibody;
Anti Mitochondrial Antibody; Thyroid Antibody; Microsomal Antibody,
T-cells antibody; Thyroglobulin Antibody, Anti SCL-70; Anti-Jo;
Anti-U.sub.1RNP; Anti-La/SSB; Anti SSA; Anti SSB; Anti Perital
Cells Antibody; Anti Histones; Anti RNP; C-ANCA; P-ANCA; Anti
centromere; Anti-Fibrillarin, and Anti GBM Antibody,
Anti-ganglioside antibody; Anti-Desmogein 3 antibody; Anti-p62
antibody; Anti-sp100 antibody; Anti-Mitochondrial (M2) antibody;
Rheumatoid factor antibody; Anti-MCV antibody; Anti-topoisomerase
antibody; Anti-neutrophil cytoplasmic (cANCA) antibody.
[0181] In certain preferred embodiments, the binding molecule for
the conjugate in the present invention, can bind to either a
receptor or a receptor complex expressed on an activated lymphocyte
which is associated with an autoimmune disease. The receptor or
receptor complex can comprise an immunoglobulin gene superfamily
member (e.g. CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD28, CD30,
CD33, CD37, CD38, CD56, CD70, CD79, CD90, CD152/CTLA-4, PD-1, or
ICOS), a TNF receptor superfamily member (e.g. CD27, CD40,
CD95/Fas, CD134/OX40, CD137/4-1BB, INF-R1, TNFR-2, RANK, TACI,
BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4,
and APO-3), an integrin, a cytokine receptor, a chemokine receptor,
a major histocompatibility protein, a lectin (C-type, S-type, or
I-type), or a complement control protein.
[0182] In another specific embodiment, useful binding ligands that
are immunospecific for a viral or a microbial antigen are humanized
or human monoclonal antibodies. As used herein, the term "viral
antigen" includes, but is not limited to, any viral peptide,
polypeptide protein (e.g. HIV gp120, HIV nef, RSV F glycoprotein,
influenza virus neuramimidase, influenza virus hemagglutinin, HTLV
tax, herpes simplex virus glycoprotein (e.g. gB, gC, gD, and gE)
and hepatitis B surface antigen) that is capable of eliciting an
immune response. As used herein, the term "microbial antigen"
includes, but is not limited to, any microbial peptide,
polypeptide, protein, saccharide, polysaccharide, or lipid molecule
(e.g. a bacteria, fungi, pathogenic protozoa, or yeast polypeptide
including, e.g., LPS and capsular polysaccharide 5/8) that is
capable of eliciting an immune response. Examples of antibodies
available 1 for the viral or microbial infection include, but are
not limited to, Palivizumab which is a humanized anti-respiratory
syncytial virus monoclonal antibody for the treatment of RSV
infection; PRO542 which is a CD4 fusion antibody for the treatment
of HIV infection; Ostavir which is a human antibody for the
treatment of hepatitis B virus; PROTVIR which is a humanized
IgG.sub.1 antibody for the treatment of cytomegalovirus; and
anti-LPS antibodies.
[0183] The binding molecules-cytotoxic agent conjugates of this
invention can be used in the treatment of infectious diseases.
These infectious diseases include, but are not limited to,
Acinetobacter infections, Actinomycosis, African sleeping sickness
(African trypanosomiasis), AIDS (Acquired immune deficiency
syndrome), Amebiasis, Anaplasmosis, Anthrax, Arcanobacterium
haemolyticum infection, Argentine hemorrhagic fever, Ascariasis,
Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereus
infection, Bacterial pneumonia, Bacterial vaginosis, Bacteroides
infection, Balantidiasis, Baylisascaris infection, BK virus
infection, Black piedra, Blastocystis hominis infection,
Blastomycosis, Bolivian hemorrhagic fever, Borrelia infection,
Botulism (and Infant botulism), Brazilian hemorrhagic fever,
Brucellosis, Burkholderia infection, Buruli ulcer, Calicivirus
infection (Norovirus and Sapovirus), Campylobacteriosis,
Candidiasis (Moniliasis; Thrush), Cat-scratch disease, Cellulitis,
Chagas Disease (American trypanosomiasis), Chancroid, Chickenpox,
Chlamydia, Chlamydophila pneumoniae infection, Cholera,
Chromoblastomycosis, Clonorchiasis, Clostridium difficile
infection, Coccidioidomycosis, Colorado tick fever, Common cold
(Acute viral rhinopharyngitis; Acute coryza), Creutzfeldt-Jakob
disease, Crimean-Congo hemorrhagic fever, Cryptococcosis,
Cryptosporidiosis, Cutaneous larva migrans, Cyclosporiasis,
Cysticercosis, Cytomegalovirus infection, Dengue fever,
Dientamoebiasis, Diphtheria, Diphyllobothriasis, Dracunculiasis,
Ebola hemorrhagic fever, Echinococcosis, Ehrlichiosis, Enterobiasis
(Pinworm infection), Enterococcus infection, Enterovirus infection,
Epidemic typhus, Erythema infectiosum (Fifth disease), Exanthem
subitum, Fasciolopsiasis, Fasciolosis, Fatal familial insomnia,
Filariasis, Food poisoning by Clostridium perfringens, Free-living
amebic infection, Fusobacterium infection, Gas gangrene
(Clostridial myonecrosis), Geotrichosis,
Gerstmann-Straussler-Scheinker syndrome, Giardiasis, Glanders,
Gnathostomiasis, Gonorrhea, Granuloma inguinale (Donovanosis),
Group A streptococcal infection, Group B streptococcal infection,
Haemophilus influenzae infection, Hand, foot and mouth disease
(HFMD), Hantavirus Pulmonary Syndrome, Helicobacter pylori
infection, Hemolytic-uremic syndrome, Hemorrhagic fever with renal
syndrome, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D,
Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infection,
Human bocavirus infection, Human ewingii ehrlichiosis, Human
granulocytic anaplasmosis, Human metapneumovirus infection, Human
monocytic ehrlichiosis, Human papillomavirus infection, Human
parainfluenza virus infection, Hymenolepiasis, Epstein-Barr Virus
Infectious Mononucleosis (Mono), Influenza, Isosporiasis, Kawasaki
disease, Keratitis, Kingella kingae infection, Kuru, Lassa fever,
Legionellosis (Legionnaires' disease), Legionellosis (Pontiac
fever), Leishmaniasis, Leprosy, Leptospirosis, Listeriosis, Lyme
disease (Lyme borreliosis), Lymphatic filariasis (Elephantiasis),
Lymphocytic choriomeningitis, Malaria, Marburg hemorrhagic fever,
Measles, Melioidosis (Whitmore's disease), Meningitis,
Meningococcal disease, Metagonimiasis, Microsporidiosis, Molluscum
contagiosum, Mumps, Murine typhus (Endemic typhus), Mycoplasma
pneumonia, Mycetoma, Myiasis, Neonatal conjunctivitis (Ophthalmia
neonatorum), (New) Variant Creutzfeldt-Jakob disease (vCJD, nvCJD),
Nocardiosis, Onchocerciasis (River blindness),
Paracoccidioidomycosis (South American blastomycosis),
Paragonimiasis, Pasteurellosis, Pediculosis capitis (Head lice),
Pediculosis corporis (Body lice), Pediculosis pubis (Pubic lice,
Crab lice), Pelvic inflammatory disease, Pertussis (Whooping
cough), Plague, Pneumococcal infection, Pneumocystis pneumonia,
Pneumonia, Poliomyelitis, Prevotella infection, Primary amoebic
meningoencephalitis, Progressive multifocal leukoencephalopathy,
Psittacosis, Q fever, Rabies, Rat-bite fever, Respiratory syncytial
virus infection, Rhinosporidiosis, Rhinovirus infection,
Rickettsial infection, Rickettsialpox, Rift Valley fever, Rocky
mountain spotted fever, Rotavirus infection, Rubella,
Salmonellosis, SARS (Severe Acute Respiratory Syndrome), Scabies,
Schistosomiasis, Sepsis, Shigellosis (Bacillary dysentery),
Shingles (Herpes zoster), Smallpox (Variola), Sporotrichosis,
Staphylococcal food poisoning, Staphylococcal infection,
Strongyloidiasis, Syphilis, Taeniasis, Tetanus (Lockjaw), Tinea
barbae (Barber's itch), Tinea capitis (Ringworm of the Scalp),
Tinea corporis (Ringworm of the Body), Tinea cruris (Jock itch),
Tinea manuum (Ringworm of the Hand), Tinea nigra, Tinea pedis
(Athlete's foot), Tinea unguium (Onychomycosis), Tinea versicolor
(Pityriasis versicolor), Toxocariasis (Ocular Larva Migrans),
Toxocariasis (Visceral Larva Migrans), Toxoplasmosis,
Trichinellosis, Trichomoniasis, Trichuriasis (Whipworm infection),
Tuberculosis, Tularemia, Ureaplasma urealyticum infection,
Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Viral
pneumonia, West Nile Fever, White piedra (Tinea blanca), Yersinia
pseudotuberculosis infection, Yersiniosis, Yellow fever,
Zygomycosis.
[0184] The binding molecules, more preferred antibodies described
in this patent that are against pathogenic strains include, but are
not limit, Acinetobacter baumannii, Actinomyces israelii,
Actinomyces gerencseriae and Propionibacterium propionicus,
Trypanosoma brucei, HIV (Human immunodeficiency virus), Entamoeba
histolytica, Anaplasma genus, Bacillus anthracis, Arcanobacterium
haemolyticum, Junin virus, Ascaris lumbricoides, Aspergillus genus,
Astroviridae family, Babesia genus, Bacillus cereus, multiple
bacteria, Bacteroides genus, Balantidium coli, Baylisascaris genus,
BK virus, Piedraia hortae, Blastocystis hominis, Blastomyces
dermatitides, Machupo virus, Borrelia genus, Clostridium botulinum,
Sabia, Brucella genus, usually Burkholderia cepacia and other
Burkholderia species, Mycobacterium ulcerans, Caliciviridae family,
Campylobacter genus, usually Candida albicans and other Candida
species, Bartonella henselae, Group A Streptococcus and
Staphylococcus, Trypanosoma cruzi, Haemophilus ducreyi, Varicella
zoster virus (VZV), Chlamydia trachomatis, Chlamydophila
pneumoniae, Vibrio cholerae, Fonsecaea pedrosoi, Clonorchis
sinensis, Clostridium difficile, Coccidioides immitis and
Coccidioides posadasii, Colorado tick fever virus, rhinoviruses,
coronaviruses, CJD prion, Crimean-Congo hemorrhagic fever virus,
Cryptococcus neoformans, Cryptosporidium genus, Ancylostoma
braziliense; multiple parasites, Cyclospora cayetanensis, Taenia
solium, Cytomegalovirus, Dengue viruses (DEN-1, DEN-2, DEN-3 and
DEN-4)-Flaviviruses, Dientamoeba fragilis, Corynebacterium
diphtheriae, Diphyllobothrium, Dracunculus medinensis, Ebolavirus,
Echinococcus genus, Ehrlichia genus, Enterobius vermicularis,
Enterococcus genus, Enterovirus genus, Rickettsia prowazekii,
Parvovirus B19, Human herpesvirus 6 and Human herpesvirus 7,
Fasciolopsis buski, Fasciola hepatica and Fasciola gigantica, FFI
prion, Filarioidea superfamily, Clostridium perfringens,
Fusobacterium genus, Clostridium perfringens; other Clostridium
species, Geotrichum candidum, GSS prion, Giardia intestinalis,
Burkholderia mallei, Gnathostoma spinigerum and Gnathostoma
hispidum, Neisseria gonorrhoeae, Klebsiella granulomatis,
Streptococcus pyogenes, Streptococcus agalactiae, Haemophilus
influenzae, Enteroviruses, mainly Coxsackie A virus and Enterovirus
71, Sin Nombre virus, Helicobacter pylori, Escherichia coli
O157:H7, Bunyaviridae family, Hepatitis A Virus, Hepatitis B Virus,
Hepatitis C Virus, Hepatitis D Virus, Hepatitis E Virus, Herpes
simplex virus 1, Herpes simplex virus 2, Histoplasma capsulatum,
Ancylostoma duodenale and Necator americanus, Hemophilus
influenzae, Human bocavirus, Ehrlichia ewingii, Anaplasma
phagocytophilum, Human metapneumovirus, Ehrlichia chaffeensis,
Human papillomavirus, Human parainfluenza viruses, Hymenolepis nana
and Hymenolepis diminuta, Epstein-Barr Virus, Orthomyxoviridae
family, Isospora belli, Kingella kingae, Klebsiella pneumoniae,
Klebsiella ozaenas, Klebsiella rhinoscleromotis, Kuru prion, Lassa
virus, Legionella pneumophila, Legionella pneumophila, Leishmania
genus, Mycobacterium leprae and Mycobacterium lepromatosis,
Leptospira genus, Listeria monocytogenes, Borrelia burgdorferi and
other Borrelia species, Wuchereria bancrofti and Brugia malayi,
Lymphocytic choriomeningitis virus (LCMV), Plasmodium genus,
Marburg virus, Measles virus, Burkholderia pseudomallei, Neisseria
meningitides, Metagonimus yokagawai, Microsporidia phylum,
Molluscum contagiosum virus (MCV), Mumps virus, Rickettsia typhi,
Mycoplasma pneumoniae, numerous species of bacteria
(Actinomycetoma) and fungi (Eumycetoma), parasitic dipterous fly
larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD
prion, Nocardia asteroides and other Nocardia species, Onchocerca
volvulus, Paracoccidioides brasiliensis, Paragonimus westermani and
other Paragonimus species, Pasteurella genus, Pediculus humanus
capitis, Pediculus humanus corporis, Phthirus pubis, Bordetella
pertussis, Yersinia pestis, Streptococcus pneumoniae, Pneumocystis
jirovecii, Poliovirus, Prevotella genus, Naegleria fowleri, JC
virus, Chlamydophila psittaci, Coxiella bumetii, Rabies virus,
Streptobacillus monilifoirnis and Spirillum minus, Respiratory
syncytial virus, Rhinosporidium seeberi, Rhinovirus, Rickettsia
genus, Rickettsia akari, Rift Valley fever virus, Rickettsia
rickettsii, Rotavirus, Rubella virus, Salmonella genus, SARS
coronavirus, Sarcoptes scabiei, Schistosoma genus, Shigella genus,
Varicella zoster virus, Variola major or Variola minor, Sporothrix
schenckii, Staphylococcus genus, Staphylococcus genus,
Staphylococcus aureus, Streptococcus pyogenes, Strongyloides
stercoralis, Treponema pallidum, Taenia genus, Clostridium tetani,
Trichophyton genus, Trichophyton tonsurans, Trichophyton genus,
Epidermophyton floccosum, Trichophyton rubrum, and Trichophyton
mentagrophytes, Trichophyton rubrum, Hortaea werneckii,
Trichophyton genus, Malassezia genus, Toxocara canis or Toxocara
cati, Toxoplasma gondii, Trichinella spiralis, Trichomonas
vaginalis, Trichuris trichiura, Mycobacterium tuberculosis,
Francisella tularensis, Ureaplasma urealyticum, Venezuelan equine
encephalitis virus, Vibrio colerae, Guanarito virus, West Nile
virus, Trichosporon beigelii, Yersinia pseudotuberculosis, Yersinia
enterocolitica, Yellow fever virus, Mucorales order (Mucormycosis)
and Entomophthorales order (Entomophthoramycosis), Pseudomonas
aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila,
Edwardsiella tarda, Yersinia pestis, Shigella dysenteriae, Shigella
flexneri, Shigella sonnei, Salmonella typhimurium, Treponema
pertenue, Treponema carateneum, Borrelia vincentii, Borrelia
burgdorferi, Leptospira icterohemorrhagiae, Pneumocystis carinii,
Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma
spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Clamydia
spp.; pathogenic fungi (Aspergillus fumigatus, Candida albicans,
Histoplasma capsulatum); protozoa (Entomoeba histolytica,
Trichomonas tenas, Trichomonas hominis, Tryoanosoma gatnbiense,
Trypanosoma rhodesiense, Leishmania donovani, Leishmania tropica,
Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax,
Plasmodium falciparum, Plasmodium malaria); or Helminiths
(Schistosoma japonicum, Schistosoma mansoni, Schistosoma
haematobium, and hookworms).
[0185] Other antibodies as a binding ligand in this invention for
treatment of viral disease include, but are not limited to,
antibodies against antigens of pathogenic viruses, including as
examples and not by limitation: Poxyiridae, Herpesviridae,
Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae,
Parvoviridae, Reoviridae, Retroviridae, influenza viruses,
parainfluenza viruses, mumps, measles, respiratory syncytial virus,
rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Non-A/Non-B
Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae,
Oncovirus [such as, HBV (Hepatocellular carcinoma), HPV (Cervical
cancer, Anal cancer), Kaposi's sarcoma-associated herpesvirus
(Kaposi's sarcoma), Epstein-Barr virus (Nasopharyngeal carcinoma,
Burkitt's lymphoma, Primary central nervous system lymphoma), MCPyV
(Merkel cell cancer), SV40 (Simian virus 40), HCV (Hepatocellular
carcinoma), HTLV-I (Adult T-cell leukemia/lymphoma)], Immune
disorders caused virus: [such as Human Immunodeficiency Virus
(AIDS)]; Central nervous system virus: [such as, JCV (Progressive
multifocal leukoencephalopathy), MeV (Subacute sclerosing
panencephalitis), LCV (Lymphocytic choriomeningitis), Arbovirus
encephalitis, Orthomyxoviridae (probable) (Encephalitis
lethargica), RV (Rabies), Chandipura virus, Herpesviral meningitis,
Ramsay Hunt syndrome type II; Poliovirus (Poliomyelitis, Post-polio
syndrome), HTLV-I (Tropical spastic paraparesis)]; Cytomegalovirus
(Cytomegalovirus retinitis, HSV (Herpetic keratitis));
Cardiovascular virus [such as CBV (Pericarditis, Myocarditis)];
Respiratory system/acute viral nasopharyngitis/viral pneumonia:
[Epstein-Barr virus (EBV infection/Infectious mononucleosis),
Cytomegalovirus; SARS coronavirus (Severe acute respiratory
syndrome) Orthomyxoviridae: Influenzavirus A/B/C (Influenza/Avian
influenza), Paramyxovirus: Human parainfluenza viruses
(Parainfluenza), RSV (Human respiratory syncytial virus), hMPV];
Digestive system virus [MuV (Mumps), Cytomegalovirus
(Cytomegalovirus esophagitis); Adenovirus (Adenovirus infection);
Rotavirus, Norovirus, Astrovirus, Coronavirus; HBV (Hepatitis B
virus), CBV, HAV (Hepatitis A virus), HCV (Hepatitis C virus), HDV
(Hepatitis D virus), HEV (Hepatitis E virus), HGV (Hepatitis G
virus)]; Urogenital virus [such as, BK virus, MuV (Mumps)].
[0186] According to a further embodiment, the present invention
also concerns pharmaceutical compositions comprising the conjugate
of the invention together with a pharmaceutically acceptable
carrier for treatment of cancer and autoimmune disorders. The
method for treatment of cancer, autoimmune disorders, infectious
diseases or viral disease can be practiced in vitro, in vivo, or ex
vivo. Examples of in vitro uses include treatments of cell cultures
in order to kill all cells except for desired variants that do not
express the target antigen; or to kill variants that express
undesired antigen. Examples of ex vivo uses include treatments of
hematopoietic stem cells (HSC) prior to the performance of the
transplantation (HSCT) into the same patient in order to kill
diseased or malignant cells. For instance, clinical ex vivo
treatment to remove tumor cells or lymphoid cells from bone marrow
prior to autologous transplantation in cancer treatment or in
treatment of autoimmune disease, or to remove T cells and other
lymphoid cells from allogeneic bone marrow or tissue prior to
transplant in order to prevent graft-versus-host disease, can be
carried out as follows. Bone marrow is harvested from the patient
or other individual and then incubated in medium containing serum
to which is added the conjugate of the invention, concentrations
range from about 1 pM to 0.1 mM, for about 30 minutes to about 48
hours at about 37.degree. C. The exact conditions of concentration
and time of incubation (=dose) are readily determined by the
skilled clinicians. After incubation the bone marrow cells are
washed with medium containing serum and returned to the patient by
i.v. infusion according to known methods. In circumstances where
the patient receives other treatment such as a course of ablative
chemotherapy or total-body irradiation between the time of harvest
of the marrow and reinfusion of the treated cells, the treated
marrow cells are stored frozen in liquid nitrogen using standard
medical equipment.
[0187] For clinical in vivo use, the cell binding agent-cytotoxic
agent conjugates of this invention will be supplied as solutions or
as a lyophilized solid that can be redissolved in sterile water for
injection. Examples of suitable protocols of conjugate
administration are as follows. Conjugates are given weekly,
biweekly, triweekly or monthly for 4-24 weeks or until disease
progression or unacceptable toxicity as an i.v. bolus. Bolus doses
are given in 10 to 500 ml of normal saline to which human serum
albumin (e.g. 0.5 to 1 mL of a concentrated solution of human serum
albumin, 100 mg/mL) can be added. Dosages will be about 50 .mu.g to
20 mg/kg of body weight weekly, biweekly, triweekly or monthly i.v.
(range of 10 .mu.g to 200 mg/kg per injection). 4-24 weeks after
treatment, the patient may receive a second course of treatment.
Specific clinical protocols with regard to route of administration,
excipients, diluents, dosages, times, etc., can be determined by
the skilled clinicians. For example, the simple excipients can be
0.002%-0.5% polysorbate (polysorbate 20, polysorbate 40,
polysorbate 60, or polysorbate 80) or the other pharmaceutical
acceptable surfactant, such as sodium lauryl sulfate, triton X-100,
0.1%.about.10% of binders, such as saccharides and their
derivatives (disaccharides: sucrose, lactose, trehalose or
maltose), sugar alcohols such as xylitol, sorbitol or maltitol, or
polyethylene glycol, and 0.1%.about.10% of phaimaceutical buffering
agents such as citrate, succinate acetate, phosphate, or borate
with a certain pH in the range of pH 4.5-9.5. In certain
applications, or a dose formula can contain other excipients, such
as polysaccharides and their derivatives: starches, cellulose or
modified cellulose such as microcrystalline cellulose and cellulose
ethers such as hydroxypropyl cellulose (HPC), hypromellose
(hydroxypropyl methylcellulose (HPMC)) or Hydroxypropyl cellulose;
a protein such as gelatin, or albumin; Synthetic polymers:
polyvinylpyrrolidone (PVP), polyethylene glycol (PEG); Antioxidants
like vitamin A, vitamin E, vitamin C, retinyl palmitate, and
selenium; The amino acids such cysteine, tyrosine or methionine;
Synthetic preservatives like the parabens: methyl paraben and
propyl paraben.
[0188] Examples of medical conditions that can be treated according
to the in vivo or ex vivo methods of killing selected cell
populations include malignancy of any types of cancer, autoimmune
diseases, graft rejections, and infections (viral, bacterial or
parasite).
[0189] The amount of a conjugate which is required to achieve the
desired biological effect, will vary depending upon a number of
factors, including the chemical characteristics, the potency, and
the bioavailability of the conjugates, the type of disease, the
species to which the patient belongs, the diseased state of the
patient, the route of administration, all factors which dictate the
required dose amounts, delivery and regimen to be administered.
[0190] In general terms, the cell binding agent-cytotoxic agent
conjugates of this invention may be provided in an aqueous
physiological buffer solution containing 0.1 to 10% w/v conjugates
for parenteral administration. Typical dose ranges are from 1
.mu.g/kg to 0.1 g/kg of body weight per day, per three-days,
weekly, bi-weekly, tri-weekly or once per four weeks. A preferred
dose range is from 0.01 mg/kg to 20 mg/kg of body weight weekly,
bi-weekly, tri-weekly or monthly, or an equivalent dose in a human
child. The preferred dosage of drug to be administered is likely to
depend on such variables as the type and extent of progression of
the disease or disorder, the overall health status of the
particular patient, the relative biological efficacy of the
compound selected, the formulation of the compound, the route of
administration (intravenous, intramuscular, or other), the
pharmacokinetic properties of the compound by the chosen delivery
route, and the speed (bolus or continuous infusion) and schedule of
administrations (number of repetitions in a given period of
time).
[0191] The cell binding agent-cytotoxic agent conjugates of the
present invention are also capable of being administered in unit
dose forms, wherein the term "unit dose" means a single dose which
is capable of being administered to a patient, and which can be
readily handled and packaged, remaining as a physically and
chemically stable unit dose comprising either the active conjugate
itself, or as a pharmaceutically acceptable composition, as
described hereinafter. As such, typical total daily dose ranges are
from 0.01 to 100 mg/kg of body weight. By way of general guidance,
unit doses for humans range from 1 mg to 3000 mg per day.
Preferably the unit dose range is from 0.1 to 500 mg administered
one to three times a day, weekly, bi-weekly, tri-weekly or monthly,
and even more preferably from 1 mg to 500 mg, weekly, bi-weekly, or
tri-weekly. Conjugates provided herein can be formulated into
pharmaceutical compositions by admixture with one or more
pharmaceutically acceptable excipients. Such unit dose compositions
may be prepared for use by oral administration, particularly in the
form of tablets, simple capsules or soft gel capsules; or
intranasally, particularly in the form of powders, nasal drops, or
aerosols; or dermally, for example, topically in ointments, creams,
lotions, gels or sprays, or via trans-dermal patches. The
compositions may conveniently be administered in unit dosage foi in
and may be prepared by any of the methods well known in the
pharmaceutical art, for example, as described in Remington: The
Science and Practice of Pharmacy, 21.sup.th ed.; Lippincott
Williams & Wilkins: Philadelphia, Pa., 2005. Preferred
formulations include pharmaceutical compositions in which a
compound of the present invention is formulated for oral or
parenteral administration. For oral administration, tablets, pills,
powders, capsules, troches and the like can contain one or more of
any of the following ingredients, or compounds of a similar nature:
a binder such as microcrystalline cellulose, or gum tragacanth; a
diluent such as starch or lactose; a disintegrant such as starch
and cellulose derivatives; a lubricant such as magnesium stearate;
a glidant such as colloidal silicon dioxide; a sweetening agent
such as sucrose or saccharin; or a flavoring agent such as
peppermint, or methyl salicylate. Capsules can be in the form of a
hard capsule or soft capsule, which are generally made from gelatin
blends optionally blended with plasticizers, as well as a starch
capsule. In addition, dosage unit forms can contain various other
materials that modify the physical fowl of the dosage unit, for
example, coatings of sugar, shellac, or enteric agents. Other oral
dosage forms syrup or elixir may contain sweetening agents,
preservatives, dyes, colorings, and flavorings. In addition, the
active compounds may be incorporated into fast dissolve,
modified-release or sustained-release preparations and
formulations, and wherein such sustained-release foi inulations are
preferably bi-modal. Preferred tablets contain lactose, cornstarch,
magnesium silicate, croscarmellose sodium, povidone, magnesium
stearate, or talc in any combination. Liquid preparations for
parenteral administration include sterile aqueous or non-aqueous
solutions, suspensions, and emulsions. The liquid compositions may
also include binders, buffers, preservatives, chelating agents,
sweetening, flavoring and coloring agents, and the like.
Non-aqueous solvents include alcohols, propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and organic
esters such as ethyl oleate. Aqueous carriers include mixtures of
alcohols and water, buffered media, and saline. In particular,
biocompatible, biodegradable lactide polymer, lactide/glycolide
copolymer, or polyoxyethylene-polyoxypropylene copolymers may be
useful excipients to control the release of the active compounds.
Intravenous vehicles can include fluid and nutrient replenishers,
electrolyte replenishers, such as those based on Ringer's dextrose,
and the like. Other potentially useful parenteral delivery systems
for these active compounds include ethylene-vinyl acetate copolymer
particles, osmotic pumps, implantable infusion systems, and
liposomes.
[0192] Alternative modes of administration include formulations for
inhalation, which include such means as dry powder, aerosol, or
drops. They may be aqueous solutions containing, for example,
polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or
oily solutions for administration in the form of nasal drops, or as
a gel to be applied intranasally. Formulations for buccal
administration include, for example, lozenges or pastilles and may
also include a flavored base, such as sucrose or acacia, and other
excipients such as glycocholate. Formulations suitable for rectal
administration are preferably presented as unit-dose suppositories,
with a solid based carrier, such as cocoa butter, and may include a
salicylate. Formulations for topical application to the skin
preferably take the form of an ointment, cream, lotion, paste, gel,
spray, aerosol, or oil. Carriers which can be used include
petroleum jelly, lanolin, polyethylene glycols, alcohols, or their
combinations. Formulations suitable for transdermal administration
can be presented as discrete patches and can be lipophilic
emulsions or buffered, aqueous solutions, dissolved and/or
dispersed in a polymer or an adhesive.
[0193] In a specific embodiment, the cell binding agent-cytotoxic
agent conjugates of this invention are administered concurrently
with the other known or will be known therapeutic agents such as
the chemotherapeutic agent, the radiation therapy, immunotherapy
agents, autoimmune disorder agents, anti-infectious agents or the
other antibody-drug conjugates, resulting in a synergistic effect.
In another specific embodiment, the synergistic drugs or radiation
therapy are administered prior or subsequent to administration of a
conjugate, in one aspect at least an hour, 12 hours, a day, a week,
bi-week, tri-week, a month, in further aspects several months,
prior or subsequent to administration of a conjugate of the
invention.
[0194] In other embodiments, the synergistic drugs include, but not
limited to:
[0195] 1). Chemotherapeutic agents: a). Alkylating agents: such as
[Nitrogen mustards: (chlorambucil, cyclophosphamide, ifosfamide,
mechlorethamine, melphalan, trofosfamide); Nitrosoureas:
(carmustine, lomustine); Alkylsulphonates: (busulfan, treosulfan);
Triazenes: (dacarbazine); Platinum containing compounds:
(carboplatin, cisplatin, oxaliplatin)]; b). Plant Alkaloids: such
as [Vinca alkaloids: (vincristine, vinblastine, vindesine,
vinorelbine); Taxoids: (paclitaxel, docetaxol)]; c). DNA
Topoisomerase Inhibitors: such as [Epipodophyllins:
(9-aminocamptothecin, camptothecin, crisnatol, etoposide, etoposide
phosphate, irinotecan, teniposide, topotecan,); Mitomycins:
(mitomycin C)]; d). Anti-metabolites: such as {[Anti-folate: DHFR
inhibitors: (methotrexate, trimetrexate); IMP dehydrogenase
Inhibitors: (mycophenolic acid, tiazofurin, ribavirin, EICAR);
Ribonucleotide reductase Inhibitors: (hydroxyurea, deferoxamine)];
[Pyrimidine analogs: Uracil analogs: (5-Fluorouracil,
doxifluridine, floxuridine, ratitrexed (Tomudex)); Cytosine
analogs: (cytarabine, cytosine arabinoside, fludarabine); Purine
analogs: (azathioprine, mercaptopurine, thioguanine)]}; e).
Hormonal therapies: such as {Receptor antagonists: [Anti-estrogen:
(megestrol, raloxifene, tamoxifen); LHRH agonists: (goserelin,
leuprolide acetate); Anti-androgens: (bicalutamide, flutamide)];
Retinoids/Deltoids: [Vitamin D3 analogs: (CB 1093, EB 1089 KH 1060,
cholecalciferol, ergocalciferol); Photodynamic therapies:
(verteporfin, phthalocyanine, photosensitizer Pc4,
demethoxy-hypocrellin A); Cytokines: (Interferon-alpha,
Interferon-gamma, tumor necrosis factor (TNFs), human proteins
containing a TNF domain)]}; f). Kinase inhibitors, such as BMW 2992
(anti-EGFR/Erb2), imatinib, gefitinib, pegaptanib, sorafenib,
dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib,
pazopanib, vandetanib, E7080 (anti-VEGFR2), mubritinib, ponatinib
(AP24534), bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib,
vismodegib, iniparib, ruxolitinib, CYT 387, axitinib, tivozanib,
sorafenib, bevacizumab, cetuximab, Trastuzumab, Ranibizumab,
Panitumumab, ispinesib; g). Others: such as gemcitabine,
epoxomicins (e. g. carfilzomib), bortezomib, thalidomide,
lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032,
STA-9090, Stimuvax, allovectin-7, Xegeva, Provenge, Yervoy,
Isoprenylation inhibitors (such as Lovastatin), Dopaminergic
neurotoxins (such as 1-methyl-4-phenylpyridinium ion), Cell cycle
inhibitors (such as staurosporine), Actinomycins (such as
Actinomycin D, dactinomycin), Bleomycins (such as bleomycin A2,
bleomycin B2, peplomycin), Anthracyclines (such as daunorubicin,
doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin,
zonibicin, mtoxantrone, MDR inhibitors (such as verapamil),
Ca.sup.2+ATPase inhibitors (such as thapsigargin), vismodegib,
Histone deacetylase inhibitors (Vorinostat, Romidepsin,
Panobinostat, Valproic acid, Mocetinostat (MGCD0103), Belinostat,
PCI-24781, Entinostat, SB939, Resminostat, Givinostat, AR-42,
CUDC-101, sulforaphane, Trichostatin A); Thapsigargin, Celecoxib,
glitazones, epigallocatechin gallate, Disulfiram, Salinosporamide
A. More detail lists of known and will be known anti-cancer drugs
that can be used as a combination therapy (a synergistic effect)
with the compounds and conjugates of the invention can be seen in
National Cancer Institute (US) website (www.cancer.gov;
www.cancer.govicancertopics/druginfo/alphalist), American Cancer
Society (www.cancer.org/treatment/index) and Cancer Research UK
(www.cancerrearchuk.org;
(www.cancerresearchuk.org/cancer-help/about-cancer/treatment/cancer-drugs-
/)
[0196] 2). An anti-autoimmune disease agent includes, but is not
limited to, cyclosporine, cyclosporine A, aminocaproic acid,
azathioprine, bromocriptine, chlorambucil, chloroquine,
cyclophosphamide, corticosteroids (e.g. amcinonide, betamethasone,
budesonide, hydrocortisone, flunisolide, fluticasone propionate,
fluocortolone danazol, dexamethasone, Triamcinolone acetonide,
beclometasone dipropionate), DHEA, enanercept, hydroxychloroquine,
infliximab, meloxicam, methotrexate, mofetil, mycophenylate,
prednisone, sirolimus, tacrolimus.
[0197] 3). An anti-infectious disease agent includes, but is not
limited to, a). Aminoglycosides: amikacin, astromicin, gentamicin
(netilmicin, sisomicin, isepamicin), hygromycin B, kanamycin
(amikacin, arbekacin, bekanamycin, dibekacin, tobramycin), neomycin
(framycetin, paromomycin, ribostamycin), netilmicin, spectinomycin,
streptomycin, tobramycin, verdamicin; b). Amphenicols:
azidamfenicol, chloramphenicol, florfenicol, thiamphenicol; c).
Ansamycins: geldanamycin, herbimycin; d). Carbapenems: biapenem,
doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem;
e). Cephems: carbacephem (loracarbef), cefacetrile, cefaclor,
cefradine, cefadroxil, cefalonium, cefaloridine, cefalotin or
cefalothin, cefalexin, cefaloglycin, cefamandole, cefapirin,
cefatrizine, cefazaflur, cefazedone, cefazolin, cefbuperazone,
cefcapene, cefdaloxime, cefepime, cefininox, cefoxitin, cefprozil,
cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren,
cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid,
cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran,
cephalexin, cefpimizole, cefpiramide, cefpirome, cefpodoxime,
cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram,
ceftibuten, ceftiolene, ceftizoxime, ceftobiprole, ceftriaxone,
cefuroxime, cefuzonam, cephamycin (cefoxitin, cefotetan,
cefmetazole), oxacephem (flomoxef, latamoxef); f). Glycopeptides:
bleomycin, vancomycin (oritavancin, telavancin), teicoplanin
(dalbavancin), ramoplanin; g). Glycylcyclines: e. g. tigecycline;
g). .beta.-Lactamase inhibitors: penam (sulbactam, tazobactam),
clavam (clavulanic acid); i). Lincosamides: clindamycin,
lincomycin; j). Lipopeptides: daptomycin, A54145, calcium-dependent
antibiotics (CDA); k). Macrolides: azithromycin, cethromycin,
clarithromycin, dirithromycin, erythromycin, flurithromycin,
josamycin, ketolide (telithromycin, cethromycin), midecamycin,
miocamycin, oleandomycin, rifamycins (rifampicin, rifampin,
rifabutin, rifapentine), rokitamycin, roxithromycin, spectinomycin,
spiramycin, tacrolimus (FK506), troleandomycin, telithromycin; l).
Monobactams: aztreonam, tigemonam; m). Oxazolidinones: linezolid;
n). Penicillins: amoxicillin, ampicillin (pivampicillin,
hetacillin, bacampicillin, metampicillin, talampicillin),
azidocillin, azlocillin, benzylpenicillin, benzathine
benzylpenicillin, benzathine phenoxymethylpenicillin,
clometocillin, procaine benzylpenicillin, carbenicillin
(carindacillin), cloxacillin, dicloxacillin, epicillin,
flucloxacillin, mecillinam (pivmecillinam), mezlocillin,
meticillin, nafcillin, oxacillin, penamecillin, penicillin,
pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin,
sulbenicillin, temocillin, ticarcillin; o). Polypeptides:
bacitracin, colistin, polymyxin B; p). Quinolones: alatrofloxacin,
balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin,
difloxacin, enoxacin, enrofloxacin, floxin, garenoxacin,
gatifloxacin, gemifloxacin, grepafloxacin, kano trovafloxacin,
levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin,
nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin,
trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin,
temafloxacin, tosufloxacin, trovafloxacin; q). Streptogramins:
pristinatnycin, quinupristin/dalfopristin); r). Sulfonamides:
mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilimide,
sulfasalazine, sulfisoxazole, trimethoprim,
trimethoprim-sulfamethoxazole (co-trimoxazole); s). Steroid
antibacterials: e.g. fusidic acid; t). Tetracyclines: doxycycline,
chlortetracycline, clomocycline, demeclocycline, lymecycline,
meclocycline, metacycline, minocycline, oxytetracycline,
penimepicycline, rolitetracycline, tetracycline, glycylcyclines
(e.g. tigecycline); u). Other types of antibiotics: annonacin,
arsphenamine, bactoprenol inhibitors (Bacitracin), DADAL/AR
inhibitors (cycloserine), dictyostatin, discodermolide,
eleutherobin, epothilone, ethambutol, etoposide, faropenem, fusidic
acid, furazolidone, isoniazid, laulimalide, metronidazole,
mupirocin, mycolactone, NAM synthesis inhibitors (e. g.
fosfomycin), nitrofurantoin, paclitaxel, platensimycin,
pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampin),
tazobactam tinidazole, uvaricin;
[0198] 4). Anti-viral drugs: a). Entry/fusion inhibitors:
aplaviroc, maraviroc, vicriviroc, gp41 (enfuvirtide), PRO 140, CD4
(ibalizumab); b). Integrase inhibitors: raltegravir, elvitegravir,
globoidnan A; c). Maturation inhibitors: bevirimat, vivecon; d).
Neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e).
Nucleosides & nucleotides: abacavir, aciclovir, adefovir,
amdoxovir, apricitabine, brivudine, cidofovir, clevudine,
dexelvucitabine, didanosine (ddI), elvucitabine, emtricitabine
(FTC), entecavir, famciclovir, fluorouracil (5-FU),
3'-fluoro-substituted 2',3'-dideoxynucleoside analogues (e.g.
3'-fluoro-2',3'-dideoxythymidine (FLT) and
3'-fluoro-2',3'-dideoxyguanosine (FLG), fomivirsen, ganciclovir,
idoxuridine, latnivudine (3TC), l-nucleosides (e.g.
.beta.-l-thymidine and .beta.-l-2'-deoxycytidine), penciclovir,
racivir, ribavirin, stampidine, stavudine (d4T), taribavirin
(viramidine), telbivudine, tenofovir, trifluridine valaciclovir,
valganciclovir, zalcitabine (ddC), zidovudine (AZT); f).
Non-nucleosides: amantadine, ateviridine, capravirine,
diarylpyrimidines (etravirine, rilpivirine), delavirdine,
docosanol, emivirine, efavirenz, foscarnet (phosphonoformic acid),
imiquimod, interferon alfa, loviride, lodenosine, methisazone,
nevirapine, NOV-205, peginterferon alfa, podophyllotoxin,
rifampicin, rimantadine, resiquimod (R-848), tromantadine; g).
Protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir,
fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril,
ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h). Other
types of anti-virus drugs: abzyme, arbidol, calanolide a,
ceragenin, cyanovirin-n, diarylpyrimidines, epigallocatechin
gallate (EGCG), foscarnet, griffithsin, taribavirin (viramidine),
hydroxyurea, KP-1461, miltefosine, pleconaril, portmanteau
inhibitors, ribavirin, seliciclib.
[0199] 5). Other immunotherapy drugs: e.g. imiquimod, interferons
(e.g. .alpha., .beta.) granulocyte colony-stimulating factors,
cytokines, Interleukins (IL-1.about.IL-35), antibodies (e. g.
trastuzumab, pertuzumab, bevacizumab, cetuximab, panitumumab,
infliximab, adalimumab, basiliximab, daclizumab, omalizumab),
Protein-bound drugs (e.g., Abraxane), an antibody conjugated with
drugs selected from calicheamicin derivative, of maytansine
derivatives (DM1 and DM4), CC-1065 and duocarmycin minor groove
binders, potent taxol derivatives, doxorubicin, auristatin
antimitotic drugs (e. g. Trastuzumab-DM1, Inotuzumab ozogamicin,
Brentuximab vedotin, Glembatumumab vedotin, lorvotuzumab
mertansine, AN-152 LMB2, TP-38, VB4-845, Cantuzumab mertansine,
AVE9633, SAR3419, CAT-8015 (anti-CD22), IMGN388,
milatuzumab-doxorubicin, SGN-75 (anti-CD70), Anti-CD22-MCC-DM1,
IMGN853, Anti-CD22-MMAE, Anti-CD22-MMAF,
Anti-CD22-calicheamicin.
[0200] The invention is further illustrated but not restricted by
the description in the following examples.
EXAMPLES
Example 1
Synthesis of (S)-3-(1H-indol-2-yl)-2-(tritylamino)propanoic acid
(1)
##STR00105##
[0202] Chlorotrimethylsilane (3.4 mL, 26.9 mmol) was added slowly
to a suspension of L-tryptophan (5.00 g, 24.5 mL) in methylene
chloride (40 mL) at r.t. The mixture was continuously stirred for
4.5 h and triethylamine (6.8 mL, 49.0 mmol) was added, followed by
a solution of triphenylmethyl chloride (7.17 g, 25.7 mmol) in
methylene chloride (20 mL). The mixture was stirred at r.t. for 20
h and then quenched with methanol (25 mL). The reaction was
concentrated to near dryness and re-dissolved in methylene
chloride, washed with 5% citric acid solution (3.times.) and brine.
The organic phase was dried over anhydrous sodium sulfate, filtered
and concentrated. The residue was further dissolved in methylene
chloride and filtered over a celite pad and the filtrate was
concentrated to give a pale white foam (11.8 g), which was used
directly in the next step. ESI MS m/z 446.5 ([M+H].sup.+).
Example 2
Synthesis of (S)-methyl 2-(3-(1H-indol-2-yl)-2-(tritylamino)propan
amido)acetate (2)
##STR00106##
[0204] To a solution of acid (9.27 g, 30.7 mmol) in THF (30 mL) was
added glycine methyl ester hydrochloride (2.85 g, 22.8 mmol) and
HOBt (3.08 g, 22.8 mmol). The mixture was cooled to 0.degree. C.
and triethylamine (7.4 mL, 51.9 mmol) was added, followed by
EDC.HCl (4.38 g, 22.8 mmol) in portions. The mixture was allowed to
warm to r.t. and stirred for 20 h and then concentrated and
redissolved in methylene chloride and washed with 5% citric acid
solution (3.times.) and brine. The organic phase was dried over
anhydrous sodium sulfate, filtered and concentrated. The residue
was triturated with ethyl acetate and a white solid was collected
by filtration (6.46 g, 65% yield over two steps). ESI MS m/z 518.2
([M+H].sup.+).
Example 3
Synthesis of Dimethyldioxirane (DMDO)
[0205] Distilled water (20 mL), acetone (30 mL), and NaHCO.sub.3
(24 g, 0.285 mol) were combined in a 1-L round-bottomed flask and
chilled in an ice/water bath with magnetic stirring for 20 min.
After 20 min, stirring was halted and Oxone (25 g, 0.0406 mol) was
added in a single portion. The flask was loosely covered and the
slurry was stirred vigorously for 15 min while still submerged in
the ice bath. The flask containing the reaction slurry was then
attached to a rotary evaporator with a bath at room temperature.
The bump bulb (250 mL) was chilled in a dry ice/acetone bath and a
vacuum of 165 mtor was applied via a benchtop diaphragm pump. After
15 min, the bath temperature was raised to 40.degree. C. over 10
min. When the bath reached 40.degree. C., the distillation was
halted immediately via releasing the vacuum and raising the flask
from the heated water bath. The pale yellow acetone solution of
DMDO was decanted from the bump bulb directly into a graduated
cylinder to measure the total volume of the solution (about 25 mL)
and then the solution was dried over Na.sub.2SO.sub.4.
[0206] The Na.sub.2SO.sub.4 is removed by filtration and rinsed
with 5 mL of acetone. Titration of the obtained DMDO solution is
then performed according to the procedure of Adam, et al (Adam, W.;
Chan, Y. Y.; Cremer, D.; Gauss, J.; Scheutzow, D.; Scheutzow, D.;
Schindler, M. J. Org. Chem. 1987, 52, 2800-2803). Results
consistently showed 2.1-2.3 mmol of DMDO in the solution. The DMDO
solution was used immediately following titration.
Example 4
Synthesis of methyl 2-(3a-hydroxy-1-trityl-1,2,3,3a,8,8a-hexahydro
pyrrolo[2,3-b]indole-2-carboxamido)acetate (3)
##STR00107##
[0208] To a solution of Trt-Trp-Gly-OMe (0.80 g, 1.54 mmol) in
methylene chloride (20 mL) at -78.degree. C. was added a solution
of DMDO in acetone (2.25 mmol). After 1 h the mixture was
concentrated to dryness under reduced pressure at room temperature.
The crude material was purified by column chromatography
(hexanes/EtOAc/Et.sub.3N 70:30:1 to 30:70:1) to give a light yellow
foam, the mixture of two diastereomers (0.58 g, 70% yield). ESI MS
m/z 534.22 ([M+H].sup.+).
Example 5
Synthesis of 2-(3a-hydroxy-1-trityl-1,2,3,3a,8,8a-hexahydropyrrolo
[2,3-b]indole-2-carboxamido)acetic acid (4)
##STR00108##
[0210] To a solution of Tr-Hpi-Gly-OMe (mixture of diastereomers)
(0.80 g, 1.50 mmol) in dioxane/water (30 mL, v/v 2:1) was added
LiOH (0.63 g, 15.0 mmol) and the reaction was stirred at r.t. for
30 min (following consumption of the starting material by TLC
(CH.sub.2Cl.sub.2/MeOH, 9:1)). The reaction mixture was evaporated
to dryness and the residue was purified by a short silica gel plug,
eluting with CH.sub.2Cl.sub.2/MeOH/Et.sub.3N (90:10:1). Fractions
were combined to yield a light yellow solid as the triethylamine
salt of the two diastereomers (0.89 g, 95% yield).
Example 6
Synthesis of (5S,11R)-methyl 5-((S)-sec-butyl)-1-(9H-fluoren
-9-yl)-3,6,9-trioxo-11-((tritylthio)methyl)-2-oxa-4,7,10-triazadodecan-12-
-oate (5)
##STR00109##
[0212] To a solution of Fmoc-Ile-Gly-OH (2.50 g, 6.09 mmol),
H-Cys(Trt)-OMe (2.76 g, 7.30 mmol), HOBt (1.11g, 7.30 mmol) in THF
(40 mL) was added DIPEA (2.6 mL, 15.3 mmol) at 0.degree. C.,
followed by EDC.HCl (1.40 g, 7.30 mmol) in portions. The reaction
was warmed to r.t. and stirred for 16 h. The reaction was
concentrated to dryness and diluted with ethyl acetate and washed
with 5% citric acid (3.times.), saturated NaHCO.sub.3 (3.times.)
and brine. The organic phase was dried over anhydrous
Na.sub.2SO.sub.4 and concentrated. The residue was purified by
column chromatography (0-20% ethyl acetate/hexanes) to give a white
solid (4.45 g, 95% yield). ESI MS m/z 770.2 ([M+H].sup.+).
Example 7
Synthesis of (6S,12R)-methyl
6-((S)-sec-butyl)-1-(3a-hydroxy-1-trityl-1,2,3,3a,8,8a-hexahydropyrrolo[2-
,3-b]indol-2-yl)-1,4,7,10-tetraoxo-12-((tritylthio)methyl)-2,5,8,11-tetraa-
zatridecan-13-oate (6)
##STR00110##
[0214] Fmoc-Ile-Gly-Cys(Trt)-OMe (4.45 g, 5.78 mmol) was mixed with
piperidine/DMF (20%, 10 mL) and stirred at r.t. for 30 min. The
reaction mixture was diluted with dichloromethane (50 mL) and
washed with water and brine. The organic phase was dried over
anhydrous Na.sub.2SO.sub.4 and concentrated. The residue was
purified by column chromatography (0-10% MeOH/CH.sub.2Cl.sub.2 with
1% Et.sub.3N) to give a white solid (3.12 g, 99% yield). ESI MS m/z
548.2 ([M+H].sup.+).
[0215] The above solid (1.04 g, 1.90 mmol) was mixed with
Trt-Hpi-Gly-OH.NEt.sub.3 (0.98 g, 1.58 mmol) in CH.sub.2Cl.sub.2
(10 mL), to which HOBt (0.29 g, 1.90 mmol) and DIPEA (0.7 mL, 4.00
mmol) were added at 0.degree. C. EDC.HCl (0.36 g, 1.90 mmol) was
added at last in portions. The reaction was warmed to r.t. and
stirred for 16 h and then diluted with ethyl acetate and washed
with 5% citric acid (3.times.), saturated NaHCO.sub.3 (3.times.)
and brine. The organic phase was dried over anhydrous
Na.sub.2SO.sub.4 and concentrated. The residue was purified by
column chromatography (20-70% ethyl acetate/hexanes) to give a
white solid (0.75 g, 45% yield). ESI MS m/z 1049.4
([M+H].sup.+).
Example 8
Synthesis of
(6S,12R)-6-((S)-sec-butyl)-1-(3a-hydroxy-1-trityl-1,2,3,3a,8,8a-hexahydro-
pyrrolo[2,3-b]indol-2-yl)-1,4,7,10-tetraoxo-12-((tritylthio)methyl)-2,5,8,-
11-tetraazatridecan-13-oic acid (7)
##STR00111##
[0217] Trt-Hpi-Gly-Ile-Gly-Cys(Trt)-OMe (0.75 g, 0.71 mmol) was
dissolved in dioxane/water (v/v 2:1, 30 mL) and treated with
LiOH.H.sub.2O (0.30 g, 7.1 mmol) at r.t. for 30 min. The reaction
mixture was evaporated to dryness and the residue was purified by a
short silica gel plug, eluting with CH.sub.2Cl.sub.2/MeOH/Et.sub.3N
(90:10:1). Fractions were combined to yield a white solid as the
triethylamine salt of Trt-Hpi-Gly-Ile-Gly-Cys(Trt)-OH (0.77 g, 95%
yield).
Example 9
Synthesis of (2S,4R)-(9H-fluoren-9-yl)methyl
4-(tert-butoxy)-2-(((2S,3S)-1-(tert-butoxy)-3-methyl-1-oxopentan-2-yl)car-
bamoyl)pyrrolidine-1-carboxylate (8)
##STR00112##
[0219] To a solution of Fmoc-Pro(O.sup.tBu)-OH (2.50 g, 6.10 mmol),
H-Ile-O.sup.tBu (1.37 g, 7.32 mmol), HOBt (1.12g, 7.32 mmol) in THF
(40 mL) was added DIPEA (2.6 mL, 15.3 mmol) at 0.degree. C.,
followed by EDC.HCl (1.40 g, 7.32 mmol) in portions. The reaction
was warmed to r.t. and stirred for 16 h. After concentration, the
residue was diluted with ethyl acetate and washed with 5% citric
acid (3.times.), saturated NaHCO.sub.3 (3.times.) and brine. The
organic phase was dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. The residue was purified by column chromatography
(0-20% ethyl acetate/hexanes) to give a white solid (2.55 g, 85%
yield). ESI MS m/z 579.3 ([M+H].sup.+).
Example 10
Synthesis of (2S,3S)-tert-butyl
2-((2S,4R)-1-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methoxy-
-4-oxobutanoyl)-4-(tert-butoxy)pyrrolidine-2-carboxamido)-3-methylpentanoa-
te (9)
##STR00113##
[0221] Fmoc-Pro(O.sup.tBu)-Ile-O.sup.tBu (2.55 g, 4.40 mmol) was
treated with 20% piperidine in DMF (20 mL) for 30 min. The reaction
mixture was concentrated and purified by column chromatography
(0-10% MeOH/CH.sub.2Cl.sub.2 with 1% Et.sub.3N) to give a white
solid (1.41 g, 90% yield). ESI MS m/z 357.2 ([M+H].sup.+).
[0222] The above solid (1.41 g, 3.96 mmol) was mixed with
Fmoc-Asp(OMe)-OH (1.22 g, 3.30 mmol) in DMF (20 mL), to which HOBt
(0.61 g, 3.96 mmol) and DIPEA (1.4 mL, 8.25 mmol) were added at
0.degree. C. EDC.HCl (0.76 g, 3.96 mmol) was added at last in
portions. The reaction was warmed to r.t. and stirred for 16 h and
then diluted with ethyl acetate and washed with H.sub.2O, 5% citric
acid (3.times.), saturated NaHCO.sub.3 (3.times.) and brine. The
organic phase was dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. The residue was purified by column chromatography
(20-70% ethyl acetate/hexanes) to give a white solid (1.78 g, 76%
yield). ESI MS m/z 708.4 ([M+H].sup.+).
Example 11
Synthesis of ((6S,12R,15S)-methyl
15-((2S,4R)-4-(tert-butoxy)-2-(((2S,3S)-1-(tert-butoxy)-3-methyl-1-oxopen-
tan-2-yl)carbamoyl)pyrrolidine-1-carbonyl)-6-((S)-sec-butyl)-1-(3a-hydroxy-
-1-trityl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indol-2-yl)-1,4,7,10,13-pen-
taoxo-12-((tritylthio)methyl)-2,5,8,11,14-pentaazaheptadecan-17-oate
(10)
##STR00114##
[0224] Fmoc-Asp(OMe)-Pro(O.sup.tBu)-Ile-O.sup.tBu (0.59 g, 0.90
mmol) was mixed with piperidine/DMF (20%, 10 mL) and stirred at
r.t. for 30 min. The reaction mixture was diluted with
dichloromethane (50 mL) and washed with water and brine. The
organic phase was dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. The residue was purified by column chromatography
(0-10% MeOH/CH.sub.2Cl.sub.2 with 1% Et.sub.3N) to give a white
solid (0.43 g, 99% yield). ESI MS m/z 486.6 ([M+H].sup.+).
[0225] The above solid (0.43 g, 0.89 mmol) was mixed with
Trt-Hpi-Gly-Ile-Gly-Cys(Trt)-OH.NEt.sub.3 (0.77 g, 0.67 mmol) in
CH.sub.2Cl.sub.2 (5 mL), to which HOBt (0.12 g, 0.80 mmol) and
DIPEA (0.33 mL, 1.88 mmol) were added at 0.degree. C. EDC.HCl (0.15
g, 0.80 mmol) was added at last in portions. The reaction was
warmed to r.t. and stirred for 18 h and then diluted with ethyl
acetate and washed with 5% citric acid (3.times.), saturated
NaHCO.sub.3 (3.times.) and brine. The organic phase was dried over
anhydrous Na.sub.2SO.sub.4 and concentrated. The residue was
purified by column chromatography (30-80% ethyl acetate/hexanes) to
give a white solid (0.50 g, 50% yield). ESI MS m/z 1502.7
([M+H].sup.+).
Example 12
Synthesis of
(2S,3S)-2-((2S,4R)-1-((S)-2-((3R,9S,15S)-15-amino-9-((S)-sec-butyl)-5,8,1-
1,14-tetraoxo-2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,21-hexadecahydro-[1,4,7-
,10,13]thiatetraazacyclooctadecino[18,17-b]indole-3-carboxamido)-4-methoxy-
-4-oxobutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-methylpentanoic
acid (11)
##STR00115##
[0227]
Trt-Hpi-Gly-Ile-Gly-Cys(Trt)-Asp(OMe)-Pro(O.sup.tBu)-Ile-O.sup.tBu
(0.50 g, 0.33 mmol) was treated with neat TFA at r.t. for 5 h.
Methanol was added and the reaction was concentrated. This was
repeated twice and the residue was purified by prep-HPLC
(H.sub.2O/MeCN) to afford a white solid (99.6 mg, 34% yield).
[0228] Fmoc-Ile-OH was Attached on the 2-Chlorotrityl Chloride
Resin According to the Following Protocol:
[0229] Fmoc-Ile-OH (0.35 g, 1.0 mmol) and DIPEA (0.70 mL, 4.0 mmol)
were dissolved in dry methylene chloride (10 mL). The resulting
solution was added to chlorotrityl resin (1.0 g, 0.911 mmol/g, GL
Biochem) and the mixture was shaken under nitrogen for 1.5 h.
Subsequently methanol (2 mL) was added and shaking continued for 30
min. The liquid was drained under vacuum and resin washed with
methylene chloride (15 mL), DMF (10 mL) and methanol (10 mL) and
dried under vacuum.
[0230] Coupling was Performed According to the Following
Protocol:
[0231] Resin was placed in a column and swollen in DMF (10 mL) for
30 min. The solvent was drained under vacuum and the N-terminal
Fmoc protecting group was cleaved by shaking with 20% piperidine in
DMF for 30 min. Following deprotection, the resin was washed with
DMF (3.times.10 mL), followed by CH.sub.2Cl.sub.2 (3.times.10 mL)
and again with DMF (3.times.10 mL). The next Fmoc protected amino
acid (Fmoc-Xaa-OH, 5 eq.) was coupled to the resin with coupling
reagent HBTU (5 eq.) and DIPEA (10 eq.) in DMF (10 mL) with shaking
for 2 h. The resin was then washed extensively with DMF (3.times.10
mL), followed by CH.sub.2Cl.sub.2 (3.times.10 mL) and DMF
(3.times.10 mL). A small sample was taken and treated with
hexafluoroisopropanol (HFIP) in CH.sub.2Cl.sub.2 for 5 min to
cleave the peptide from the resin and checked by mass spectrometry.
In case of coupling of non-commercially available amino acid, such
as Trt-Hpi-Gly-OH, fewer equivalents (3 eq.) and longer time (3 h)
were employed.
[0232] When all the couplings were completed, the resin-bounded
peptide was transferred to a round bottom flask and TFA (10 mL) was
added and stirred at r.t. for 5 h. The acid labile protecting
groups were concomitantly removed during TFA treatment. The resin
was filtered and washed with CH.sub.2Cl.sub.2 (10 mL) and methanol
(10 mL). The filtrate was concentrated and partitioned between
water and ethyl acetate. The aqueous layer was purified by
prep-HPLC (H.sub.2O/MeCN) to yield a white solid of monocyclic
octapeptide (40.3 mg, 5% yield). ESI MS m/z 888.38
([M+H].sup.+).
Example 13
Synthesis of Ile.sup.3-S-deoxo-amanitin (12)
##STR00116##
[0234] To a solution of monocyclic octapeptide (25.7 mg, 0.0289
mmol) in dry DMF (5 mL) was added EDC.HCl (27.7 mg, 0.145 mmol),
HOBt (39.0 mg, 0.289 mmol) and DIPEA (0.025 mL, 0.145 mmol). The
reaction was stirred at r.t. for 20 h and then concentrated and
purified by prep-HPLC (H.sub.2O/MeCN) to give a white solid
compound 12 (9.0 mg, 36% yield). ESI MS m/z 870.40
([M+H].sup.+).
Example 14
Synthesis of Compound 13
##STR00117##
[0236] To a solution of compound 12 (5.0 mg, 0.00575 mmol, 1.0 eq.)
in THF (1 mL) was added t-BuONO (7 .mu.L, 0.0575 mmol) at 0.degree.
C. The reaction was stirred at 0.degree. C. for 1 h then room
temperature 20 h. After water (5 mL) was added, the reaction
mixture was concentrated and purified by prep-HPLC (H.sub.2O/MeCN)
to give a white solid (2.6 mg, 50% yield). ESI MS m/z 915.38
([M+H].sup.+).
Example 15
Synthesis of Compound 14
##STR00118##
[0238] A mixture of nitro compound (2.6 mg, 0.00284 mmol) and Pd/C
(10 wt %, 10 mg) in methanol (2 mL) was hydrogenated (1 atm
H.sub.2) at r.t. for 1 h, and then filtered through Celite (filter
aid). The filtrate was concentrated to afford a white solid (2.5
mg, 99% yield). ESI MS m/z 885.38 ([M+H].sup.+).
Example 16
Synthesis of Compound 15
##STR00119##
[0240] To a solution of compound 14 (2.5 mg, 0.00283 mmol) and
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid (2.6 mg,
0.0141 mmol) in dry DMF (1 mL) was added HATU (5.4 mg, 0.0141 mmol)
and DIPEA (0.05 mL, 0.283 mmol). The reaction was stirred at r.t.
for 20 h and then diluted with ethyl acetate and washed with brine.
The organic phase was concentrated and purified by prep-HPLC
(H.sub.2O/MeCN) to give a white solid Ile.sup.3-S-deoxo-amanitin
(1.7 mg, 56% yield). ESI MS m/z 1050.41 ([M+H].sup.+).
Example 17
Synthesis of 2-(2-(dibenzylamino)ethoxy)ethanol (16)
##STR00120##
[0242] 2-(2-aminoethoxy)ethanol (21.00 g, 200 mmol, 1.0 eq.) and
K.sub.2CO.sub.3 (83.00 g, 600 mmol, 3.0 eq.) in acetonitrile (350
mL) was added BnBr(57.0 mL, 480 mmol, 2.4 eq.). The mixture was
refluxed overnight. Water (1 L) was added and extracted with EtOAc
(3.times.300 mL). The combined organic layers were washed with
brine (1000 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered,
concentrated and purified by SiO.sub.2 column chromatography (4:1
hexanes/EtOAc) to give a colorless oil (50.97 g, 89.2% yield). MS
ESI m/z [M+Na].sup.+ 309.1967.
Example 18
Synthesis of tert-butyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)
propanoate (17)
##STR00121##
[0244] To a mixture of 2-(2-(dibenzylamino)ethoxy)ethanol (47.17 g,
165.3 mmol, 1.0 eq.), tert-butyl acrylate (72.0 mL, 495.9 mmol, 3.0
eq.) and n-Bu.sub.4NI (6.10 g, 16.53 mmol, 0.1 eq.) in DCM (560 mL)
was added sodium hydroxide solution (300 mL, 50%). The mixture was
stirred overnight. The organic layer was separated and the water
layer was extracted with EtOAc (3.times.100 mL). The organic layers
were washed with water(3.times.300 mL) and brine (300 mL), dried
over anhydrous Na.sub.2SO.sub.4, filtered, concentrated and
purified by SiO.sub.2 column chromatography (7:1 hexanes/EtOAc) to
give a colorless oil (61.08 g, 89.4% yield). MS ESI m/z [M+H].sup.+
414.2384.
Example 19
Synthesis of tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate
(18)
##STR00122##
[0246] To a solution of tert-butyl
3-(2-(2-(dibenzylamino)ethoxy)ethoxy) propanoate (20.00 g, 48.36
mmol, 1.0 eq.) in THF (30 mL) and MeOH (60 mL) was added Pd/C (2.00
g, 10 wt %, 50% wet) in a hydrogenation bottle. The mixture was
shaken overnight, filtered through Celite (filter aid), and the
filtrate was concentrated to afford a colorless oil (10.58 g, 93.8%
yield). MS ESI m/z [M+H].sup.+ 234.1810.
Example 20
Synthesis of
(E)-16-bromo-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa-13-azaheptadec-15-en-1-
7-oic acid (19)
##STR00123##
[0248] To a solution of 3-bromofuran-2,5-dione (89 mg, 0.5 mmol) in
THF (5 mL), tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate (117
mg, 0.5 mmol) was added. The resulting solution was stirred at r.t.
for 4 h. The solvent was removed under vacuum to afford compound 19
(205 mg, theoretical yield). MS ESI m/z [M+H].sup.+ 410.03.
Example 21
Synthesis of (E)-1-tert-butyl 18-methyl
13-bromo-11,14-dioxo-4,7-dioxa-10,15-diazaoctadec-12-ene-1,18-dioate
(20)
##STR00124##
[0250] Compound 19 (205 mg, 0.5 mmol) and methyl 3-aminopropanoate
hydrochloride (70 mg, 0.5 mmol) were dissolved in DCM (20 mL), to
which DIPEA (0.26 mL, 1.5 mmol) and EDC.HCl (144 mg, 0.75 mmol)
were added. The resulting solution was stirred at r.t. overnight,
and then washed with brine (50 mL), dried over anhydrous
Na.sub.2SO.sub.4. Concentration and purification by column
chromatography (0 to 10% MeOH/DCM) yielded compound 20 (88 mg, 36%
yield). MS ESI m/z [M+H].sup.+ 495.25.
Example 22
Synthesis of (E)-8-bromo-3,7,10-trioxo-2,14,17-trioxa-6,11-diaza
icos-8-en-20-oic acid (21)
##STR00125##
[0252] Compound 20 (88 mg, 0.18 mmol) in DCM (3 mL) was treated
with formic acid (6 ml) at 38.degree. C. overnight. All volatiles
were removed under vacuum to yield compound 21 (78 mg, theoretical
yield).
Example 23
Synthesis of Compound 22
##STR00126##
[0254] Compound 14 (2.0 mg, 0.00226 mmol) and compound 21 (5.0 mg,
0.0113 mmol) were dissolved in DMF (1 mL), to which HATU (4.3 mg,
0.0113 mmol) and DIPEA (2.0 .mu.L, 0.0113 mmol) were added. The
resulting solution was stirred at r.t. overnight, diluted with
ethyl acetate and then washed with brine, dried over anhydrous
Na.sub.2SO.sub.4. Concentration and purification by prep-HPLC
(H.sub.2O/MeCN) gave a white solid (1.2 mg, 44% yield). MS ESI m/z
[M+H].sup.+ 1227.50.
Example 24
Synthesis of 4-(((benzyloxy)carbonyl)amino)butanoic acid (23)
##STR00127##
[0256] A solution of 4-aminobutyric acid (7.5 g, 75 mmol) and NaOH
(6 g, 150 mmol) in H.sub.2O (40 mL) was cooled to 0.degree. C. and
treated with a solution of CbzCl (16.1 g, 95 mmol) in THF (32 ml)
dropwise. After 1 h, the reaction was allowed to warm to r.t. and
stirred for 3 h. THF was removed under vacuum, the pH of the
aqueous solution was adjusted to 1.5 by addition of 6 N HCl.
Extracted with ethyl acetate, and the organic layer was washed with
brine, dried and concentrated to give compound 23 (16.4 g, 92%
yield). MS ESI m/z [M+H].sup.+ 238.08.
Example 25
Synthesis of tert-butyl 4-(((benzyloxy)carbonyl)amino)butanoate
(24)
##STR00128##
[0258] DMAP (0.8 g, 6.56 mmol) and DCC (17.1 g, 83 mmol) were added
to a solution of 4-(((benzyloxy)carbonyl)amino)butanoic acid (16.4
g, 69.2 mmol) and t-BuOH (15.4 g, 208 mmol) in DCM (100 mL). After
stirring at r.t. overnight, the reaction was filtered and filtrate
concentrated. The residue was dissolved in ethyl acetate and the
washed with 1N HCl, brine and dried over Na.sub.2SO.sub.4.
Concentration and purification by column chromatography (10 to 50%
EtOAc/hexanes) yielded compound 24 (7.5 g, 37% yield). MS ESI m/z
[M+Na].sup.+ 316.13.
Example 26
Synthesis of tert-butyl 4-aminobutanoate (25)
##STR00129##
[0260] tert-Butyl 4-(((benzyloxy)carbonyl)amino)butanoate (560 mg,
1.91 mmol) was dissolved in MeOH (50 mL), and mixed with Pd/C
catalyst (10 wt %, 100 mg) then hydrogenated (1 atm) at r.t. for 3
h. The catalyst was filtered off and all volatiles were removed
under vacuum to afford compound 25 (272 mg, 90% yield). MS ESI m/z
[M+H].sup.+ 160.13.
Example 27
Synthesis of
(E)-2-bromo-4-((4-(tert-butoxy)-4-oxobutyl)amino)-4-oxobut-2-enoic
acid (26)
##STR00130##
[0262] 3-Bromofuran-2,5-dione (300 mg, 1.71 mmol) was dissolved in
THF (20 mL), to which tert-butyl 4-aminobutanoate(272 mg, 1.71
mmol) was added and the resulting solution was stirred at r.t. for
3 h. The solvent was removed under vacuum to afford compound 26
(572 mg, theoretical yield). MS ESI m/z [M+H].sup.+ 338.04.
Example 28
Synthesis of (E)-tert-butyl 4-(3-bromo-4-((2-methoxyethyl)
amino)-4-oxobut-2-enamido)butanoate (27)
##STR00131##
[0264]
2-Bromo-4-((4-(tert-butoxy)-4-oxobutyl)amino)-4-oxobut-2-enoic
acid(286 mg, 0.85 mmol) and 2-methoxyethanamine (128 mg, 1.7 mmol)
were dissolved in DCM (40 mL), to which DIPEA (329 mg, 2.55 mmol)
and EDC.HCl (490 mg, 2.55 mmol) were added. The resulting solution
was stirred at r.t. for 24 h and then washed with brine, dried over
Na.sub.2SO.sub.4. Concentration and purification by column
chromatography (0 to 10% MeOH/DCM) yielded compound 27 (102 mg, 31%
yield). MS ESI m/z [M+H].sup.+ 393.11.
Example 29
Synthesis of
(E)-4-(3-bromo-4-((2-methoxyethyl)amino)-4-oxobut-2-enamido)butanoic
acid (28)
##STR00132##
[0266] Compound 27 (52 mg, 0.132 mmol) was dissolved in DCM (3 mL),
to which formic acid (6 ml) was added. The resulting solution was
stirred at 38.degree. C. overnight then concentrated to afford
compound 28 (45 mg, theoretical yield). MS ESI m/z [M+H].sup.+
339.05.
Example 30
Synthesis of (E)-2,5-dioxopyrrolidin-1-yl
4-(3-bromo-4-((2-methoxyethyl)amino)-4-oxobut-2-enamido)butanoate
(29)
##STR00133##
[0268] To a solution of compound 28 (45 mg, 0.132 mmol) in DCM (10
mL) NHS (23 mg, 0.199 mmol) and EDC.HCl (38 mg, 0.199 mmol) were
added. After stirring at r.t. for 3 h, the reaction was
concentrated and purified by column chromatography (10 to 50%
EtOAc/hexanes) to yield compound 29 (57 mg, 99% yield). MS ESI m/z
[M+H].sup.+ 436.06.
Example 31
Synthesis of Compound 30
##STR00134##
[0270] To a solution of compound 14 (2.0 mg, 0.00226 mmol) in
ethanol (1 mL) and phosphate buffer solution (pH 7.2, 1 mL) was
added compound 29 (4.9 mg, 0.0113 mmol) in ethanol (1 mL) over 30
min. After stirring at r.t. for 3 h, the reaction was concentrated
and purified by prep-HPLC (H.sub.2O/MeCN) to yield compound 30 (2.7
mg, 30% yield). MS ESI m/z [M+H].sup.+ 1203.40.
Example 32
Synthesis of tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)
propanoate (31)
##STR00135##
[0272] To a solution of 2,2'-(ethane-1,2-diylbis(oxy))diethanol
(55.0 mL, 410.75 mmol, 3.0 eq.) in anhydrous THF (200 mL) was added
sodium (0.1 g). The mixture was stirred until Na disappeared and
then tert-butyl acrylate (20.0 mL, 137.79 mmol, 1.0 eq.) was added
dropwise. The mixture was stirred overnight and then quenched by
HCl solution (20.0 mL, 1 N) at 0.degree. C. THF was removed by
rotary evaporation, brine (300 mL) was added and the resulting
mixture was extracted with EtOAc (3.times.100 mL). The organic
layers were washed with brine (3.times.300 mL), dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated to afford a
colorless oil (30.20 g, 79.0% yield), which was used without
further purification. MS ESI m/z [M+H].sup.+ 278.17.
Example 33
Synthesis of tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)
propanoate (32)
##STR00136##
[0274] To a solution of tert-butyl
3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy) propanoate (30.20 g, 108.5
mmol, 1.0 eq.) and TsCl (41.37 g, 217.0 mmol, 2.0 eq.) in anhydrous
DCM (220 mL) at 0.degree. C. was added TEA (30.0 mL, 217.0 mmol,
2.0 eq.). The mixture was stirred at room temperature overnight,
and then washed with water (3.times.300 mL) and brine (300 mL),
dried over anhydrous Na.sub.2SO.sub.4, filtered, concentrated and
purified by SiO.sub.2 column chromatography (3:1 hexanes/EtOAc) to
give a colorless oil (39.4 g, 84.0% yield). MS ESI m/z [M+H].sup.+
433.28.
Example 34
Synthesis of tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)
propanoate (33)
##STR00137##
[0276] To a solution of tert-butyl
3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy) propanoate (39.4 g, 91.1
mmol, 1.0 eq.) in anhydrous DMF (100 mL) was added NaN.sub.3 (20.67
g, 316.6 mmol, 3.5 eq.). The mixture was stirred at room
temperature overnight. Water (500 mL) was added and extracted with
EtOAc (3.times.300 mL). The combined organic layers were washed
with water (3.times.900 mL) and brine (900 mL), dried over
anhydrous Na.sub.2SO.sub.4, filtered, concentrated and purified by
SiO.sub.2 column chromatography (5:1 hexanes/EtOAc) to give a light
yellow oil (23.8 g, 85.5% yield). MS ESI m/z [M+Na].sup.+
326.20.
Example 35
Synthesis of tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)
propanoate (34)
##STR00138##
[0278] Raney-Ni (7.5 g, suspended in water) was washed with water
(three times) and isopropyl alcohol (three times) and mixed with
compound 33 (5.0 g, 16.5 mmol) in isopropyl alcohol. The mixture
was stirred under a H.sub.2 balloon at r.t. for 16 h and then
filtered over a Celite pad, with washing of the pad with isopropyl
alcohol. The filtrate was concentrated and purified by column
chromatography (5-25% MeOH/DCM) to give a light yellow oil (2.60 g,
57% yield). MS ESI m/z [M+H].sup.+ 279.19.
Example 36
Synthesis of di-tert-butyl
14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacont-15-yne-1,30-dioat-
e (35)
##STR00139##
[0280] Acetylenedicarboxylic acid (0.35 g, 3.09 mmol, 1.0 eq.) was
dissolved in NMP (10 mL) and cooled to 0.degree. C., to which
compound 34 (2.06 g, 7.43 mmol, 2.4 eq.) was added, followed by
DMTMM (2.39 g, 8.65 mmol, 2.8 eq.) in portions. The reaction was
stirred at 0.degree. C. for 6 h and then diluted with ethyl acetate
and washed with water and brine. The organic solution was
concentrated and triturated with a mixture solvent of ethyl acetate
and petroleum ether. The solid was filtered off and the filtrate
was concentrated and purified by column chromatography (80-90%
EA/PE) to give a light yellow oil (2.26 g, >100% yield), which
was used without further purification. MS ESI m/z [M+H].sup.+
633.30.
Example 37
Synthesis of 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diaza
triacont-15-yne-1,30-dioic acid (36)
##STR00140##
[0282] Compound 35 (2.26 g) was dissolved in dichloromethane (15
mL) and cooled to 0.degree. C. then treated with TFA (15 mL). The
reaction was warmed to r.t. and stirred for 45 min, and then the
solvent and residual TFA was removed on rotovap. The crude product
was purified by column chromatography (0-15% MeOH/DCM) to give a
light yellow oil (1.39 g, 86% yield for two steps). MS ESI m/z
[M+H].sup.+ 521.24.
Example 38
Synthesis of Compound 37
##STR00141##
[0284] Compound 14 (2.0 mg, 0.00226 mmol) and compound 36 (5.9 mg,
0.0113 mmol) were dissolved in DMF (1 mL), to which HATU (4.3 mg,
0.0113 mmol) and DIPEA (2.0 .mu.L, 0.0113 mmol) were added. The
resulting solution was stirred at r.t. overnight, and then
concentrated and purified by prep-HPLC (H.sub.2O/MeCN) to give a
white solid (0.94 mg, 30% yield). MS ESI m/z [M+H].sup.+
1387.50.
Example 39
Synthesis of methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate
(38)
##STR00142##
[0286] To a solution of maleimide (6.35 g, 65.4 mmol) in ethyl
acetate (120 mL) at 0.degree. C. was added NMM (8.6 mL, 78.5 mmol),
followed by methyl chloroformate (6.0 mL, 78.5 mmol). The reaction
was stirred at 0.degree. C. for 30 min and r.t. 1 h. The solid was
filtered off and filtrate was concentrated. The residue was
dissolved in methylene chloride and filtered through a silica gel
plug and eluted with methylene chloride to remove the red color.
After concentration, the solid was triturated with ethyl acetate
and petroleum ether to give a white solid (9.0 g, 88% yield).
Example 40
Synthesis of tert-butyl (2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)
ethyl)carbamate (39)
##STR00143##
[0288] A mixture of tert-butyl (2-aminoethyl)carbamate (11.2 mL)
and saturated NaHCO.sub.3 (120 mL) was stirred vigorously at
0.degree. C., to which compound 38 (10.0 g, 64.4 mmol) was added in
portions. After stirring at 0.degree. C. for 30 min the reaction
was warmed to r.t. and stirred for 1.0 h. The solid was then
collected by vacuum filtration and then dissolved in ethyl acetate
and washed with brine, dried over anhydrous Na.sub.2SO.sub.4,
filtered and concentrated to give a white foam (13.6 g, 87%
yield).
Example 41
Synthesis of tert-butyl
(2-(1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindol-2(3H)-yl)ethyl)ca-
rbamate (40)
##STR00144##
[0290] A mixture of compound 39 (6.0 g, 25.0 mmol) and furan (18
mL) in toluene (120 mL) was heated to 100.degree. C. in a high
pressure flask. The reaction was stirred for 16 h and then the
solvent removed. A solid was formed which was triturated with ethyl
ether to give a white solid (6.5 g, 84% yield).
Example 42
Synthesis of
2-(2-aminoethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione
hydrochloride (41)
##STR00145##
[0292] To an ice cooled solution of compound 40 (7.90 g, 25.6 mmol)
in methylene chloride (60 mL) was added HCl/dioxane (10 mL of
concentrated HCl dissolved in 30 mL of dioxane) slowly. The
reaction mixture was then warmed to r.t. and stirred for 2 h. The
process of reaction was monitored by TLC. Once completed, the
reaction was concentrated and triturated with ethyl acetate. A
white solid was collected (6.32 g, theoretical yield) by vacuum
filtration.
Example 43
Synthesis of Compound 42
##STR00146##
[0294] Compound 14 (2.0 mg, 0.00226 mmol) and
3-((tert-butoxycarbonyl)amino) propanoic acid (2.1 mg, 0.0113 mmol)
were dissolved in DMF (1 mL), to which HATU (4.3 mg, 0.0113 mmol)
and DIPEA (2.0 .mu.L, 0.0113 mmol) were added. The resulting
solution was stirred at r.t. overnight, and then concentrated and
purified by prep-HPLC
References