U.S. patent application number 14/117125 was filed with the patent office on 2016-06-02 for bio-orthogonal drug activation.
This patent application is currently assigned to KONINKLIJKE PHILIPS N.V.. The applicant listed for this patent is Freek Johannes Maria Hoeben, Marc Stefan Robillard, Raffaella Rossin, Wolter Ten Hoeve, Ronny Mathieu Versteegen. Invention is credited to Freek Johannes Maria Hoeben, Marc Stefan Robillard, Raffaella Rossin, Wolter Ten Hoeve, Ronny Mathieu Versteegen.
Application Number | 20160151505 14/117125 |
Document ID | / |
Family ID | 47176369 |
Filed Date | 2016-06-02 |
United States Patent
Application |
20160151505 |
Kind Code |
A1 |
Robillard; Marc Stefan ; et
al. |
June 2, 2016 |
BIO-ORTHOGONAL DRUG ACTIVATION
Abstract
The invention relates to a Prodrug activation method, for
therapeutics, wherein use is made of abiotic reactive chemical
groups that exhibit bio-orthogonal reactivity towards each other.
The invention also relates to a Prodrug kit comprising at least one
Prodrug and at least one Activator, wherein the Prodrug comprises a
Drug and a first Bio-orthogonal Reactive Group (the Trigger), and
wherein the Activator comprises a second Bio-orthogonal Reactive
Group. The invention also relates to targeted therapeutics used in
the above-mentioned method and kit. The invention particularly
pertains to antibody-drug conjugates and to bi- and trispecific
antibody derivatives.
Inventors: |
Robillard; Marc Stefan;
(Eindhoven, NL) ; Ten Hoeve; Wolter; (Groningen,
NL) ; Versteegen; Ronny Mathieu; (Hegelsom, NL)
; Rossin; Raffaella; (Eindhoven, NL) ; Hoeben;
Freek Johannes Maria; (Beegden, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Robillard; Marc Stefan
Ten Hoeve; Wolter
Versteegen; Ronny Mathieu
Rossin; Raffaella
Hoeben; Freek Johannes Maria |
Eindhoven
Groningen
Hegelsom
Eindhoven
Beegden |
|
NL
NL
NL
NL
NL |
|
|
Assignee: |
KONINKLIJKE PHILIPS N.V.
EINDHOVEN
NL
|
Family ID: |
47176369 |
Appl. No.: |
14/117125 |
Filed: |
May 16, 2012 |
PCT Filed: |
May 16, 2012 |
PCT NO: |
PCT/IB12/52447 |
371 Date: |
December 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61515432 |
Aug 5, 2011 |
|
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61515458 |
Aug 5, 2011 |
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Current U.S.
Class: |
424/181.1 ;
514/34; 530/391.9; 536/6.4; 544/95; 548/523 |
Current CPC
Class: |
A61K 39/3955 20130101;
C07D 237/26 20130101; Y02A 50/30 20180101; A61K 47/22 20130101;
Y02A 50/471 20180101; A61K 47/558 20170801; A61K 47/6897 20170801;
A61K 47/555 20170801; A61K 39/39558 20130101; A61K 31/435 20130101;
A61P 43/00 20180101; A61K 47/6803 20170801; A61K 31/444 20130101;
A61K 31/704 20130101; A61K 47/545 20170801; A61K 38/05 20130101;
B82Y 5/00 20130101; C07C 33/16 20130101; A61P 35/00 20180101; C07D
257/08 20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 39/395 20060101 A61K039/395; A61K 38/05 20060101
A61K038/05; A61K 31/704 20060101 A61K031/704; A61K 47/22 20060101
A61K047/22 |
Foreign Application Data
Date |
Code |
Application Number |
May 16, 2011 |
EP |
11166241.7 |
May 20, 2011 |
EP |
11166942.0 |
Aug 5, 2011 |
EP |
11176736.4 |
Aug 5, 2011 |
EP |
11176741.4 |
Dec 8, 2011 |
EP |
11192572.3 |
Dec 8, 2011 |
EP |
11192577.2 |
Claims
1. A kit for the administration and activation of a Prodrug, the
kit comprising a Drug D.sup.D linked, directly or indirectly, to a
Trigger moiety T.sup.R, and an Activator for the Trigger moiety,
wherein the Trigger moiety comprises a dienophile and the Activator
comprises a diene, the dienophile, including said Drug linked
thereto, satisfying the following formula (1a): ##STR00127##
wherein T, F each independently denotes H, or a substituent
selected from the group consisting of alkyl, F, Cl, Br or I; the
meaning of the letters A,P,Q,X,Y, and Z is selected from the group
consisting of the following (1) to (6): (1) one of the bonds PQ,
QP, QX, XQ, XZ, ZX, ZY, YZ, YA, AY consists of
--CR.sup.aX.sup.D--CR.sup.aY.sup.D--, the remaining groups
constituted by A,Y,Z,X,Q,P being independently from each other
CR.sup.a.sub.2, S, O, SiR.sup.b.sub.2, such that P and A are
CR.sup.a.sub.2, and no adjacent pairs of atoms are present selected
from the group consisting of O--O, O--S, and S--S, and such that
Si, if present, is adjacent to CR.sup.a.sub.2 or O; X.sup.D is
O--C(O)-(L.sup.D).sub.n-(D.sup.D),
S--C(O)-(L.sup.D).sub.n-(D.sup.D),
O--C(S)-(L.sup.D).sub.n-(D.sup.D),
S--C(S)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(O)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(S)-(L.sup.D).sub.n-(D.sup.D); and Y.sup.D is NHR.sup.c,
OH, SH; or X.sup.D is C(O)-(L.sup.D).sub.n-(D.sup.D),
C(S)-(L.sup.D)-(D.sup.D); and Y.sup.D is CR.sup.c.sub.2NHR.sup.c,
CR.sup.c.sub.2OH, CR.sup.c.sub.2SH, NH--NH.sub.2, O--NH.sub.2, or
NH--OH; (2) A is CR.sup.aX.sup.D and Z is CR.sup.aY.sup.D, or Z is
CR.sup.aX.sup.D and A is CR.sup.aY.sup.D, or P is CR.sup.aX.sup.D
and X is CR.sup.aY.sup.D, or X is CR.sup.aX.sup.D and P is
CR.sup.aY.sup.D, such that X.sup.D and Y.sup.D are positioned in a
trans conformation with respect to one another; the remaining
groups constituted by A,Y,Z,X,Q, and P being independently from
each other CR.sup.a.sub.2, S, O, SiR.sup.b.sub.2, such that P and A
are CR.sup.a.sub.2, and no adjacent pairs of atoms are present
selected from the group consisting of O--O, O--S, and S--S, and
such that Si, if present, is adjacent to CR.sup.a.sub.2 or O;
X.sup.D is O--C(O)-(L.sup.D)-(D.sup.D),
S--C(O)-(L.sup.D)-(D.sup.D), O--C(S)-(L.sup.D).sub.n-(D.sup.D),
S--C(S)-(L.sup.D)-(D.sup.D),
NR.sup.c--C(O)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(S)-(L.sup.D)-(D.sup.D), and Y.sup.D is NHR.sup.c, OH,
SH, CR.sup.c.sub.2NHR.sup.c, CR.sup.c.sub.2OH, CR.sup.c.sub.2SH,
NH--NH.sub.2, O--NH.sub.2, or NH--OH; or X.sup.D is
CR.sup.c.sub.2--O--C(O)-(L.sup.D).sub.n-(D.sup.D),
CR.sup.c.sub.2--C(O)-(L.sup.D).sub.n-(D.sub.D),
CR.sup.c.sub.2--O--C(S)-(L.sup.D).sub.n-(D.sup.D),
CR.sup.c.sub.2--S--C(S)-(L.sup.D).sub.n-(D.sup.D),
CR.sup.c.sub.2--NR.sup.c--C(O)- (L.sup.D).sub.n-(D.sup.D),
CR.sup.c.sub.2--NR.sup.c--C(S)-(L.sup.D).sub.n-(D.sup.D), and
Y.sup.D is NHR.sup.c, OH, SH; or X.sup.D is
C(O)-(L.sup.D).sub.n-(D.sup.D), C(S)-(L.sup.D).sub.n-(D.sup.D); and
Y.sup.D is CR.sup.c.sub.2NHR.sup.c, CR.sup.c.sub.2OH,
CR.sup.c.sub.2SH, NH--NH.sub.2, O--NH.sub.2, NH--OH; (3) A is
CR.sup.aY.sup.D and one of P, Q, X, Z is CR.sup.aX.sup.D, or P is
CR.sup.aY.sup.D and one of A, Y, Z, X is CR.sup.aX.sup.D, or Y is
CR.sup.aY.sup.D and X or P is CR.sup.aX.sup.D, or Q is
CR.sup.aY.sup.D and Z or A is CR.sup.aX.sup.D, or either Z or X is
CR.sup.aY.sup.D and A or P is CR.sup.aX.sup.D, such that X.sup.D
and Y.sup.D are positioned in a trans conformation with respect to
one another; the remaining groups constituted by A,Y,Z,X,Q, and P
being independently from each other CR.sup.a.sub.2, S, O,
SiR.sup.b.sub.2, such that P and A are CR.sup.a.sub.2, and no
adjacent pairs of atoms are present selected from the group
consisting of O--O, O--S, and S--S, and such that Si, if present,
is adjacent to CR.sup.a.sub.2 or O; X.sup.D is
(O--C(O)).sub.p-(L.sup.D).sub.n-(D.sup.D),
S--C(O)-(L.sup.D).sub.n-(D.sup.D),
O--C(S)-(L.sup.D).sub.n-(D.sup.D),
S--C(S)-(L.sup.D).sub.n-(D.sup.D), and Y.sup.D is
CR.sup.c.sub.2NHR.sup.c, CR.sup.c.sub.2OH, CR.sup.c.sub.2SH,
NH--NH.sub.2, O--NH.sub.2, NH--OH; with p being 0 or 1; (4) P is
CR.sup.aY.sup.D and Y is CR.sup.aX.sup.D, or A is CR.sup.aY.sup.D
and Q is CR.sup.aX.sup.D, or Q is CR.sup.aY.sup.D and A is
CR.sup.aX.sup.D, or Y is CR.sup.aY.sup.D and P is CR.sup.aX.sup.D,
such that X.sup.D and Y.sup.D are positioned in a trans
conformation with respect to one another; the remaining groups
constituted from A,Y,Z,X,Q, and P being independently from each
other CR.sup.a.sub.2, S, O, SiR.sup.b.sub.2, such that P and A are
CR.sup.a.sub.2, and no adjacent pairs of atoms are present selected
from the group consisting of O--O, O--S, and S--S, and such that
Si, if present, is adjacent to CR.sup.a.sub.2 or O; X.sup.D is
(O--C(O)).sub.p-(L.sup.D).sub.n-(D.sup.D),
S--C(O)-(L.sup.D).sub.n-(D.sup.D),
O--C(S)-(L.sup.D).sub.n-(D.sup.D),
S--C(S)-(L.sup.D).sub.n-(D.sup.D); Y.sup.D is NHR.sup.c, OH, SH;
p=0 or 1; (5) Y is Y.sup.D and P is CR.sup.aX.sup.D, or Q is
Y.sup.D and A is CR.sup.aX.sup.D; the remaining groups constituted
by A,Y,Z,X,Q, and P being independently from each other
CR.sup.a.sub.2, S, O, SiR.sup.b.sub.2, such that P and A are
CR.sup.a.sub.2, and no adjacent pairs of atoms are present selected
from the group consisting of O--O, O--S, and S--S, and such that
Si, if present, is adjacent to CR.sup.a.sub.2 or O; X.sup.D is
(O--C(O)).sub.p-(L.sup.D).sub.n-(D.sup.D),
S--C(O)-(L.sup.D).sub.n-(D.sup.D),
O--C(S)-(L.sup.D).sub.n-(D.sup.D),
S--C(S)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(O)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(S)-(L.sup.D).sub.n-(D.sup.D),
C(O)-(L.sup.D).sub.n-(D.sup.D), C(S)-(L.sup.D).sub.n-(D.sup.D);
Y.sup.D is NH; p=0 or 1; (6) Y is Y.sup.D and P or Q is X.sup.D, or
Q is Y.sup.D and A or Y is X.sup.D; the remaining groups
constituted by A,Y,Z,X,Q, and P being independently from each other
CR.sup.a.sub.2, S, O, SiR.sup.b.sub.2, such that P and A are
CR.sup.a.sub.2, and no adjacent pairs of atoms are present selected
from the group consisting of O--O, O--S, and S--S, and such that
Si, if present, is adjacent to CR.sup.a.sub.2 or O; X.sup.D is
N--C(O)-(L.sup.D).sub.n-(D.sup.D),
N--C(S)-(L.sup.D).sub.n-(D.sup.D); Y.sup.D is NH; wherein each
R.sup.a independently is selected from the group consisting of H,
alkyl, aryl, OR', SR', S(.dbd.O)R''', S(.dbd.O).sub.2R''',
S(.dbd.O).sub.2NR'R'', Si--R''', Si--O--R''', OC(.dbd.O)R''',
SC(.dbd.O)R''', OC(.dbd.S)R''', SC(.dbd.S)R''', F, Cl, Br, I,
N.sub.3, SO.sub.2H, SO.sub.3H, SO.sub.4H, PO.sub.3H, PO.sub.4H, NO,
NO.sub.2, CN, OCN, SCN, NCO, NCS, CF.sub.3, CF.sub.2--R', NR'R'',
C(.dbd.O)R', C(.dbd.S)R', C(.dbd.O)O--R', C(.dbd.S)O--R',
C(.dbd.O)S--R', C(.dbd.S)S--R', C(.dbd.O)NR'R'', C(.dbd.S)NR'R'',
NR'C(.dbd.O)--R''', NR'C(.dbd.S)--R''', NR'C(.dbd.O)O--R''',
NR'C(.dbd.S)O--R''', NR'C(.dbd.O)S--R''', NR'C(.dbd.S)S--R''',
OC(.dbd.O)NR'--R''', SC(.dbd.O)NR'--R''', OC(.dbd.S)NR'--R''',
SC(.dbd.S)NR'--R''', NR'C(.dbd.O)NR''--R'', NR'C(.dbd.S)NR''--R'',
CR'NR'', with each R' and each R'' independently being H, aryl or
alkyl and R''' independently being aryl or alkyl; wherein each
R.sup.b is independently selected from the group consisting of H,
alkyl, aryl, O-alkyl, O-aryl, OH; wherein each R.sup.c is
independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 aryl;
wherein two or more R.sup.a,b,c moieties together may form a ring;
and wherein (L.sup.D).sub.n is an optional linker with n=0 or 1,
preferably linked to T.sup.R via S, N, NH, or O, wherein these
atoms are part of the linker, which may consist of multiple units
arranged linearly and/or branched; D.sup.D is one or more
therapeutic moieties or drugs, preferably linked via S, N, NH, or
O, wherein these atoms are part of the therapeutic moiety.
2. A kit according to claim 1, wherein A,P,Q,X,Y, and Z are
selected such that one of the bonds PQ, QP, QX, XQ, XZ, ZX, ZY, YZ,
YA, AY consists of --CR.sup.aX.sup.D--CR.sup.aY.sup.D-, the
remaining groups constituted by A,Y,Z,X,Q,P being independently
from each other CR.sup.a.sub.2, S, O, SiR.sup.b.sub.2, such that P
and A are CR.sup.a.sub.2, and no adjacent pairs of atoms are
present selected from the group consisting of O--O, O--S, and S--S,
and such that Si, if present, is adjacent to CR.sup.a.sub.2 or O;
X.sup.D is O--C(O)-(L.sup.D).sub.n-(D.sup.D),
S--C(O)-(L.sup.D).sub.n-(D.sup.D),
O--C(S)-(L.sup.D).sub.n-(D.sup.D),
S--C(S)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(O)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(S)-(L.sup.D).sub.n-(D.sup.D); and Y.sup.D is NHR.sup.c,
OH, SH; or X.sup.D is C(O)-(L.sup.D).sub.n-(D.sup.D),
C(S)-(L.sup.D).sub.n-(D.sup.D); and Y.sup.D is
CR.sup.c.sub.2NHR.sup.c, CR.sup.c.sub.2OH, CR.sup.c.sub.2SH,
NH--NH.sub.2, O--NH.sub.2, or NH--OH.
3. A kit according to claim 2, wherein PQ, QP, AY or YA is
--CR.sup.aX.sup.D--CR.sup.aY.sup.D-, and X.sup.D and Y.sup.D are
positioned trans relative to each other.
4. A kit according to claim 2, wherein ZX or XZ is
--CR.sup.aX.sup.D--CR.sup.aY.sup.D-, and X.sup.D and Y.sup.D are
positioned cis relative to each other.
5. A kit according to claim 1, wherein X.sup.D is
NR.sup.c--C(O)-(L.sup.D).sub.n-(D.sup.D), and Y.sup.D is
NHR.sup.c.
6. A kit according to claim 5, wherein the dienophile is a compound
selected from the following structures: ##STR00128## =rest of
attached D.sup.D, L.sup.D-D.sup.D, optionally comprising T.sup.T or
S.sup.P-T.sup.T or M.sup.M or S.sup.P-M.sup.M.
7. A kit according to claim 1, wherein the activator comprises a
diene selected from the dienes, according to formulae (2)-(4):
##STR00129## wherein R.sup.1 is selected from the group consisting
of H, alkyl, aryl, CF.sub.3, CF.sub.2--R', OR', SR', C(.dbd.O)R',
C(.dbd.S)R', C(.dbd.O)O--R', C(.dbd.O)S--R', C(.dbd.S)O--R',
C(.dbd.S)S--R'', C(.dbd.O)NR'R'', C(.dbd.S)NR'R'', NR'R'',
NR'C(.dbd.O)R'', NR'C(.dbd.S)R'', NR'C(.dbd.O)OR'',
NR'C(.dbd.S)OR'', NR'C(.dbd.O)SR'', NR'C(.dbd.S)SR'',
NR'C(.dbd.O)NR''R'', NR'C(.dbd.S)NR''R'' with each R' and each R''
independently being H, aryl or alkyl; A and B each independently
are selected from the group consisting of alkyl-substituted carbon,
aryl substituted carbon, nitrogen, N.sup.+O.sup.-, N.sup.+R with R
being alkyl, with the proviso that A and B are not both carbon; X
is selected from the group consisting of O, N-alkyl, and C.dbd.O,
and Y is CR with R being selected from the group consisting of H,
alkyl, aryl, C(.dbd.O)OR', C(.dbd.O)SR', C(.dbd.S)OR',
C(.dbd.S)SR', C(.dbd.O)NR'R'' with R' and R'' each independently
being H, aryl or alkyl; ##STR00130## wherein R.sup.1 and R.sup.2
each independently are selected from the group consisting of H,
alkyl, aryl, CF.sub.3, CF.sub.2--R', NO.sub.2, OR', SR',
C(.dbd.O)R', C(.dbd.S)R', OC(.dbd.O)R''', SC(.dbd.O)R''',
OC(.dbd.S)R''', SC(.dbd.S)R''', S(.dbd.O)R', S(.dbd.O).sub.2R''',
S(.dbd.O).sub.2NR'R'', C(.dbd.O)O--R', C(.dbd.O)S--R',
C(.dbd.S)O--R', C(.dbd.S)S--R', C(.dbd.O)NR'R'', C(.dbd.S)NR'R'',
NR'R'', NR'C(.dbd.O)R'', NR'C(.dbd.S)R'', NR'C(.dbd.O)OR'',
NR'C(.dbd.S)OR'', NR'C(.dbd.O)SR'', NR'C(.dbd.S)SR'',
OC(.dbd.O)NR'R'', SC(.dbd.O)NR'R'', OC(.dbd.S)NR'R'',
SC(.dbd.S)NR'R'', NR'C(.dbd.O)NR''R'', NR'C(.dbd.S)NR''R'' with
each R' and each R'' independently being H, aryl or alkyl, and R'''
independently being aryl or alkyl; A is selected from the group
consisting of N-alkyl, N-aryl, C.dbd.O, and CN-alkyl; B is O or S;
X is selected from the group consisting of N, CH, C-alkyl, C-aryl,
CC(.dbd.O)R', CC(.dbd.S)R', CS(.dbd.O)R', CS(.dbd.O).sub.2R''',
CC(.dbd.O)O--R', CC(.dbd.O)S--R', CC(.dbd.S)O--R', CC(.dbd.S)S--R',
CC(.dbd.O)NR'R'', CC(.dbd.S)NR'R'', R' and R'' each independently
being H, aryl or alkyl and R''' independently being aryl or alkyl;
Y is selected from the group consisting of CH, C-alkyl, C-aryl, N,
and N.sup.+O.sup.-; ##STR00131## wherein R.sup.1 and R.sup.2 each
independently are selected from the group consisting of H, alkyl,
aryl, CF.sub.3, CF.sub.2--R', NO, NO.sub.2, OR', SR', CN,
C(.dbd.O)R', C(.dbd.S)R', OC(.dbd.O)R''', SC(.dbd.O)R''',
OC(.dbd.S)R''', SC(.dbd.S)R''', S(.dbd.O)R', S(.dbd.O).sub.2R''',
S(.dbd.O).sub.2OR', PO.sub.3R'R'', S(.dbd.O).sub.2NR'R'',
C(.dbd.O)O--R', C(.dbd.O)S--R', C(.dbd.S)O--R', C(.dbd.S)S--R',
C(.dbd.O)NR'R'', C(.dbd.S)NR'R'', NR'R'', NR'C(.dbd.O)R'',
NR'C(.dbd.S)R'', NR'C(.dbd.O)OR'', NR'C(.dbd.S)OR'',
NR'C(.dbd.O)SR'', NR'C(.dbd.S)SR'', OC(.dbd.O)NR'R'',
SC(.dbd.O)NR'R'', OC(.dbd.S)NR'R'', SC(.dbd.S)NR'R'',
NR'C(.dbd.O)NR''R'', NR'C(.dbd.S)NR''R'' with each R' and each R''
independently being H, aryl or alkyl, and R''' independently being
aryl or alkyl; A is selected from the group consisting of N,
C-alkyl, C-aryl, and N.sup.+O.sup.-; B is N; X is selected from the
group consisting of N, CH, C-alkyl, C-aryl, CC(.dbd.O)R',
CC(.dbd.S)R', CS(.dbd.O)R', CS(.dbd.O).sub.2R''', CC(.dbd.O)O--R',
CC(.dbd.O)S--R', CC(.dbd.S)O--R', CC(.dbd.S)S--R',
CC(.dbd.O)NR'R'', CC(.dbd.S)NR'R'', R' and R'' each independently
being H, aryl or alkyl and R''' independently being aryl or alkyl;
Y is selected from the group consisting of CH, C-alkyl, C-aryl, N,
and N.sup.+O.sup.-.
8. A kit according to claim 7, wherein the diene satisfies formula
(7) as given in the description, ##STR00132## being a tetrazine
para substituted with R.sub.1 and R.sub.2, wherein R.sub.1 and
R.sub.2 each independently denote a substituent selected from the
group consisting of H, alkyl, NO.sub.2, F, Cl, CF.sub.3, CN, COOR,
CONHR, CONR.sub.2, COR, SO.sub.2R, SO.sub.2OR, SO.sub.2NR.sub.2,
PO.sub.3R.sub.2, NO, 2-pyridyl, 3-pyridyl, 4-pyridyl,
2,6-pyrimidyl, 3,5-pyrimidyl, 2,4-pyrimidyl, 2,4 imidazyl, 2,5
imidazyl and phenyl, optionally substituted with one or more
electron-withdrawing groups selected from the group consisting of
NO.sub.2, F, Cl, CF.sub.3, CN, COOR, CONHR, CONR, COR, SO.sub.2R,
SO.sub.2OR, SO.sub.2NR.sub.2, PO.sub.3R.sub.2, NO, and Ar, wherein
R is H or C.sub.1-C.sub.6 alkyl, and Ar stands for phenyl, pyridyl,
or naphthyl.
9. A kit according to claim 7, wherein the diene is selected from
the group consisting of: ##STR00133## ##STR00134## ##STR00135##
##STR00136## ##STR00137## ##STR00138##
10. A kit according to claim 7, wherein the diene is selected from
the group consisting of: ##STR00139## ##STR00140## ##STR00141##
##STR00142##
11. A kit according to claim 1, wherein at least one of the drug
D.sup.D or the linker L.sup.D or the trigger moiety T.sup.R
comprises a targeting agent T.sup.T, preferably an antibody.
12. A kit according to claim 1, wherein at least one of the L.sup.D
or the trigger moiety T.sup.R comprises a masking moiety M.sup.M,
preferably a peptide.
13. A kit according to claim 1, wherein the drug is a T-cell
engaging antibody construct.
14. A kit according to claim 1, wherein the Prodrug comprises an
antibody-toxin or antibody-drug conjugate.
15. A prodrug comprising a drug compound linked, directly or
indirectly, to a dienophile moiety of formula (1a), as defined in
claim 1.
16. A method of modifying a drug compound into a prodrug that can
be triggered by an abiotic, bio-orthogonal reaction, comprising
providing a drug and chemically linking the drug to a dienophile
moiety, so as to form a prodrug of formula (1a) as defined in claim
1.
17. A method of treatment wherein a patient suffering from a
disease that can be modulated by a drug, is treated by
administering, to said patient, a prodrug comprising a trigger
moiety after activation of which the drug will be released, wherein
the trigger moiety comprises a trans-cyclooctene ring, the ring
optionally including one or more hetero-atoms, and the diene being
selected so as to be capable of reacting with the dienophile in an
inverse electron-demand Diels-Alder reaction, the trigger moiety
satisfying the formula (1a) as defined in claim 1.
18. A compound satisfying the formula (1a) as defined in claim 1:
##STR00143## said compound comprising a linkage to a drug, for use
in prodrug therapy in an animal or a human being.
19. The use of a tetrazine as an activator for the release, in a
physiological environment, of a substance linked to a compound
satisfying formula (1a) as defined in claim 1.
20. The use of the inverse electron-demand Diels-Alder reaction
between a compound satisfying formula (1a) as defined in claim 1
and a tetrazine as a chemical tool for the release, in a
physiological environment, of a substance administered in a
chemically bound form, wherein the substance is bound to a compound
satisfying formula (1a).
21. The use of a trans-cyclooctene satisfying formula (1a), as
defined in claim 1, as a carrier for a therapeutic compound.
Description
FIELD OF THE INVENTION
[0001] The invention relates to therapeutical methods on the basis
of inactivated drugs, such as prodrugs, that are activated by means
of an abiotic, bio-orthogonal chemical reaction.
BACKGROUND OF THE INVENTION
[0002] In the medical arena the use of inactive compounds such as
prodrugs which are activated in a specific site in the human or
animal body is well known. Also targeted delivery of inactives such
as prodrugs has been studied extensively. Much effort has been
devoted to drug delivery systems that effect drug release
selectivity at a target site and/or at a desired moment in time.
One way is to selectively activate a (systemic) prodrug
specifically by local and specific enzymatic activity. However, in
many cases a target site of interest lacks a suitable overexpressed
enzyme. An alternative is to transport an enzyme to target tissue
via a technique called antibody-directed enzyme prodrug therapy
(ADEPT). In this approach an enzyme is targeted to a tumor site by
conjugation to an antibody that binds a tumor-associated antigen.
After systemic administration of the conjugate, its localization at
the target and clearance of unbound conjugate, a designed prodrug
is administered systemically and locally activated. This method
requires the catalysis of a reaction that must not be accomplished
by an endogenous enzyme. Enzymes of non-mammalian origin that meet
these needs are likely to be highly immunogenic, a fact that makes
repeated administration impossible. Alternatively, prodrugs can be
targeted to a disease site followed by disease-specific or
-non-specific endogenous activation processes (eg pH, enzymes,
thiol-containing compounds).
[0003] Targeted anticancer therapeutics are designed to reduce
nonspecific toxicities and increase efficacy relative to
conventional cancer chemotherapy. This approach is embodied by the
powerful targeting ability of monoclonal antibodies (mAbs) to
specifically deliver highly potent, conjugated small molecule
therapeutics to a cancer cell. In an attempt to address the issue
of toxicity, chemotherapeutic agents (drugs) have been coupled to
targeting molecules such as antibodies or protein receptor ligands
that bind with a high degree of specificity to tumor cell to form
compounds referred to as antibody-drug conjugates (ADC) or
immunoconjugates. Immunoconjugates in theory should be less toxic
because they direct the cytotoxic drug to tumors that express the
particular cell surface antigen or receptor. This strategy has met
limited success in part because cytotoxic drugs tend to be inactive
or less active when conjugated to large antibodies, or protein
receptor ligands. Promising advancements with immunoconjugates has
seen cytotoxic drugs linked to antibodies through a linker that is
cleaved at the tumor site or inside tumor cells (Senter et al,
Current Opinion in Chemical Biology 2010, 14:529-537). Ideally, the
mAb will specifically bind to an antigen with substantial
expression on tumor cells but limited expression on normal tissues.
Specificity allows the utilization of drugs that otherwise would be
too toxic for clinical application. Most of the recent work in this
field has centered on the use of highly potent cytotoxic agents.
This requires the development of linker technologies that provide
conditional stability, so that drug release occurs after tumor
binding, rather than in circulation.
[0004] As a conjugate the drug is inactive but upon target
localization the drug is released by eg pH or an enzyme, which
could be target specific but may also be more generic. The drug
release may be achieved by an extracellular mechanism such as low
pH in tumor tissue, hypoxia, certain enzymes, but in general more
selective drug release can be achieved through intracellular,
mostly lysosomal, release mechanisms (e.g. glutathione, proteases,
catabolism) requiring the antibody conjugate to be first
internalized. Specific intracellular release mechanisms (eg
glutathione, cathepsin) usually result in the parent drug, which
depending on its properties, can escape the cell and attack
neighboring cells. This is viewed as an important mechanism of
action for a range of antibody-drug conjugates, especially in
tumors with heterogeneous receptor expression, or with poor mAb
penetration. Examples of cleavable linkers are: hydrazones (acid
labile), peptide linkers (cathepsin B cleavable), hindered
disulfide moieties (thiol cleavable). Also non-cleavable linkers
can be used in mAb-drug conjugates. These constructs release their
drug upon catabolism, presumably resulting in a drug molecule still
attached to one amino acid. Only a subset of drugs will regain
their activity as such a conjugate. Also, these aminoacid-linked
drugs cannot escape the cells. Nevertheless, as the linker is
stable, these constructs are generally regarded as the safest and
depending on the drug and target, can be very effective.
[0005] The current antibody-drug conjugate release strategies have
their limitations. The extracellular drug release mechanisms are
usually too unspecific (as with pH sensitive linkers) resulting in
toxicity. Intracellular release depends on efficient (e.g
receptor-mediated internalization) of the mAb-drug, while several
cancers lack cancer-specific and efficiently internalizing targets
that are present in sufficiently high copy numbers. Intracellular
release may further depend on the presence of an activating enzyme
(proteases) or molecules (thiols such as glutathione) in
sufficiently high amount. Following intracellular release, the drug
may, in certain cases, escape from the cell to target neighbouring
cells. This effect is deemed advantageous in heterogeneous tumors
where not every cell expresses sufficiently high amounts of target
receptor. It is of further importance in tumors that are difficult
to penetrate due e.g. to elevated interstitial pressure, which
impedes convectional flow. This is especially a problem for large
constructs like mAb (conjugates). This mechanism is also essential
in cases where a binding site barrier occurs. Once a targeted agent
leaves the vasculature and binds to a receptor, its movement within
the tumor will be restricted. The likelihood of a mAb conjugate
being restricted in the perivascular space scales with its affinity
for its target. The penetration can be improved by increasing the
mAb dose, however, this approach is limited by dose limiting
toxicity in e.g. the liver. Further, antigens that are shed from
dying cells can be present in the tumor interstitial space where
they can prevent mAb-conjugates of binding their target cell. Also,
many targets are hampered by ineffective internalization, and
different drugs cannot be linked to a mAb in the same way. Further,
it has been proven cumbersome to design linkers to be selectively
cleavable by endogenous elements in the target while stable to
endogenous elements en route to the target (especially the case for
slow clearing full mAbs). As a result, the optimal drug, linker,
mAb, and target combination needs to be selected and optimized on a
case by case basis.
[0006] Another application area that could benefit from an
effective prodrug approach is the field of T-cell engaging antibody
constructs (e.g., bi- or trispecific antibody fragments), which act
on cancer by engaging the immunesystem. It has long been considered
that bringing activated T-cells into direct contact with cancer
cells offers a potent way of killing them (Thompson et al.,
Biochemical and Biophysical Research Communications 366 (2008)
526-531). Of the many bispecific antibodies that have been created
to do this, the majority are composed of two antibody binding
sites, one site targets the tumor and the other targets a T-cell
(Thakur et al. Current Opinion in Molecular Therapeutics 2010,
12(3), 340-349). However, with bispecific antibodies containing an
active T-cell binding site, peripheral T-cell binding will occur.
This not only prevents the conjugate from getting to the tumor but
can also lead to cytokine storms and T-cell depletion.
Photo-activatable anti-T-cell antibodies, in which the anti-T-cell
activity is only restored when and where it is required (i.e. after
tumor localization via the tumor binding arm), following
irradiation with UV light, has been used to overcome these
problems. Anti-human CD3 (T-cell targeting) antibodies could be
reversibly inhibited with a photocleavable 1-(2-nitrophenyl)ethanol
(NPE) coating (Thompson et al., Biochemical and Biophysical
Research Communications 366 (2008) 526-531). However, light based
activation is limited to regions in the body where light can
penetrate, and is not easily amendable to treating systemic disease
such as metastatic cancer. Strongly related constructs that could
benefit from a prodrug approach are trispecific T-cell engaging
antibody constructs with for example a CD3- and a CD28 T-cell
engaging moiety in addition to a cancer targeting agent. Such
constructs are too toxic to use as such and either the CD3 or the
CD28 or both binding domains need to be masked.
[0007] It is desirable to be able to activate targeted drugs
selectively and predictably at the target site without being
dependent on homogenous penetration and targeting, and on
endogenous parameters which may vary en route to and within the
target, and from indication to indication and from patient to
patient.
[0008] In order to avoid the drawbacks of current prodrug
activation, it has been proposed in Bioconjugate Chem 2008, 19,
714-718, to make use of an abiotic, bio-orthogonal chemical
reaction, viz. the Staudinger reaction, to provoke activation of
the prodrug. Briefly, in the introduced concept, the Prodrug is a
conjugate of a Drug and a Trigger, and this Drug-Trigger conjugate
is not activated endogeneously by e.g. an enzyme or a specific pH,
but by a controlled administration of the Activator, i.e. a species
that reacts with the Trigger moiety in the Prodrug, to induce
release of the Drug from the Trigger (or vice versa, release of the
Trigger from the Drug, however one may view this release process).
The presented Staudinger approach for this concept, however, has
turned out not to work well, and its area of applicability is
limited in view of the specific nature of the release mechanism
imposed by the Staudinger reaction. Other drawbacks for use of
Staudinger reactions are their limited reaction rates, and the
oxidative instability of the phosphine components of these
reactions. Therefore, it is desired to provide reactants for an
abiotic, bio-orthogonal reaction that are stable in physiological
conditions, that are more reactive towards each other, and that are
capable of inducing release of a bound drug by means of a variety
of mechanisms, thus offering a greatly versatile activated drug
release method.
[0009] The use of a biocompatible chemical reaction that does not
rely on endogenous activation mechanisms (eg pH, enzymes) for
selective Prodrug activation would represent a powerful new tool in
cancer therapy. Selective activation of Prodrugs when and where
required allows control over many processes within the body,
including cancer. Therapies, such as anti-tumor antibody therapy,
may thus be made more specific, providing an increased therapeutic
contrast between normal cells and tumour to reduce unwanted side
effects. In the context of T-cell engaging anticancer antibodies,
the present invention allows the systemic administration and tumor
targeting of an inactive antibody construct (i.e. this is then the
Prodrug), diminishing off-target toxicity. Upon sufficient tumor
uptake and clearance from non target areas, the tumor-bound
antibody is activated by administration of the Activator, which
reacts with the Trigger or Triggers on the antibody or particular
antibody domain, resulting in removal of the Trigger and
restoration of the T-cell binding function. This results in T-cell
activation and anticancer action (i.e. this is then the Drug
release).
SUMMARY OF THE INVENTION
[0010] In order to better address one or more of the foregoing
desires, the present invention provides a kit for the
administration and activation of a Prodrug, the kit comprising a
Drug linked, directly or indirectly, to a Trigger moiety, and an
Activator for the Trigger moiety, wherein the Trigger moiety
comprises a dienophile and the Activator comprises a diene, the
dienophile satisfying the following formula (1a):
##STR00001##
wherein T, F each independently denotes H, or a substituent
selected from the group consisting of alkyl, F, Cl, Br or I; the
meaning of the letters A,P,Q,X,Y, and Z is selected from the group
consisting of the following Embodiments:
[0011] (1) one of the bonds PQ, QP, QX, XQ, XZ, ZX, ZY, YZ, YA, AY
consists of --CR.sup.aX.sup.D--CR.sup.aY.sup.D-, the remaining
groups constituted by A,Y,Z,X,Q,P being independently from each
other CR.sup.a.sub.2, S, O, SiR.sup.b.sub.2, such that P and A are
CR.sup.a.sub.2, and no adjacent pairs of atoms are present selected
from the group consisting of O--O, O--S, and S--S, and such that
Si, if present, is adjacent to CR.sup.a.sub.2 or O; X.sup.D is
O--C(O)-(L.sup.D).sub.n-(D.sup.D),
S--C(O)-(L.sup.D).sub.n-(D.sup.D),
O--C(S)-(L.sup.D).sub.n-(D.sup.D),
S--C(S)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(O)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(S)-(L.sup.D).sub.n-(D.sup.D); and Y.sup.D is NHR.sup.c,
OH, SH; or X.sup.D is C(O)-(L.sup.D).sub.n-(D.sup.D),
C(S)-(L.sup.D).sub.n-(D.sup.D); and Y.sup.D is
CR.sup.c.sub.2NHR.sup.c, CR.sup.c.sub.2OH, CR.sup.c.sub.2SH,
NH--NH.sub.2, O--NH.sub.2, or NH--OH;
[0012] (2) A is CR.sup.aX.sup.D and Z is CR.sup.aY.sup.D, or Z is
CR.sup.aX.sup.D and A is CR.sup.aY.sup.D, or P is CR.sup.aX.sup.D
and X is CR.sup.aY.sup.D, or X is CR.sup.aX.sup.D and P is
CR.sup.aY.sup.D, such that X.sup.D and Y.sup.D are positioned in a
trans conformation with respect to one another; the remaining
groups constituted by A,Y,Z,X,Q, and P being independently from
each other CR.sup.a.sub.2, S, O, SiR.sup.b.sub.2, such that P and A
are CR.sup.a.sub.2, and no adjacent pairs of atoms are present
selected from the group consisting of O--O, O--S, and S--S, and
such that Si, if present, is adjacent to CR.sup.a.sub.2 or O;
X.sup.D is O--C(O)-(L.sup.D).sub.n-(D.sup.D),
S--C(O)-(L.sup.D).sub.n-(D.sup.D), O--C(S)-(L.sup.D)-(D.sup.D),
S--C(S)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(O)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(S)-(L.sup.D).sub.n-(D.sup.D), and Y.sup.D is NHR.sup.c,
OH, SH, CR.sup.c.sub.2NHR.sup.c, CR.sup.c.sub.2OH,
CR.sup.c.sub.2SH, NH--NH.sub.2, O--NH.sub.2, or NH--OH; or X.sup.D
is CR.sup.c--O--C(O)-(L.sup.D).sub.n-(D.sup.D),
CR.sup.c.sub.2--S--C(O)-(L.sup.D).sub.n-(D.sup.D),
CR.sup.c.sub.2--O--C(S)-(L.sup.D).sub.n-(D.sup.D),
CR.sup.c.sub.2--S--C(S)-(L.sup.D).sub.n-(D.sup.D),
CR.sup.c.sub.2--NR.sup.c--C(O)-(L.sup.D).sub.n-(D.sup.D),
CR.sup.c.sub.2--NR.sup.c--C(S)-(L.sup.D).sub.n-(D.sup.D), and
Y.sup.D is NHR.sup.c, OH, SH; or X.sup.D is
C(O)-(L.sup.D).sub.n-(D.sup.D), C(S)-(L.sup.D).sub.n-(D.sup.D); and
Y.sup.D is CR.sup.c.sub.2NHR.sup.c, CR.sup.c.sub.2OH,
CR.sup.c.sub.2SH, NH--NH.sub.2, O--NH.sub.2, NH--OH.
[0013] (3) A is CR.sup.aY.sup.D and one of P, Q, X, Z is
CR.sup.aX.sup.D, or P is CR.sup.aY.sup.D and one of A, Y, Z, X is
CR.sup.aX.sup.D, or Y is CR.sup.aY.sup.D and X or P is
CR.sup.aX.sup.D, or Q is CR.sup.aY.sup.D and Z or A is
CR.sup.aX.sup.D, or either Z or X is CR.sup.aY.sup.D and A or P is
CR.sup.aX.sup.D, such that X.sup.D and Y.sup.D are positioned in a
trans conformation with respect to one another; the remaining
groups constituted by A,Y,Z,X,Q, and P being independently from
each other CR.sup.a.sub.2, S, O, SiR.sup.b.sub.2, such that P and A
are CR.sup.a.sub.2, and no adjacent pairs of atoms are present
selected from the group consisting of O--O, O--S, and S--S, and
such that Si, if present, is adjacent to CR.sup.a.sub.2 or O;
X.sup.D is (O--C(O)).sub.p-(L.sup.D)-(D.sup.D),
S--C(O)-(L.sup.D).sub.n-(D.sup.D), O--C(S)-(L.sup.D)-(D.sup.D),
S--C(S)-(L.sup.D).sub.n-(D.sup.D), and Y.sup.D is
CR.sup.c.sub.2NHR.sup.c, CR.sup.c.sub.2OH, CR.sup.c.sub.2SH,
NH--NH.sub.2, O--NH.sub.2, NH--OH; with p being 0 or 1;
[0014] (4) P is CR.sup.aY.sup.D and Y is CR.sup.aX.sup.D, or A is
CR.sup.aY.sup.D and Q is CR.sup.aX.sup.D, or Q is CR.sup.aY.sup.D
and A is CR.sup.aX.sup.D, or Y is CR.sup.aY.sup.D and P is
CR.sup.aX.sup.D, such that X.sup.D and Y.sup.D are positioned in a
trans conformation with respect to one another; the remaining
groups constituted from A,Y,Z,X,Q, and P being independently from
each other CR.sup.a.sub.2, S, O, SiR.sup.b.sub.2, such that P and A
are CR.sup.a.sub.2 and no adjacent pairs of atoms are present
selected from the group consisting of O--O, O--S, and S--S, and
such that Si, if present, is adjacent to CR.sup.a.sub.2 or O;
X.sup.D is (O--C(O)).sub.p-(L.sup.D).sub.n-(D.sup.D),
S--C(O)-(L.sup.D).sub.n-(D.sup.D),
O--C(S)-(L.sup.D).sub.n-(D.sup.D),
S--C(S)-(L.sup.D).sub.n-(D.sup.D); Y.sup.D is NHR.sup.c, OH, SH;
p=0 or 1.
[0015] (5) Y is Y.sup.D and P is CR.sup.aX.sup.D, or Q is Y.sup.D
and A is CR.sup.aX.sup.D; the remaining groups constituted by
A,Y,Z,X,Q, and P being independently from each other
CR.sup.a.sub.2, S, O, SiR.sup.b.sub.2, such that P and A are
CR.sup.a.sub.2, and no adjacent pairs of atoms are present selected
from the group consisting of O--O, O--S, and S--S, and such that
Si, if present, is adjacent to CR.sup.a.sub.2 or O; X.sup.D is
(O--C(O)).sub.p-(L.sup.D).sub.n-(D.sup.D),
S--C(O)-(L.sup.D).sub.n-(D.sup.D), O--C(S)-(L.sup.D)-(D.sup.D),
S--C(S)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(O)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(S)-(L.sup.D).sub.n-(D.sup.D),
C(O)-(L.sup.D).sub.n-(D.sup.D), C(S)-(L.sup.D).sub.n-(D.sup.D);
Y.sup.D is NH; p=0 or 1;
[0016] (6) Y is Y.sup.D and P or Q is X.sup.D, or Q is Y.sup.D and
A or Y is X.sup.D; the remaining groups constituted by A,Y,Z,X,Q,
and P being independently from each other CR.sup.a.sub.2, S, O,
SiR.sup.b.sub.2, such that P and A are CR.sup.a.sub.2, and no
adjacent pairs of atoms are present selected from the group
consisting of O--O, O--S, and S--S, and such that Si, if present,
is adjacent to CR.sup.a.sub.2 or O; X.sup.D is
N--C(O)-(L.sup.D).sub.n-(D.sup.D),
N--C(S)-(L.sup.D).sub.n-(D.sup.D); Y.sup.D is NH;
wherein each R.sup.a independently is selected from the group
consisting of H, alkyl, aryl, OR', SR', S(.dbd.O)R''',
S(.dbd.O).sub.2R''', S(.dbd.O).sub.2NR'R'', Si--R''', Si--O--R''',
OC(.dbd.O)R''', SC(.dbd.O)R''', OC(.dbd.S)R''', SC(.dbd.S)R''', F,
Cl, Br, I, N.sub.3, SO.sub.2H, SO.sub.3H, SO.sub.4H, PO.sub.3H,
PO.sub.4H, NO, NO.sub.2, CN, OCN, SCN, NCO, NCS, CF.sub.3,
CF.sub.2--R', NR'R'', C(.dbd.O)R', C(.dbd.S)R', C(.dbd.O)O--R',
C(.dbd.S)O--R', C(.dbd.O)S--R', C(.dbd.S)S--R', C(.dbd.O)NR'R'',
C(.dbd.S)NR'R'', NR'C(.dbd.O)--R''', NR'C(.dbd.S)--R''',
NR'C(.dbd.O)O--R''', NR'C(.dbd.S)O--R''', NR'C(.dbd.O)S--R''',
NR'C(.dbd.S)S--R''', OC(.dbd.O)NR'--R''', SC(.dbd.O)NR'--R''',
OC(.dbd.S)NR'--R''', SC(.dbd.S)NR'--R''', NR'C(.dbd.O)NR''--R'',
NR'C(.dbd.S)NR''--R'', CR'NR'', with each R' and each R''
independently being H, aryl or alkyl and R''' independently being
aryl or alkyl; wherein each R.sup.b independently is selected from
the group consisting of H, alkyl, aryl, O-alkyl, O-aryl, OH;
wherein each Re is independently selected from H, C.sub.1-6 alkyl
and C.sub.1-6 aryl; wherein two or more R.sup.a,b,c moieties
together may form a ring; and wherein (L.sup.D).sub.n is an
optional linker with n=0 or 1, preferably linked to T.sup.R via S,
N, NH, or O, wherein these atoms are part of the linker, which may
consist of multiple units arranged linearly and/or branched;
D.sup.D is one or more therapeutic moieties or drugs, preferably
linked via S, N, NH, or O, wherein these atoms are part of the
therapeutic moiety.
[0017] In another aspect, the invention presents a Prodrug
comprising a Drug compound linked, directly or indirectly, to a
trans-cyclooctene moiety satisfying the above formula (1a).
[0018] In yet another aspect, the invention provides a method of
modifying a Drug compound into a Prodrug that can be triggered by
an abiotic, bio-orthogonal reaction, the method comprising the
steps of providing a Drug and chemically linking the Drug to a
cyclic moiety satisfying the above formula (1a).
[0019] In a still further aspect, the invention provides a method
of treatment wherein a patient suffering from a disease that can be
modulated by a drug, is treated by administering, to said patient,
a Prodrug comprising a Trigger moiety after activation of which by
administration of an Activator the Drug will be released, wherein
the Trigger moiety comprises a ring structure satisfying the above
formula (1a).
[0020] In a still further aspect, the invention is a compound
comprising an eight-membered non-aromatic cyclic mono-alkenylene
moiety (preferably a cyclooctene moiety, and more preferably a
trans-cyclooctene moiety), said moiety comprising a linkage to a
Drug, for use in prodrug therapy in an animal or a human being.
[0021] In another aspect, the invention is the use of a diene,
preferably a tetrazine, as an activator for the release, in a
physiological environment, of a substance linked to a compound
satisfying formula (1a). In connection herewith, the invention also
pertains to a tetrazine for use as an activator for the release, in
a physiological environment, of a substance linked to a a compound
satisfying formula (1a), and to a method for activating, in a
physiological environment, the release of a substance linked to a
compound satisfying formula (1a), wherein a tetrazine is used as an
activator.
[0022] In another aspect, the invention presents the use of the
inverse electron-demand Diels-Alder reaction between a compound
satisfying formula (1a) and a diene, preferably a tetrazine, as a
chemical tool for the release, in a physiological environment, of a
substance administered in a covalently bound form, wherein the
substance is bound to a compound satisfying formula (1a).
The Inverse Electron Demand ("Retro") Diels-Alder Reaction
[0023] The dienophile of formula (1a) and the diene are capable of
reacting in an inverse electron-demand Diels-Alder reaction.
Activation of the Prodrug by the retro Diels-Alder reaction of the
Trigger with the Activator leads to release of the Drug.
[0024] Below a reaction scheme is given for a [4+2] Diels-Alder
reaction between the (3,6)-di-(2-pyridyl)-s-tetrazine diene and a
trans-cylcooctene dienophile, followed by a retro Diels Alder
reaction in which the product and dinitrogen is formed. The
reaction product may tautomerize, and this is also shown in the
scheme. Because the trans cyclooctene derivative does not contain
electron withdrawing groups as in the classical Diels Alder
reaction, this type of Diels Alder reaction is distinguished from
the classical one, and frequently referred to as an "inverse
electron demand Diels Alder reaction". In the following text the
sequence of both reaction steps, i.e. the initial Diels-Alder
cyclo-addition (typically an inverse electron demand Diels Alder
cyclo-addition) and the subsequent retro Diels Alder reaction will
be referred to in shorthand as "retro Diels Alder reaction" or
"retro-DA". It will sometimes be abbreviated as "rDA" reaction. The
product of the reaction is then the retro Diels-Alder adduct, or
the rDA adduct.
##STR00002##
DETAILED DESCRIPTION OF THE INVENTION
[0025] In a general sense, the invention is based on the
recognition that a drug can be released from trans-cyclooctene
derivatives satisfying formula (1a) upon cyclooaddition with
compatible dienes, such as tetrazine derivatives. The dienophiles
of formula (1a) have the advantage that they react (and effectuate
drug release) with substantially any diene.
[0026] Without wishing to be bound by theory, the inventors believe
that the molecular structure of the retro Diels-Alder adduct is
such that a spontaneous elimination or cyclization reaction within
this rDA adduct releases the drug. Particularly, the inventors
believe that appropriately modified rDA components, i.e. according
to the present invention, lead to rDA adducts wherein the bond to
the drug on the part originating from the dienophile is broken by
the reaction with a nucleophile on the part originating from the
dienophile, while such an intramolecular reaction within the part
originating from the dienophile is precluded prior to rDA reaction
with the diene.
[0027] The general concept of using the retro-Diels Alder reaction
in Prodrug activation is illustrated in Scheme 1.
##STR00003##
[0028] In this scheme "TCO" stands for trans-cyclooctene. The term
trans-cyclooctene is used here as possibly including one or more
hetero-atoms, and particularly refers to a structure satisfying
formula (1a). In a broad sense,
the inventors have found that--other than the attempts made on the
basis of the Staudinger reaction--the selection of a TCO as the
trigger moiety for a prodrug, provides a versatile tool to render
drug (active) moieties into prodrug (activatable) moieties, wherein
the activation occurs through a powerful, abiotic, bio-orthogonal
reaction of the dienophile (Trigger) with the diene (Activator),
viz the aforementioned retro Diels-Alder reaction, and wherein the
Prodrug is a Drug-dienophile conjugate.
[0029] It will be understood that in Scheme 1 in the retro
Diels-Alder adduct as well as in the end product, the indicated TCO
group and the indicated diene group are the residues of,
respectively, the TCO and diene groups after these groups have been
converted in the retro Diels-Alder reaction.
[0030] A requirement for the successful application of an abiotic
bio-orthogonal chemical reaction is that the two participating
functional groups have finely tuned reactivity so that interference
with coexisting functionality is avoided. Ideally, the reactive
partners would be abiotic, reactive under physiological conditions,
and reactive only with each other while ignoring their
cellular/physiological surroundings (bio-orthogonal). The demands
on selectivity imposed by a biological environment preclude the use
of most conventional reactions.
[0031] The inverse electron demand Diels Alder reaction, however,
has proven utility in animals at low concentrations and
semi-equimolar conditions (R. Rossin et al, Angewandte Chemie Int
Ed 2010, 49, 3375-3378). The reaction partners subject to this
invention are strained trans-cyclooctene (TCO) derivatives and
suitable dienes, such as tetrazine derivatives. The cycloaddition
reaction between a TCO and a tetrazine affords an intermediate,
which then rearranges by expulsion of dinitrogen in a
retro-Diels-Alder cycloaddition to form a dihydropyridazine
conjugate. This and its tautomers is the retro Diels-Alder
adduct.
[0032] Reflecting the suitability of the rDA reaction, the
invention provides, in one aspect, the use of a tetrazine as an
activator for the release, in a physiological environment, of a
substance linked to a trans-cyclooctene. In connection herewith,
the invention also pertains to a tetrazine for use as an activator
for the release, in a physiological environment, of a substance
linked to a trans-cyclooctene, and to a method for activating, in a
physiological environment, the release of a substance linked to a
trans-cyclooctene, wherein a tetrazine is uses as an activator.
[0033] The present inventors have further come to the non-obvious
insight, that the structure of the TCO of formula (1a), par
excellence, is suitable to provoke the release of a drug linked to
it, as a result of the reaction involving the double bond available
in the TCO dienophile, and a diene. The feature believed to enable
this is the change in nature of the eight membered ring of the TCO
in the dienophile reactant as compared to that of the eight
membered ring in the rDA adduct. The eight membered ring in the rDA
adduct has significantly more conformational freedom and has a
significantly different conformation as compared to the eight
membered ring in the highly strained TCO prior to rDA reaction. The
nucleophilic site in the dienophile prior to rDA reaction is locked
within the specific conformation of the dienophile and is therefore
not properly positioned to react intramolecularly and to thereby
release the drug species. In contrast, and due to the changed
nature of the eight membered ring, this nucleophilic site is
properly positioned within the rDA adduct and will react
intramolecularly, thereby releasing the drug. According to the
above, but without being limited by theory, we believe that the
drug release is mediated by strain-release of the TCO-dienophile
after and due to the rDA reaction with the diene Activator.
[0034] It is to be emphasized that the invention is thus of a scope
well beyond specific chemical structures. In a broad sense, the
invention puts to use the recognition that the rDA reaction using a
dienophile of formula (1a) as well as the rDA adduct embody a
versatile platform for enabling provoked drug release in a
bioorthogonal reaction.
[0035] Reflecting on this, the invention also presents the use of
the inverse electron-demand Diels-Alder reaction between a
trans-cyclooctene and a tetrazine as a chemical tool for the
release, in a physiological environment, of a bound substance.
[0036] The fact that the reaction is bio-orthogonal, and that many
structural options exist for the reaction pairs, will be clear to
the skilled person. E.g., the rDA reaction is known in the art of
pre-targeted medicine. Reference is made to, e.g., WO 2010/119382,
WO 2010/119389, and WO 2010/051530. Whilst the invention presents
an entirely different use of the reaction, it will be understood
that the various structural possibilities available for the rDA
reaction pairs as used in pre-targeting, are also available in the
field of the present invention.
[0037] The dienophile trigger moiety used in the present invention
comprises a trans-cyclooctene ring, the ring optionally including
one or more hetero-atoms. Hereinafter this eight-membered ring
moiety will be defined as a trans-cyclooctene moiety, for the sake
of legibility, or abbreviated as "TCO" moiety. It will be
understood that the essence resides in the possibility of the
eight-membered ring to act as a dienophile and to be released from
its conjugated drug upon reaction. The skilled person is familiar
with the fact that the dienophile activity is not necessarily
dependent on the presence of all carbon atoms in the ring, since
also heterocyclic monoalkenylene eight-membered rings are known to
possess dienophile activity.
[0038] Thus, in general, the invention is not limited to strictly
drug-substituted trans-cyclooctene. The person skilled in organic
chemistry will be aware that other eight-membered ring-based
dienophiles exist, which comprise the same endocyclic double bond
as the trans-cyclooctene, but which may have one or more
heteroatoms elsewhere in the ring. I.e., the invention generally
pertains to eight-membered non-aromatic cyclic alkenylene moieties,
preferably a cyclooctene moiety, and more preferably a
trans-cyclooctene moiety, comprising a conjugated drug.
[0039] Other than is the case with e.g. medicinally active
substances, where the in vivo action is often changed with minor
structural changes, the present invention first and foremost
requires the right chemical reactivity combined with an appropriate
design of the drug-conjugate. Thus, the possible structures extend
to those of which the skilled person is familiar with that these
are reactive as dienophiles.
[0040] It should be noted that, depending on the choice of
nomenclature, the TCO dienophile may also be denoted E-cyclooctene.
With reference to the conventional nomenclature, it will be
understood that, as a result of substitution on the cyclooctene
ring, depending on the location and molecular weight of the
substituent, the same cyclooctene isomer may formally become
denoted as a Z-isomer. In the present invention, any substituted
variants of the invention, whether or not formally "E" or "Z," or
"cis" or "trans" isomers, will be considered derivatives of
unsubstituted trans-cyclooctene, or unsubstituted E-cyclooctene.
The terms "trans-cyclooctene" (TCO) as well as E-cyclooctene are
used interchangeably and are maintained for all dienophiles
according to the present invention, also in the event that
substituents would formally require the opposite nomenclature.
I.e., the invention relates to cyclooctene in which carbon atoms 1
and 6 as numbered below are in the E (entgegen) or trans
position.
##STR00004##
[0041] The present invention will further be described with respect
to particular embodiments and with reference to certain drawings
but the invention is not limited thereto but only by the claims.
Any reference signs in the claims shall not be construed as
limiting the scope. The drawings described are only schematic and
are non-limiting. In the drawings, the size of some of the elements
may be exaggerated and not drawn on scale for illustrative
purposes. Where an indefinite or definite article is used when
referring to a singular noun e.g. "a" or "an", "the", this includes
a plural of that noun unless something else is specifically
stated.
[0042] It is furthermore to be noticed that the term "comprising",
used in the description and in the claims, should not be
interpreted as being restricted to the means listed thereafter; it
does not exclude other elements or steps. Thus, the scope of the
expression "a device comprising means A and B" should not be
limited to devices consisting only of components A and B. It means
that with respect to the present invention, the only relevant
components of the device are A and B.
[0043] In several chemical formulae below reference is made to
"alkyl" and "aryl." In this respect "alkyl", each independently,
indicates an aliphatic, straight, branched, saturated, unsaturated
and/or or cyclic hydrocarbyl group of up to ten carbon atoms,
possibly including 1-10 heteroatoms such as O, N, or S, and "aryl",
each independently, indicates an aromatic or heteroaromatic group
of up to twenty carbon atoms, that possibly is substituted, and
that possibly includes 1-10 heteroatoms such as O, N, P or S.
"Aryl" groups also include "alkylaryl" or "arylalkyl" groups
(simple example: benzyl groups). The number of carbon atoms that an
"alkyl", "aryl", "alkylaryl" and "arylalkyl" contains can be
indicated by a designation preceding such terms (i.e.
C.sub.1-10alkyl means that said alkyl may contain from 1 to 10
carbon atoms). Certain compounds of the invention possess chiral
centers and/or tautomers, and all enantiomers, diasteriomers and
tautomers, as well as mixtures thereof are within the scope of the
invention. In several formulae, groups or substituents are
indicated with reference to letters such as "A", "B", "X", "Y", and
various (numbered) "R" groups. The definitions of these letters are
to be read with reference to each formula, i.e. in different
formulae these letters, each independently, can have different
meanings unless indicated otherwise.
[0044] In all embodiments of the invention as described herein,
alkyl is preferably lower alkyl (C.sub.1-4 alkyl), and each aryl
preferably is phenyl.
[0045] Earlier work (R. Rossin et al, Angewandte Chemie Int Ed
2010, 49, 3375-3378) demonstrated the utility of the
inverse-electron-demand Diels Alder reaction for pretargeted
radioimmunoimaging. This particular cycloaddition example occurred
between a (3,6)-di-(2-pyridyl)-s-tetrazine derivative and a
E-cyclooctene, followed by a retro Diels Alder reaction in which
the product and nitrogen is formed. Because the trans cyclooctene
derivative does not contain electron withdrawing groups as in the
classical Diels Alder reaction, this type of Diels Alder reaction
is distinguished from the classical one, and frequently referred to
as an "inverse electron demand Diels Alder reaction". In the
following text the sequence of both reaction steps, i.e. the
initial Diels-Alder cyclo-addition (typically an inverse electron
demand Diels Alder cyclo-addition) and the subsequent retro Diels
Alder reaction will be referred to in shorthand as "retro Diels
Alder reaction."
Retro Diels-Alder Reaction
[0046] The Retro Diels-Alder coupling chemistry generally involves
a pair of reactants that couple to form an unstable intermediate,
which intermediate eliminates a small molecule (depending on the
starting compounds this may be e.g. N.sub.2, CO.sub.2, RCN), as the
sole by-product through a retro Diels-Alder reaction to form the
retro Diels-Alder adduct. The paired reactants comprise, as one
reactant (i.e. one Bio-orthogonal Reactive Group), a suitable
diene, such as a derivative of tetrazine, e.g. an
electron-deficient tetrazine and, as the other reactant (i.e. the
other Bio-orthogonal Reactive Group), a suitable dienophile, such
as a strained cyclooctene (TCO).
[0047] The exceptionally fast reaction of e.g. electron-deficient
(substituted) tetrazines with a TCO moiety results in a ligation
intermediate that rearranges to a dihydropyridazine retro
Diels-Alder adduct by eliminating N.sub.2 as the sole by-product in
a [4+2] Retro Diels-Alder cycloaddition. In aqueous environment,
the initially formed 4,5-dihydropyridazine product may tautomerize
to a 1,4-dihydropyridazine product.
[0048] The two reactive species are abiotic and do not undergo fast
metabolism or side reactions in vivo. They are bio-orthogonal, e.g.
they selectively react with each other in physiologic media. Thus,
the compounds and the method of the invention can be used in a
living organism. Moreover, the reactive groups are relatively small
and can be introduced in biological samples or living organisms
without significantly altering the size of biomolecules therein.
References on the Inverse electron demand Diels Alder reaction, and
the behavior of the pair of reactive species include: Thalhammer,
F; Wallfahrer, U; Sauer, J, Tetrahedron Letters, 1990, 31 (47),
6851-6854; Wijnen, .mu.W; Zavarise, S; Engberts, JBFN, Journal Of
Organic Chemistry, 1996, 61, 2001-2005; Blackman, M L; Royzen, M;
Fox, J M, Journal Of The American Chemical Society, 2008, 130 (41),
13518-19), R. Rossin, P. Renart Verkerk, Sandra M. van den Bosch,
R. C. M. Vulders, 1. Verel, J. Lub, M. S. Robillard, Angew Chem Int
Ed 2010, 49, 3375, N. K. Devaraj, R. Upadhyay, J. B. Haun, S. A.
Hilderbrand, R. Weissleder, Angew Chem Int Ed 2009, 48, 7013, and
Devaraj et al., Angew. Chem. Int. Ed., 2009, 48, 1-5.
[0049] It will be understood that, in a broad sense, according to
the invention the aforementioned retro Diels-Alder coupling and
subsequent drug activation chemistry can be applied to basically
any pair of molecules, groups, or moieties that are capable of
being used in Prodrug therapy. I.e. one of such a pair will
comprise a drug linked to a dienophile (the Trigger). The other one
will be a complementary diene for use in reaction with said
dienophile.
Trigger
[0050] The Prodrug comprises a Drug denoted as D.sup.D linked,
directly or indirectly, to a Trigger moiety denoted as T.sup.R,
wherein the Trigger moiety is a dienophile. The dienophile, in a
broad sense, is an eight-membered non-aromatic cyclic alkenylene
moiety (preferably a cyclooctene moiety, and more preferably a
trans-cyclooctene moiety).
[0051] In this invention, the release of the drug or drugs is
caused by an intramolecular cyclization/elimination reaction within
the part of the Retro Diels-Alder adduct that originates from the
TCO dienophile. A nucleophilic site present on the TCO (i.e. the
dienophile, particularly from the Y.sup.D group in this Trigger,
vide supra) reacts with an electrophilic site on the same TCO
(particularly from the X.sup.D group in this Trigger, vide supra)
after this TCO reacts with the Activator to form an rDA adduct. The
part of the rDA adduct that originates from the TCO, i.e. the eight
membered ring of the rDA adduct, has a different conformation and
has an increased conformational freedom compared to the eight
membered ring in the TCO prior to the rDA reaction, allowing the
nucleophilic reaction to occur, thereby releasing the drug as a
leaving group. The intramolecular cyclization/elimination reaction
takes place, as the nucleophilic site and the electrophilic site
have been brought together in close proximity within the Retro
Diels-Alder adduct, and produce a favorable structure with a low
strain. Additionally, the formation of the cyclic structure may
also be a driving force for the intramolecular reaction to take
place, and thus may also contribute to an effective release of the
leaving group, i.e. release of the drug. Reaction between the
nucleophilic site and the electrophilic site does not take place or
is relatively inefficient prior to the Retro Diels-Alder reaction,
as both sites are positioned unfavorably for such a reaction, due
to the relatively rigid, conformationally restrained TCO ring. The
Prodrug itself is relatively stable as such and elimination is
favored only after the Activator and the Prodrug have reacted and
have been assembled in a retro Diels-Alder adduct that is subject
to intramolecular reaction. In a preferred embodiment the TCO ring
is in the crown conformation. The example below illustrates the
release mechanism pertaining to this invention.
##STR00005##
[0052] The above example illustrates how the intramolecular
cyclization/elimination reaction within the retro Diels-Alder
adduct can result in release of a drug species. The rDA reaction
produces A, which may tautomerize to product B and C. Structures B
and C may also tautomerize to one another (not shown). rDA products
A, B, and C may intramolecularly cyclize, releasing the bound drug,
and affording structures D, E, and F, which optionally may oxidise
to form product G. As the tautomerization of A into B and C in
water is very fast (in the order of seconds) it is the inventors'
belief, that drug release occurs predominantly from structures B
and C. It may also be possible that the nucleophilic site assists
in expelling the drug species by a nucleophilic attack on the
electrophilic site with subsequent drug release, but without
actually forming a (stable) cyclic structure. In this case, no ring
structure is formed and the nucleophilic site remains intact, for
example because the ring structure is shortlived and unstable and
breaks down with reformation of the nucleophilic site. In any case,
and in whatever way the process is viewed, the drug species (here
the alcohol `drug-OH`) is effectively expelled from the retro
Diels-Alder adduct, while it does not get expelled from the Prodrug
alone.
[0053] Without wishing to be bound by theory, the above example
illustrates how the conformational restriction and the resulting
unfavorable positioning of the nucleophilic and electrophilic site
in the TCO trigger is relieved following rDA adduct formation,
leading to an elimination/cyclization reaction and release of the
drug.
[0054] With respect to the nucleophilic site on the TCO, one has to
consider that the site must be able to act as a nucleophile under
conditions that may exist inside the (human) body, so for example
at physiological conditions where the pH=ca. 7.4, or for example at
conditions that prevail in malignant tissue where pH-values may be
lower than 7.4. Preferred nucleophiles are amine, thiol or alcohol
groups, as these are generally most nucleophilic in nature and
therefore most effective.
[0055] In this invention, the TCO satisfies the following formula
(1a):
##STR00006##
[0056] In Embodiment 1, one of the bonds PQ, QP, QX, XQ, XZ, ZX,
ZY, YZ, YA, AY consists of --CR.sup.aX.sup.D--CR.sup.aY.sup.D-, the
remaining groups (from A,Y,Z,X,Q,P) being independently from each
other CR.sup.a.sub.2, S, O, SiR.sup.b.sub.2, such that P and A are
CR.sup.a.sub.2, and no adjacent pairs of atoms are present selected
from the group consisting of O--O, O--S, and S--S, and such that
Si, if present, is adjacent to CR.sup.a.sub.2 or O.
[0057] X.sup.D is O--C(O)-(L.sup.D).sub.n-(D.sup.D),
S--C(O)-(L.sup.D).sub.n-(D.sup.D),
O--C(S)-(L.sup.D).sub.n-(D.sup.D),
S--C(S)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(O)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(S)-(L.sup.D).sub.n-(D.sup.D), and then Y.sup.D is
NHR.sup.c, OH, SH; or X.sup.D is C(O)-(L.sup.D).sub.n-(D.sup.D),
C(S)-(L.sup.D).sub.n-(D.sup.D); and then Y.sup.D is
CR.sup.c.sub.2NHR.sup.c, CR.sup.c.sub.2OH, CR.sup.c.sub.2SH,
NH--NH.sub.2, O--NH.sub.2, NH--OH.
[0058] Preferably X.sup.D is
NR.sup.c--C(O)-(L.sup.D).sub.n-(D.sup.D), and Y.sup.D is
NHR.sup.c.
[0059] In this Embodiment 1, the X.sup.D and Y.sup.D groups may be
positioned cis or trans relative to each other, where depending on
the positions on the TCO, cis or trans are preferred: if PQ, QP, AY
or YA is --CR.sup.aX.sup.D--CR.sup.aY.sup.D-, then X.sup.D and
Y.sup.D are preferably positioned trans relative to each other; if
ZX or XZ is --CR.sup.aX.sup.D--CR.sup.aY.sup.D-, then X.sup.D and
Y.sup.D are preferably positioned cis relative to each other.
[0060] In Embodiment 2, A is CR.sup.aX.sup.D and Z is
CR.sup.aY.sup.D, or Z is CR.sup.aX.sup.D and A is CR.sup.aY.sup.D,
or P is CR.sup.aX.sup.D and X is CR.sup.aY.sup.D, or X is
CR.sup.aX.sup.D and P is CR.sup.aY.sup.D, such that X.sup.D and
Y.sup.D are positioned in a trans conformation with respect to one
another; the remaining groups (from A,Y,Z,X,Q,P) being
independently from each other CR.sup.a.sub.2, S, O,
SiR.sup.b.sub.2, such that P and A are CR.sup.a.sub.2, and no
adjacent pairs of atoms are present selected from the group
consisting of O--O, O--S, and S--S, and such that Si, if present,
is adjacent to CR.sup.a.sub.2 or O; X.sup.D is
O--C(O)-(L.sup.D).sub.n-(D.sup.D),
S--C(O)-(L.sup.D).sub.n-(D.sup.D),
O--C(S)-(L.sup.D).sub.n-(D.sup.D),
S--C(S)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(O)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(S)-(L.sup.D).sub.n-(D.sup.D), and then Y.sup.D is
NHR.sup.c, OH, SH, CR.sup.c.sub.2NHR.sup.c, CR.sup.c.sub.2OH,
CR.sup.c.sub.2SH, NH--NH.sub.2, O--NH.sub.2, NH--OH; or X.sup.D is
CR.sup.c.sub.2--O--C(O)-(L.sup.D).sub.n-(D.sup.D),
CR.sup.c.sub.2--S--C(O)-(L.sup.D).sub.n-(D.sup.D),
CR.sup.c.sub.2--O--C(S)-(L.sup.D).sub.n-(D.sup.D),
CR.sup.c.sub.2--S--C(S)-(L.sup.D).sub.n-(D.sup.D),
CR.sup.c.sub.2--NR.sup.c--C(O)-(L.sup.D).sub.n-(D.sup.D),
CR.sup.c.sub.2--NR.sup.c--C(S)-(L.sup.D).sub.n-(D.sup.D); and then
Y.sup.D is NHR.sup.c, OH, SH; or X.sup.D is
C(O)-(L.sup.D).sub.n-(D.sup.D), C(S)-(L.sup.D).sub.n-(D.sup.D); and
then Y.sup.D is CR.sup.c.sub.2NHR.sup.c, CR.sup.c.sub.2OH,
CR.sup.c.sub.2SH, NH--NH.sub.2, O--NH.sub.2, NH--OH.
[0061] Preferably X.sup.D is
NR.sup.c--C(O)-(L.sup.D).sub.n-(D.sup.D), and Y.sup.D is
NHR.sup.c.
[0062] In Embodiment 3, A is CR.sup.aY.sup.D and one of P, Q, X, Z
is CR.sup.aX.sup.D, or P is CR.sup.aY.sup.D and one of A, Y, Z, X
is CR.sup.aX.sup.D, or Y is CR.sup.aY.sup.D and X or P is
CR.sup.aX.sup.D, or Q is CR.sup.aY.sup.D and Z or A is
CR.sup.aX.sup.D, or either Z or X is CR.sup.aY.sup.D and A or P is
CR.sup.aX.sup.D, such that X.sup.D and Y.sup.D are positioned in a
trans conformation with respect to one another; the remaining
groups (from A,Y,Z,X,Q,P) being independently from each other
CR.sup.a.sub.2, S, O, SiR.sup.b.sub.2, such that P and A are
CR.sup.a.sub.2, and no adjacent pairs of atoms are present selected
from the group consisting of O--O, O--S, and S--S, and such that
Si, if present, is adjacent to CR.sup.a.sub.2 or O.
[0063] X.sup.D is (O--C(O)).sub.p-(L.sup.D)-(D.sup.D),
S--C(O)-(L.sup.D)-(D.sup.D), O--C(S)-(L.sup.D).sub.n-(D.sup.D),
S--C(S)-(L.sup.D).sub.n-(D.sup.D); Y.sup.D is
CR.sup.c.sub.2NHR.sup.c, CR.sup.c.sub.2OH, CR.sup.c.sub.2SH,
NH--NH.sub.2, O--NH.sub.2, NH--OH; p=0 or 1.
[0064] Preferably X.sup.D is
(O--C(O)).sub.p-(L.sup.D).sub.n-(D.sup.D), with p=1, and Y.sup.D is
CR.sup.c.sub.2NHR.sup.c.
In Embodiment 4, P is CR.sup.aY.sup.D and Y is CR.sup.aX.sup.D, or
A is CR.sup.aY.sup.D and Q is CR.sup.aX.sup.D, or Q is
CR.sup.aY.sup.D and A is CR.sup.aX.sup.D, or Y is CR.sup.aY.sup.D
and P is CR.sup.aX.sup.D, such that X.sup.D and Y.sup.D are
positioned in a trans conformation with respect to one another; the
remaining groups (from A,Y,Z,X,Q,P) being independently from each
other CR.sup.a.sub.2, S, O, SiR.sup.b.sub.2, such that P and A are
CR.sup.a.sub.2, and no adjacent pairs of atoms are present selected
from the group consisting of O--O, O--S, and S--S, and such that
Si, if present, is adjacent to CR.sup.a.sub.2 or O.
[0065] X.sup.D is (O--C(O))-(L.sup.D).sub.n-(D.sup.D),
S--C(O)-(L.sup.D).sub.n-(D.sup.D),
O--C(S)-(L.sup.D).sub.n-(D.sup.D),
S--C(S)-(L.sup.D).sub.n-(D.sup.D); Y.sup.D is NHR.sup.c, OH, SH;
p=0 or 1.
[0066] Preferably X.sup.D is
(O--C(O)).sub.n-(L.sup.D).sub.n-(D.sup.D), with p=1, and Y.sup.D is
NHR.sup.c.
[0067] In Embodiment 5, Y is Y.sup.D and P is CR.sup.aX.sup.D, or Q
is Y.sup.D and A is CR.sup.aX.sup.D; the remaining groups (from
A,Y,Z,X,Q,P) being independently from each other CR.sup.a.sub.2, S,
O, SiR.sup.b.sub.2, such that P and A are CR.sup.a.sub.2, and no
adjacent pairs of atoms are present selected from the group
consisting of O--O, O--S, and S--S, and such that Si, if present,
is adjacent to CR.sup.a.sub.2 or O.
[0068] X.sup.D is (O--C(O)).sub.n-(L.sup.D)-(D.sup.D),
S--C(O)-(L.sup.D)-(D.sup.D), O--C(S)-(L.sup.D).sub.n-(D.sup.D),
S--C(S)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(O)-(L.sup.D).sub.n-(D.sup.D),
NR.sup.c--C(S)-(L.sup.D).sub.n-(D.sup.D),
C(O)-(L.sup.D).sub.n-(D.sup.D), C(S)-(L.sup.D).sub.n-(D.sup.D);
Y.sup.D is NH; p=0 or 1.
[0069] Preferably X.sup.D is
NR.sup.c--C(O)-(L.sup.D).sub.n-(D.sup.D) or
(O--C(O)).sub.p-(L.sup.D).sub.n-(D.sup.D), with p=0 or 1.
[0070] In Embodiment 6, Y is Y.sup.D and P or Q is X.sup.D, or Q is
Y.sup.D and A or Y is X.sup.D; the remaining groups (from
A,Y,Z,X,Q,P) being independently from each other CR.sup.a.sub.2, S,
O, SiR.sup.b.sub.2, such that P and A are CR.sup.a.sub.2, and no
adjacent pairs of atoms are present selected from the group
consisting of O--O, O--S, and S--S, and such that Si, if present,
is adjacent to CR.sup.a.sub.2 or O.
[0071] X.sup.D is N--C(O)-(L.sup.D).sub.n-(D.sup.D),
N--C(S)-(L.sup.D).sub.n-(D.sup.D); Y.sup.D is NH;
[0072] Preferably X.sup.D is N--C(O)-(L.sup.D).sub.n-(D.sup.D)
[0073] T, F each independently denotes H, or a substituent selected
from the group consisting of alkyl, F, Cl, Br, or I. (L.sup.D) is
an optional linker with n=0 or 1, preferably linked to T.sup.R via
S, N, NH, or O, wherein these atoms are part of the linker, which
may consist of multiple units arranged linearly and/or branched.
D.sup.D is one or more therapeutic moieties or drugs, preferably
linked via S, N, NH, or O, wherein these atoms are part of the
therapeutic moiety.
In a preferred embodiment, the TCO of formula (1a) is an all-carbon
ring. In another preferred embodiment, the TCO of formula (1a) is a
heterocyclic carbon ring, having of one or two oxygen atoms in the
ring, and preferably a single oxygen atom.
[0074] It is preferred that when D.sup.D is bound to T.sup.R or
L.sup.D via NH, this NH is a primary amine (--NH.sub.2) residue
from D.sup.D, and when D.sup.D is bound via N, this N is a
secondary amine (--NH--) residue from D.sup.D. Similarly, it is
preferred that when D.sup.D is bound via O or S, said O or S are,
respectively, a hydroxyl (--OH) residue or a sulfhydryl (--SH)
residue from D.sup.D. It is further preferred that said S, N, NH,
or O moieties comprised in D.sup.D are bound to an aliphatic or
aromatic carbon of D.sup.D.
[0075] It is preferred that when L.sup.D is bound to T.sup.R via
NH, this NH is a primary amine (--NH.sub.2) residue from L.sup.D,
and when L.sup.D is bound via N, this N is a secondary amine
(--NH--) residue from L.sup.D. Similarly, it is preferred that when
L.sup.D is bound via O or S, said O or S are, respectively, a
hydroxyl (--OH) residue or a sulfhydryl (--SH) residue from
L.sup.D.
It is further preferred that said S, N, NH, or O moieties comprised
in L.sup.D are bound to an aliphatic or aromatic carbon of
L.sup.D.
[0076] Where reference is made in the invention to a linker L.sup.D
this can be self-immolative or not, or a combination thereof, and
which may consist of multiple self-immolative units. By way of
further clarification, if p=0 and n=0 the drug species D.sup.D
directly constitutes the leaving group of the elimination reaction,
and if p=0 and n=1, the self-immolative linker constitutes the
leaving group of the elimination. The position and ways of
attachment of linkers L.sup.D and drugs D.sup.D are known to the
skilled person (see for example Papot et al, Anti-Cancer Agents in
Medicinal Chemistry, 2008, 8, 618-637). Nevertheless, typical but
non-limiting examples of self-immolative linkers L.sup.D are
benzyl-derivatives, such as those drawn below. On the right, an
example of a self-immolative linker with multiple units is shown;
this linker will degrade not only into CO.sub.2 and one unit of
4-aminobenzyl alcohol, but also into one
1,3-dimethylimidazolidin-2-one unit.
##STR00007##
[0077] By substituting the benzyl groups of aforementioned
self-immolative linkers L.sup.D, preferably on the 2- and/or
6-position, it may be possible to tune the rate of release of the
drug species D.sup.D, caused by either steric and/or electronic
effects on the intramolecular cyclization/elimination reaction.
Synthetic procedures to prepare such substituted benzyl-derivatives
are known to the skilled person (see for example Greenwald et al,
J. Med. Chem., 1999, 42, 3657-3667 and Thomthwaite et al, Polym.
Chem., 2011, 2, 773-790). Some examples of substituted
benzyl-derivatives with different release rates are drawn
below.
##STR00008##
[0078] Each R.sup.a as above-indicated can independently be H,
alkyl, aryl, OR', SR', S(.dbd.O)R''', S(.dbd.O).sub.2R''',
S(.dbd.O).sub.2NR'R'', Si--R''', Si--O--R''', OC(.dbd.O)R''',
SC(.dbd.O)R''', OC(.dbd.S)R''', SC(.dbd.S)R''', F, Cl, Br, I,
N.sub.3, SO.sub.2H, SO.sub.3H, SO.sub.4H, PO.sub.3H, PO.sub.4H, NO,
NO.sub.2, CN, OCN, SCN, NCO, NCS, CF.sub.3, CF.sub.2--R', NR'R'',
C(.dbd.O)R', C(.dbd.S)R', C(.dbd.O)O--R', C(.dbd.S)O--R',
C(.dbd.O)S--R', C(.dbd.S)S--R', C(.dbd.O)NR'R'', C(.dbd.S)NR'R'',
NR'C(.dbd.O)--R''', NR'C(.dbd.S)--R''', NR'C(.dbd.O)O--R''',
NR'C(.dbd.S)O--R''', NR'C(.dbd.O)S--R''', NR'C(.dbd.S)S--R''',
OC(.dbd.O)NR'--R''', SC(.dbd.O)NR'--R''', OC(.dbd.S)NR'--R''',
SC(.dbd.S)NR'--R''', NR'C(.dbd.O)NR''--R'', NR'C(.dbd.S)NR''--R'',
CR'NR'', with each R' and each R'' independently being H, aryl or
alkyl and R''' independently being aryl or alkyl;
[0079] Each R.sup.b as above indicated is independently selected
from the group consisting of H, alkyl, aryl, O-alkyl, O-aryl,
OH;
[0080] Each R.sup.c as above indicated is independently selected
from H, C.sub.1-6alkyl and C.sub.1-6 aryl;
wherein two or more R.sup.a,b,c moieties together may form a
ring;
[0081] Preferably, each R.sup.a is independently selected from the
group consisting of H, alkyl, O-alkyl, O-aryl, OH, C(.dbd.O)NR'R'',
NR'C(.dbd.O)--R''', with R' and R'' each independently being H,
aryl or alkyl, and with R''' independently being alkyl or aryl.
[0082] In all of the above embodiments, optionally one of A, P, Q,
Y, X, and Z, or the substituents, or the self-immolative linker
L.sup.D, or the drug D.sup.D, is bound, optionally via a spacer or
spacers S.sup.P, to one or more targeting agents T.sup.T or masking
moieties M.sup.M.
[0083] The synthesis of TCO's as described above is well available
to the skilled person. This expressly also holds for TCO's having
one or more heteroatoms in the strained cycloalkene rings.
References in this regard include Cere et al. Journal of Organic
Chemistry 1980, 45, 261 and Prevost et al. Journal of the American
Chemical Society 2009, 131, 14182.
[0084] In a further preferred embodiment, the dienophile is a
compound selected from the following structures:
##STR00009##
[0085] =rest of attached D.sup.D, L.sup.D-D.sup.D, optionally
comprising T.sup.T or S.sup.P-T.sup.T or M.sup.M or
S.sup.P-M.sup.M
[0086] In alternative embodiments, the dienophile is a compound
selected from the following structures:
##STR00010##
[0087] =rest of attached D.sup.D, L.sup.D-D.sup.D, optionally
comprising T.sup.T or S.sup.P-T.sup.T or M.sup.M or
S.sup.P-M.sup.M
Use of TCO as a Carrier
[0088] The invention also pertains to the use of a
trans-cyclooctene satisfying formula (1a), in all its embodiments,
as a carrier for a therapeutic compound. The trans-cyclooctene is
to be read as a TCO in a broad sense, as discussed above,
preferably an all-carbon ring or including one or two hetero-atoms.
A therapeutic compound is a drug or other compound or moiety
intended to have therapeutic application. The use of TCO as a
carrier according to this aspect of the invention does not relate
to the therapeutic activity of the therapeutic compound. In fact,
also if the therapeutic compound is a drug substance intended to be
developed as a drug, many of which will fail in practice, the
application of TCO as a carrier still is useful in testing the
drug. In this sense, the TCO in its capacity of a carrier is to be
regarded in the same manner as a pharmaceutical excipient, serving
as a carrier when introducing a drug into a subject.
[0089] The use of a TCO as a carrier has the benefit that it
enables the administration, to a subject, of a drug carried by a
moiety that is open to a bioorthogonal reaction, with a diene,
particularly a tetrazine. This provides a powerful tool not only to
affect the fate of the drug carried into the body, but also to
follow its fate (e.g. by allowing a labeled diene to react with
it), or to change its fate (e.g. by allowing pK modifying agents to
bind with it). This is all based on the possibility to let a diene
react with the TCO in the above-discussed rDA reaction. The carrier
is preferably reacted with an Activator as discussed below, so as
to provoke the release of the therapeutic compound from the TCO, as
amply discussed herein.
Activator
[0090] The Activator comprises a Bio-orthogonal Reactive Group,
wherein this Bio-orthogonal Reactive Group of the Activator is a
diene. This diene reacts with the other Bio-orthogonal Reactive
Group, the Trigger, and that is a dienophile (vide supra). The
diene of the Activator is selected so as to be capable of reacting
with the dienophile of the Trigger by undergoing a Diels-Alder
cycloaddition followed by a retro Diels-Alder reaction, giving the
Retro Diels-Alder adduct. This intermediate adduct then releases
the drug or drugs, where this drug release can be caused by various
circumstances or conditions that relate to the specific molecular
structure of the retro Diels-Alder adduct.
Dienes
[0091] The person skilled in the art is aware of the wealth of
dienes that are reactive in the Retro Diels-Alder reaction. The
diene comprised in the Activator can be part of a ring structure
that comprises a third double bond, such as a tetrazine (which is a
preferred Activator according to the invention).
[0092] Generally, the Activator is a molecule comprising a
heterocyclic moiety comprising at least 2 conjugated double
bonds.
[0093] Preferred dienes are given below, with reference to formulae
(2)-(4).
##STR00011##
[0094] In formula (2) R.sup.1 is selected from the group consisting
of H, alkyl, aryl, CF.sub.3, CF.sub.2--R', OR', SR', C(.dbd.O)R',
C(.dbd.S)R', C(.dbd.O)O--R', C(.dbd.O)S--R', C(.dbd.S)O--R',
C(.dbd.S)S--R'', C(.dbd.O)NR'R'', C(.dbd.S)NR'R'', NR'R'',
NR'C(.dbd.O)R'', NR'C(.dbd.S)R'', NR'C(.dbd.O)OR'',
NR'C(.dbd.S)OR'', NR'C(.dbd.O)SR'', NR'C(.dbd.S)SR'',
NR'C(.dbd.O)NR'R'', NR'C(.dbd.S)NR'R'' with each R' and each R''
independently being H, aryl or alkyl; A and B each independently
are selected from the group consisting of alkyl-substituted carbon,
aryl substituted carbon, nitrogen, N.sup.+O.sup.-, N.sup.+R with R
being alkyl, with the proviso that A and B are not both carbon; X
is selected from the group consisting of O, N-alkyl, and C.dbd.O,
and Y is CR with R being selected from the group consisting of H,
alkyl, aryl, C(.dbd.O)OR', C(.dbd.O)SR', C(.dbd.S)OR',
C(.dbd.S)SR', C(.dbd.O)NR'R'' with R' and R'' each independently
being H, aryl or alkyl.
##STR00012##
[0095] A diene particularly suitable as a reaction partner for
cyclooctene is given in formula (3), wherein R.sup.1 and R.sup.2
each independently are selected from the group consisting of H,
alkyl, aryl, CF.sub.3, CF.sub.2--R', NO.sub.2, OR', SR',
C(.dbd.O)R', C(.dbd.S)R', OC(.dbd.O)R''', SC(.dbd.O)R''',
OC(.dbd.S)R''', SC(.dbd.S)R''', S(.dbd.O)R', S(.dbd.O).sub.2R''',
S(.dbd.O).sub.2NR'R'', C(.dbd.O)O--R', C(.dbd.O)S--R',
C(.dbd.S)O--R', C(.dbd.S)S--R', C(.dbd.O)NR'R'', C(.dbd.S)NR'R'',
NR'R'', NR'C(.dbd.O)R'', NR'C(.dbd.S)R'', NR'C(.dbd.O)OR'',
NR'C(.dbd.S)OR'', NR'C(.dbd.O)SR'', NR'C(.dbd.S)SR'',
OC(.dbd.O)NR'R'', SC(.dbd.O)NR'R'', OC(.dbd.S)NR'R'',
SC(.dbd.S)NR'R''', NR'C(.dbd.O)NR'R'', NR'C(.dbd.S)NR'R'' with each
R' and each R'' independently being H, aryl or alkyl, and R'''
independently being aryl or alkyl; A is selected from the group
consisting of N-alkyl, N-aryl, C.dbd.O, and CN-alkyl; B is O or S;
X is selected from the group consisting of N, CH, C-alkyl, C-aryl,
CC(.dbd.O)R', CC(.dbd.S)R', CS(.dbd.O)R', CS(.dbd.O).sub.2R''',
CC(.dbd.O)O--R', CC(.dbd.O)S--R', CC(.dbd.S)O--R', CC(.dbd.S)S--R',
CC(.dbd.O)NR'R'', CC(.dbd.S)NR'R'', R' and R'' each independently
being H, aryl or alkyl and R''' independently being aryl or alkyl;
Y is selected from the group consisting of CH, C-alkyl, C-aryl, N,
and N.sup.+O.sup.-.
##STR00013##
[0096] Another diene particularly suitable as a reaction partner
for cyclooctene is diene (4), wherein R.sup.1 and R.sup.2 each
independently are selected from the group consisting of H, alkyl,
aryl, CF.sub.3, CF.sub.2--R', NO, NO.sub.2, OR', SR', CN,
C(.dbd.O)R', C(.dbd.S)R', OC(.dbd.O)R''', SC(.dbd.O)R''',
OC(.dbd.S)R''', SC(.dbd.S)R''', S(.dbd.O)R', S(.dbd.O).sub.2R''',
S(.dbd.O).sub.2OR', PO.sub.3R'R'', S(.dbd.O).sub.2NR'R'',
C(.dbd.O)O--R', C(.dbd.O)S--R', C(.dbd.S)O--R', C(.dbd.S)S--R',
C(.dbd.O)NR'R'', C(.dbd.S)NR'R'', NR'R'', NR'C(.dbd.O)R'',
NR'C(.dbd.S)R'', NR'C(.dbd.O)OR'', NR'C(.dbd.S)OR'',
NR'C(.dbd.O)SR'', NR'C(.dbd.S)SR'', OC(.dbd.O)NR'R'',
SC(.dbd.O)NR'R'', OC(.dbd.S)NR'R'', SC(.dbd.S)NR'R'',
NR'C(.dbd.O)NR''R'', NR'C(.dbd.S)NR''R'' with each R' and each R''
independently being H, aryl or alkyl, and R''' independently being
aryl or alkyl; A is selected from the group consisting of N,
C-alkyl, C-aryl, and N.sup.+O.sup.-; B is N; X is selected from the
group consisting of N, CH, C-alkyl, C-aryl, CC(.dbd.O)R',
CC(.dbd.S)R', CS(.dbd.O)R', CS(.dbd.O).sub.2R''', CC(.dbd.O)O--R',
CC(.dbd.O)S--R', CC(.dbd.S)O--R', CC(.dbd.S)S--R',
CC(.dbd.O)NR'R'', CC(.dbd.S)NR'R'', R' and R'' each independently
being H, aryl or alkyl and R''' independently being aryl or alkyl;
Y is selected from the group consisting of CH, C-alkyl, C-aryl, N,
and N.sup.+O.sup.-.
##STR00014##
[0097] According to the invention, particularly useful dienes are
1,2-diazine, 1,2,4-triazine and 1,2,4,5-tetrazine derivatives, as
given in formulas (5), (6) and (7), respectively.
[0098] The 1,2-diazine is given in (5), wherein R.sup.1 and R.sup.2
each independently are selected from the group consisting of H,
alkyl, aryl, CF.sub.3, CF.sub.2--R', NO.sub.2, OR', SR',
C(.dbd.O)R', C(.dbd.S)R', OC(.dbd.O)R''', SC(.dbd.O)R''',
OC(.dbd.S)R''', SC(.dbd.S)R''', S(.dbd.O)R', S(.dbd.O).sub.2R''',
S(.dbd.O).sub.2NR'R'', C(.dbd.O)O--R', C(.dbd.O)S--R',
C(.dbd.S)O--R', C(.dbd.S)S--R', C(.dbd.O)NR'R'', C(.dbd.S)NR'R'',
NR'R'', NR'C(.dbd.O)R'', NR'C(.dbd.S)R'', NR'C(.dbd.O)OR'',
NR'C(.dbd.S)OR'', NR'C(.dbd.O)SR'', NR'C(.dbd.S)SR'',
OC(.dbd.O)NR'R'', SC(.dbd.O)NR'R'', OC(.dbd.S)NR'R'',
SC(.dbd.S)NR'R'', NR'C(.dbd.O)NR''R'', NR'C(.dbd.S)NR''R'' with
each R' and each R'' independently being H, aryl or alkyl, and R'''
independently being aryl or alkyl; X and Y each independently are
selected from the group consisting of O, N-alkyl, N-aryl, C.dbd.O,
CN-alkyl, CH, C-alkyl, C-aryl, CC(.dbd.O)R', CC(.dbd.S)R',
CS(.dbd.O)R', CS(.dbd.O).sub.2R''', CC(.dbd.O)O--R',
CC(.dbd.O)S--R', CC(.dbd.S)O--R', CC(.dbd.S)S--R',
CC(.dbd.O)NR'R'', CC(.dbd.S)NR'R'', with R' and R'' each
independently being H, aryl or alkyl and R''' independently being
aryl or alkyl, where X--Y may be a single or a double bond, and
where X and Y may be connected in a second ring structure apart
from the 6-membered diazine. Preferably, X--Y represents an ester
group (X.dbd.O and Y.dbd.C.dbd.O; X--Y is a single bond) or X--Y
represents a cycloalkane group (X.dbd.CR' and Y.dbd.CR''; X--Y is a
single bond; R' and R'' are connected), preferably a cyclopropane
ring, so that R' and R'' are connected to each other at the first
carbon atom outside the 1,2-diazine ring.
[0099] The 1,2,4-triazine is given in (6), wherein R.sup.1 and
R.sup.2 each independently are selected from the group consisting
of H, alkyl, aryl, CF.sub.3, CF.sub.2--R', NO.sub.2, OR', SR',
C(.dbd.O)R', C(.dbd.S)R', OC(.dbd.O)R''', SC(.dbd.O)R''',
OC(.dbd.S)R''', SC(.dbd.S)R''', S(.dbd.O)R', S(.dbd.O).sub.2R''',
S(.dbd.O).sub.2NR'R'', C(.dbd.O)O--R', C(.dbd.O)S--R',
C(.dbd.S)O--R', C(.dbd.S)S--R', C(.dbd.O)NR'R'', C(.dbd.S)NR'R'',
NR'R'', NR'C(.dbd.O)R'', NR'C(.dbd.S)R'', NR'C(.dbd.O)OR'',
NR'C(.dbd.S)OR'', NR'C(.dbd.O)SR'', NR'C(.dbd.S)SR'',
OC(.dbd.O)NR'R'', SC(.dbd.O)NR'R'', OC(.dbd.S)NR'R'',
SC(.dbd.S)NR'R'', NR'C(.dbd.O)NR''R'', NR'C(.dbd.S)NR''R'' with
each R' and each R'' independently being H, aryl or alkyl, and R'''
independently being aryl or alkyl; X is selected from the group
consisting of CH, C-alkyl, C-aryl, CC(.dbd.O)R', CC(.dbd.S)R',
CS(.dbd.O)R', CS(.dbd.O).sub.2R''', CC(.dbd.O)O--R',
CC(.dbd.O)S--R', CC(.dbd.S)O--R', CC(.dbd.S)S--R',
CC(.dbd.O)NR'R'', CC(.dbd.S)NR'R'', R' and R'' each independently
being H, aryl or alkyl and R''' independently being aryl or
alkyl.
[0100] The 1,2,4,5-tetrazine is given in (7), wherein R.sup.1 and
R.sup.2 each independently are selected from the group consisting
of H, alkyl, aryl, CF.sub.3, CF.sub.2--R', NO, NO.sub.2, OR', SR',
CN, C(.dbd.O)R', C(.dbd.S)R', OC(.dbd.O)R''', SC(.dbd.O)R''',
OC(.dbd.S)R''', SC(.dbd.S)R''', S(.dbd.O)R', S(.dbd.O).sub.2R''',
S(.dbd.O).sub.2OR', PO.sub.3R'R'', S(.dbd.O).sub.2NR'R'',
C(.dbd.O)O--R', C(.dbd.O)S--R', C(.dbd.S)O--R', C(.dbd.S)S--R',
C(.dbd.O)NR'R'', C(.dbd.S)NR'R'', NR'R'', NR'C(.dbd.O)R'',
NR'C(.dbd.S)R'', NR'C(.dbd.O)OR'', NR'C(.dbd.S)OR'',
NR'C(.dbd.O)SR'', NR'C(.dbd.S)SR'', OC(.dbd.O)NR'R'',
SC(.dbd.O)NR'R'', OC(.dbd.S)NR'R'', SC(.dbd.S)NR'R'',
NR'C(.dbd.O)NR'R'', NR'C(.dbd.S)NR'R'' with each R' and each R''
independently being H, aryl or alkyl, and R''' independently being
aryl or alkyl.
[0101] Electron-deficient 1,2-diazines (5), 1,2,4-triazines (6) or
1,2,4,5-tetrazines (7) are especially interesting as such dienes
are generally more reactive towards dienophiles. Di-tri- or
tetra-azines are electron deficient when they are substituted with
groups or moieties that do not generally hold as electron-donating,
or with groups that are electron-withdrawing.
[0102] For example, R.sup.1 and/or R.sup.2 may denote a substituent
selected from the group consisting of H, alkyl, NO.sub.2, F, Cl,
CF.sub.3, CN, COOR, CONHR, CONR.sub.2, COR, SO.sub.2R, SO.sub.2OR,
SO.sub.2NR.sub.2, PO.sub.3R.sub.2, NO, 2-pyridyl, 3-pyridyl,
4-pyridyl, 2,6-pyrimidyl, 3,5-pyrimidyl, 2,4-pyrimidyl, 2,4
imidazyl, 2,5 imidazyl or phenyl, optionally substituted with one
or more electron-withdrawing groups such as NO.sub.2, F, Cl,
CF.sub.3, CN, COOR, CONHR, CONR, COR, SO.sub.2R, SO.sub.2OR,
SO.sub.2NR.sub.2, PO.sub.3R.sub.2, NO, Ar, wherein R is H or
C.sub.1-C.sub.6 alkyl, and Ar stands for an aromatic group,
particularly phenyl, pyridyl, or naphthyl.
[0103] The 1,2,4,5-tetrazines of formula (7) are most preferred as
Activator dienes, as these molecules are generally most reactive in
retro Diels-Alder reactions with dienophiles, such as the preferred
TCO dienophiles, even when the R.sup.1 and/or R.sup.2 groups are
not necessarily electron withdrawing, and even when R.sup.1 and/or
R.sup.2 are in fact electron donating. Electron donating groups are
for example OH, OR', SH, SR', NH.sub.2, NHR', NR'R'',
NHC(.dbd.O)R'', NR'C(.dbd.O)R'', NHC(.dbd.S)R'', NR'C(.dbd.S)R'',
NHSO.sub.2R'', NR'SO.sub.2R'' with R' and R'' each independently
being alkyl or aryl groups. Examples of other electron donating
groups are phenyl groups with attached to them one or more of the
electron donating groups as mentioned in the list above, especially
when substituted in the 2-, 4- and/or 6-position(s) of the phenyl
group.
[0104] According to the invention, 1,2,4,5-tetrazines with two
electron withdrawing residues, or those with one electron
withdrawing residue and one residue that is neither electron
withdrawing nor donating, are called electron deficient. In a
similar way, 1,2,4,5-tetrazines with two electron donating
residues, or those with one electron donating residue and one
residue that is neither electron withdrawing nor donating, are
called electron sufficient. 1,2,4,5-Tetrazines with two residues
that are both neither electron withdrawing nor donating, or those
that have one electron withdrawing residue and one electron
donating residue, are neither electron deficient nor electron
sufficient.
[0105] The 1,2,4,5-tetrazines can be asymmetric or symmetric in
nature, i.e. the R.sup.1 and R.sup.2 groups in formula (7) may be
different groups or may be identical groups, respectively.
Symmetric 1,2,4,5-tetrazines are more convenient as these
Activators are more easily accessible via synthetic procedures.
[0106] We have tested several 1,2,4,5-tetrazines with respect to
their ability as Activator to release a model drug compound (e.g.
phenol) from a Prodrug via an elimination process, and we have
found that tetrazines that are electron deficient, electron
sufficient or neither electron deficient nor electron sufficient
are capable to induce the drug release. Furthermore, both symmetric
as well as asymmetric tetrazines were effective.
[0107] Electron deficient 1,2,4,5-tetrazines and 1,2,4,5-tetrazines
that are neither electron deficient nor electron sufficient are
generally more reactive in retro Diels-Alder reactions with
dienophiles (such as TCOs), so these two classes of
1,2,4,5-tetrazines are preferred over electron sufficient
1,2,4,5-tetrazines, even though the latter are also capable of
inducing drug release in Prodrugs.
[0108] Therefore, particularly useful tetrazine derivatives are
electron-deficient tetrazines, i.e. tetrazines substituted with
groups or moieties that do not generally hold as electron-donating,
and preferably carrying electron-withdrawing substituents. With
reference to formula (7), for electron-deficient tetrazines,
R.sup.1 and R.sup.2 each independently denote a substituent
selected from the group consisting of 2-pyridyl, 3, pyridyl,
4-pyridyl, 2,6-pyrimidyl, 3,5-pyrimidyl, 2,4-pyrimidyl, or phenyl,
optionally substituted with one or more electron-withdrawing groups
such as NO.sub.2, F, Cl, CF.sub.3, CN, COOH, COOR, CONH.sub.2,
CONHR, CONR.sub.2, CHO, COR, SO.sub.2R, SO.sub.2O R, NO, Ar,
wherein R is C.sub.1-C.sub.6 alkyl and Ar stands for an aromatic
group, particularly phenyl, pyridyl, or naphthyl.
[0109] In the compounds according to each of the formulae (2)-(7),
the R.sup.1 and R.sup.2 groups can further be provided with
suitable linker or spacer moieties as discussed herein.
[0110] In the following paragraphs specific examples of
1,2,4,5-tetrazine Activators will be highlighted by defining the
R.sup.1 and R.sup.2 residues in formula (7).
[0111] Symmetric electron deficient 1,2,4,5-tetrazines with
electron withdrawing residues are for example those with
R.sup.1.dbd.R.sup.2.dbd.H, 2-pyridyl, 3-pyridyl, 4-pyridyl,
2,4-pyrimidyl, 2,6-pyrimidyl, 3,5-pyrimidyl, 2,3,4-triazyl or
2,3,5-triazyl. Other examples are those with
R.sup.1.dbd.R.sup.2=phenyl with COOH or COOMe carboxylate, or with
CN nitrile, or with CONH.sub.2, CONHCH.sub.3 or
CON(CH.sub.3).sub.2amide, or with SO.sub.3H or SO.sub.3Na
sulfonate, or with SO.sub.2NH.sub.2, SO.sub.2NHCH.sub.3 or
SO.sub.2N(CH.sub.3).sub.2sulfonamide, or with PO.sub.3H.sub.2 or
PO.sub.3Na.sub.2 phosphonate substituents in the 2-, 3- or
4-position of the phenyl group, or in the 3- and 5-positions, or in
the 2- and 4-positions, or in the 2-, and 6-positions of the phenyl
group. Other substitution patterns are also possible, including the
use of different substituents, as long as the tetrazine remains
symmetric. See below for some examples of these structures.
##STR00015##
[0112] Symmetric electron sufficient 1,2,4,5-tetrazines with
electron donating residues are for example those with
R.sup.1.dbd.R.sup.2.dbd.OH, OR', SH, SR', NH.sub.2, NHR',
NR'.sub.2, NH--CO--R', NH--SO--R', NH--SO.sub.2--R', 2-pyrryl,
3-pyrryl, 2-thiophene, 3-thiophene, where R' represents a methyl,
ethyl, phenyl or tolyl group. Other examples are those with
R.sup.1.dbd.R.sup.2=phenyl with OH, OR', SH, SR', NH.sub.2, NHR',
NR'.sub.2, NH--CO--R', NR''--CO--R', NH--SO--R' or NH--SO.sub.2--R'
substituent(s), where R' represents a methyl, ethyl, phenyl or
tolyl group, where R'' represents a methyl or ethyl group, and
where the substitution is done on the 2- or 3- or 4- or 2- and 3-
or 2- and 4- or 2- and 5- or 2- and 6- or 3- and 4- or 3- and 5- or
3-, 4- and 5-position(s). See below for some examples of these
structures.
##STR00016##
[0113] Symmetric 1,2,4,5-tetrazines with neither electron
withdrawing nor electron donating residues are for example those
with R.sup.1.dbd.R.sup.2=phenyl, methyl, ethyl, (iso)propyl,
2,4-imidazyl, 2,5-imidazyl, 2,3-pyrazyl or 3,4-pyrazyl. Other
examples are those where R.sup.1.dbd.R.sup.2=a hetero(aromatic)
cycle such as a oxazole, isoxazole, thiazole or oxazoline cycle.
Other examples are those where R.sup.1.dbd.R.sup.2=a phenyl with
one electron withdrawing substituent selected from COOH, COOMe, CN,
CONH.sub.2, CONHCH.sub.3, CON(CH.sub.3).sub.2, SO.sub.3H,
SO.sub.3Na, SO.sub.2NH.sub.2, SO.sub.2NHCH.sub.3,
SO.sub.2N(CH.sub.3).sub.2, PO.sub.3H.sub.2 or PO.sub.3Na.sub.2 and
one electron donating substituent selected from OH, OR', SH, SR',
NH.sub.2, NHR', NR'.sub.2, NH--CO--R', NR''--CO--R', NH--SO--R' or
NH--SO.sub.2--R' substituent(s), where R' represents a methyl,
ethyl, phenyl or tolyl group and where R'' represents a methyl or
ethyl group. Substitutions can be done on the 2- and 3-, 2- and 4-,
2-, and 5-, 2- and 6, 3- and 4-, and the 3- and 5-positions. Yet
other examples are those where R.sup.1.dbd.R.sup.2=a pyridyl or
pyrimidyl moiety with one electron donating substituent selected
from OH, OR', SH, SR', NH.sub.2, NHR', NR'.sub.2, NH--CO--R',
NR''--CO--R', NH--SO--R' or NH--SO.sub.2--R' substituents, where R'
represents a methyl, ethyl, phenyl or tolyl group and where R''
represents a methyl or ethyl group. See below for some
examples.
##STR00017##
[0114] In case asymmetric 1,2,4,5-tetrazines are considered, one
can choose any combination of given R.sup.1 and R.sup.2 residues
that have been highlighted and listed above for the symmetric
tetrazines according to formula (7), provided of course that
R.sup.1 and R.sup.2 are different. Preferred asymmetric
1,2,4,5-tetrazines are those where at least one of the residues
R.sup.1 or R.sup.2 is electron withdrawing in nature. Find below
some example structures drawn.
##STR00018##
Further Considerations Regarding the Activator
[0115] In the above the Activator has been described and defined
with respect to either of two preferred embodiments of this
invention, and for both embodiments 1,2-diazines, 1,2,4-triazines
and 1,2,4,5-tetrazines, particularly 1,2,4,5-tetrazines, are the
preferred diene Activators. In the below, some relevant features of
the Activator will be highlighted, where it will also become
apparent that there are plentiful options for designing the right
Activator formulation for every specific application.
[0116] According to the invention, the Activator, e.g. a
1,2,4,5-tetrazine, has useful and beneficial pharmacological and
pharmaco-kinetic properties, implying that the Activator is
non-toxic or at least sufficiently low in toxicity, produces
metabolites that are also sufficiently low in toxicity, is
sufficiently soluble in physiological solutions, can be applied in
aqueous or other formulations that are routinely used in
pharmaceutics, and has the right log D value where this value
reflects the hydrophilic/hydrophobic balance of the Activator
molecule at physiological pH. As is known in the art, log D values
can be negative (hydrophilic molecules) or positive (hydrophobic
molecules), where the lower or the higher the log D values become,
the more hydrophilic or the more hydrophobic the molecules are,
respectively. Log D values can be predicted fairly adequately for
most molecules, and log D values of Activators can be tuned by
adding or removing polar or apolar groups in their designs. Find
below some Activator designs with their corresponding calculated
log D values (at pH=7.4). Note that addition of methyl,
cycloalkylene, pyridine, amine, alcohol or sulfonate groups or
deletion of phenyl groups modifies the log D value, and that a very
broad range of log D values is accessible.
##STR00019##
[0117] The given log D numbers have been calculated from a weighed
method, with equal importance of the `VG` (Viswanadhan, V. N.;
Ghose, A. K.; Revankar, G. R.; Robins, R. K., J. Chem. Inf. Comput.
Sci., 1989, 29, 163-172), `KLOP` (according to Klopman, G.; Li,
Ju-Yun.; Wang, S.; Dimayuga, M.: J. Chem. Inf. Comput. Sci., 1994,
34, 752) and `PHYS` (according to the PHYSPROP.COPYRGT. database)
methods, based on an aqueous solution in 0.1 M in
Na.sup.+/K.sup.+Cl.sup.-.
[0118] The Activator according to the invention has an appropriate
reactivity towards the Prodrug, and this can be regulated by making
the diene, particularly the 1,2,4,5-tetrazines, sufficiently
electron deficient. Sufficient reactivity will ensure a fast retro
Diels-Alder reaction with the Prodrug as soon as it has been
reached by the Activator.
[0119] The Activator according to the invention has a good
bio-availability, implying that it is available inside the (human)
body for executing its intended purpose: effectively reaching the
Prodrug at the Primary Target. Accordingly, the Activator does not
stick significantly to blood components or to tissue that is
non-targeted. The Activator may be designed to bind to albumin
proteins that are present in the blood (so as to increase the blood
circulation time, as is known in the art), but it should at the
same time be released effectively from the blood stream to be able
to reach the Prodrug. Accordingly, blood binding and blood
releasing should then be balanced adequately. The blood circulation
time of the Activator can also be increased by increasing the
molecular weight of the Activator, e.g. by attaching polyethylene
glycol (PEG) groups to the Activator (`pegylation`). Alternatively,
the PK/PD of the activator may be modulated by conjugating the
activator to another moiety such as a polymer, protein, (short)
peptide, carbohydrate.
[0120] The Activator according to the invention may be multimeric,
so that multiple diene moieties may be attached to a molecular
scaffold, particularly to e.g. multifunctional molecules,
carbohydrates, polymers, dendrimers, proteins or peptides, where
these scaffolds are preferably water soluble. Examples of scaffolds
that can be used are (multifunctional) polyethylene glycols,
poly(propylene imine) (PPI) dendrimers, PAMAM dendrimers, glycol
based dendrimers, heparin derivatives, hyaluronic acid derivatives
or serum albumine proteins such as HSA.
[0121] For applications where the prodrug activation is to occur in
the extracellular domain, the diene is relatively hydrophilic.
[0122] Preferably, the Activator is a tetrazine selected from the
following formulae:
##STR00020## ##STR00021## ##STR00022## ##STR00023##
[0123] Depending on the position of the Prodrug (e.g. inside the
cell or outside the cell; specific organ that is targeted) the
Activator is designed to be able to effectively reach this Prodrug.
Therefore, the Activator can for example be tailored by varying its
log D value, its reactivity or its charge. The Activator may even
be engineered with a targeting agent (e.g. a protein, a peptide
and/or a sugar moiety), so that the Primary Target can be reached
actively instead of passively. In case a targeting agent is
applied, it is preferred that it is a simple moiety (i.e. a short
peptide or a simple sugar).
[0124] According to the invention, a mixture of different
Activators can be applied. This may be relevant for regulation of
the release profile of the drug.
[0125] The Activator that according to the invention will cause and
regulate drug release at the Primary Target may additionally be
modified with moieties giving extra function(s) to the Activator,
either for in-vitro and/or for in-vivo studies or applications. For
example, the Activator may be modified with dye moieties or
fluorescent moieties (see e.g. S. Hilderbrand et al., Bioconjugate
Chem., 2008, 19, 2297-2299 for 3-(4-benzylamino)-1,2,4,5-tetrazine
that is amidated with the near-infrared (NIR) fluorophore VT680),
or they may be functionalized with imaging probes, where these
probes may be useful in imaging modalities, such as the nuclear
imaging techniques PET or SPECT. In this way, the Activator will
not only initiate drug release, but can also be localized inside
the (human) body, and can thus be used to localize the Prodrug
inside the (human) body. Consequently, the position and amount of
drug release can be monitored. For example, the Activator can be
modified with DOTA (or DTPA) ligands, where these ligands are
ideally suited for complexation with .sup.111In.sup.3+-ions for
nuclear imaging. In other examples, the Activator may be linked to
.sup.123I or .sup.18F moieties, that are well established for use
in SPECT or PET imaging, respectively. Furthermore, when used in
combination with e.g. beta-emitting isotopes, such as Lu-177, or
Y-90, prodrug activation can be combined with localized
radiotherapy in a pretargeted format.
[0126] Synthesis routes to the above activators are readily
available to the skilled person, based on standard knowledge in the
art. References to tetrazine synthesis routes include Lions et al,
J. Org. Chem., 1965, 30, 318-319; Horwitz et al, J. Am. Chem. Soc.,
1958, 80, 3155-3159; Hapiot et al, New. J. Chem., 2004, 28,
387-392, Kaim et al, Z. Naturforsch., 1995, 50b, 123-127.
Prodrug A Prodrug is a conjugate of the Drug D.sup.D and the
Trigger T.sup.R and thus comprises a Drug that is capable of
therapeutic action after its release from the Trigger. Such a
Prodrug may optionally have specificity for disease targets.
[0127] The general formula of the Prodrug is shown below in Formula
(8a) and (8b).
##STR00024##
[0128] The moiety Y.sup.M can either be a targeting agent T.sup.T
or a masking moiety M.sup.M; S.sup.P is spacer; T.sup.R is Trigger,
L.sup.D is linker, and D.sup.D is drug.
[0129] For applications where drugs are released from a targeting
agent: Y.sup.M is a targeting agent T.sup.T;
[0130] Formula (8a): k=1; m,r.gtoreq.1; t,n.gtoreq.0.
[0131] Formula (8b): k=1; m,n,r.gtoreq.1; t.gtoreq.0.
[0132] For applications where masked drugs are unmasked: Y.sup.M is
a masking moiety M.sup.M;
[0133] Formula (8a) and (8b): r=1; m.gtoreq.1; k,n,t.gtoreq.0.
[0134] Although it has been omitted for the sake of clarity in the
above formula, D.sup.D can further comprise T.sup.T and/or M.sup.M,
optionally via S.sup.P.
[0135] Drugs that can be used in a Prodrug relevant to this
invention include but are not limited to: antibodies, antibody
derivatives, antibody fragments, e.g. Fab2, Fab, scFV, diabodies,
triabodies, antibody (fragment) fusions (eg bi-specific and
trispecific mAb fragments), proteins, aptamers, oligopeptides,
oligonucleotides, oligosaccharides, as well as peptides, peptoids,
steroids, organic drug compounds, toxins, hormones, viruses, whole
cells, phage. Typical drugs for which the invention is suitable
include, but are not limited to: bi-specific and trispecific mAb
fragments, immunotoxins, comprising eg ricin A, diphtheria toxin,
cholera toxin. Other embodiments use auristatins, maytansines,
calicheamicin, Duocarmycins, maytansinoids DM1 and DM4, auristatin
MMAE, CC1065 and its analogs, camptothecin and its analogs, SN-38
and its analogs; antiproliferative/antitumor agents, antibiotics,
cytokines, anti-inflammatory agents, anti-viral agents,
antihypertensive agents, chemosensitizing and radiosensitizing
agents. In other embodiments the released Drug D.sup.D is itself a
prodrug designed to release a further drug D.sup.D. Drugs
optionally include a membrane translocation moiety (adamantine,
poly-lysine/argine, TAT) and/or a targeting agent (against eg a
tumor cel receptor) optionally linked through a stable or labile
linker.
[0136] Exemplary drugs for use as conjugates to the TCO derivative
and to be released upon retro Diels Alder reaction with the
Activator include but are not limited to: cytotoxic drugs,
particularly those which are used for cancer therapy. Such drugs
include, in general, DNA damaging agents, anti-metabolites, natural
products and their analogs. Exemplary classes of cytotoxic agents
include the enzyme inhibitors such as dihydrofolate reductase
inhibitors, and thymidylate synthase inhibitors, DNA alkylators,
radiation sensitizers, DNA intercalators, DNA cleavers,
anti-tubulin agents, topoisomerases inhibitors, platinum-based
drugs, the anthracycline family of drugs, the vinca drugs, the
mitomycins, the bleomycins, the cytotoxic nucleosides, taxanes,
lexitropsins, the pteridine family of drugs, diynenes, the
podophyllotoxins, dolastatins, maytansinoids, differentiation
inducers, and taxols. Particularly useful members of those classes
include, for example, duocarmycin, methotrexate, methopterin,
dichloromethotrexate, 5-fluorouracil DNA minor groove binders,
6-mercaptopurine, cytosine arabinoside, melphalan, leurosine,
leurosideine, actinomycin, daunorubicin, doxorubicin, mitomycin C,
mitomycin A, caminomycin, aminopterin, tallysomycin,
podophyllotoxin and podophyllotoxin derivatives such as etoposide
or etoposide phosphate, vinblastine, vincristine, vindesine, taxol,
taxotere retinoic acid, butyric acid, N8-acetyl spermidine,
camptothecin, calicheamicin, esperamicin, ene-diynes, and their
analogues.
[0137] Exemplary drugs include the dolastatins and analogues
thereof including: dolastatin A (U.S. Pat. No. 4,486,414),
dolastatin B (U.S. Pat. No. 4,486,414), dolastatin 10 (U.S. Pat.
Nos. 4,486,444, 5,410,024, 5,504,191, 5,521,284, 5,530,097,
5,599,902, 5,635,483, 5,663,149, 5,665,860, 5,780,588, 6,034,065,
6,323,315), dolastatin 13 (U.S. Pat. No. 4,986,988), dolastatin 14
(U.S. Pat. No. 5,138,036), dolastatin 15 (U.S. Pat. No. 4,879,278),
dolastatin 16 (U.S. Pat. No. 6,239,104), dolastatin 17 (U.S. Pat.
No. 6,239,104), and dolastatin 18 (U.S. Pat. No. 6,239,104), each
patent incorporated herein by reference in their entirety.
[0138] In exemplary embodiments of the invention, the drug moiety
is a mytomycin, vinca alkaloid, taxol, anthracycline, a
calicheamicin, maytansinoid or an auristatin.
[0139] It will be understood that chemical modifications may also
be made to the desired compound in order to make reactions of that
compound more convenient for purposes of preparing conjugates of
the invention. Drugs containing an amine functional group for
coupling to the TCO include mitomycin-C, mitomycin-A, daunorubicin,
doxorubicin, aminopterin, actinomycin, bleomycin, 9-amino
camptothecin, N8-acetyl spermidine, 1-(2
chloroethyl)1,2-dimethanesulfonyl hydrazide, tallysomycin,
cytarabine, dolastatins (including auristatins) and derivatives
thereof.
[0140] Drugs containing a hydroxyl function group for coupling to
the TCO include etoposide, camptothecin, taxol, esperamicin,
1,8-dihydroxy-bicyclo[7.3.1]trideca-4-9-diene-2,6-diyne-13-one
(U.S. Pat. No. 5,198,560), podophyllotoxin, anguidine, vincristine,
vinblastine, morpholine-doxorubicin,
n-(5,5-diacetoxy-pentyl)doxorubicin, and derivatives thereof.
[0141] Drugs containing a sulfhydryl functional group for coupling
to the TCO include esperamicin and 6-mecaptopurine, and derivatives
thereof.
[0142] It will be understood that the drugs can optionally be
attached to the TCO derivative through a linker L.sup.D or a
self-immolative linker L.sup.D, or a combination thereof, and which
may consist of multiple (self-immolative, or non immolative)
units.
[0143] It will further be understood that one or more targeting
agents T.sup.T or masking moieties M.sup.M may optionally be
attached to the Drug D.sup.D, Trigger T.sup.R, or Linker L.sup.D,
optionally via a spacer or spacers S.sup.P.
[0144] Several drugs may be replaced by an imageable label to
measure drug targeting and release.
[0145] According to a further particular embodiment of the
invention, the Prodrug is selected so as to target and or address a
disease, such as cancer, an inflammation, an infection, a
cardiovascular disease, e.g. thrombus, atherosclerotic lesion,
hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain
disorder, apoptosis, angiogenesis, an organ, and reporter
gene/enzyme.
[0146] According to one embodiment, the Prodrug and/or the
Activator can be multimeric compounds, comprising a plurality of
Drugs and/or bioorthogonal reactive moieties. These multimeric
compounds can be polymers, dendrimers, liposomes, polymer
particles, or other polymeric constructs.
[0147] In the Prodrug, the Drug D.sup.D and the Trigger
T.sup.R--the TCO derivative--can be directly linked to each other.
They can also be bound to each other via a linker or a
self-immolative linker L.sup.D. It will be understood that the
invention encompasses any conceivable manner in which the
dienophile Trigger is attached to the Drug. The same holds for the
attachment of an optional targeting agent T.sup.T or masking moiety
M.sup.M to the Prodrug. Methods of affecting conjugation to these
drugs, e.g. through reactive amino acids such as lysine or cysteine
in the case of proteins, are known to the skilled person.
[0148] It will be understood that the drug moiety is linked to the
TCO in such a way that the drug is eventually capable of being
released after formation of the retro Diels-Alder adduct.
Generally, this means that the bond between the drug and the TCO,
or in the event of a linker, the bond between the TCO and the
linker L.sup.D, or in the event of a self-immolative linker
L.sup.D, the bond between the linker and the TCO and between the
drug and the linker, should be cleavable. Predominantly, the drug
and the optional linker is linked via a hetero-atom, preferably via
O, N, NH, or S. The cleavable bond is preferably selected from the
group consisting of carbamate, thiocarbamate, carbonate, ether,
ester, amine, amide, thioether, thioester, sulfoxide, and
sulfonamide bonds.
[0149] Thus, in the invention, linker concepts can be applied
analogously to those known to the skilled person. Most reported
prodrugs consist of three components: a trigger, a linker, and a
parent drug, optionally a targeting molecule is attached to either
the linker or the trigger. The trigger, which can e.g. be a
substrate for a site-specific enzyme, or pH labile group, is often
connected to the parent drug via a self-elimination linker. This
linker is incorporated to facilitate enzymatic cleavage of the
trigger, increasing active site accessibility and decreasing steric
hindrance from the attached drug. Also the linker facilitates the
straightforward use of a broad range of prodrugs in combination
with the same trigger. Furthermore, the linker modulates prodrug
stability, pharmacokinetics, organ distribution, enzyme
recognition, and release kinetics. After trigger
activation/removal, the linker must spontaneously eliminate to
release the parent drug. Depending on the attached drug the linker
or parts thereof can remain on the drug without impairing its
action. The general concept is depicted in Scheme 2.
##STR00025##
[0150] Two types of self-elimination linkers can be distinguished
a) the electronic cascade linker b) the cyclization linker. The
most prominent example of a cascade linker is the 1,6 elimination
spacer shown in Scheme 3 in a 3-glucuronide prodrug of anticancer
agent 9-aminocamptothecin. After unmasking of the aromatic hydroxyl
function by the enzyme 3-glucuronidase (present in certain necrotic
tumor areas), this group becomes electron-donating and initiates an
electronic cascade that leads to expulsion of the leaving group,
which releases the free drug after elimination of CO.sub.2. This
cascade, based on a quinone-methide rearrangement, can also be
initiated by the lone pair of an unmasked amine or thiol instead of
the hydroxyl. The formed quinone-methide species is trapped by
water to form a phenol derivative.
##STR00026##
[0151] Some other trigger-linker concepts are depicted in Scheme 4.
The trigger in A is activated by plasmatic esterases. Hydrolysis of
the tert-butyl ester affords the free aromatic hydroxyl group,
which starts the quinone-methide cascade. This construct has been
targeted by conjugation to an antibody (R). In B, the hydrolysis of
cephalosporins by beta-lactamase enzymes is used as a trigger.
Hydrolysis of the lactam ring can to lead expulsion of the drug
substituent depending on its leaving group nature. Drugs have been
conjugated via an ester, amide, sulfide, amine and carbamate link.
Two examples of aromatic cyclization-based linkers are C and D. In
C cleavage by penicillin G-amidase leads to intramolecular attack
of the amine on the carbonyl, releasing the drug. D shows a
phosphatase-sensitive prodrug. Cleavage of the phosphate by human
alkaline phosphatase affords a hydroxyl that reacts to a lactam by
releasing the drug. In E an example is shown of a prodrug that it
triggered by the reduction of a nitro group to an amine. This
reduction can be performed by nitroreductase in the presence of
NADPH. Furthermore, a number of heterocyclic nitro constructs are
known (F) that are reduced in hypoxic (tumor) tissue and, hence,
can initiate a cascade without the assistance of an enzyme. Other
triggers used in prodrug therapy are sensitive to plasmin, tyrosine
hydroxylase (highly expressed in neuroblastoma), tyrosinase or
cathepsin B.
##STR00027## ##STR00028##
The Combination of and Reaction Between the TCO-Trigger and the
Activator
[0152] The following schemes depict non-limiting examples
illustrative for the various mechanisms that can be made to apply
on the basis of the choice for the rDA reaction for activating a
prodrug. Note that in cases of release of amine functional drugs
these can be e.g. primary or secondary amine, aniline, imidazole or
pyrrole type of drugs, so that the drug may be varying in leaving
group character. Release of drugs with other functionalities may
also be possible (e.g. thiol functionalized drugs), in case
corresponding hydrolytically stable TCO prodrugs are applied. The
drawn fused ring products may or may not tautomerize to other more
favorable tautomers.
##STR00029## ##STR00030## ##STR00031##
[0153] In a preferred embodiment, the drug is provided in the form
of an antibody-toxin conjugate. The conjugate is provided with a
TCO moiety as identified above, so as to enable bio-orthogonal
chemically activated toxin release. In another embodiment, the drug
is a bi- or trispecific antibody derivative that serves to bind to
tumor cells and recruit and activate T-cells, the T-cell binding
function of which is inactivated by being linked to a TCO moiety as
described above. The latter, again, serving to enable
bio-orthogonal chemically activated drug activation.
Targeting
[0154] The kits and method of the invention are very suitable for
use in targeted delivery of drugs.
[0155] A "primary target" as used in the present invention relates
to a target for a targeting agent for therapy. For example, a
primary target can be any molecule, which is present in an
organism, tissue or cell. Targets include cell surface targets,
e.g. receptors, glycoproteins; structural proteins, e.g. amyloid
plaques; abundant extracullular targets such as stroma;
extracellular matrix targets such as growth factors, and proteases;
intracellular targets, e.g. surfaces of Golgi bodies, surfaces of
mitochondria, RNA, DNA, enzymes, components of cell signaling
pathways; and/or foreign bodies, e.g. pathogens such as viruses,
bacteria, fungi, yeast or parts thereof. Examples of primary
targets include compounds such as proteins of which the presence or
expression level is correlated with a certain tissue or cell type
or of which the expression level is up regulated or down-regulated
in a certain disorder. According to a particular embodiment of the
present invention, the primary target is a protein such as a
(internalizing or non-internalizing) receptor.
[0156] According to the present invention, the primary target can
be selected from any suitable targets within the human or animal
body or on a pathogen or parasite, e.g. a group comprising cells
such as cell membranes and cell walls, receptors such as cell
membrane receptors, intracellular structures such as Golgi bodies
or mitochondria, enzymes, receptors, DNA, RNA, viruses or viral
particles, antibodies, proteins, carbohydrates, monosacharides,
polysaccharides, cytokines, hormones, steroids, somatostatin
receptor, monoamine oxidase, muscarinic receptors, myocardial
sympatic nerve system, leukotriene receptors, e.g. on leukocytes,
urokinase plasminogen activator receptor (uPAR), folate receptor,
apoptosis marker, (anti-)angiogenesis marker, gastrin receptor,
dopaminergic system, serotonergic system, GABAergic system,
adrenergic system, cholinergic system, opoid receptors, GPIIb/IIIa
receptor and other thrombus related receptors, fibrin, calcitonin
receptor, tuftsin receptor, integrin receptor, fibronectin,
VEGF/EGF and VEGF/EGF receptors, TAG72, CEA, CD19, CD20, CD22,
CD40, CD45, CD74, CD79, CD105, CD138, CD174, CD227, CD326, CD340,
MUC1, MUC16, GPNMB, PSMA, Cripto, Tenascin C, Melanocortin-1
receptor, CD44v6, G250, HLA DR, ED-B, TMEFF2, EphB2, EphA2, FAP,
Mesothelin, GD2, CAIX, 5T4, matrix metalloproteinase (MMP),
P/E/L-selectin receptor, LDL receptor, P-glycoprotein, neurotensin
receptors, neuropeptide receptors, substance P receptors, NK
receptor, CCK receptors, sigma receptors, interleukin receptors,
herpes simplex virus tyrosine kinase, human tyrosine kinase. In
order to allow specific targeting of the above-listed primary
targets, the targeting agent T.sup.T can comprise compounds
including but not limited to antibodies, antibody fragments, e.g.
Fab2, Fab, scFV, diabodies, triabodies, VHH, antibody (fragment)
fusions (eg bi-specific and trispecific mAb fragments), proteins,
peptides, e.g. octreotide and derivatives, VIP, MSH, LHRH,
chemotactic peptides, bombesin, elastin, peptide mimetics,
carbohydrates, monosacharides, polysaccharides, viruses, whole
cells, drugs, polymers, liposomes, chemotherapeutic agents,
receptor agonists and antagonists, cytokines, hormones, steroids.
Examples of organic compounds envisaged within the context of the
present invention are, or are derived from, estrogens, e.g.
estradiol, androgens, progestins, corticosteroids, methotrexate,
folic acid, and cholesterol. In a preferred embodiment, the
targeting agent T.sup.T is an antibody. According to a particular
embodiment of the present invention, the primary target is a
receptor and a targeting agent is employed, which is capable of
specific binding to the primary target. Suitable targeting agents
include but are not limited to, the ligand of such a receptor or a
part thereof which still binds to the receptor, e.g. a receptor
binding peptide in the case of receptor binding protein ligands.
Other examples of targeting agents of protein nature include
interferons, e.g. alpha, beta, and gamma interferon, interleukins,
and protein growth factor, such as tumor growth factor, e.g. alpha,
beta tumor growth factor, platelet-derived growth factor (PDGF),
uPAR targeting protein, apolipoprotein, LDL, annexin V, endostatin,
and angio statin. Alternative examples of targeting agents include
DNA, RNA, PNA and LNA which are e.g. complementary to the primary
target.
[0157] According to a further particular embodiment of the
invention, the primary target and targeting agent are selected so
as to result in the specific or increased targeting of a tissue or
disease, such as cancer, an inflammation, an infection, a
cardiovascular disease, e.g. thrombus, atherosclerotic lesion,
hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain
disorder, apoptosis, angiogenesis, an organ, and reporter
gene/enzyme. This can be achieved by selecting primary targets with
tissue-, cell- or disease-specific expression. For example,
membrane folic acid receptors mediate intracellular accumulation of
folate and its analogs, such as methotrexate. Expression is limited
in normal tissues, but receptors are overexpressed in various tumor
cell types.
Masking Moieties
[0158] Masking moieties M.sup.M can be a protein, peptide, polymer,
polyethylene glycol, carbohydrate, organic construct, that further
shield the bound drug D.sup.D or Prodrug. This shielding can be
based on eg steric hindrance, but it can also be based on a non
covalent interaction with the drug D.sup.D. Such masking moiety may
also be used to affect the in vivo properties (eg blood clearance;
recognition by the immunesystem) of the drug D.sup.D or
Prodrug.
Spacers
[0159] Spacers S.sup.P include but are not limited to polyethylene
glycol (PEG) chains varying from 2 to 200, particularly 3 to 113
and preferably 5-50 repeating units. Other examples are biopolymer
fragments, such as oligo- or polypeptides or polylactides. Further
preferred examples are shown in Example 9.
Administration
[0160] In the context of the invention, the Prodrug is usually
administered first, and it will take a certain time period before
the Prodrug has reached the Primary Target. This time period may
differ from one application to the other and may be minutes, days
or weeks. After the time period of choice has elapsed, the
Activator is administered, will find and react with the Prodrug and
will thus activate Drug release at the Primary Target.
[0161] The compositions of the invention can be administered via
different routes including intravenous injection, intraperatonial,
oral administration, rectal administration and inhalation.
Formulations suitable for these different types of administrations
are known to the skilled person. Prodrugs or Activators according
to the invention can be administered together with a
pharmaceutically acceptable carrier. A suitable pharmaceutical
carrier as used herein relates to a carrier suitable for medical or
veterinary purposes, not being toxic or otherwise unacceptable.
Such carriers are well known in the art and include saline,
buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof. The formulation should suit the mode of
administration.
[0162] It will be understood that the chemical entities
administered, viz. the prodrug and the activator, can be in a
modified form that does not alter the chemical functionality of
said chemical entity, such as salts, hydrates, or solvates
thereof.
[0163] After administration of the Prodrug, and before the
administration of the Activator, it is preferred to remove excess
Prodrug by means of a Clearing Agent in cases when prodrug
activation in circulation is undesired and when natural prodrug
clearance is insufficient. A Clearing Agent is an agent, compound,
or moiety that is administered to a subject for the purpose of
binding to, or complexing with, an administered agent (in this case
the Prodrug) of which excess is to be removed from circulation. The
Clearing Agent is capable of being directed to removal from
circulation. The latter is generally achieved through liver
receptor-based mechanisms, although other ways of secretion from
circulation exist, as are known to the skilled person. In the
invention, the Clearing Agent for removing circulating Prodrug,
preferably comprises a diene moiety, e.g. as discussed above,
capable of reacting to the TCO moiety of the Prodrug.
EXAMPLES
[0164] The following examples demonstrate the invention or aspects
of the invention, and do not serve to define or limit the scope of
the invention or its claims.
[0165] Methods.
[0166] .sup.1H-NMR and .sup.13C-NMR spectra were recorded on a
Varian Mercury (400 MHz for .sup.1H-NMR and 100 MHz for
.sup.13C-NMR) spectrometer at 298 K. Chemical shifts are reported
in ppm downfield from TMS at room temperature. Abbreviations used
for splitting patterns are s=singlet, t=triplet, q=quartet,
m=multiplet and br=broad. IR spectra were recorded on a Perkin
Elmer 1600 FT-IR (UATR). LC-MS was performed using a Shimadzu LC-10
AD VP series HPLC coupled to a diode array detector (Finnigan
Surveyor PDA Plus detector, Thermo Electron Corporation) and an
Ion-Trap (LCQ Fleet, Thermo Scientific). Analyses were performed
using a Alltech Alltima HP C.sub.18 3.mu. column using an injection
volume of 1-4 .mu.L, a flow rate of 0.2 mL min.sup.-1 and typically
a gradient (5% to 100% in 10 min, held at 100% for a further 3 min)
of CH.sub.3CN in H.sub.2O (both containing 0.1% formic acid) at
25.degree. C. Preparative RP-HPLC (CH.sub.3CN/H.sub.2O with 0.1%
formic acid) was performed using a Shimadzu SCL-1A VP coupled to
two Shimadzu LC-8A pumps and a Shimadzu SPD-10AV VP UV-vis detector
on a Phenomenex Gemini 5.mu. C.sub.18 110A column. Size exclusion
(SEC) HPLC was carried out on an Agilent 1200 system equipped with
a Gabi radioactive detector. The samples were loaded on a
Superdex-200 10/300 GL column (GE Healthcare Life Sciences) and
eluted with 10 mM phosphate buffer, pH 7.4, at 0.35-0.5 mL/min. The
UV wavelength was preset at 260 and 280 nm. The concentration of
antibody solutions was determined with a NanoDrop 1000
spectrophotometer (Thermo Fisher Scientific) from the absorbance at
322 nm and 280 nm, respectively.
[0167] Materials.
[0168] All reagents, chemicals, materials and solvents were
obtained from commercial sources, and were used as received:
Biosolve, Merck and Cambridge Isotope Laboratories for (deuterated)
solvents; and Aldrich, Acros, ABCR, Merck and Fluka for chemicals,
materials and reagents. All solvents were of AR quality.
4-(t-Butyldimethylsilyloxymethyl)-2,6-dimethylphenol was
synthesized according to a literature procedure (Y. H. Choe, C. D.
Conover, D. Wu, M. Royzen, Y. Gervacio, V. Borowski, M. Mehlig, R.
B. Greenwald, J. Controlled Release 2002, 79, 55-70). Doxorubicin
hydrochloride was obtained from Avachem Scientific.
Example 1
Synthesis of Tetrazine Activators
General Procedures
[0169] Apart from the tetrazines described in detail below, a
series of other tetrazines has been prepared. Pinner-type reactions
have been used, where the appropriate nitriles have been reacted
with hydrazine to make the dihydro 1,2,4,5-tetrazine intermediates.
Instead of nitriles, amidines have also been used as reactants, as
it is known in the art. The use of sulfur in this reaction is also
known, as in some cases this aids the formation of the dihydro
1,2,4,5-tetrazine. Oxidation of this intermediate results in the
tetrazine diene Activators. The below reactions describe some of
the prepared tetrazines and illustrate some of the possibilities
(e.g. use of solvent, concentrations, temperature, equivalents of
reactants, options for oxidation, etc.) to make and isolate
tetrazines. Other methods known in the art may also be used to
prepare tetrazines or other Activators.
Synthesis of 3,6-bis(2-pyridyl)-1,2,4,5-tetrazine (2)
##STR00032##
[0171] 2-Cyanopyridine (10.00 g, 96.0 mmol) and hydrazine hydrate
(15.1 g; 300 mmol) were stirred overnight at 90.degree. C. in an
inert atmosphere. The turbid mixture was cooled to room
temperature, filtered, and the residue was subsequently washed with
water (20 mL) and ethanol (20 mL), and dried in vacuo to yield the
crude dihydrotetrazine 1 as an orange solid (7.35 g; 65%).
[0172] The dihydrotetrazine (1, 100 mg; 0.419 mmol) was suspended
in acetic acid (3 mL), and sodium nitrite (87 mg; 1.26 mmol) was
added. An immediate color change from orange to dark red was
observed, and the oxidized product was isolated by filtration. The
residue was washed with water (10 mL) and dried in vacuo to yield
the title compound as a purple solid (2, 92 mg; 93%).
[0173] .sup.1H NMR (CDCl.sub.3): .delta.=9.00 (d, 2H), 8.76 (d,
2H), 8.02 (t, 2H), 7.60 (dd, 2H) ppm. .sup.13C NMR (CDCl.sub.3):
.delta.=163.9, 151.1, 150.1, 137.5, 126.6, 124.5 ppm. HPLC-MS/PDA:
one peak in chromatogram, m/z=237.00 (M+H.sup.+),
.lamda..sub.max=296 and 528 nm.
Synthesis of
3-(5-acetamido-2-pyridyl)-6-(2-pyridyl)-1,2,4,5-tetrazine (5)
##STR00033##
[0175] 2-Cyanopyridine (5.00 g, 48.0 mmol), 5-amino-2-cyanopyridine
(5.72 g; 48.0 mmol) and hydrazine hydrate (15.1 g; 300 mmol) were
stirred overnight at 90.degree. C. in an inert atmosphere. The
turbid mixture was cooled to room temperature, filtered, and the
residue was subsequently washed with water (20 mL) and ethanol (20
mL), and dried in vacuo. The orange solid was suspended in acetone
(200 mL), impregnated onto silica gel (20 g), and chromatographed
using a gradient (0% to 70%) of acetone and heptane, to yield
dihydrotetrazine 3 as an orange solid (1.46 g; 12% yield).
[0176] The dihydrotetrazine (3, 90 mg; 0.355 mmol) was dissolved in
THF (1 mL), and acetic anhydride (54.4 mg; 0.533 mmol) was added.
The solution was heated to reflux in an inert atmosphere for 18 hr.
The orange precipitate was isolated by filtration, and washed with
THF (3 mL) to give the acetamide of the dihydrotetrazine (4, 90 mg;
86% yield).
[0177] Acetamide 4 (50 mg, 0.169 mmol) was suspended in acetic acid
(1 mL), and sodium nitrite (35 mg; 0.508 mmol) was added. An
immediate color change from orange to dark red was observed, and
the oxidized product was isolated by filtration. The residue was
washed with water (5 mL) and dried in vacuo to yield the title
compound as a purple solid (5, 42 mg; 84%).
[0178] .sup.1H NMR (DMSO-d.sub.6): .delta.=9.03 (d, 1H), 8.93 (d,
1H), 8.61 (dd, 2H), 8.42 (dd, 1H), 8.16 (dt, 1H), 7.73 (dd, 1H),
2.17 (s, 3H) ppm. .sup.13C NMR (DMSO-d.sub.6): .delta.=169.5,
163.0, 162.8, 150.6, 150.2, 143.8, 141.2, 138.5, 137.8, 126.6,
126.1, 124.9, 124.2, 24.1 ppm. HPLC-MS/PDA: one peak in
chromatogram, m/z=293.9 (M+H.sup.+), .lamda..sub.max=323 and 529
nm.
Synthesis of 3-(2-pyridyl)-6-methyl-1,2,4,5-tetrazine (7)
##STR00034##
[0180] 2-Cyanopyridine (500 mg, 4.8 mmol), acetamidine
hydrochloride (2.00 g, 21.2 mmol) and sulfur (155 mg, 4.8 mmol)
were stirred in ethanol (5 mL) under an inert atmosphere of argon.
Hydrazine hydrate (2.76 g; 55.2 mmol) was added and the mixture was
stirred overnight at 20.degree. C. The turbid mixture was filtered
and the filtrate was evaporated to dryness, to yield 2.9 g of
orange colored crude product 6.
[0181] Subsequently, 6 (800 mg) was suspended in a mixture of THF
(3 mL) and acetic acid (4 mL). A solution of NaNO.sub.2 (2.0 g;
29.0 mmol) in water (3 mL) was added at 0.degree. C. Instantaneous
coloration to a red/purple suspension was observed. After 5 min of
stirring at 0.degree. C., chloroform and water were added. The
purple chloroform layer was washed twice with water and then
concentrated. The solid residue was stirred in a 1:1 mixture of
chloroform and hexane, and then filtered. The filtrate was
concentrated and the crude product was purified by silica column
chromatography applying chloroform/acetone mixtures as eluent,
yielding pure product 7 (48 mg, 21% yield overall, as calculated
from 2-cyanopyridine).
[0182] .sup.1H NMR (CDCl.sub.3): .delta.=8.96 (d, 1H), 8.65 (d,
1H), 7.99 (t, 1H), 7.56 (dd, 1H), 3.17 (s, 3H) ppm. .sup.13C NMR
(CDCl.sub.3): .delta.=168.1, 163.6, 150.9, 150.3, 137.4, 126.3,
123.9, 21.4 ppm. HPLC-MS/PDA: one peak in chromatogram, m/z=174.3
(M+H.sup.+), .lamda..sub.max=274 and 524 nm.
Synthesis of 3,6-bis(2-aminophenyl)-1,2,4,5-tetrazine (9)
##STR00035##
[0184] 2-Aminobenzonitrile (1.00 g; 8.46 mmol) was dissolved in
ethanol (3 mL) and hydrazine hydrate (2.06 g; 41.2 mmol) was added.
The mixture was cooled to 0.degree. C. and sulfur (0.17 g; 5.30
mmol) was added. Stirring was continued for 15 min, and
subsequently the mixture was heated at 90.degree. C. After 3 hr,
the yellow precipitate was isolated by filtration, washed with
ethanol (10 mL), and subsequently triturated twice with chloroform
(2 times 10 mL), to yield the yellow intermediate 8 (343 mg,
30%).
Intermediate 8 (105 mg; 0.394 mmol) was dissolved in ethanol (15
mL), and oxygen was bubbled through this solution at 50.degree. C.
Within minutes, the color changed from yellow to dark orange/red,
and a precipitate was formed. After 2 hr, the precipitate was
filtered, washed with ethanol and dried to give the product 9 as
dark red crystals (89 mg, 86%).
[0185] .sup.1H NMR (DMSO-d.sub.6): .delta.=8.39 (d, 2H), 7.32 (t,
2H), 7.04 (s, 4H), 6.93 (d, 2H), 6.75 (t, 2H) ppm. .sup.13C NMR
(DMSO-d.sub.6): .delta.=162.7, 149.6, 133.0, 129.0, 117.1, 115.8,
111.6 ppm. HPLC-MS/PDA: one peak in chromatogram, m/z=265.4
(M+H.sup.+), .lamda..sub.max=237, 293, 403 and 535 nm.
Synthesis of 3,6-bis(4-hydroxyphenyl)-1,2, 4,5-tetrazine (11)
##STR00036##
[0187] 4-Hydroxybenzonitrile (1.06 g; 8.90 mmol) was dissolved in
hydrazine hydrate (3.09 g; 61.7 mmol), and the mixture was heated
to 90.degree. C. for 16 hr. The yellow precipitate was filtered and
washed with water (25 mL) and ethanol (10 mL), to yield crude
intermediate 10 as a yellow powder (870 mg; 62%).
[0188] The intermediate (10, 173 mg; 0.645 mmol) was suspended in
ethanol (10 mL), and oxygen was bubbled through this mixture at
50.degree. C. Within minutes, the color changed from yellow to dark
orange/red. After 6 hr, the precipitate was filtered, washed with
ethanol and dried, to give the product 11 as dark red crystals (136
mg, 80%).
[0189] .sup.1H NMR (DMSO-d.sub.6): .delta.=10.35 (br. s, 2H), 8.36
(d, 4H), 7.02 (d, 4H) ppm. .sup.13C NMR (DMSO-d.sub.6):
.delta.=162.6, 161.5, 129.2, 122.6, 116.3 ppm. HPLC-MS/PDA: one
peak in chromatogram, m/z=267.1 (M+H.sup.+), .lamda..sub.max=235,
330 and 535 nm.
Synthesis of 3,6-bis(4-aminophenyl)-1,2,4,5-tetrazine (13)
##STR00037##
[0191] 4-Aminobenzonitrile (1.00 g; 8.46 mmol) was dissolved in
ethanol (3 mL), and subsequently hydrazine hydrate (2.12 g; 42.2
mmol) and sulfur (0.176 g; 5.5 mmol) were added. The mixture was
heated at 90.degree. C. for 90 min, and the yellow precipitate was
isolated by filtration, washed with ethanol (10 mL), and
subsequently triturated with acetone (12 mL) to yield the yellow
intermediate 12 (190 mg, 17%).
[0192] Intermediate 12 (50 mg; 0.188 mmol) was dissolved in DMSO (1
mL), and oxygen was bubbled through this solution at 20.degree. C.
After 5 hr, the reaction mixture was poured in brine (13 mL), and
the red precipitate was filtered off, washed with water (10 mL),
and dried in vacuo. The red powder was further purified by
trituration with acetone (15 mL), to yield product 13 as a red
solid (13.7 mg, 27%).
[0193] .sup.1H NMR (DMSO-d.sub.6): .delta.=8.17 (d, 2H), 7.75 (d,
2H), 6.02 (s, 4H) ppm. .sup.13C NMR (DMSO-d.sub.6): .delta.=162.3,
152.8, 128.5, 118.3, 113.8 ppm. HPLC-MS/PDA: one peak in
chromatogram, m/z=265.2 (M+H.sup.+), .lamda..sub.max=241, 370 and
530 nm.
Synthesis of 3,6-bis(3-aminophenyl)-1,2,4,5-tetrazine (15)
##STR00038##
[0195] 3-Aminobenzonitrile (1.00 g; 8.460 mmol) was dissolved in
hydrazine hydrate (2.50 mL; 51.4 mmol), and the mixture was heated
to 90.degree. C. for 3 days. Water (5 mL) was added, and the yellow
precipitate was filtered off and washed with water (15 mL) and
ethanol (10 mL), to yield the crude intermediate 14 as a orange
powder (910 mg; 81%).
[0196] Intermediate 14 (50 mg; 0.188 mmol) was suspended in ethanol
(4 mL), and oxygen was bubbled through this mixture at 50.degree.
C. Within minutes, the color changed from yellow to red. After 16
hr, the precipitate was filtered off, and washed with ethanol, to
give the product 15 as a red powder (31 mg, 62%).
[0197] .sup.1H NMR (DMSO-d.sub.6): .delta.=7.77 (s, 2H), 7.66 (d,
2H), 7.30 (t, 2H), 6.85 (d, 2H), 5.53 (s, 4H) ppm. HPLC-MS/PDA: one
peak in chromatogram, m/z=265.2 (M+H.sup.+), .lamda..sub.max=240,
296 and 527 nm.
Synthesis of 3,6-bis(aminomethyl)-1,2, 4,5-tetrazine (20)
##STR00039##
[0199] Boc-amino acetonitrile (1.00 g; 6.40 mmol) was dissolved in
methanol (10 mL) and sodium methoxide (0.145 mL 25% in MeOH; 0.64
mmol) was added. The mixture was stirred at 20.degree. C. for 18
hr, and subsequently ammonium chloride (0.34 g; 6.40 mmol) was
added, and the mixture was stirred at 20.degree. C. for 3 days. The
solution was precipitated in diethyl ether (40 mL), and the
precipitate was collected by filtration, washed, and dried to yield
the amidine hydrochloride 17.
[0200] The amidine hydrochloride (17, 241 mg; 1.15 mmol) was
dissolved in hydrazine hydrate (3 mL; 61.9 mmol), and the solution
was stirred at 20.degree. C. for 16 hr. Then it was diluted with
water (10 mL), and the precipitate was collected by centrifugation,
and dried. The colorless solid was dissolved in acetic acid (1.5
mL) and sodium nitrite (28 mg; 0.41 mmol) was added. The pink
mixture was stirred for 15 min and subsequently chloroform (15 mL)
and saturated sodium bicarbonate (30 mL) were added. The organic
layer was isolated and washed with water (15 mL), dried over sodium
sulfate, and evaporated to dryness, to yield the Boc-protected
tetrazine as a pink solid (19, 70 mg; 35%). This compound (12 mg;
0.035 mmol) was dissolved in chloroform (1 mL), and TFA (1 mL) was
added. The mixture was stirred for 15 min, and the precipitated in
diethyl ether (15 mL). The pink precipitate was filtered off,
washed, and dried to give the title compound as its TFA salt (20,
10 mg, 78%).
[0201] .sup.1H NMR (D.sub.2O): .delta.=5.06 (s, 4H) ppm. .sup.13C
NMR (D.sub.2O): .delta.=164.5, 41.1 ppm. HPLC-MS/PDA: one peak in
chromatogram, m/z=141 (M+H.sup.+), .lamda..sub.max=267 and 517
nm.
Synthesis of 2,2'
2''-(10-(2-oxo-2-(6-oxo-6-(6-(6-(pyridin-2-yl)-1,2,24,5-tetrazin-3-yl)pyr-
idin-3-ylamino)hexylamino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triy-
l)triacetic acid (27) and
2,2',2''-(10-(2-oxo-2-(11-oxo-11-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3--
yl)pyridin-3-ylamino)
undecylamino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic
acid (28)
##STR00040## ##STR00041##
[0203] 5-Amino-2-cyanopyridine 21 (1.02 g; 8.60 mmol),
N-Boc-6-amino-hexanoic acid 22 (0.99 g; 4.30 mmol), DCC (1.77 g;
8.60 mmol), DMAP (1.05 g; 8.60 mmol), and PPTS (0.37 g; 1.47 mmol)
were suspended in chloroform (15 mL). The mixture was stirred at
room temperature for 18 hr, and then evaporated to dryness, and
stirred in acetonitrile (20 mL). The precipitate was removed by
filtration, and the filtrate was evaporated to dryness, dissolved
in chloroform (20 mL), and washed with respectively aqueous citric
acid (15 mL 0.5 M), aqueous potassium hydrogencarbonate (15 mL, 1
M), and water (15 mL). The organic phase was dried over sodium
sulfate and evaporated to dryness. The crude product was purified
by column chromatography (silica, hexane/ethylacetate=1:1) to yield
the product 23 as a white solid (0.95 g; 61%).
[0204] MS (ESI, m/z): Calcd for
C.sub.17H.sub.25N.sub.4O.sub.3.sup.+ ([M+H].sup.+): 333.19. Found:
333.17.
[0205] Tert-butyl 6-(6-cyanopyridin-3-ylamino)-6-oxohexylcarbamate
23 (0.70 g; 2.1 mmol), 2-cyanopyridine (0.87 g; 8.4 mmol),
hydrazine hydrate (1.25 g; 20 mmol) were dissolved in ethanol (2
mL), and sulfur (0.22 g; 7 mmol) was added. The mixture was stirred
at 70.degree. C. under an inert atmosphere of argon for 2 hr, and
then at 50.degree. C. for 16 hr. The orange suspension was diluted
with chloroform (10 mL), and the resulting solution was washed with
water (2 times 15 mL). The organic phase was dried over sodium
sulfate and evaporated to dryness. The crude product was purified
by column chromatography (silica, chloroform/acetone=4:1) to yield
the product 24 as an orange solid (0.65 g; 66%). MS (ESI, m/z):
Calcd for C.sub.23H.sub.31N.sub.8O.sub.3.sup.+ ([M+H].sup.+):
467.25. Found: 467.33.
[0206] Tert-butyl
6-oxo-6-(6-(6-(pyridin-2-yl)-1,2-dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3--
ylamino)hexylcarbamate 24 (0.30 g; 0.64 mmol) was dissolved in THF
(1.5 mL), and acetic acid (2 mL) was added. Sodium nitrite (0.25 g;
3.62 mmol) was dissolved in water (1 mL) and added dropwise. The
red solution was poured in aqueous potassium hydrogencarbonate (50
mL; 1 M), and the product was extracted with chloroform (50 mL).
The organic layer was washed with water (50 mL), and dried over
sodium sulfate and evaporated to dryness, to yield the product 25
as a purple solid (0.25 g; 83%).
[0207] MS (ESI, m/z): Calcd for
C.sub.23H.sub.29N.sub.8O.sub.3.sup.+ ([M+H].sup.+): 465.23. Found:
465.42.
[0208] tert-Butyl
6-oxo-6-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-ylamino)
hexylcarbamate 25 (66 mg; 0.14 mmol) was dissolved in chloroform (6
mL), and TFA (6 mL) was added. The solution was stirred at room
temperature for 2 hr, and subsequently evaporated to dryness, to
yield the product 26 as its TFA salt (52 mg; 100%).
[0209] MS (ESI, m/z): Calcd for C.sub.18H.sub.21N.sub.8O.sup.+
([M+H].sup.+): 365.19. Found: 365.33.
[0210]
6-Amino-N-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)h-
exanamide 26 (52 mg; 0.14 mmol) was dissolved in DMF (2.5 mL), and
DIPEA was added (320 mg; 2.0 mmol). N-Hydroxysuccinimide activated
DOTA (161 mg; 0.2 mmol) was added, and the mixture was stirred at
room temperature for 5 hr. The solution was evaporated to dryness,
and the crude product was dissolved in a mixture of acetonitrile
and water, and purified by preparative RP-HPLC. After
lyophilisation the pure product 27 was obtained as a pink fluffy
solid (80 mg, 76% yield).
[0211] .sup.1H-NMR (30% acetonitrile-d.sub.3 in D.sub.2O):
.delta.=8.90 (m, 2H, ArH), 8.68 (d, 1H, ArH), 8.60 (dd, 1H, ArH),
8.31 (m, 1H, ArH), 8.24 (t, 1H, ArH), 7.82 (t, 1H, ArH), 3.80 (br
s, 6H, NCH.sub.2COOH), 3.72 (br s, 2H, NCH.sub.2CONH), 3.34-3.23
(br m, 18H, NCH.sub.2CH.sub.2N, CH.sub.2NHCO), 2.49 (t, 2H,
NHCOCH.sub.2), 1.70 (m, 2H, NHCOCH.sub.2CH.sub.2), 1.59 (m, 2H,
CH.sub.2CH.sub.2NHCO), 1.41 (m, 2H, CH.sub.2CH.sub.2CH.sub.2NHCO)
ppm. .sup.13C-NMR (30% acetonitrile-d.sub.3 in D.sub.2O):
.delta.=175.5, 171.5 (br), 162.6, 162.5, 150.1, 148.1, 142.9,
141.6, 139.6, 138.4, 128.0, 127.9, 125.4, 124.8, 55.4, 54.3 (br),
49.4 (br), 39.4, 36.5, 28.2, 25.9, 24.6 ppm. ESI-MS: m/z for
C.sub.34H.sub.47N.sub.12O.sub.8.sup.+ ([M+H].sup.+): 751.37; Obs.
[M+H].sup.+ 751.58, [M+Na].sup.+ 773.50, [M+2H].sup.2+ 376.42,
[M+3H].sup.3+251.33. FT-IR (ATR): v=3263, 3094, 2941, 2862, 1667,
1637, 1582, 1540, 1460, 1431, 1395, 1324, 1296, 1272, 1251, 1226,
1198, 1128, 1087, 1060, 1020, 992, 977, 920, 860, 831, 798, 782,
742, 718, 679, 663 cm.sup.-1.
[0212] For 28, a procedure was used comparable to the described
synthesis of
2,2',2''-(10-(2-oxo-2-(6-oxo-6-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-
-yl)pyridin-3-ylamino)
hexylamino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic
acid (27). After lyophilisation the pure product 28 was obtained as
a pink fluffy solid (90 mg, 78% yield).
[0213] .sup.1H-NMR (DMSO-d.sub.6): .delta.=10.65 (s, 1H, NH), 9.06
(d, 1H, ArH), 8.93 (d, 1H, ArH), 8.61 (t, 2H, ArH), 8.44 (dd, 1H,
ArH), 8.16 (t, 2H, ArH, NH), 7.73 (dd, 1H, ArH), 3.51 (br s, 6H,
NCH.sub.2COOH), 3.28 (br s, 2H, NCH.sub.2CONH), 3.06 (q, 2H,
CH.sub.2NHCO), 3.34-3.23 (br m, 16H, NCH.sub.2CH.sub.2N), 2.43 (t,
2H, NHCOCH.sub.2), 1.64 (m, 2H, NHCOCH.sub.2CH.sub.2), 1.42 (m, 2H,
CH.sub.2CH.sub.2NHCO), 1.38-1.22 (m, 12H, CH.sub.2) ppm.
.sup.13C-NMR (DMSO-d.sub.6): .delta.=173.0, 171.0 (br), 169.1 (br),
163.5, 163.2, 151.0, 150.6, 144.2, 141.7, 139.1, 138.2, 127.0,
126.5, 125.3, 124.6, 57.3 (br), 55.2 (br), 50.7, 39.0, 36.8, 29.5,
29.4, 29.3, 29.19, 29.17, 29.1, 26.9, 25.3 ppm. ESI-MS: m/z Calcd
for C.sub.39H.sub.57N.sub.12O.sub.8.sup.+([M+H].sup.+): 821.44;
Obs. [M+Na].sup.+ 843.58, [M+H].sup.+ 821.58, [M+2H].sup.2+ 411.42,
[M+3H].sup.3+ 274.67. FT-IR (ATR): v=3261, 3067, 2925, 2851, 1633,
1583, 1541, 1458, 1433, 1394, 1324, 1298, 1270, 1249, 1228, 1200,
1165, 1128, 1088, 1059, 1016, 991, 920, 885, 860, 832, 798, 782,
764, 742, 719, 687, 661 cm.sup.-1.
DOTA-Tetrazine Activator 29
##STR00042##
[0215] The tetrazine 29 above has been described in detail in
Robillard et al., Angew. Chem., 2010, 122, 3447-3450. It also
serves as an example a structure that can be used as an Activator
according to this invention. The amide function on one of the
2-pyridyl groups of the 1,2,4,5-tetrazine moiety is an electron
donating group, while both pyridine groups can be viewed as
electron withdrawing. The tetrazine can therefore be seen as
slightly electron deficient.
[0216] Activator 29 displays suitable and favorable pharmacological
properties: 29 is rather stable in PBS solution with little
degradation within 2 hrs and most of the material still intact
after overnight incubation; it has a 10 min blood clearance
half-life in mice; its partial volume of distribution (V.sub.d) in
mice corresponds to the total extracellular water compartment, as
it does not significantly enter cells. Activator 29 contains a DOTA
ligand, and such ligands are instrumental in a variety of imaging
modalities (e.g. MRI, SPECT, PET). Consequently, Activator 29 is
not only suitable for drug release, but it can simultaneously be
used for imaging purposes. In fact, Activator 29 has been employed
as a SPECT/CT imaging probe after complexation with
.sup.111In.sup.3+. See Robillard et al., Angew. Chem., 2010, 122,
3447-3450 for further details.
[0217] Note that the amino-1,2,4,5-tetrazine moieties comprised in
Activators 27-29 can be used for conjugation to a range additional
functional groups such as sugars, PEG, polymers, peptides (such as
RGD or c-RGD), proteins, fluorescent molecules or dye
molecules.
Example 2
Synthesis of (E)-cyclooctene Model Prodrugs and Prodrugs
Synthesis cis-(E)-cyclooct-5-ene-1,2diamine (35)
##STR00043##
[0219] Epoxycyclooctene was prepared by reaction of
1,5-cyclooctadiene with sodium perborate in acetic acid and
dichloromethane, cf. Giines, Y.; Senocak, E.; Tosun, C.;
Taskesenligil, Y., Org. Commun. 2009, 2:3, 79-83. The crude product
was used as such. A solution of epoxycyclooctene (80.0 g, 0.645
mol) in 140 mL acetone was added over a 45 min period to a solution
of sodium azide (80 g, 1.23 mol) in 200 mL water. The addition
funnel was flushed with 20 mL acetone and the mixture was heated
under reflux for 76 hrs. Most of the acetone was removed by rotary
evaporation, 100 mL water was added to the residue and the mixture
was extracted with 3.times.250 mL TBME. The organic layers were
washed with 100 mL water, then dried and rotary evaporated to yield
crude trans-(Z)-8-azidocyclooct-4-enol (30) (mixed with the
epoxide) which was used as such in the next step.
[0220] .sup.1H-NMR (CDCl.sub.3): .delta.=1.6-2.6 (m, 8H), 3.65-3.8
(m, 2H), 5.5-5.65 (m, 2H) ppm.
[0221] Toluene (200 mL) was added to all of 30 and about 150 mL of
solvent was removed by rotary evaporation. 300 mL toluene was added
to the remainder (a ca. 1/1 mixture of the azido alcohol 30 and the
starting epoxide) and the solution was cooled in ice. Triethylamine
(86.1 g, 0.852 mol) was added, followed by the addition of
methanesulfonyl chloride (93.8 g, 0.819 mol) in 100 mL toluene over
a 1 hr period and with mechanical stirring. The suspension was
stirred for 2 days, then 200 mL water was added. The layers were
separated and the organic layer was washed with 2.times.50 mL
water. The successive aqueous layers were extracted with 250 mL
toluene. Drying and rotary evaporation yielded a residue which was
a mixture of the azido mesylate and the starting epoxide.
[0222] .sup.1H-NMR (CDCl.sub.3): .delta.=1.95-2.6 (m, 8H), 3.95
(dt, 1H), 4.8 (dt, 1H), 5.5-5.65 (m, 2H) ppm.
[0223] Half of the residue was warmed for 44 hrs at 75.degree. C.
with 100 mL DMF and sodium azide (20 g, 0.307 mol), then for 3 hrs
at 85.degree. C. The mixture was poured into 200 mL water and then
extracted with 3.times.200 mL TBME. The organic layers were washed
with 3.times.50 mL water, then dried and rotary evaporated to yield
a residue which was a mixture of the
cis-(Z)-5,6-diazidocyclooct-1-ene (31), epoxycyclooctene and
impurities.
[0224] .sup.1H-NMR (CDCl.sub.3): .delta.=1.8 (m, 2H), 1.95-2.1 (m,
4H), 2.5-2.65 (m, 2H), 3.8 (dt, 2H), 5.6-5.7 (m, 2H) ppm.
[0225] The crude diazide 31 obtained above was dissolved in 150 mL
THF and added over a 90 min period to lithium aluminium hydride
(12.0 g, 0.315 mol) in 200 mL THF, cooling being done with cold
water. The reaction mixture was heated under reflux for 8 hrs, then
it was cooled and slowly quenched with 6 mL water and 12 mL 30%
sodium hydroxide solution. Filtration, washing with THF and rotary
evaporation yielded a residue (32), which was dissolved in 150 mL
dichloromethane, then cooled in ice. Trifluoroacetic anhydride
(69.0 g, 0.328 mol) was added over a 30 min period. The solution
was stirred for 4 hrs then rotary evaporated. The residue was
chromatographed on a 250 g silicagel column, elution being
performed with heptane containing increasing amounts of ethyl
acetate. The first fractions were the trifluoroacetate of
cyclooct-2-en-1-ol. The fractions with the desired
cis-(Z)--N,N'-(cyclooct-5-ene-1,2-diyl)bis(2,2,2-trifluoroacetamide)
(33) were combined and recrystallized from a mixture of TBME and
heptane to give 18.85 g of the product (33, 56.74 mmol, 18% based
on epoxycyclooctene).
[0226] .sup.1H-NMR of diamine 32 (CDCl.sub.3): .delta.=1.65 (m,
2H), 1.8 (m, 2H), 2.0 (m, 2H), 2.4 (m, 2H), 3.0 (dt, 2H), 5.6 (m,
2H) ppm.
[0227] .sup.1H-NMR of bisamide 33 (CDCl.sub.3): .delta.=1.65 (m,
2H), 2.0 (m, 2H), 2.2 (m, 2H), 2.35 (m, 2H), 4.15 (dt, 2H), 5.9 (m,
2H), 7.5 (m, 2H) ppm. .sup.13C-NMR (CDCl.sub.3): .delta. 23
(CH.sub.2), 32 (CH.sub.2), 54 (CH), 110-122 (q, CF.sub.3), 132
(CH), 157-159 (q, C.dbd.O) ppm. .sup.19F-NMR (CDCl.sub.3):
.delta.=-76 ppm.
[0228] The crude trifluoroacetamide 33 obtained after evaporation
of the reaction product from 3.50 g (25.0 mmol)
cis-(Z)-cyclooct-5-ene-1,2-diamine 32 and trifluoroacetic anhydride
(12.3 g, 58.6 mmol) was mixed with 4.0 g methyl benzoate and ca.
500 mL heptane/ether (ca. 2:1). The mixture was irradiated for 42
hrs while the solution was continuously flushed through a 41 g
silver nitrate impregnated silicagel column (containing ca. 4.1 g
silver nitrate). The column was flushed with 150 mL TBME, then with
150 mL TBME containing some methanol. The fractions were washed
with 100 mL 15% ammonia, dried and rotary evaporated. The first
fraction yielded a 1:2 mixture of the Z and E alkene, the second
fraction yielded a small amount of the E alkene. The column
material was stirred with TBME and ammonia, then filtered and the
layers were separated. The solid was treated once more with the
aqueous layer and TBME, then filtered and the layers were
separated. The organic layers were dried and rotary evaporated to
yield 3.07 g of the E alkene
cis-(E)-N,N'-(cyclooct-5-ene-1,2-diyl)bis(2,2,2-trifluoroacetamide-
) (34, 9.25 mmol, 37% based on the amine).
[0229] .sup.1H-NMR (CDCl.sub.3): .delta.=1.6-1.9 (m, 4H), 2.1-2.5
(m, 4H), 3.8 (m, 1H), 4.1 (t, 1H), 5.4-5.55 (m, 1H), 5.65-5.8 (m,
1H), 6.4 (bs, 1H), 7.9 (bs, 1H) ppm.
[0230] The amide 34 obtained above was mixed with 40 mL methanol,
5.0 g sodium hydroxide and 10 mL water. The mixture was warmed for
90 min at near reflux, then it was rotary evaporated and the
residue was diluted with 30 mL water. Extraction with 3.times.50 mL
dichloromethane, drying and rotary evaporation yielded the desired
diamine cis-(E)-cyclooct-5-ene-1,2-diamine (35), containing a small
amount of solvent (1.38 g, ca. 100%).
[0231] .sup.1H-NMR (CDCl.sub.3): .delta.=1.4-2.5 (m, 8H), 2.8 (bs,
1H), 2.9 (d, 1H), 5.4-5.6 (m, 2H) ppm.
Synthesis of cis-(E)-phenyl(8-aminocyclooct-4-en-1-yl)carbamate
(36)
##STR00044##
[0233] Diphenylcarbonate (500 mg, 2.33 mmol) was added to a
solution of the diamine 35 (300 mg, 2.14 mmol) in 10 mL
dichloromethane and the solution was stirred for 4 days at room
temperature. The solution was chromatographed on 25 g silica,
eluting with dichloromethane containing increasing amounts of
methanol. The product fractions were combined and stirred with 15
mL TBME for 2 hrs. 15 mL heptane was added and the mixture was
filtered. The solid was stirred with 15 mL TBME then filtered. The
combined filtrates were rotary evaporated and the residue was
stirred overnight with heptane to give a solid. Filtration yielded
the desired product
cis-(E)-phenyl(8-aminocyclooct-4-en-1-yl)carbamate (36).
[0234] .sup.1H-NMR (CDCl.sub.3): .delta.=1.5 (bs, 4H), 1.8-2.35 (m,
6H), 3.1 (bs, 1H), 3.6 (t, 1H), 5.5 (bd, 1H), 5.6 (m, 2H), 7.05-7.4
(m, 5H) ppm. .sup.13C-NMR (CDCl.sub.3): .delta.=28.4 (CH.sub.2),
33.0 (CH.sub.2), 36.7 (CH.sub.2), 40.7 (CH.sub.2), 57.7 (CH), 58.4
(CH), 121.8 (CH), 125.4 (CH), 129.5 (CH), 133.2 (CH), 133.3 (CH),
151.3 (C), 154.0 (C) ppm.
Synthesis of
cis-(E)-3,4,5-trimethoxybenzyl(8-aminocyclooct-4-en-1-yl)carbamate
(37)
##STR00045##
[0236] A solution of 3,4,5-trimethoxybenzyl alcohol (6.20 g, 31.3
mmol) in THE (20 mL) was added in 15 min to a solution of CDI (5.44
g, 33.58 mmol) in THF (25 mL), cooled in an ice-water bath. The
mixture was then stirred for 3 days at room temperature. The
solvent was removed under vacuum and TBME (100 mL) was added. The
mixture was stirred for 1 hr, decanted and filtered. The residue
was stirred with TBME (25 mL) for 5 min, decanted and filtered. The
combined TBME filtrates were concentrated to give the CDI-adduct
(10.45 g, 35.78 mmol, not corrected for free imidazole) as an oil
which slowly solidified. By NMR it contains one equivalent of free
imidazole.
[0237] .sup.1H-NMR (CDCl.sub.3): .delta.=3.85 (s, 3H), 3.9 (s, 6H),
5.35 (s, 2H), 6.65 (s, 2H), 7.05 (s, 1H), 7.45 (s, 1H), 8.2 (s, 1H)
ppm.
[0238] The CDI-derivative (705 mg, 2.41 mmol) was added to a
solution of the diamine 35 (330 mg, 2.35 mmol) in dichloromethane
(15 mL) and the mixture was stirred for 1 hr at room temperature.
The mixture was concentrated, ethyl acetate (20 mL) was added to
the residue and the mixture was filtered while warm. The residue
was washed with warm ethyl acetate and the combined filtrates were
cooled in ice until a precipitate started to appear, then it was
stirred for 1 hr. The mixture was cooled for 30 min at -15.degree.
C. and the precipitate collected by filtration, washed with cold
ethyl acetate and dried to give
cis-(E)-3,4,5-trimethoxybenzyl(8-aminocyclooct-4-en-1-yl)carbamate
37 (170 mg, 0.47 mmol, 20%).
[0239] .sup.1H-NMR (CDCl.sub.3): .delta.=1.4-2.3 (m, 10H), 3.05
(bs, 1H), 3.55 (t, 1H), 3.8 (s, 3H), 3.85 (s, 6H), 5.0 (s, 2H), 5.2
(m, 1H), 5.6 (m, 2H), 6.6 (s, 2H), 7.45 (s, 1H), 8.2 (s, 1H) ppm.
.sup.13C-NMR (CDCl.sub.3): .delta.=28 (CH.sub.2), 33 (CH.sub.2), 37
(CH.sub.2), 41 (CH.sub.2), 56 (CH), 58 (CH), 58.5 (CH), 61 (CH), 67
(CH.sub.2), 106 (CH), 132.5 (C), 133 (CH), 133.5 (CH), 138 (C),
153.5 (C), 155.5 (C) ppm.
Synthesis of cis
2-methylphenyl((E)-8-aminocyclooct-4-en-1-yl)carbamate (39)
##STR00046##
[0241] Cis-(E)-cyclooct-5-ene-1,2-diamine (35, 9.7 mg; 0.069 mmol)
was dissolved in acetonitrile (1 mL) and cooled to 0.degree. C.
NHS-activated o-cresol 38 (17.2 mg; 0.069 mmol) was dissolved in
acetonitrile (1 mL) and was added. The mixture was stirred for 30
min at 0.degree. C. The precipitate was removed by filtration, and
the filtrate was evaporated to dryness. The oily residue was
dissolved in dichloromethane (2 mL) and washed with water (1 mL).
The product was extracted with 0.5 M citric acid (1.5 mL) and the
aqueous phase was isolated and neutralized with brine (2 mL). The
product was extracted with dichloromethane (two times 2 mL). The
combined organic layers were dried over sodium sulfate and
evaporated to dryness, to give the product 39 as a viscous oil (6.1
mg, 32%).
[0242] .sup.1H NMR (CDCl.sub.3): .delta.=7.2-7.0 (m, 4H), 5.62 (m,
2H), 5.57 (s, 1H), 3.59 (t, 1H), 3.14 (d, 1H), 2.19 (s, 3H),
2.3-1.8 (m, 8H), 1.6 (br. s, 2H) ppm. HPLC-MS/PDA: one peak in
chromatogram, m/z=275 (M+H.sup.+), .lamda..sub.max=261 nm.
Synthesis of cis
2-methoxyphenyl((E)-8-aminocyclooct-4-en-1-yl)carbamate (41)
##STR00047##
[0244] Cis-(E)-cyclooct-5-ene-1,2-diamine (35, 9.7 mg; 0.069 mmol)
was dissolved in acetonitrile (1 mL) and cooled to 0.degree. C.
NHS-activated 2-methoxyphenol (40, 18.3 mg; 0.069 mmol) was
dissolved in acetonitrile (1 mL) and was added. The mixture was
stirred for 45 min at 0.degree. C. The precipitate was removed by
filtration, and the filtrate was evaporated to dryness. The oily
residue was dissolved in dichloromethane (2 mL) and washed with
water (1 mL). The product was extracted with 0.5 M citric acid (1.5
mL) and the aqueous phase was isolated and neutralized with brine
(2 mL). The product was extracted with dichloromethane (two times 2
mL). The combined organic layers were dried over sodium sulfate and
evaporated to dryness, to give the product 41 as a viscous oil (9.0
mg, 45%).
[0245] .sup.1H NMR (CDCl.sub.3): .delta.=7.22 (m, 2H), 6.97 (m,
2H), 5.62 (m, 2H), 5.71 (s, 1H), 3.89 (s, 3H), 3.59 (t, 1H), 3.14
(d, 1H), 2.3-1.8 (m, 8H), 1.6 (br. s, 2H) ppm. HPLC-MS/PDA: one
peak in chromatogram, m/z=291 (M+H.sup.+), .lamda..sub.max=269
nm.
Synthesis of cis
2,6-dimethylphenyl((E)-8-aminocyclooct-4-en-1-yl)carbamate (43)
##STR00048##
[0247] Cis-(E)-cyclooct-5-ene-1,2-diamine (35, 26.5 mg; 0.189 mmol)
was dissolved in acetonitrile (1.5 mL) and cooled to 0.degree. C.
NHS-activated 2,6-dimethylphenol (42, 50 mg; 0.189 mmol) was added,
and the mixture was stirred for 1 hr. The precipitate was removed
by filtration, and the filtrate was evaporated to dryness. The oily
residue was dissolved in dichloromethane (3 mL) and washed with
water (1.5 mL). The product was extracted with 0.5 M citric acid (2
mL) and the aqueous phase was isolated and neutralized with brine
(3 mL). The product was extracted with dichloromethane (two times 3
mL). The combined organic layers were dried over sodium sulfate and
evaporated to dryness, to give the product 43 as a viscous oil (32
mg, 60%).
[0248] .sup.1H NMR (CDCl.sub.3): .delta.=7.02 (m, 3H), 5.57 (m,
2H), 5.49 (s, 1H), 3.58 (t, 1H), 3.10 (d, 1H), 2.17 (s, 6H),
2.3-1.8 (m, 8H), 1.6 (br. s, 2H) ppm. HPLC-MS/PDA: one peak in
chromatogram, m/z=289 (M+H.sup.+), .lamda..sub.max=259 nm.
Synthesis of cis
2-tert-butylphenyl((E)-8-aminocyclooct-4-en-1-yl)carbamate (45)
##STR00049##
[0250] Cis-(E)-cyclooct-5-ene-1,2-diamine (35, 26.5 mg; 0.189 mmol)
was dissolved in acetonitrile (1.5 mL) and cooled to 0.degree. C.
NHS-activated 2-tert-butylphenol (44, 55 mg; 0.189 mmol) was added,
and the mixture was stirred for 1 hr, and then evaporated to
dryness. The oily residue was dissolved in dichloromethane (3 mL)
and washed with water (1.5 mL). The product was extracted with 0.5
M citric acid (2 mL) and the aqueous phase was isolated and
neutralized with brine (3 mL). The product was extracted with
dichloromethane (two times 3 mL). The combined organic layers were
dried over sodium sulfate and evaporated to dryness, to give the
product 45 as a viscous oil (15 mg, 25%).
[0251] HPLC-MS/PDA: one peak in chromatogram, m/z=317 (M+H.sup.+),
.lamda..sub.max=259 nm.
Synthesis of cis phenyl((E)-8-aminocyclooct-4-en-1-yl)urea (47)
##STR00050##
[0253] Cis-(E)-cyclooct-5-ene-1,2-diamine (35, 23.4 mg; 0.167 mmol)
was dissolved in chloroform (1 mL). 3,5-Dimethylphenylisocyanate
(46, 24.5 mg; 0.167 mmol) was dissolved in chloroform (1 mL) and
was added slowly at 20.degree. C. The mixture was stirred for 30
min at 20.degree. C., and subsequently concentrated in vacuo. The
crude material was subsequently purified by prep-HPLC, yielding two
isomeric products 47 with m/z=288 (M+H.sup.+), .lamda..sub.max=243
nm: 10.9 mg of major isomer (23% yield) and 1.9 mg of minor isomer
(4% yield).
Synthesis of trans phenyl(E)-2-aminocyclooct-3-en-1-yl carbamate
(56)
##STR00051##
[0255] A mixture of 1,3-cyclooctadiene (21.72 g, 0.201 mol), 200 mL
dichloromethane, 75 mL acetic acid and 35.16 g sodium perborate
tetrahydrate (0.228 mol) was stirred for 2 days at room
temperature, then for 28 hrs at 35.degree. C. The mixture was
poured into 150 mL water and 200 mL dichloromethane. The layers
were separated and the organic layer was washed with 50 mL water
and with 100 mL 20% sodium hydroxide solution. The successive
aqueous layers were extracted with 200 mL dichloromethane
(filtration over Celite being necessary). The organic layers were
dried and rotary evaporated. The residue comprising
(Z)-9-oxabicyclo[6.1.0]non-2-ene (48) was used as such.
[0256] A solution of the epoxycyclooctene 48 obtained above in 65
mL acetone was added over a 30 min period to a solution of sodium
azide (24.0 g, 0.369 mol) in 60 mL water. The mixture was heated
under reflux for 7 days, distilling off ca. 30 mL acetone after 4
days (Note). 50 mL water was added to the residue and the mixture
was extracted with 2.times.200 mL TBME. The organic layers were
washed with 25 mL water, then dried and rotary evaporated to yield
19.6 g of residue comprising trans-(Z)-2-azidocyclooct-3-enol (49).
Note: The ring opening with sodium azide was described in Organic
Synthesis 2010, 87, 161 for cyclohexene oxide. We have our doubts
about the reported use of acetone as the organic solvent, because
the Organic Synthesis procedure reports a reflux temperature of
85.degree. C., which seems to correspond better with acetonitrile
as solvent than acetone.
[0257] .sup.1H-NMR (CDCl.sub.3): .delta.=1.2-2.4 (m, 8H), 3.55 (m,
1H), 4.35 (t, 1H), 5.55 (m, 1H), 5.85 (m, 1H) ppm.
[0258] The crude 49 obtained above was dissolved in 130 mL TBME and
28 mL triethylamine (0.202 mol). The solution was cooled with
ethanol--dry ice and methanesulfonyl chloride (21.2 g, 0.185 mol)
in 30 mL TBME was added over a 30 min period at -10 to 0.degree. C.
The resulting suspension was stirred overnight, then 100 mL water
was added. The layers were separated and the organic layer was
washed with 50 mL water. The successive aqueous layers were
extracted with 200 mL TBME. Drying and rotary evaporation yielded
20.3 g azidomesylate residue which was dissolved in 75 mL DMF.
Potassium acetate (19.65 g, 0.20 mol) was added and the mixture was
heated for 2 hrs at 80.degree. C. Another 50 mL DMF was added and
heating was continued for 17 hrs at 90.degree. C. After cooling,
the mixture was poured into 150 mL diluted ammonia and the product
was extracted with 3.times.200 mL TBME. The successive organic
layers were washed with 3.times.25 mL water, then dried and rotary
evaporated. The residue was chromatographed on 150 g silica,
elution being done with heptane/ethyl acetate, yielding cis
(Z)-2-azidocyclooct-3-en-1-yl acetate (50).
[0259] .sup.1H-NMR of the azidomesylate (CDCl.sub.3):
.delta.=1.2-2.4 (m, 8H), 3.1 (s, 3H), 4.5-4.65 (m, 2H), 5.4 (m,
1H), 6.0 (m, 1H) ppm.
[0260] .sup.1H-NMR of the azido acetate 50 (CDCl.sub.3):
.delta.=1.2-2.0 (m, 8H), 2.05 (s, 3H), 4.35 (m, 1H), 5.45-5.7 (m,
3H) ppm.
[0261] The azidoacetate 50 was stirred for 1 hr with 50 mL methanol
and 15 mL 30% sodium hydroxide solution. Most of the methanol was
then removed by rotary evaporation. The residue was extracted with
2.times.100 mL TBME. The organic layers were dried and rotary
evaporated to leave 10.5 g of azidoalcohol.
[0262] This residue was dissolved in 100 mL TBME and triethylamine
(21 mL, 0.151 mol) was added. The mixture was cooled in ice and
methanesulfonyl chloride (13.75 g, 0.120 mol) in 50 mL TBME was
added over a 1 hr period. The resulting suspension was stirred
overnight then 100 mL water was added. The layers were separated
and the organic layer was washed with 50 mL water. The successive
aqueous layers were extracted with 100 mL TBME. Drying and rotary
evaporation yielded the azidomesylate derivative which was
dissolved in 45 mL DMF. Sodium azide (11.0 g, 0.169 mol) was added
and the mixture was heated for 18 hrs at 70.degree. C., then for 3
hrs at 90.degree. C. After cooling, the mixture was poured into 150
mL water and the product was extracted with 3.times.150 mL TBME.
The successive organic layers were washed with 2.times.50 mL water,
then dried and rotary evaporated. The residue comprising
trans-(Z)-3,4-diazidocyclooct-1-ene (51, 11.28 g) was used as such
in the next step.
[0263] .sup.1H-NMR of the azido alcohol (CDCl.sub.3):
.delta.=1.2-2.2 (m, 8H), 4.25 (m, 1H), 4.55 (m, 1H), 5.45 (m, 1H),
5.7 (m, 1H) ppm.
[0264] .sup.1H-NMR of the azidomesylate (CDCl.sub.3):
.delta.=1.2-2.3 (m, 8H), 3.0 (s, 3H), 4.25 (m, 1H), 5.4 (m, 1H),
5.55-5.8 (m, 1H) ppm.
[0265] .sup.1H-NMR of the diazide 51 (CDCl.sub.3): .delta.=1.2-2.4
(m, 8H), 3.45 (m, 1H), 4.35 (m, 1H), 5.45 (m, 1H), 5.95 (m, 1H)
ppm.
[0266] The crude diazide 51 obtained above was dissolved in 100 mL
THF and added over a 30 min period to lithium aluminium hydride
(5.4 g, 0.142 mol) in 100 mL THF, cooling being done with cold
water. The reaction mixture was heated under reflux for 18 hrs,
then it was cooled and slowly quenched with 6 mL water and 6 mL 30%
sodium hydroxide solution.
[0267] Filtration, washing with THF and rotary evaporation yielded
7.8 g crude diamine 52 which was dissolved in 100 mL
dichloromethane, then cooled in ice. Trifluoroacetic anhydride
(20.87 g, 0.099 mol) was added over a 30 min period. The solution
was stirred for 30 min, heated under reflux for 1 hr, and rotary
evaporated. The residue was chromatographed on 100 g silica,
elution being performed with heptane containing increasing amounts
of ethyl acetate. The product fractions were combined and the
residue was stirred with a mixture of TBME and heptanes to give a
suspension. Filtration yielded 5.86 g of trans
N,N'--((Z)-cyclooct-3-ene-1,2-diyl)bis(2,2,2-trifluoroacetamide)
(53, 17.64 mmol, 9% based on 1,3-cyclooctadiene).
[0268] .sup.1H-NMR of the diamine 52 (CDCl.sub.3): .delta.=1.2-2.3
(m, 8H), 2.55 (m, 1H), 3.4 (t, 1H), 5.35 (m, 1H), 5.6 (m, 1H)
ppm.
[0269] .sup.1H-NMR of bisamide 53 (CDCl.sub.3): .delta.=1.3-2.0 (m,
6H), 2.25 (m, 2H), 4.05 (m, 1H), 5.0 (q, 1H), 5.4 (t, 1H), 5.9 (q,
1H), 7.0 (bs, 1H), 7.1 (bs, 1H) ppm.
[0270] The trifluoroacetamide 53 (5.86 g, 17.64 mmol) was mixed
with 6.25 g methyl benzoate and ca. 500 mL heptane/ether (ca. 1:2).
The suspension was irradiated for 78 hrs while the mixture was
continuously flushed through a 32.2 g silver nitrate impregnated
silicagel column (containing ca. 3.2 g silver nitrate). The
undissolved amide 53 was collected on top of the column and
dissolved very slowly during the irradiation and flushing process
and was not yet completely dissolved at the end of the irradiation.
The column material was flushed with 300 mL heptane/TBME (1:1),
then with 300 mL TBME. The fractions were washed with 100 mL 15%
ammonia, dried and rotary evaporated, affording a mixture of 53 and
54 from which 54 could be purified by stirring with heptane: TBME.
The column material was stirred with dichloromethane and ammonia,
then filtered and the layers were separated. The solid was treated
once more with the aqueous layer and dichloromethane, then filtered
and the layers were separated. The combined organic layers were
dried and rotary evaporated to yield the trans alkene 54 (0.91 g,
16%).
[0271] .sup.1H-NMR of 54 (CDCl.sub.3): .delta.=0.8-2.5 (m, 8H),
4.35 (m, 1H), 4.55 (m, 1H), 5.7-6.0 (m, 2H) ppm. .sup.19F-NMR
(CDCl.sub.3): .delta.=-75.9, -76.1 ppm (in addition, there are two
small signals at -76.4 and -76.6 ppm, possibly another
E-isomer).
[0272] Amide 54 (430 mg, 1.29 mmol) was mixed with 10 mL methanol
and 1.65 g 50% sodium hydroxide solution was added. The mixture was
warmed for 90 min at near reflux, then it was rotary evaporated and
the residue was diluted with 15 mL water. Extraction with
4.times.30 mL dichloromethane, drying and rotary evaporation
yielded the desired trans (E)-cyclooct-3-ene-1,2-diamine (55, 128
mg, 0.91 mmol, 71%).
[0273] .sup.1H-NMR (CDCl.sub.3): .delta.=1.1-2.1 (m, 9H), 2.45 (m,
1H), 3.15 (d, 1H), 3.45 (s, 1H), 5.95 (m, 1H) ppm. .sup.13C-NMR
(CDCl.sub.3): .delta.=20.0 (CH.sub.2), 30.6 (CH.sub.2), 35.9
(CH.sub.2), 36.2 (CH.sub.2), 59.2 (CH), 63.7 (CH), 130.8 (CH),
133.4 (CH) ppm.
[0274] Diphenylcarbonate (200 mg, 0.93 mmol) was added to a
solution of the diamine 55 (95 mg, 0.68 mmol) in 10 mL
dichloromethane and the solution was stirred for 3 days at room
temperature (reaction not yet being complete). The solution was
rotary evaporated and the residue was chromatographed on 13 g
silica, eluting with dichloromethane with increasing amounts of
methanol. This yielded 52 mg of the desired product
trans-phenyl(E)-2-aminocyclooct-3-en-1-yl)carbamate 56 (0.2 mmol,
30%). A fraction with a slightly lower R.sub.f value was assumed to
be the carbamate at the 2-amino position. This product was not
obtained in a completely pure form (26 mg, 0.1 mmol, 15%).
[0275] .sup.1H-NMR (CDCl.sub.3): .delta.=0.8-2.2 (m, 9H), 2.45 (m,
1H), 3.8-3.95 (m, 2H), 5.35 (bd, 1H, amide NH), 5.75 (dd, 1H),
6.0-6.15 (m, 1H), 7.1-7.4 (m, 5H) ppm. .sup.13C-NMR (CDCl.sub.3):
.delta.=21.9 (CH.sub.2), 28.1 (CH.sub.2), 36.1 (CH.sub.2), 36.2
(CH.sub.2), 56.4 (CH), 63.2 (CH), 121.8 (CH), 125.6 (CH), 129.5
(CH), 131.7 (CH), 132.7 (CH), 151.2 (C), 154.3 (C.dbd.O) ppm. MS:
261.0 (M+1). The compound which is assumed to be the other isomer
has .sup.1H-NMR signals at .delta. 1.0-2.2 (m, 7H), 2.45 (m, 1H),
3.6 (bs, 1H), 3.8 (b, 2H), 4.25 (bs, 1H), 5.6 (bs, 1H), 5.7 (m,
1H), 6.0 (d, 1H), 7.1-7.4 (m, 5H) ppm. MS 261.0 (M+1).
Synthesis of (E)-phenyl
2,3,4,5-tetrahydro-1,4-diazocine-1(8H)-carboxylate (61)
##STR00052##
[0277] Trifluoroacetic anhydride (92.69 g, 0.441 mol) was added
over a 1 hr period to an ice-cooled solution of ethylenediamine
(12.09 g, 0.20 mol) in 250 mL dichloromethane. The mixture was
warmed to reflux for 1 hr, then rotary evaporated. Water (100 mL)
and TBME (250 mL) were added and the mixture was stirred for 1 hr.
Filtration and washing with TBME gave the product. The filtrate
layers were separated, the organic layer was rotary evaporated and
the residue stirred with some TBME. Filtration gave an additional
amount of N,N'-(ethane-1,2-diyl)bis(2,2,2-trifluoroacetamide) (57)
for a total yield of 47.23 g (0.187 mol, 93%).
[0278] Product 57 obtained above was stirred for 15 min with 400 mL
acetonitrile, 100 g potassium carbonate, and 3.2 g
benzyltriethylammonium chloride. Cis-1,4-dichloro-2-butene (26.97
g, 95%, 0.205 mol), dissolved in 50 mL acetonitrile, was added over
a 30 min period. The mixture was warmed to 71.degree. C. over a 3
hrs period and kept at that temperature for 64 hrs, then heated for
24 hrs at 77.degree. C. The mixture was filtered while warm and the
solid was washed with acetonitrile. Rotary evaporation left a
residue which was chromatographed on 250 g silica gel using
dichloromethane and dichloromethane containing some triethylamine
as the eluent. This yielded 23.25 g of
(Z)-1,1'-(2,3-dihydro-1,4-diazocine-1,4(5H,8H)-diyl)bis(2,2,2-trifluoroet-
hanone) (58, 76.43 mmol, 41%).
[0279] .sup.1H-NMR (CDCl.sub.3): .delta.=3.65-3.95 (m, 5.4H),
4.05-4.2 (m, 1.1H), 4.25 (bs, 1.5H), 5.6 (m, 0.8H), 5.8 (bs, 0.5H),
6.15 (m, 0.7H) ppm. .sup.13C-NMR (CDCl.sub.3): .delta.=45 (2
CH.sub.2), 47 (CH.sub.2), 48 (CH.sub.2), 48.5 (CH.sub.2), 49
(CH.sub.2), 50 (CH.sub.2), 53 (CH.sub.2), 110-122 (2q, CF.sub.3),
126 (CH), 127 (CH), 128 (CH), 128.5 (CH), 155-158 (2q, C.dbd.O)
ppm. .sup.19F-NMR (CDCl.sub.3): .delta.=-69.2, -69.4, -69.8, -70.0
ppm. MS: 305.0 (M+1), 303.0 (M-1).
[0280] The trifluoroacetamide 58 (14.0 g, 46.0 mmol) was mixed with
8.0 g methyl benzoate and ca. 500 mL heptane/ether (ca. 10:1). The
mixture was irradiated for 92 hrs while the solution was
continuously flushed through a 70 g silver nitrate impregnated
silica gel column (containing ca. 7.0 g silver nitrate). The column
material was then flushed with 300 mL portions of heptane/TBME in
the ratios 5:1, 3:1, 2:1, 1:1 and then with 300 mL TBME, each
fraction being washed with 200 mL 10% ammonia, dried and rotary
evaporated. The remaining column material was stirred with TBME and
ammonia, then filtered and the layers were separated. The solid was
treated once more with the aqueous layer and TBME, then filtered
and the layers were separated. The combined organic layers were
dried and rotary evaporated to yield 3.48 g of
(E)-1,1'-(2,3-dihydro-1,4-diazocine-1,4(5H,8H)-diyl)bis(2,2,2-trifluoroet-
hanone) (59) as a solidifying oil (11.45 mmol, 25%).
[0281] .sup.1H-NMR (CDCl.sub.3): .delta.=2.6 (t, 1H), 2.95 (t, 1H),
3.4-3.55 (m, 1H), 3.75-3.9 (m, 1H), 4.0-4.3 (m, 1H), 4.35-4.6 (m,
1H), 4.65 (d, 1H), 5.25 (d, 1H), 5.8-6.0 (m, 2H) ppm. .sup.13C-NMR
(CDCl.sub.3): .delta.=49.4 (CH.sub.2), 49.6 (CH.sub.2), 50.0
(CH.sub.2), 50.1 (CH.sub.2), 51.9 (CH.sub.2), 52.0 (CH.sub.2), 53.9
(CH.sub.2), 54.1 (CH.sub.2), 110-122 (2q, CF.sub.3), 136.0 (CH),
136.1 (CH), 155-158 (2q, C.dbd.O) ppm. .sup.19F-NMR (CDCl.sub.3):
.delta.=-69.2, -69.25, -69.4, -69.45 ppm.
[0282] The amide 59 obtained above (520 mg, 1.71 mmol) was mixed
with 10 mL methanol and 1.60 g 50% sodium hydroxide solution, then
warmed for 1 hr at 55.degree. C. Most of the methanol was removed
by rotary evaporation and the residue was diluted with 20 mL water.
Extraction with 5.times.25 mL dichloromethane, drying and rotary
evaporation yielded the desired
(E)-1,2,3,4,5,8-hexahydro-1,4-diazocine (60, 150 mg, 1.34 mmol,
78%).
[0283] .sup.1H-NMR (CDCl.sub.3): .delta.=2.45 (d, 2H), 3.15 (d,
2H), 3.3 (m, 2H), 3.55 (dd, 2H), 6.0 (m, 2H) ppm. .sup.13C-NMR:
8=53.0 (CH.sub.2), 54.0 (CH.sub.2), 140.0 (CH) ppm.
[0284] Diphenylcarbonate (266 mg, 1.24 mmol) was added to a
solution of the diamine 60 obtained above in 10 mL dichloromethane
and the solution was stirred for 2 days at 30.degree. C. The
solution was chromatographed on 17 g silica, eluting with
dichloromethane containing increasing amounts of methanol. The
product fractions were combined and rotary evaporated. The residue
was chromatographed on 20 g silica, eluting with TBME containing
increasing amounts of methanol. Further elution with
dichloromethane-methanol yielded the product (E)-phenyl
2,3,4,5-tetrahydro-1,4-diazocine-1(8H)-carboxylate (61).
[0285] .sup.1H-NMR (CDCl.sub.3): .delta.=2.5-2.85 (m, 2H), 3.25-3.4
(m, 2H), 3.4-3.55 (m, 2H), 4.25 (m, 1H), 4.9 (m, 1H), 5.8-6.1 (m,
2H), 7.0-7.4 (m, 5H) ppm. .sup.13C-NMR (CDCl.sub.3): .delta.=50.2
(CH.sub.2), 50.8 (CH.sub.2), 52.5 (CH.sub.2), 52.6 (CH.sub.2), 53.5
(CH.sub.2), 121.9 (CH), 125.5 (CH), 129.5 (CH), 135.3 (CH), 135.4
(CH), 141.3 (CH), 141.6 (CH), 151.6 (C), 155.0 (C.dbd.O) ppm.
[0286] MS: 232.9 (M+1).
Synthesis of (E)-cyclooctene-doxorubicin conjugate 64
##STR00053##
[0288] 4-(Hydroxymethyl)phenyl 4-nitrophenyl carbonate (62) was
synthesized via a modified literature procedure (K. Haba, M.
Popkov, M. Shamis, R. A. Lerner, C. F. Barbas III, and D. Shabat,
Angew. Chem. Int. Ed. 2005, 44, 716-720). In a 25 mL round-bottom
flask, 4-hydroxybenzyl alcohol (0.3 g, 2.3 mmol) and DIPEA (400
.mu.L, 0.3 g, 2.3 mmol, 1 eq) were dissolved in dry THF (3 mL). The
flask was put in an ice bath, 4-nitrophenyl chloroformate (0.52 g,
2.5 mmol, 1.05 eq) in dry THF (2 mL) was added dropwise and the
mixture was stirred at room temperature for 1 hr. After filtration
of the formed white precipitate (DIPEA.HCl salt), the solvent was
removed in vacuo and the residue was redissolved in EtOAc (50 mL).
The organic layer was washed with water and brine (both 20 mL),
dried with MgSO.sub.4, filtrated and the solvent was removed in
vacuo. Purification was achieved using column chromatography (flash
silica, 10% THF in chloroform, the compound was added to the top of
the column in 30 mL eluent) and precipitation
(acetone.fwdarw.pentane). This yielded pure 62 (0.57 g, 2.0 mmol,
84%) as a white solid. Its spectral characteristics match the
reported data.
[0289] In a 10 mL round-bottom flask, 62 (50 mg, 0.17 mmol) was
dissolved in dry THF (1 mL) under an Ar atmosphere. After the
addition of phosgene (179 .mu.L of a 1.9 M solution in toluene,
0.34 mmol, 2 eq) the flask was sealed and the mixture was stirred
at room temperature for 15 hrs. The solvent was removed in vacuo
and the resulting oil was flushed with toluene (3.times.) and
chloroform. This yielded the analogous chloroformate as a colorless
oil which was used immediately without further purification.
[0290] .sup.1H-NMR (CDCl.sub.3): .delta.=8.33 (d, 2H, ArH), 7.49
(d, 2H, ArH), 7.48 (d, 2H, ArH), 7.33 (d, 2H, ArH), 5.31 (s, 2H,
CH.sub.2) ppm.
[0291] Subsequently, in a 25 mL round-bottom flask, doxorubicin
hydrochloride (86 mg, 0.15 mmol) was dissolved in dry THF (2 mL)
and a solution of the chloroformate (61 mg, 0.17 mmol, 1.2 eq) in
dry THF (4 mL) was added. After the addition of DIPEA (115 .mu.L,
0.65 mmol, 4.4 eq) the mixture was stirred at room temperature for
23 hrs. The solution was filtered over Celite and the solvent was
removed in vacuo. The residue was redissolved in chloroform (60 mL)
and washed with water (2.times.) and brine (all 20 mL). The organic
layer was dried with MgSO.sub.4, filtrated and the solvent was
removed in vacuo. Purification was achieved using column
chromatography (flash silica) using a gradient of 2% MeOH in
chloroform to 12% MeOH in chloroform. This yielded pure 63 (70 mg,
82 .mu.mol, 56%) as an orange solid.
[0292] .sup.1H-NMR (CDCl.sub.3): .delta.=13.97 (s, 1H, ArOH),
13.22, (s, 1H, ArOH), 8.30 (d, 2H, ArH), 8.03 (d, 1H, ArH), 7.78
(t, 1H, ArH), 7.46 (d, 2H, ArH), 7.39 (m, 3H, ArH), 7.23 (d, 2H,
ArH), 5.50 (d, 1H, OCHO), 5.28 (s, 1H, CCHHCH), 5.17 (d, 1H, NH),
5.04 (s, 2H, ArCH.sub.2O), 4.75 (s, 2H, CH.sub.2OH), 4.54 (s, 1H,
COH), 4.14 (m, 1H, CHCH.sub.3), 4.08 (s, 3H, OCH.sub.3), 3.87 (m,
1H, NHCH), 3.66 (s, 1H, CHOH), 3.27 (d, 1H, ArCHH), 3.02 (s, 1H,
CH.sub.2OH), 3.00 (d, 1H, ArCHH), 2.33 (d, 1H, CCHH), 2.17 (d, 1H,
CCHH), 1.98 (br, 1H, CHOH), 1.88 (m, 1H, NHCHCHH), 1.77 (m, 1H,
NHCHCHH), 1.29 (d, 3H, CHCH.sub.3) ppm. The assignments were
confirmed by 2D (.sup.1H--.sup.1H) correlation spectroscopy
(gCOSY). .sup.13C-NMR (CDCl.sub.3): .delta.=213.8, 187.1, 186.6,
161.0, 156.2, 155.6, 155.3, 155.2, 150.9, 150.3, 145.6, 135.8,
135.4, 135.1, 133.6, 133.5, 129.5, 125.4, 125.3, 121.7, 120.8,
119.9, 118.5, 111.6, 111.4, 100.7, 69.7, 69.6, 67.2, 65.8, 65.5,
56.7, 47.0, 35.6, 34.0, 30.2, 16.8 ppm. ESI-MS: m/z Calc. 858.21;
Obs. [M+Na].sup.+ 881.42.
[0293] In a 25 mL round-bottom flask,
cis-5,6-diamino-trans-cyclooctene* 35 (16.5 mg, 71 .mu.mol, 1.1 eq)
was dissolved in dry THF (4 mL) under an Ar atmosphere. A solution
of 63 (55 mg, 64 mol) in dry THF (4 mL) was added, the flask was
sealed and the mixture was stirred at room temperature for 1 hr.
The solvent was removed in vacuo and the residue was purified using
RP-HPLC (CH.sub.3CN/H.sub.2O with 0.1% formic acid) while
monitoring at .lamda.=253 and 317 nm. The gradient comprised, %
CH.sub.3CN (min): 28 (1-11), 28 to 100 (11-12), 100 (12-13), 100 to
28 (13-14), 28 (14-15) This yielded pure
(E)-cyclooctene-doxorubicin conjugate 64 (19 mg, 22 mol, 35%) as an
orange solid after freeze-drying.
[0294] .sup.1H-NMR (CDCl.sub.3/MeOD-d.sub.4 95:5): .delta.=7.98 (d,
1H, ArH), 7.74 (t, 1H, ArH), 7.35 (d, 1H, ArH), 7.23 (d, 2H, ArH),
6.99 (d, 2H, ArH), 5.75 (m, 1H, CH.dbd.CH), 5.64 (m, 1H,
CH.dbd.CH), 5.43 (s, 1H, OCHO), 5.23 (s, 1H, CCHHCH), 4.97 (d, 1H,
ArCHHO), 4.91 (d, 1H, ArCHHO), 4.71 (s, 2H, CH.sub.2OH), 4.09 (m,
1H, CHCH.sub.3), 4.08 (s, 3H, OCH.sub.3), 3.80 (m, 1H, NHCH), 3.70
(m, 1H, NHCHCHNH.sub.2), 3.56 (s, 1H, CHOH), 3.40 (m, 1H,
CHNH.sub.2), 3.22 (d, 1H, ArCHH), 2.98 (d, 1H, ArCHH), 2.38-1.96
(m, 10H, trans-cyclooctene CH.sub.2, CCHH), 1.77 (m, 2H, NHCHCHH),
1.22 (d, 3H, CHCH.sub.3) ppm. .sup.13C-NMR (MeOD-d.sub.4):
.delta.=213.2, 186.1, 185.9, 160.8, 156.5, 155.7, 155.1, 154.5,
150.6, 135.7, 134.6, 134.2, 134.1, 133.6, 133.1, 132.8, 128.6,
121.2, 119.7, 119.0, 118.7, 110.8, 110.6, 100.8, 78.0, 76.0, 69.7,
68.7, 67.2, 65.4, 64.3, 57.3, 55.9, 55.6, 35.9, 35.7, 35.5, 32.6,
31.6, 29.4, 27.6, 15.9 ppm. ESI-MS: m/z Calc. 859.32; Obs.
[M+H].sup.+ 860.50. FT-IR (ATR): v=3347, 2972, 2940, 2872, 1676,
1617, 1579, 1525, 1503, 1444, 1429, 1413, 1346, 1285, 1261, 1201,
1134, 1069, 1016, 985, 952, 916, 894, 875, 836, 821, 799, 765, 737,
721, 706, 693, 673 cm.sup.-1.
[0295] * estimated batch purity 60%
Synthesis of (E)-cyclooctene-doxorubicin conjugate 68
##STR00054##
[0297] In a 25 mL round-bottom flask,
4-(t-butyldimethylsilyloxymethyl)-2,6-dimethylphenol (84 mg, 0.32
mmol; Y. H. Choe, C. D. Conover, D. Wu, M. Royzen, Y. Gervacio, V.
Borowski, M. Mehlig, R. B. Greenwald, J. Controlled Release 2002,
79, 55-70) and DIPEA (111 .mu.L, 82 mg, 0.63 mmol, 2 eq) were
dissolved in dry THF (0.5 mL) and the mixture was cooled on an ice
bath. 4-Nitrophenyl chloroformate (132 mg, 0.63 mmol, 2 eq) in dry
THF (0.5 mL) was added dropwise and the mixture was stirred at
45.degree. C. for 2 hrs. Since .sup.1H-NMR indicated .eta.=78%,
additional DIPEA (55 .mu.L, 41 mg, 0.31 mmol, 1 eq) and
4-nitrophenyl chloroformate (66 mg, 0.31 mmol, 1 eq) were added and
the mixture was stirred at 45.degree. C. for 30 min. After
filtration of the formed white precipitate (DIPEA.HCl salt) over
Celite, the solvent was removed in vacuo and the residue was
redissolved in EtOAc (140 mL). The organic layer was washed with
water and brine (both 45 mL), dried with MgSO.sub.4, filtrated and
the solvent was removed in vacuo. Purification was achieved using
column chromatography (flash silica) using a gradient of 1:1
chloroform/pentane to chloroform. This yielded pure
2,6-dimethyl-4-(t-butyldimethylsilyloxymethyl)phenyl 4-nitrophenyl
carbonate (65) (75 mg, 0.17 mmol, 55%) as a colorless solid.
[0298] .sup.1H-NMR (CDCl.sub.3): .delta.=8.31 (d, 2H, ArH), 7.48
(d, 2H, ArH), 7.06 (s, 2H, ArH), 4.68 (s, 2H, CH.sub.2), 2.28 (s,
6H, ArCH.sub.3), 0.95 (s, 9H, CCH.sub.3), 0.11 (s, 6H, SiCH.sub.3)
ppm. .sup.13C-NMR (CDCl.sub.3): .delta.=155.5, 150.4, 146.9, 145.6,
139.8, 129.5, 126.6, 125.4, 121.6, 64.3, 26.0, 18.4, 16.2, -5.3
ppm. ESI-MS: m/z Calc. 431.18; Obs. [M+2H-TBDMS-H.sub.2O].sup.+
300.17. FT-IR (ATR): v=2954, 2929, 2885, 2857, 1777, 1616, 1594,
1526, 1491, 1471, 1462, 1444, 1407, 1346, 1323, 1292, 1220, 1177,
1164, 1128, 1100, 1003, 947, 910, 883, 834, 815, 775, 736, 700,
673, 657 cm.sup.-1.
[0299] In a 25 mL round-bottom flask, 65 (166 mg, 0.38 mmol) was
dissolved in ethanol (6 mL) and the solution was cooled on an ice
bath. Conc. HCl in ethanol (1% v/v, 4.5 mL) was added and the
mixture was stirred at room temperature for 75 min. The solvent was
removed in vacuo and the residue was flushed with chloroform. This
yielded pure 2,6-dimethyl-4-(hydroxymethyl)phenyl 4-nitrophenyl
carbonate (66) (136 mg, max. 0.38 mmol, 100%) as a colorless
oil.
[0300] .sup.1H-NMR (CDCl.sub.3): .delta.=8.32 (d, 2H, ArH), 7.47
(d, 2H, ArH), 7.13 (s, 2H, ArH), 4.65 (s, 2H, CH.sub.2), 2.30 (s,
6H, ArCH.sub.3) ppm. .sup.13C-NMR (CDCl.sub.3): .delta.=155.4,
150.4, 147.3, 145.6, 139.3, 130.0, 127.5, 125.4, 121.6, 64.6, 16.1
ppm. ESI-MS: m/z Calc. 317.09; Obs. [M+H-H.sub.2O].sup.+ 300.17.
FT-IR (ATR): v=3555, 3366, 3119, 3086, 2924, 2867, 1772, 1616,
1593, 1523, 1490, 1454, 1380, 1346, 1324, 1311, 1293, 1220, 1176,
1164, 1126, 1056, 1035, 1003, 955, 941, 910, 884, 857, 845, 764,
732, 702, 679, 664 cm.sup.1.
[0301] In a 10 mL round-bottom flask, 66 (51 mg, 0.16 mmol) was
dissolved in dry THF (1 mL) under an Ar atmosphere. After the
addition of phosgene (180 .mu.L of a 1.9 M solution in toluene,
0.34 mmol, 2 eq) the flask was sealed and the mixture was stirred
at room temperature for 15 hrs. The solvent was removed in vacuo
and the resulting oil was flushed with toluene (3.times.) and
chloroform. This yielded the analogous chloroformate as a colorless
oil which was used immediately without further purification.
[0302] .sup.1H-NMR (CDCl.sub.3): .delta.=8.33 (d, 2H, ArH), 7.48
(d, 2H, ArH), 7.16 (s, 2H, ArH), 5.24 (s, 2H, CH.sub.2), 2.31 (s,
6H, CH.sub.3) ppm.
[0303] Subsequently, in a 10 mL round-bottom flask, doxorubicin
hydrochloride (89 mg, 0.15 mmol) was dissolved in dry THF (2 mL)
and a solution of the chloroformate (max. 0.16 mmol, 1.1 eq) in dry
THF (4 mL) was added. After the addition of DIPEA (118 .mu.L, 88
mg, 0.67 mmol, 4.4 eq) the mixture was stirred at room temperature
for 24 hrs. The solution was filtered over Celite and the solvent
was removed in vacuo. The residue was redissolved in chloroform
(120 mL) and washed with water (2.times.) and brine (all 40 mL).
The organic layer was dried with MgSO.sub.4, filtered and the
solvent was removed in vacuo. Purification was achieved using
column chromatography (flash silica) using a gradient of 2% MeOH in
chloroform to 3% MeOH in chloroform. This yielded pure 67 (82 mg,
92 mol, 61%) as an orange solid.
[0304] .sup.1H-NMR (CDCl.sub.3): .delta.=13.98 (s, 1H, ArOH),
13.25, (s, 1H, ArOH), 8.30 (d, 2H, ArH), 8.05 (d, 1H, ArH), 7.79
(t, 1H, ArH), 7.45 (d, 2H, ArH), 7.40 (d, 1H, ArH), 7.06 (s, 2H,
ArH), 5.51 (s, 1H, OCHO), 5.30 (s, 1H, CCHHCH), 5.11 (d, 1H, NH),
4.97 (s, 2H, ArCH.sub.2O), 4.75 (s, 2H, CH.sub.2OH), 4.53 (s, 1H,
COH), 4.14 (m, 1H, CHCH.sub.3), 4.08 (s, 3H, OCH.sub.3), 3.87 (m,
1H, NHCH), 3.67 (d, 1H, CHOH), 3.28 (d, 1H, ArCHH), 3.03 (d, 1H,
ArCHH), 2.98 (t, 1H, CH.sub.2OH), 2.33 (d, 1H, CCHH), 2.25 (s, 6H,
ArCH.sub.3), 2.17 (d, 1H, CCHH), 1.87 (m, 2H, NHCHCHH, CHOH), 1.77
(m, 1H, NHCHCHH), 1.29 (d, 3H, CHCH.sub.3) ppm. .sup.13C-NMR
(CDCl.sub.3): .delta.=213.8, 186.9, 186.5, 161.0, 156.1, 155.5,
155.4, 155.3, 150.2, 147.7, 145.6, 135.8, 135.4, 134.8, 133.6,
133.5, 130.1, 128.8, 125.4, 121.6, 120.7, 119.8, 118.5, 111.5,
111.3, 100.7, 77.2, 76.6, 69.7, 69.5, 67.3, 66.0, 65.5, 56.6, 47.0,
35.6, 33.9, 30.1, 16.8, 16.0 ppm. ESI-MS: m/z Calc. 886.24; Obs.
[M+Na].sup.+ 909.33. FT-IR (ATR): v=3492, 3431, 3058, 2937, 1777,
1719, 1616, 1579, 1525, 1491, 1444, 1429, 1412, 1381, 1347, 1325,
1283, 1225, 1209, 1183, 1132, 1071, 1015, 982, 948, 917, 879, 858,
847, 820, 791, 765, 734, 702, 681 cm.sup.-1.
[0305] In a 25 mL round-bottom flask,
cis-5,6-diamino-trans-cyclooctene* (35, 14.6 mg, 104 .mu.mol, 1.1
eq) was dissolved in dry THF (4 mL) under an Ar atmosphere. A
solution of 67 (82 mg, 92 .mu.mol) in dry THF (2 mL) was added, the
flask was sealed and the mixture was stirred at room temperature
for 90 min. Since .sup.1H-NMR indicated .eta.=75%, additional
5,6-diamino-trans-cyclooctene (4.4 mg, 31 mol, 0.3 eq) was added
and the mixture was stirred at room temperature for 1 hr
(eventually .eta..apprxeq.90% based on .sup.1H-NMR). The solvent
was removed in vacuo and part (22 mg) of the residue (100 mg) was
purified using RP-HPLC (CH.sub.3CN/H.sub.2O with 0.1% formic acid)
while monitoring at .lamda.=253 and 317 nm. The gradient comprised,
% CH.sub.3CN (min): 25 to 45 (1-11), 45 to 100 (11-12), 100
(12-13), 100 to 25 (13-14), 25 (14-15) This yielded pure
cis-(E)-cyclooctene-doxorubicin conjugate 68 (10 mg, 11 .mu.mol,
55%) as an orange solid after freeze-drying.
[0306] .sup.1H-NMR (CDCl.sub.3/MeOD-d.sub.4 95:5): .delta.=8.02 (d,
1H, ArH), 7.80 (t, 1H, ArH), 7.41 (d, 1H, ArH), 6.98 (s, 2H, ArH),
5.82 (m, 1H, CH.dbd.CH), 5.66 (m, 1H, CH.dbd.CH), 5.49 (s, 1H,
OCHO), 5.32 (s, 1H, CCHHCH), 4.95 (d, 1H, ArCHHO), 4.88 (d, 1H,
ArCHHO), 4.77 (s, 2H, CH.sub.2OH), 4.14 (m, 1H, CHCH.sub.3), 4.08
(s, 3H, OCH.sub.3), 3.84 (m, 1H, NHCH), 3.74 (m, 1H,
NHCHCHNH.sub.2), 3.60 (s, 1H, CHOH), 3.40 (m, 1H, CHNH.sub.2), 3.26
(d, 1H, ArCHH), 3.02 (d, 1H, ArCHH), 2.38-1.96 (m, 10H,
trans-cyclooctene CH.sub.2, CCHH), 2.11 (s, 6H, ArCH.sub.3), 1.81
(m, 2H, NHCHCHH), 1.28 (d, 3H, CHCH.sub.3) ppm. .sup.13C-NMR
(CDCl.sub.3/MeOD-d.sub.4 9:1): .delta.=213.9, 187.1, 186.7, 161.0,
155.8, 155.3, 153.9, 147.7, 135.8, 135.4, 133.8, 133.6, 133.2,
132.8, 130.9, 128.3, 120.8, 119.7, 118.5, 111.5, 111.3, 100.6,
76.4, 69.3, 69.0, 67.9, 67.4, 66.2, 65.2, 57.8, 56.6, 55.9, 46.8,
36.5, 36.1, 35.7, 33.7, 32.6, 29.9, 28.0, 25.5, 16.6, 15.9 ppm.
ESI-MS: m/z Calc. 887.35; Obs. [M+H].sup.+ 888.58. FT-IR (ATR):
v=3432, 2940, 1678, 1617, 1582, 1515, 1412, 1350, 1285, 1236, 1201,
1140, 1075, 1017, 983, 949, 912, 873, 836, 820, 799, 765, 729, 672
cm.sup.-1.
[0307] * estimated batch purity 98%
Example 3
Stability and Reactivity of Tetrazine Activators
Hydrolytic Stability Tests of Tetrazines
[0308] 10 .mu.L of a solution of the specific tetrazine in DMSO (25
mM) was diluted with PBS buffer (3 mL) (or a mixture of PBS and
acetonitrile in case the aqueous solubility was too low). This
solution was filtered and, the decrease of the absorption band at
525 nm was monitored using UV spectroscopy. The rate of hydrolysis
and half-life time was determined from these data.
Reactivity of Tetrazines Towards trans-cyclooct-4-ene-1-ol (Axial
Isomer)
[0309] A competition experiment was performed to determine the
reactivity ratio of a specific tetrazine and
3-(5-acetamido-2-pyridyl)-6-(2-pyridyl)-1,2,4,5-tetrazine (that was
chosen as the reference tetrazine), in the inverse-electron demand
Diels-Alder reaction with trans-cyclooct-4-ene-1-ol ("minor" isomer
with OH in axial position, see: Whitham et al. J. Chem. Soc. (C),
1971, 883-896)).
[0310] To acetonitrile (0.100 mL) was added 5 .mu.L of a solution
of the specific tetrazine in DMSO (25 mM) and 5 .mu.L of a solution
of the reference tetrazine in DMSO (25 mM). This mixture was
diluted with water (0.9 mL), and the absolute amounts of both
tetrazines were determined by HPLC-MS/PDA analysis. Subsequently, a
solution of trans-cyclooct-4-ene-1-ol (axial isomer) in DMSO (25
.mu.L 2.5 mM) was slowly added, and the mixture was stirred for 5
min. Again, the absolute amounts of both tetrazines were determined
by HPLC-MS/PDA analysis, and conversions for both tetrazines was
calculated. From these conversions, the reactivity ratio
(R=k.sub.2,TCO/k.sub.2,Ref) of both tetrazines was calculated using
the mathematical procedure from Ingold and Shaw (J. Chem. Soc.,
1927, 2918-2926).
[0311] The table below demonstrates how the reactivity and
stability profile of tetrazines can be tailored to certain
specifications by varying the substituents.
TABLE-US-00001 stability in PBS at 20.degree. C. Reactivity ratio
tetrazine t.sub.1/2 (hrs) (R = k.sub.2,TZ/k.sub.2,Ref) ##STR00055##
44 1.17 2 ##STR00056## 340 0.4 ##STR00057## 80 1 5 ##STR00058## 24
1.6 ##STR00059## >300* <0.01* ##STR00060## 115 1.07
##STR00061## 3.6* 5.3* ##STR00062## 35* 1.84* ##STR00063## 3.2 2.7
##STR00064## 117 0.95 ##STR00065## 0.68 1.5 ##STR00066## >150
0.19 ##STR00067## 2.4 0.83 ##STR00068## >300* <0.01*
##STR00069## 183 0.77 ##STR00070## >300* <0.01* ##STR00071##
>300* <0.01* ##STR00072## 4 1.76 ##STR00073## >300*
<0.01* ##STR00074## >300* <0.01* ##STR00075## 2.7 3.06
##STR00076## 10.3 2.8 ##STR00077## 230 0.25 7 ##STR00078## 300 0.18
##STR00079## 0.42 2 ##STR00080## >300* <0.01* ##STR00081##
n.d. 1.2 ##STR00082## >300* <0 .01* 9 ##STR00083## >300*
<0.01* 11 ##STR00084## >300* <0.01* 13 ##STR00085## 16
n.d. 20 *This value was determined in a 50:50 mixture of PBS and
acetonitrile.
Example 4
Stability and Reactivity of Trans-Cyclooctene Model Prodrugs and
Prodrugs
Stability
[0312] 10 .mu.L of a solution of the specific trans-cyclooctene
derivative in dioxane (25 mM) was diluted with PBS buffer (3 mL),
and this solution was stored at 20.degree. C. in the dark.
[0313] The fate of the TCO compound was monitored by HPLC-MS
analysis, and an estimation of the half-life time was made based on
the release of the model prodrug.
##STR00086##
Reactivity of Trans-Cyclooctene Derivatives Towards
bis(2-pyridyl)-1,2,4,5-tetrazine: Second-Order Rate Constant
Determination
[0314] The kinetics of the inverse-electron demand Diels-Alder
reaction of a trans-cyclooctene derivative with
3-(5-acetamido-2-pyridyl)-6-(2-pyridyl)-1,2,4,5-tetrazine,
performed in acetonitrile at 20.degree. C., was determined using
UV-visible spectroscopy. A cuvette was filled with acetonitrile (3
mL) and equilibrated at 20.degree. C.
3-(5-Acetamido-2-pyridyl)-6-(2-pyridyl)-1,2,4,5-tetrazine
(2.50.times.10.sup.-7 mol) was added, followed by the
trans-cyclooctene derivative (2.50.times.10.sup.-7 mol). The decay
of the absorption at .lamda.=540 nm was monitored, and from this
curve the second-order rate constant, k.sub.2, was determined
assuming second order rate kinetics.
Reactivity of Trans-Cyclooctene Derivatives Towards
bis(2-pyridyl)-1,2,4,5-tetrazine: Competition Experiment
[0315] A competition experiment was performed to determine the
reactivity ratio of a specific trans-cyclooctene derivative and
trans-cyclooct-4-ene-1-ol (axial isomer) (that was chosen as the
reference), in the inverse-electron demand Diels-Alder reaction
with bis(2-pyridyl)-1,2,4,5-tetrazine.
[0316] To acetonitrile (0.05 mL) was added a solution of the
specific trans-cyclooctene derivative in dioxane (5 .mu.L 25 mM;
1.25.times.10.sup.-7 mol) and a solution of the reference
trans-cyclooctene in dioxane (5 .mu.L 25 mM; 1.25.times.10.sup.-7
mol). This mixture was diluted with water (0.45 mL). Subsequently,
a solution of bis(2-pyridyl)-1,2,4,5-tetrazine
(6.25.times.10.sup.-8 mol) in a mixture of acetonitrile (0.05 mL)
and water (0.45 mL) was slowly added while stirring vigorously.
After addition, the mixture was stirred for an additional 5 min.
The conversion of both trans-cyclooctene derivatives was determined
by HPLC-MS/PDA analysis, and from these conversions, the reactivity
ratio (R=k.sub.2,TCO/k.sub.2,Ref) of the specific trans-cyclooctene
derivative was calculated using the mathematical procedure from
Ingold and Shaw (J. Chem. Soc., 1927, 2918-2926).
TABLE-US-00002 reactivity ratio** (R = stability in PBS rate
contant* k.sub.2,TCO/ trans-cyclooctene derivative at 20.degree. C.
t.sub.1/2 k.sub.2 (M.sup.-1 s.sup.-1) k.sub.2,Ref) ##STR00087##
>3 days 577 1 axial isomer ##STR00088## >10 days 300 37
##STR00089## 27 hrs 240 36 ##STR00090## 24 hrs@20.degree. C. 3.6
hrs@37.degree. C. 64 ##STR00091## 26 days@20.degree. C. 6.2
days@37.degree. C. 68 ##STR00092## >>20 days 47 61
##STR00093## 5 days 44.4 56 ##STR00094## 8 days 39 ##STR00095## 21
hrs 41 ##STR00096## >>20 days 43 ##STR00097## >>20 days
45 ##STR00098## >>20 days 106 47 major ##STR00099##
>>20 days 56 47 minor *determined by UV-visible spectroscopy
in acetonitrile at 20.degree. C. **determined by a competition
experiment
Example 5
Activation of Model Prodrugs
3,6-Bis(2-pyridinyl)-1,2,4,5-tetrazine (2) and
phenyl((E)-8-aminocyclooct-4-en-1-yl)carbamate (36)
##STR00100##
[0318] 3,6-Bis(2-pyridinyl)-1,2,4,5-tetrazine (2,
2.50.times.10.sup.7 mol) was dissolved in PBS buffer (1 mL). Next,
phenyl((E)-8-aminocyclooct-4-en-1-yl)carbamate (36,
2.50.times.10.sup.-7 mol) was added. The solution was stirred at
20.degree. C., and the reaction progress was monitored by HPLC-MS
analysis, demonstrating nearly instantaneous formation of the
rDA-adduct, followed by the formation of the cyclic urea with
m/z=+375 Da (M+H.sup.+), and release of phenol: .lamda..sub.max=270
nm. The half-life time of this release was 40 min.
3,6-Bis(2-pyridinyl)-1,2,4,5-tetrazine (2) and
phenyl((E)-2-aminocyclooct-3-en-1-yl)carbamate (56)
##STR00101##
[0320] 3,6-Bis(2-pyridinyl)-1,2,4,5-tetrazine (2,
2.50.times.10.sup.-7 mol) was dissolved in PBS buffer (1 mL). Next,
phenyl((E)-2-aminocyclooct-3-en-1-yl)carbamate (56,
2.50.times.10.sup.-7 mol) was added. The solution was stirred at
20.degree. C., and the reaction progress was monitored by HPLC-MS
analysis, proving almost instantaneous formation of the rDA-adduct,
followed by the formation of the cyclic urea with m/z=+375 Da
(M+H.sup.+), and release of phenol: .lamda..sub.max=270 nm. The
half-life time of this release was 40 min.
Example 6
Activation of Doxorubicin Prodrugs
3,6-Bis(2-pyridinyl)-1,2,4,5-tetrazine (2) and
(E)-cyclooctene-doxorubicin conjugate (64)
##STR00102##
[0322] 3,6-Bis(2-pyridinyl)-1,2,4,5-tetrazine (2,
1.18.times.10.sup.-5 g; 5.00.times.10.sup.-8 mol) was dissolved in
PBS buffer (1 mL). Next, cis-(E)-cyclooctene-doxorubicin conjugate
(64, 2.67.times.10.sup.-5 g; 2.50.times.10.sup.-8 mol) was added.
The solution was stirred at 20.degree. C., and the reaction
progress was monitored by HPLC-MS analysis, proving the formation
of the cyclic urea with m/z=+375 Da (M+H.sup.+), and release of
doxorubicin: m/z=+544 Da (M+H.sup.+) and .lamda..sub.max=478 nm.
The half-life time of this release was 2 hrs.
Performing this reaction at 37.degree. C. yielded a doxorubicin
release half-life time of 40 min.
3-(5-Acetamido-2-pyridyl)-6-(2-pyridyl)-1,2,4,5-tetrazine (5) and
(E)-cyclooctene-doxorubicin conjugate (64)
##STR00103##
[0324] Same procedure as previous reaction.
[0325] After 1 hr at 20.degree. C., 30% doxorubicin was
released.
3-(5-Butyramido-2-pyridyl)-6-(2-pyrimidyl)-1,2,4,5-tetrazine and
(E)-cyclooctene-doxorubicin conjugate (64)
##STR00104##
[0327] Same procedure as previous reaction.
[0328] After 1 hr at 20.degree. C., 20% doxorubicin was
released.
4-(1,2,4,5-Tetrazin-3-yl)phenylmethanamine and
(E)-cyclooctene-doxorubicin conjugate (64)
##STR00105##
[0330] Same procedure as previous reaction.
[0331] After 1 hr at 20.degree. C., 50% doxorubicin was
released.
3-Methyl-6-(2-pyridyl)-1,2,4,5-tetrazine (7) and
(E)-cyclooctene-doxorubicin conjugate (64)
##STR00106##
[0333] Same procedure as previous reaction.
[0334] After 1 hr at 20.degree. C., 52% doxorubicin was
released.
3,6-Diphenyl-1,2,4,5-tetrazine and (E)-cyclooctene-doxorubicin
conjugate (64)
##STR00107##
[0336] Same procedure as previous reaction.
[0337] After 1 hr at 20.degree. C., 48% doxorubicin was
released.
3,6-Bis(2-pyridinyl)-1,2,4,5-tetrazine (2) and
(E)-cyclooctene-doxorubicin conjugate (68)
##STR00108##
[0339] 3,6-Bis(2-pyridinyl)-1,2,4,5-tetrazine (2,
1.18.times.10.sup.-5 g; 5.00.times.10.sup.-8 mol) was dissolved in
PBS buffer (1 mL). Next, (E)-cyclooctene-doxorubicin conjugate (68,
2.67.times.10.sup.-5 g; 2.50.times.10.sup.-8 mol) was added. The
solution was stirred at 20.degree. C., and the reaction progress
was monitored by HPLC-MS analysis, proving the formation of the
cyclic urea with m/z=+375 Da (M+H.sup.+), and release of
doxorubicin: m/z=+544 Da (M+H.sup.+) and .lamda..sub.max=478 nm.
The half-life time of this release was 4 days.
[0340] Performing this reaction at 37.degree. C. yielded a
half-life time of 16 hrs.
Example 7
Cell Proliferation Assay with Doxorubicin Prodrug 64 and Tetrazine
29
[0341] A431 squamous carcinoma cells were maintained in a
humidified CO.sub.2 (5%) incubator at 37.degree. C. in DMEM
(Invitrogen) supplemented with 10% heat-inactivated fetal bovine
serum and 0.05% glutamax (Invitrogen) in the presence of penicillin
and streptomycin. The cells were plated in 96-well plates (Nunc) at
a 2000 cells/well density 24 hrs prior to the experiment.
Doxorubicin (Dox) and the prodrug 64 (1 mM in DMSO) were serially
diluted in pre-warmed culture medium immediately before the
experiment and added to the wells (200 .mu.L final volume; t=0).
The prodrug was either added alone or in combination with 10 M
tetrazine 29. After 6 hrs incubation at 37.degree. C. the medium
was gently aspirated, 200 .mu.L fresh culture medium was added to
each well and the cells were incubated for 66 hrs more. In a
parallel experiment, a solution of tetrazine 29 (2 mM in PBS) was
serially diluted (from 1 mM to 1 nM) in pre-warmed culture medium
and added to A431 cells in a 96-well plate, which was incubated at
at 37.degree. C. for 72 hrs. At the end of each experiment, the
cell proliferation was assessed by an MTT assay. Briefly,
methylthiazolyldiphenyltetrazolium bromide (MTT) was dissolved in
PBS at 5 mg/ml, filtered through 0.22 m and 25 .mu.l was added to
each well. After 120 min incubation at 37.degree. C., the medium
was gently aspirated. The formed formazan crystals were dissolved
in 100 .mu.l DMSO and the absorbance was measured with a plate
reader (BMG Labtech) at 560 nm. IC.sub.50 values (.+-.standard
error; see table) were derived from the normalized cell growth
curves (see figure) generated with GraphPad Prism (version 5.01).
The cell proliferation assays shows a significant toxicity increase
when A431 cells are exposed to a combination of the prodrug 64 and
tetrazine 29 (IC.sub.50=49.+-.4 nM) compared to the prodrug alone
(IC.sub.50=128+17 nM) or the tetrazine alone (IC.sub.50>100
.mu.M). This confirms that doxorubicin is released following the
retro Diels-Alder reaction between the trans-cyclooctene of the
prodrug and the tetrazine Activator.
IC.sub.50 Values for Doxorubicin (Dox) and Prodrug 64 with and
without Activation by Tetrazine 29 (10 .mu.M) Determined in A431
Cell Line.
TABLE-US-00003 Compound IC.sub.50 (.mu.M) Dox 0.038 .+-.
0.003.sup.a Prodrug 64 0.128 .+-. 0.017.sup.a Prodrug 64 +
tetrazine 29 (10 .mu.M) 0.049 .+-. 0.004.sup.a Tetrazine 29
>100.sup.b .sup.a6 h incubation at 37.degree. C. followed by
medium replacement; .sup.b72 h incubation at 37.degree. C.
Example 8
Structures of Exemplary L.sup.D Moieties
##STR00109##
[0343] The linkers L.sup.D are so-called self-immolative linkers,
meaning that upon reaction of the trigger with the activator the
linker will degrade via intramolecular reactions thereby releasing
the drug D.sup.D. Some of the above also contain a S.sup.P.
Example 9
Structures of Exemplary S.sup.P Moieties
##STR00110##
[0345] =rest of attached Prodrug
[0346] Note that the maleimide, active ester and bromo acetamide
groups are active groups to which targeting moieties T.sup.T and
masking moieties M.sup.M, optionally via further spacers S.sup.P,
can be coupled. Maleimides and bromo acetamide groups typically
react with thiols, while active esters are typically suitable for
coupling to primary or secondary amines.
Example 10
Structures of TCO Triggers with Depicted Exemplary L.sup.D
Moieties
[0347] The T.sup.T featured in this example can optionally be
replaced by M.sup.M.
##STR00111##
Example 11
Structures of TCO Triggers with Depicted Exemplary L.sup.D and/or
S.sup.P Moieties
[0348] Trigger conjugated to T.sup.T via amine or thiol of T.sup.T.
The T.sup.T featured in this example can optionally be replaced by
M.sup.M.
##STR00112## ##STR00113##
Example 12
Structures of Antibody-Drug Conjugates
[0349] Auristatin E (MMAE) toxin is attached via a self immolative
linker L.sup.D to a TCO trigger and via S.sup.P to a targeting
antibody or fragment (conjugated through cysteine or lysine
residue). Ab=antibody or antibody fragment; q=Ab modification # and
is typically between 1 and 10.
##STR00114## ##STR00115##
Example 13
Structures of Antibody-Drug Conjugates
[0350] Maytansine toxin is attached via a self immolative linker
L.sup.D to a TCO trigger and via S.sup.P to a targeting antibody or
fragment (conjugated through cysteine or lysine residue).
Ab=antibody or antibody fragment; q=Ab modification # and is
typically between 1 and 10.
##STR00116## ##STR00117## ##STR00118##
Example 14
Structures of Trigger-Drug Constructs that can be Conjugated to a
Targeting Agent T.sup.T Eg Via an Amine or Thiol Moiety
[0351] Auristatin E (MMAE) toxin is attached via a self immolative
linker L.sup.D to a TCO trigger and via S.sup.P to a reactive
moiety for T.sup.T conjugation.
##STR00119## ##STR00120##
Example 15
Structures of Trigger-Drug Constructs that can be Conjugated to a
Targeting Agent T.sup.T Eg Via an Amine or Thiol Moiety
[0352] Maytansine toxin is attached via a self immolative linker
L.sup.D to a TCO trigger and via S.sup.P to a reactive moiety for
T.sup.T conjugation.
##STR00121## ##STR00122## ##STR00123##
Example 16
Activation of Tumor Bound CC49-Auristatin E Conjugate
[0353] CC49 as mAb or mAb fragment binds the non-internalizing
pan-solid tumor marker TAG72. After Prodrug administration, tumor
binding and clearance from blood, the Activator is injected. The
reaction of the Activator with the TCO trigger in the Prodrug
results in release of Auristatin E from CC49 (antibody, or antibody
fragment), allowing it to penetrate the cancer cell inside which it
has its anticancer action.
##STR00124## ##STR00125##
Example 17
Activation of Tumor-Bound T-Cell Engaging Triabody
[0354] The triabody comprises a tumor-binding moiety, a CD3 T-cell
engaging moiety, and a CD28 T-cell co-stimulatory moiety. As the
CD3 and CD28 combined in one molecule will result in unacceptable
toxic effect off target, the anti-CD28 domain is blocked by a
Masking Moiety M.sup.M, a peptide resembling the CD28 binding
domain and which has affinity for the anti-CD28 moiety. This
peptide is linked through a further peptide or a PEG chain S.sup.P
to the TCO trigger which is itself conjugated to a site
specifically engineered cysteine. After Prodrug administration,
tumor binding and clearance from blood, the Activator is injected.
The reaction of the Activator with the TCO trigger in the Prodrug
results in release of the Masking Moiety from the anti-CD28 domain
enabling CD28 co-stimulation of T-cells, boosting the T-cell
mediated anticancer effect, while avoiding off target toxicity.
##STR00126##
* * * * *