U.S. patent application number 13/923544 was filed with the patent office on 2014-06-05 for conjugates of pyrrolo[1,4]benzodiazepine dimers as anticancer agents.
The applicant listed for this patent is Sanofi. Invention is credited to Alain Commercon, Laurence Gauzy-Lazo.
Application Number | 20140155590 13/923544 |
Document ID | / |
Family ID | 41720559 |
Filed Date | 2014-06-05 |
United States Patent
Application |
20140155590 |
Kind Code |
A1 |
Commercon; Alain ; et
al. |
June 5, 2014 |
Conjugates of Pyrrolo[1,4]Benzodiazepine Dimers As Anticancer
Agents
Abstract
The present invention relates to pyrrolo[1,4]benzodiazepine
(PBD) dimer conjugates, to the compositions comprising them and to
their therapeutic application, in particular as anticancer agents.
The invention also relates to the process for the preparation of
the conjugates, to their application as anticancer agents and to
the dimers themselves.
Inventors: |
Commercon; Alain;
(Vitry-Sur-Seine, FR) ; Gauzy-Lazo; Laurence;
(Paris, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sanofi |
Paris |
|
FR |
|
|
Family ID: |
41720559 |
Appl. No.: |
13/923544 |
Filed: |
June 21, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13404872 |
Feb 24, 2012 |
8481042 |
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13923544 |
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PCT/FR2010/051709 |
Aug 12, 2010 |
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13404872 |
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Current U.S.
Class: |
540/496 |
Current CPC
Class: |
A61P 35/00 20180101;
C07D 487/04 20130101; C07D 519/00 20130101 |
Class at
Publication: |
540/496 |
International
Class: |
C07D 487/04 20060101
C07D487/04 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 25, 2009 |
FR |
0904043 |
Sep 11, 2009 |
FR |
0904368 |
Claims
1.-9. (canceled)
10. A process for the preparation of a conjugate, said process
comprising: (i) bringing into contact and allowing to react an
optionally buffered aqueous solution of a binding agent and a
solution of a compound according to formula (I): ##STR00103##
wherein: represents a single bond or a double bond; R.sub.1 and
R.sub.2, which are identical or different, represent, independently
of one another: H, Hal or a (C.sub.1-C.sub.6)alkyl group optionally
substituted by one or more substituent(s) chosen from: Hal, CN,
NRR', CF.sub.3, OR, an aryl or heteroaryl group, or S(O).sub.qR
with q=0, 1 or 2; or R.sub.1 and R.sub.2 together form a double
bond .dbd.CH.sub.2 or .dbd.CH--CH.sub.3; R.sub.1' and R.sub.2',
which are identical or different, represent, independently of one
another: H, Hal or a (C.sub.1-C.sub.6)alkyl group optionally
substituted by one or more substituent(s) chosen from: Hal, CN,
NRR', CF.sub.3, OR, an aryl or heteroaryl group, or S(O).sub.qR
with q=0, 1 or 2; or R.sub.1' and R.sub.2' together form a double
bond .dbd.CH.sub.2 or .dbd.CH--CH.sub.3; Y and Y', which are
identical or different, represent, independently of one another, H
or OR; M represents CH or N; ALK and ALK', which are identical or
different, represent, independently of one another, a
(C.sub.1-C.sub.6)alkylene group; R and R' represent, independently
of one another, H or a (C.sub.1-C.sub.6)alkyl or aryl group
optionally substituted by one or more substituent(s) chosen from:
Hal, CN, NRR', CF.sub.3, or an aryl or heteroaryl group; L
represents an -L.sub.1-L.sub.2- group, wherein: L.sub.1 is attached
to the aromatic ring comprising M via the ALK or OALK group and
represents one of the following groups: -ALK-S--; ##STR00104##
--O-ALK-NR.sub.3-ALK-S--; ##STR00105## and L.sub.2 represents the
--CH.sub.2C(.dbd.O)--NR.sub.3--(CH.sub.2CH.sub.2O).sub.i-ALK- group
attached to L.sub.1 via --CH.sub.2C(.dbd.O)--; or L represents the
--O-ALK-NR.sub.3-ALK-S--(CH.sub.2CH.sub.2O).sub.i-ALK- group
attached to the aromatic ring comprising M via the OALK group;
R.sub.3 represents H or a (C.sub.1-C.sub.6)alkyl group; i
represents an integer ranging from 1 to 40, rather from 1 to 20,
preferably from 1 to 10; and Z.sub.b represents a single bond,
--O-- or --NH-- and R.sub.b represents H or a
(C.sub.1-C.sub.6)alkyl, (C.sub.3-C.sub.7)cycloalkyl, aryl,
heteroaryl or (C.sub.3-C.sub.7)heterocycloalkyl group; or Z.sub.b
represents a single bond and R.sub.b represents Hal; provided that
when represents a single bond, then: represents a single bond; U
and U' each is H; and W and W', which are identical or different,
each represents, independently of one another, --OH, --OR, --OCOR,
--COOR, --OCOOR, --OCONRR', a cyclic carbamate such that N10 and
C11 are included in a ring, --NRCONRR', --OCSNHR, a cyclic
thiocarbamate such that N10 and C11 are included in a ring, --SH,
--SR, --SOR, --SOOR, --SO.sub.3.sup.-, --NRSOOR', --NRR', a cyclic
amine such that N10 and C11 are included in a ring, --NROR',
--NRCOR', --N.sub.3, --CN, Hal or a trialkylphosphonium or
triarylphosphonium group; and (ii) optionally separating the
conjugate formed in stage (i) from the compound of formula (I)
and/or from the binding agent not having reacted and/or from the
aggregates which might be formed.
11. The process according to claim 10, in which the
--C(.dbd.O)Z.sub.bR.sub.b group of said compound of formula (I) is
reactive with regard to the chemical groups present on the binding
agent, in particular with regard to the amino groups present on an
antibody, so as to ensure the attachment of the compound of formula
(I) to the binding agent by formation of a covalent bond.
12. The process according to claim 10, in which step (ii)
comprises: separating the conjugate which has formed in stage (i)
from the unreacted binding agent and from the aggregates possibly
present in the solution; or separating the conjugate of stage (i)
only from the unreacted compound of formula (I) and from the
aggregates which might be formed and leaving in the solution the
binding agent which might not have reacted.
13. The process according to claim 10, in which the binding agent
is a ligand, a protein, an antibody, a protein or antibody
fragment, a peptide, an oligonucleotide or an oligosaccharide.
14. The process according to claim 10, in which the reaction takes
place at a temperature of between 20 and 40.degree. C. and/or the
duration of the reaction varies between 1 and 24 h.
15. The process according to claim 10, in which, after stage (i) or
(ii), the solution of the conjugate is subjected to a stage (iii)
of ultrafiltration and/or of diafiltration.
16.-38. (canceled)
39. The process according to claim 10, wherein the compound of
formula (I) is according to formula: ##STR00106##
40. The process according to claim 10, wherein the compound of
formula (I) is according to formula (IA) or (IB): ##STR00107##
41. The process according to claim 10, wherein each of Y and Y'
represent, independently, a (C.sub.1-C.sub.4)alkoxy group.
42. The process according to claim 41, wherein each of Y and Y'
represent a methoxy group.
43. The process according to claim 10, wherein L is:
-ALK-S--CH.sub.2C(.dbd.O)--NH--(CH.sub.2CH.sub.2O).sub.i--CH.sub.2CH.sub.-
2--; ##STR00108##
--O-ALK-NR.sub.3-ALK-S--CH.sub.2C(.dbd.O)--NH--(CH.sub.2CH.sub.2O).sub.i--
-CH.sub.2CH.sub.2; ##STR00109## or
--O-ALK-NR.sub.3-ALK-S--(CH.sub.2CH.sub.2O).sub.i--CH.sub.2CH.sub.2.
44. The process according to claim 10, wherein L is:
##STR00110##
45. The process according to claim 10, wherein L is: ##STR00111##
##STR00112##
46. The process according to claim 10, wherein --COZ.sub.bR.sub.b
represents: --COOH; --COO(C.sub.1-C.sub.6)alkyl, --COOCH.sub.3,
--COOCH.sub.2CH.dbd.CH.sub.2; ##STR00113## in which IG represents
one or more inductive group,
47. The process according to claim 46, in which IG represents:
##STR00114##
48. The process according to claim 10, wherein the compound of
formula (I) is ##STR00115## ##STR00116##
Description
[0001] The present invention relates to pyrrolo[1,4]benzodiazepine
(PBD) dimer conjugates, to the compositions comprising them and to
their therapeutic application, in particular as anticancer agents.
The invention also relates to the process for the preparation of
the conjugates, to their application as anticancer agents and to
the dimers themselves.
TECHNICAL FIELD
[0002] Pyrrolo[1,4]benzodiazepine dimers are anticancer agents
which act by covalently bonding to the DNA of the cells. These
derivatives have been described in Applications WO 00/12508 and WO
2005/085260, and in the following publications: Eur. J. Med. Chem.,
2005, 40, 641-654; Tetrahedron Letters, 1988, 29(40),
5105-5108.
[0003] The chemistry of conjugates has been known for many years
and has been applied to several families of cytotoxic agents, such
as, for example, the maytansinoids (WO 04103272), the taxans (WO
06061258), the leptomycins (WO 07144709), or CC-1065 and its
analogues (WO 2007102069); see also, with regard to the conjugates,
Monneret C. et al., Bulletin du Cancer, 2000, 87(11), 829-38;
Ricart A. D. et al., Nature Clinical Practice Oncology, 2007, 4,
245-255; Singh R. and Rickson H. K., Therapeutic Antibodies:
Methods and Protocols, 2009, 525, 445-467.
PRIOR ART
[0004] Pyrrolo[1,4]benzodiazepine dimer conjugates have already
been described in Applications WO 07085930 or WO 2009/016516. The
dimers used have more particularly the formulae:
##STR00001##
in which T can represent an aryl or heteroaryl group substituted by
G-D--(Z).sub.p--SZ.sub.a or
G-D--(Z).sub.p--C(.dbd.O)Z.sub.bR.sub.b. G represents a single or
double bond or else --O--, --S-- or --NR--. D represents a single
bond or else one of the following groups: -.alpha.-, -.alpha.-NR--,
-.alpha.-NR--F--, -.alpha.-O--, -.alpha.-O--F--, -.alpha.-NR--CO--,
-.alpha.-, NR--CO--F, -.alpha.-CO--, --CO-.alpha.-, -.alpha.-CO--F,
-.alpha.-S--, -.alpha.-S--F--, -.alpha.-NR--CS--,
-.alpha.-NR--CS--F--, for which E and F are chosen from
--(OCH.sub.2CH.sub.2).sub.ialkyl(OCH.sub.2CH.sub.2).sub.i--,
-alkyl(OCH.sub.2CH.sub.2).sub.i-alkyl-, CH.sub.2CH.sub.2).sub.i--,
--(OCH.sub.2CH.sub.2).sub.icycloalkyl(OCH.sub.2CH.sub.2).sub.j--,
--(OCH.sub.2CH.sub.2).sub.iheterocyclyl(OCH.sub.2CH.sub.2).sub.i--,
--(OCH.sub.2CH.sub.2).sub.iaryl(OCH.sub.2CH.sub.2).sub.j--,
--(OCH.sub.2CH.sub.2).sub.iheteroaryl(OCH.sub.2CH.sub.2).sub.j--,
-alkyl-(OCH.sub.2CH.sub.2).sub.ialkyl(OCH.sub.2CH.sub.2).sub.j--,
-alkyl-(OCH.sub.2CH.sub.2).sub.i--,
-alkyl-(OCH.sub.2CH.sub.2).sub.icycloalkyl(OCH.sub.2CH.sub.2).sub.j,
-alkyl(OCH.sub.2CH.sub.2).sub.iheterocyclyl(OCH.sub.2CH.sub.2).sub.j,
-alkyl-(OCH.sub.2CH.sub.2).sub.iaryl(OCH.sub.2CH.sub.2).sub.j--,
-alkyl(OCH.sub.2CH.sub.2).sub.iheteroaryl(OCH.sub.2CH.sub.2).sub.j--,
-cycloalkyl-alkyl-, -alkyl-cycloalkyl-, -heterocyclyl-alkyl-,
-alkyl-heterocyclyl-, -alkyl-aryl-, -aryl-alkyl-,
-alkyl-heteroaryl-, -heteroaryl-alkyl-. i and j represent integers
ranging from 0 to 2000. Z represents an alkyl group and p is an
integer having the value 0 or 1.
[0005] The group
L.sub.2=--CH.sub.2C(.dbd.O)NR.sub.3--(CH.sub.2CH.sub.2O).sub.i-ALK-
which characterizes some of the compounds of the present invention
comprises the amide unit (--CONR.sub.3--) and can correspond in WO
07085930 or in WO 2009/016516 only to the unit -.alpha.-CONR--F--
with E=alkyl and F=--(CH.sub.2CH.sub.2O).sub.i-alkyl-. However, the
group L.sub.1 which is attached to the phenyl or pyridinyl ring and
which is attached to L.sub.2 is not described or suggested in these
two patent applications. Specifically, it could correspond only to
the unit G. In point of fact, G can only be a bond (single, double,
triple) or else --O--, --S-- or --NR--. As regards the other
compounds of the present invention which are characterized by the
linker --O-ALK-NR.sub.3-ALK-S--(CH.sub.2CH.sub.2O).sub.i-ALK-, none
of the units D of WO 07085930 or of WO 2009/016516 provides for the
combination of an amine group NR.sub.3 and of a bond --S--. The
following dimers are described in WO 2009/016516:
##STR00002##
but none of these dimers comprises a linker similar to those
described in the present invention (in particular, no -ALK-S--
unit).
[0006] Thus, the two applications WO 07085930 and WO 2009/016516
neither describe nor suggest the compounds of the present
invention.
TECHNICAL PROBLEM
[0007] The technical problem which the present invention intends to
solve is that of providing novel pyrrolo[1,4]benzodiazepine dimer
conjugates.
DEFINITIONS
[0008] The following terms have the accompanying meanings: [0009]
conjugate: a cell binding agent to which is covalently attached at
least one molecule of a cytotoxic compound; [0010] cell binding
agent: a molecule having an affinity for a biological target: it
can, for example, be a ligand, a protein, an antibody, more
particularly a monoclonal antibody, a protein or antibody fragment,
a peptide, an oligonucleotide or an oligosaccharide. The role of
the binding agent is to direct the biologically active compound,
such as a cytotoxic agent, towards the biological target; [0011]
biological target: an antigen (or group of antigens) preferentially
located at the surface of the cancerous cells or stromal cells
associated with this tumour; it being possible for these antigens
to be, for example, a growth factor receptor, an oncogene product
or mutated "tumour suppressant" gene product, an
angiogenesis-related molecule or an adhesion molecule; [0012] alkyl
group: a saturated aliphatic hydrocarbon group obtained by removing
a hydrogen atom from an alkane. The alkyl group can be linear or
branched. Mention may be made, as examples, of the methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl,
2,2-dimethylpropyl or hexyl groups; [0013] cycloalkyl group: a
cyclic alkyl group comprising between 3 and 8 carbon atoms involved
in the cyclic structure. Mention may be made, as examples, of the
cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl groups; [0014]
aryl group: a mono- or bicyclic aromatic group not comprising a
heteroatom. The phenyl and naphthyl groups are more particularly
concerned; [0015] heteroaryl group: a mono- or bicyclic aromatic
group comprising at least one heteroatom. (O, S, N) involved in the
ring and connected to the carbon atoms forming the ring. The
pyridinyl, pyrrolyl, thienyl, furanyl, pyrimidinyl or triazolyl
groups are more particularly concerned; [0016] heterocycloalkyl
group: a cycloalkyl group comprising at least one heteroatom (O, S,
N) involved in the ring and connected to the carbon atoms forming
the ring; [0017] alkoxy group: an --O-alkyl group, where the alkyl
group is as defined above; alkanoyloxy group: an --O--CO-alkyl
group, where the alkyl group is as defined above; [0018] alkylene
group: a saturated divalent group of empirical formula
C.sub.mH.sub.2m--, obtained by removing two hydrogen atoms from an
alkane. The alkane can be linear or branched. Mention may be made,
as examples, of the methylene (--CH.sub.2--), ethylene
(--CH.sub.2CH.sub.2--), propylene (--CH.sub.2CH.sub.2CH.sub.2--),
butylene (--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), isobutylene
##STR00003##
[0018] or hexylene
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--) groups. A
linear alkylene group can more particularly be of formula
--(CH.sub.2).sub.m--, m representing an integer; [0019] in the
ranges of values, the limits are included (for example, a range of
the type "i ranging from 1 to 6" includes the limits 1 and 6.
ABBREVIATIONS USED
[0020] AcOEt: ethyl acetate; ALK: (C.sub.1-C.sub.12)alkylene group,
more particularly (C.sub.1-C.sub.6)alkylene group; TLC: thin layer
chromatography; DAR: drug antibody ratio; DBU:
1,8-diazabicyclo[5.4.0]undec-7-ene; DCC:
N,N'-dicyclohexylcarbodiimide; DCM: dichloromethane; DEAD: diethyl
azodicarboxylate; DIC: N,N'-diisopropylcarbodiimide; DIPEA:
N,N-diisopropylethylamine; DMA: dimethylacetamide; DMAP:
4-dimethylaminopyridine; DME: dimethoxyethane; DMF:
dimethylformamide; DMSO: dimethyl sulphoxide; e: molar extinction
coefficient; EEDQ: 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline;
EDCI: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide; EDTA:
ethylenediaminetetraacetic acid; Fmoc: fluorenylmethoxycarbonyl;
PG: protecting group; Hal: halogen atom; HOBt:
1-hydroxybenzotriazole; HEPES:
4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid; LG: leaving
group; NHS: N-hydroxysuccinimide; NMP: N-methylpyrrolidinone; RP:
reduced pressure; Rf: retention factor; SEC: steric exclusion
chromatography; AT: ambient temperature; TBDMS:
tert-butyldimethylsilyl; TEA: triethylamine; TFA: trifluoroacetic
acid; TIPS: triisopropylsilyl; THF: tetrahydrofuran; rt: retention
time.
FIGURES
[0021] FIG. 1: high resolution mass spectrum of the conjugate of
Ex. 1 after deglycosylation;
[0022] FIG. 2: high resolution mass spectrum of the conjugate of
Ex. 2 after deglycosylation;
[0023] FIG. 3: high resolution mass spectrum of the conjugate of
Ex. 3 after deglycosylation;
[0024] FIG. 4: high resolution mass spectrum of the conjugate of
Ex. 4 after deglycosylation;
[0025] FIG. 5: high resolution mass spectrum of the
non-deglycosylated conjugate of Ex. 6.
[0026] These figures show, for each conjugate after deconvolution,
the distribution of the entities carrying from 0 to 8 tomaymycin
dimers (D.sub.0: no dimer; D.sub.x: x dimers).
DESCRIPTION OF THE INVENTION
[0027] The invention relates to compounds of formula:
##STR00004##
in which: [0028] represents a single bond or a double bond with the
condition that, if represents a single bond, then: [0029]
represents a single bond; [0030] U and/or U', which are identical
or different, represent(s), independently of one another, H; [0031]
W and/or W', which are identical or different, represent(s),
independently of one another: OH, --OR, --OCOR, --COOR, --OCOOR,
--OCONRR', a cyclic carbamate such that N10 and C11 are included in
a ring, NRCONRR', --OCSNHR, a cyclic thiocarbamate such that N10
and C11 are included in a ring, --SH, --SR, --SOR, --SOOR,
--SO.sub.3.sup.-, --NRSOOR', NRR', a cyclic amine such that N10 and
C11 are included in a ring, --NROR', --NRCOR', --N.sub.3, --CN, Hal
or a trialkylphosphonium or triarylphosphonium group; [0032]
R.sub.1, R.sub.2, R.sub.1' and R.sub.2', which are identical or
different, represent, independently of one another: H, Hal or a
(C.sub.1-C.sub.6)alkyl group optionally substituted by one or more
substituent(s) chosen from: Hal, CN, NRR', CF.sub.3, OR, an aryl or
heteroaryl group, or S(O).sub.qR with q=0, 1 or 2; or else [0033]
R.sub.1 and R.sub.2 and/or R.sub.1' and R.sub.2' together form
respectively a double bond .dbd.CH.sub.2 or .dbd.CH--CH.sub.3;
[0034] Y and Y', which are identical or different, represent,
independently of one another, H or OR; [0035] M represents CH or N;
[0036] ALK and ALK', which are identical or different, represent,
independently of one another, a (C.sub.1-C.sub.6)alkylene group;
[0037] R and R' represent, independently of one another, H or a
(C.sub.1-C.sub.6)alkyl or aryl group optionally substituted by one
or more substituent(s) chosen from: Hal, CN, NRR', CF.sub.3, OR or
an aryl or heteroaryl group; [0038] L represents: [0039] the
-L.sub.1-L.sub.2- group in which L.sub.1 is attached to the
aromatic ring comprising M via the ALK or OALK group and represents
one of the following groups: [0040] -ALK-S--
[0040] ##STR00005## [0041] and L.sub.2 represents the
--CH.sub.2C(.dbd.O)--NR.sub.3--(CH.sub.2CH.sub.2O).sub.i-ALK- group
attached to L.sub.1 via --CH.sub.2C(.dbd.O)--; [0042] or else
[0043] the --O-ALK-NR.sub.3-ALK-S--(CH.sub.2CH.sub.2O).sub.i-ALK-
group attached to the aromatic ring comprising M via the OALK
group; [0044] R.sub.3 represents H or a (C.sub.1-C.sub.6)alkyl
group; [0045] i represents an integer ranging from 1 to 40, rather
from 1 to 20, preferably from 1 to 10; [0046] Z.sub.b represents a
single bond, --O-- or --NH-- and R.sub.b represents H or a
(C.sub.1-C.sub.6)alkyl, (C.sub.3-C.sub.7)cycloalkyl, aryl,
heteroaryl or (C.sub.3-C.sub.7)heterocycloalkyl group or else
Z.sub.b represents a single bond and R.sub.b represents Hal.
[0047] The compounds of formula (I), including those exemplified,
can exist in the form of bases or of addition salts with
pharmaceutically acceptable acids or also of hydrates or solvates
of these bases or of these salts.
[0048] More particularly, the two ALK and ALK' groups attached to
the phenyl or pyridinyl nucleus both denote a methylene group:
##STR00006##
[0049] More particularly, among the compounds of formula (I), those
of formula (IA) or (IB):
##STR00007##
are singled out.
[0050] Y et Y' more particularly represent a
(C.sub.1-C.sub.4)alkoxy group, in particular the methoxy group. R
and R' can more particularly represent, independently of one
another, H or a (C.sub.1-C.sub.6)alkyl group. According to a
particular form, U.dbd.U' and/or W.dbd.W and/or R.sub.1=R.sub.1'
and/or R.sub.2=R.sub.2' and/or Y.dbd.Y' and/or the two ALK and ALK'
groups attached to the phenyl or pyridinyl nucleus are
identical.
[0051] L can more particularly be chosen from one of the
following:
##STR00008##
[0052] Among these, ALK more particularly represents a
(C.sub.1-C.sub.4)alkylene group. In particular, ALK can be one of
the following: --CH.sub.2CH.sub.2--, --CH.sub.2CMe.sub.2- or
--CH.sub.2CH.sub.2CMe.sub.2--. L can also be one of those described
in Table I below or in Table II.
[0053] i represents an integer ranging from 1 to 40, rather from 1
to 20, preferably from 1 to 10. i can take each of the values
ranging from 1 to 40; in particular, i can have the value 3, 4, 5,
6, 7, 8, 9 or 10.
[0054] Table I describes representative examples of compounds
according to the formula (IA). Each compound of this table can
exist in the form with M=CH (benzene) or M=N (pyridine). The
compounds with M=N are more soluble in water.
TABLE-US-00001 TABLE I Compound of formula (IA) ##STR00009##
##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014##
##STR00015##
[0055] The compounds according to the invention comprise the
chemical group --C(C.dbd.O)Z.sub.bR.sub.b (RCG1) which is reactive
with regard to a reactive chemical group (RCG2) present on the
binding agent. The reaction between RCG1 and RCG2 ensures the
attachment of the compound to the binding agent by formation of a
covalent bond. Thus, the compound is capable of being conjugated to
a binding agent. More particularly, Z.sub.b represents O; in this
case, RCG1 represents an acid functional group (R.sub.b=H) or an
ester functional group. More particularly,
--C(C.dbd.O)Z.sub.bR.sub.b represents --COOH,
--COO(C.sub.1-C.sub.6)alkyl, in particular --COOCH.sub.3, or
--COOCH.sub.2CH.dbd.CH.sub.2. Preference is given, among the ester
functional groups, to the "activated" esters which exhibit a good
reactivity with regard to the RCG2 groups, in particular with
regard to the amino groups present on antibodies. Examples of
activated esters are the following:
##STR00016##
in which IG represents at least one inductive group, such as
--NO.sub.2 or -Hal, in particular --F. The following groups may be
concerned, for example:
##STR00017##
Another type of --C(.dbd.O)Z.sub.bR.sub.b group is the
following:
##STR00018##
[0056] Mention may be made, as examples of RCG2, of the
epsilon-amino groups of the lysines carried by the side chains of
the lysine residues which are present at the surface of an
antibody, the saccharide groups of the hinge region or the thiols
of cysteines by reduction of intrachain disulphide bonds (Garnett
M. C. et al., Advanced Drug Delivery Reviews, 2001, 53, 171-216).
More recently, other approaches have been considered, such as the
introduction of cysteins by mutation (Junutula J. R. et al., Nature
Biotechnology, 2008, 26, 925-932; WO 09026274) or the introduction
of non-natural amino acids, making possible other types of
chemistry (de Graaf A. J. et al., Bioconjugate Chem., 2009, Feb. 3,
2009 (Review); DOI: 10.1021/bc800294a; WO 2006/069246, and
according to Chin J. W. et al., JACS, 2002, 124, 9026-9027
(ReCode.RTM. technology)). These methods of attachment used with
the antibodies can be applied to all known binding agents according
to their structure.
[0057] The compounds according to the invention can thus be used in
the preparation of a binding agent to which is covalently attached,
in the position para to M, the dimer of formula:
##STR00019##
[0058] More particularly, the binding agent is an antibody. More
particularly, the dimer has the formula:
##STR00020##
Process for the Preparation of the Compounds of Formula (I)
[0059] The compounds of formula (I) can be prepared according to
Scheme 1:
##STR00021##
[0060] The compounds P.sub.1, P'.sub.1 and P.sub.2 are reacted
together to result in P.sub.3. LG and LG' denote a leaving group.
L.sub.3 can represent the L-C(C.dbd.O)Z.sub.bR.sub.b group; in this
case, P.sub.3 thus represents a compound of formula (I). In the
case where P.sub.3 does not represent the
L-C(C.dbd.O)Z.sub.bR.sub.b group, it is necessary to convert
L.sub.3 to the L-C(C.dbd.O)Z.sub.bR.sub.b group using one or more
reactions. In particular, in the case where
##STR00022## [0061] it is possible to introduce the L.sub.3 group
terminated by the --C(C.dbd.O)Z.sub.bR.sub.b
group=--C(C.dbd.O)O--(C.sub.1-C.sub.4)alkyl or --C(C.dbd.O)O-allyl,
which is subsequently converted to the --C(C.dbd.O)OH group, which
finally reacts with N,N'-disuccinimidyl carbonate or NHS. The
conversion of --COOalkyl/allyl to COOH can be carried out by
treatment with lithium hydroxide. It is particularly advantageous
to use in particular a methyl ester. The reaction with
N,N'-disuccinimidyl carbonate is carried out in the presence of a
base, for example DIPEA; that with the NHS is carried out in the
presence of a coupling agent, for example DCC. Likewise, in the
case where
##STR00023##
[0061] it is possible to introduce a --C(.dbd.O)Z.sub.bR.sub.b
group=--COOH which subsequently reacts with
N,N'-carbonyldiimidazole (JACS, 1958, 80, 4423; JACS, 1960, 82,
4596).
[0062] The compounds P.sub.1 and P'.sub.1 are described in Patent
Applications WO 00/12508, WO 00/12507, WO 2005/040170, WO
2005/085260, WO 07085930 or WO 2009/016516 or are accessible by
total synthesis (Mori M. et al. Tetrahedron, 1986, 42, 3793-3806).
In the case where P.sub.i and/or P'.sub.1 represent(s) tomaymycin
of formula:
##STR00024##
the latter can be prepared using the Streptomyces croceus strain by
following the teaching of FR 1516743 or else by total synthesis
(see J. Antibiotics, 1983, XXVI(3), 276-282, Z. Tozuka, "Studies on
tomaymycin. Total syntheses of the antitumor antibiotics E- and
Z-tomaymycins"). There also exist commercial P.sub.1/P'.sub.1
compounds. For the introduction of the W/W' groups, the imine
functional group (=double bond) is capable of adding various HW/HW'
compounds (for example H.sub.2O or alcohol ROH).
With Regard to the Compounds of Formula P.sub.2
[0063] These have the formula:
##STR00025##
in which: [0064] L, M, ALK, ALK', Z.sub.b and R.sub.b are as
defined above; [0065] E and E' represent, independently of one
another, an --OH group or a leaving group.
[0066] L can more particularly represent one of those described in
Schemes 2, 2', 3, 3', 3'', 4, 5, 5', 6, 6', 6'', 7.
[0067] The intermediates of formula:
##STR00026##
in which L* is chosen from: -ALK-SH; --O-ALK-NR.sub.3-ALK-SH;
##STR00027##
are also singled out.
[0068] In this invention, the term "leaving group" denotes an atom
or a group of atoms which, in the heterolytic reaction between
P.sub.2 and P.sub.1 or P'.sub.1, leaves while carrying away the
electron pair of the covalent bonds connecting ALK and LG or LG'.
The leaving group is chosen more particularly from a halogen atom,
in particular chlorine or bromine, or a mesylate, tosylate,
nosylate or --OPPh.sub.3.sup.+ group.
[0069] For the other Schemes below, use is made, for simplicity, of
the following abbreviations:
##STR00028##
Preparation of P.sub.2
[0070] P.sub.2 is obtained from the corresponding halogenated diol
of formula
##STR00029##
according to the teaching of Ex. 3 (Scheme 2'):
##STR00030##
i. protection of the two alcohol functional groups using a
protecting group, such as, for example, tert-butyldimethylsilyloxy
(TBS); ii. preparation of the corresponding organolithium or
organomagnesium derivative using respectively n-BuLi or magnesium;
iii. nucleophilic addition to a ketone to form an alcohol
functional group; iv. preparation of the thiol via the formation of
the corresponding thioacetate (see Ex. 3.7 and 3.8); v.
deprotection; vi. introduction of LG and LG'. In the case of a
mesylate group, use is made of methanesulphonyl chloride in the
presence of a base, such as a tertiary amine (for example, TEA or
DIPEA); see Ex. 1.4.
[0071] An example of halogenated diol and of the corresponding
protected diol is that described in Scheme 1 on page 48 of WO
2009/016516 (compounds 2 and 3 of Scheme 1). Two examples of
protected diols are those of CAS Nos. 181225-40-1 and
181225-41-2.
[0072] The halogenated diol can be obtained by reduction of the
corresponding diacid or diester compound, for example that of CAS
No. 193010-40-1. See also, in the case of a pyridine (M=N): Liebigs
Annalen der Chemie, 1991, 10, 987-988 or Tetrahedron, 2005, 61(7),
1755-1763 (compound 3 of Scheme 1).
Preparation of P.sub.4
[0073] Case where ALK=CH.sub.2CH.sub.2
[0074] Case where R.sub.3=H
##STR00031##
Stage (i):
[0075] formation of the amide and activation of the acid; the two
stages are carried out successively in a polar aprotic solvent,
such as DCM: reaction between the amine functional group and
N-hydroxysuccinimidyl haloacetate, followed by in situ addition of
a coupling agent, such as DIC.
[0076] Case where R.sub.3.noteq.H
##STR00032##
Stage (ii):
[0077] protection of the carboxylic acid in the methyl ester form
and of the amine in the trifluoroacetamide form; the reaction is
carried out in two successive stages in a polar aprotic solvent,
such as DCM: protection of the acid by treatment with
trimethylsilyldiazomethane in the presence of methanol, followed by
protection of the amine by addition of trifluoroacetic anhydride
and of a base, such as TEA;
Stage (iii):
[0078] alkylation of the amine and saponification of the ester; the
reaction is carried out in two successive stages in an anhydrous
polar aprotic solvent, such as THF: alkylation of the amine by
treatment with a base, such as NaH, in the presence of a reactant
carrying a nucleofuge group, such as an alkyl halide R.sub.3Hal,
followed by addition of lithium hydroxide and of water;
Stage (i):
[0079] subsequent to stage (iii), the reactions of stage (i) for
the case R.sub.3=H are repeated.
Case where ALK.noteq.CH.sub.2CH.sub.2
[0080] Case where R.sub.3=H
##STR00033##
[0081] Case where R.sub.3.noteq.H
##STR00034##
Stage (iv):
[0082] elongation of the PEG chain; the reaction is carried out in
an anhydrous polar aprotic solvent, such as THF or DMF, by
treatment of a halogenated ester with the alkoxide of a
benzophenone/imine/PEG alcohol generated by the action of NaH or of
potassium naphthalenide, as described in WO 2007/127440;
Stage (v):
[0083] selective cleavage of the imine by hydrogenation in the
presence of palladium-on-charcoal according to Wessjohann L. et
al., Synthesis, 1989, 5, 359-63;
Stage (vi):
[0084] protection of the amine by addition of trifluoroacetic
anhydride and of a base, such as TEA.
[0085] The amino/PEG alcohols are commercially available for, for
example, i=3,4,7,8 or can be prepared from the PEG diols,
commercially available for i=3 to 12, according to the procedure
described in U.S. Pat. No. 7,230,101. The protection of the amine
functional group by benzophenone can be carried out by azeotropic
dehydration in the presence of a Lewis acid, such as BF.sub.3
etherate.
##STR00035##
Preparation of P.sub.2
##STR00036##
[0086] i. nucleophilic reaction between one of the OH functional
groups (the other two being protected by PG and PG', which denote
protecting groups) and a bromoamine protected by Boc of formula
Br-ALK-NHBoc in the presence of a base, such as, for example,
K.sub.2CO.sub.3, in a polar solvent, such as DMF, THF or MeCN (see,
for example, the conditions on page 63 of WO 07085930).
[0087] According to an alternative form, it is possible to carry
out the nucleophilic substitution of the bromoamine by the
hydroxydiester of formula:
##STR00037##
and then to subsequently reduce the ester functional group to give
a --CH.sub.2OH functional group, for example with sodium
borohydride; it is possible to apply, for this, the conditions of
the nucleophilic substitution and of the reduction given on pages
62-63 of WO 2007/085930; ii. deprotection of protecting groups;
iii. reductive amination with the aldehyde of formula
HC(.dbd.O)-ALK-SSMe in the presence of titanium isopropoxide; the
reaction is carried out at ambient temperature in an anhydrous
polar aprotic solvent, such as THF; iv. an intermediate complex is
formed which is reduced in situ with a reducing agent, such as, for
example, sodium cyanoborohydride; v. introduction of LG and LG'. In
the case of a mesylate group, use is made of methanesulphonyl
chloride in the presence of a base, such as a tertiary amine (for
example TEA); see Ex. 1.4.
[0088] An alternative form represented in Scheme 3'' consists in
introducing the -ALK-SSMe group onto the NHBoc group, under the
inspiration in particular of the method described by Kitagawa T. et
al., JACS, 2006, 128(45), 14448-14449, used to introduce
acetylthioalkyl chains onto a secondary amine:
##STR00038##
[0089] According to this alternative form, the alkylation is
carried out using an intermediate Hal-ALK-SCOMe (Hal=I, for
example) and then the thiol is released by a treatment in a basic
medium:
vi. alkylation by an alkyl halide carrying a thioacetyl group in
the presence of caesium carbonate in a polar aprotic solvent, such
as DMF; vii. selective cleavage of the acetyl group in a weak basic
medium; viii. formation of the SSMe group by reaction of the
intermediate thiol with MeSSO.sub.2Me; ix. cleavage of the
protecting groups PG and PG'; x. conversion of the hydroxyl groups
to nucleofuge groups LG/LG', preferably mesylate groups.
##STR00039##
[0090] The introduction of an R.sub.3 group=(C.sub.1-C.sub.4)alkyl
onto an NH group is possible at different stages of the synthesis
(an example is given in Scheme 3''). It is carried out, for
example, by employing the Wallach reaction, which uses an aldehyde
(see Ex. 1.5, where the alkylation NH.fwdarw.NMe uses
formaldehyde).
With Regard to P.sub.6
[0091] Thermo Fisher Scientific, Rockford, Ill. 61105, USA, Jenkem
Technology USA Inc., 2033 W. McDermott Dr, Allen, Tex. 75013-4675,
USA, and Quanta BioDesign Ltd., 195 West Olentangy Street, Suite O,
Powell, Ohio 43065-8720, USA, sell compounds of general formula
##STR00040##
denoted by NHS-PEG-maleimide. The compound of CAS No. 756525-99-2
may more particularly be concerned.
[0092] The addition of the thiol to the maleimide unit is described
on page 721 of "Bioconjugate Techniques", Greg T. Hermanson, 2nd
Ed., Elsevier Inc. (ISBN-13: 978-0-12-370501-3;
ISBN-10:0-12-370501-0).
##STR00041##
[0093] The preparation is similar to that described in Scheme 3,
P.sub.5 being replaced by P.sub.4.
[0094] An alternative form described in Scheme 4' corresponds to a
preparation similar to that described in Schemes 2 and 2':
##STR00042##
i. deprotection by reduction of the disulphide
##STR00043##
[0095] Preparation of P.sub.2
Use is made of the compound P.sub.2 of formula:
##STR00044##
which is obtained according to Scheme 5' below:
##STR00045##
i. alkylation of the hydroxyl of the aromatic ring by a piperazine
monoprotected in the 1 position and carrying, in the 4 position, an
alkyl chain functionalized in the end position by a nucleofuge LG
group. Preferably, the nucleofuge group is a mesylate group and the
Williamson reaction is carried out in the presence of a hydride in
an anhydrous polar aprotic solvent, such as THF or DMF; ii.
deprotection of the Boc, PG and PG' groups, preferably in an acidic
medium, for example in the presence of hydrochloric acid or of TFA
when the PG and PG' groups are TBDMS.
[0096] According to an alternative form of stages i and ii, it is
possible to carry out the nucleophilic substitution of the
bromoamine by the hydroxydiester of formula:
##STR00046##
and then to subsequently reduce the ester functional group to give
a --CH.sub.2OH functional group, for example with sodium
borohydride; it is possible to apply, for this, the conditions of
the nucleophilic substitution and of the reduction given on pages
62-63 of WO 2007/085930; iii. coupling carried out after initial
activation of the acid to give an NHS ester; iv. introduction of
the leaving groups LG and LG'. In the case of a mesylate group, use
is made of methanesulphonyl chloride in the presence of a base,
such as a tertiary amine (for example TEA).
##STR00047##
Preparation of P.sub.2
[0097] Use is made of the compound P.sub.2 of formula:
##STR00048##
which is obtained by reductive amination according to Scheme 6'
below.
##STR00049##
v. introduction of the -ALK-SSMe group by reductive amination
carried out, for example, in the presence of cyanoborohydride and
of titanium isopropoxide.
[0098] An alternative form similar to that represented in Scheme
3'' and using the alkylation method described by Kitagawa T. et
al., JACS, 2006, 128(45), 14448-14449, is given in Scheme 6'':
##STR00050##
i. alkylation by monoprotected piperazine (cf. stage i. of Scheme
5'); vi. selective deprotection of the Boc group in an acidic
medium; vii. alkylation by an alkyl halide carrying a thioacetyl
group in the presence of caesium carbonate in a polar aprotic
solvent, such as DMF; viii. selective cleavage of the acetyl group
in a weak basic medium and formation of the SSMe group by reaction
of the intermediate thiol with MeSSO.sub.2Me in the presence of a
base, such as TEA; ix. cleavage of the protecting groups PG and PG'
and conversion of the hydroxyl groups, preferably to mesylates with
methanesulphonyl chloride.
##STR00051##
[0099] This Scheme draws its inspiration from the preceding Scheme
3. The compound P.sub.6 can, for example, be the compound of CAS
No. 564476-32-0, which is prepared according to WO 03068144 (cf.
compound 10a of FIG. 7), or else the compound of CAS No.
309916-91-4. Analogous compounds with different chain lengths i can
be prepared according to the same principle of FIG. 7 of WO
03068144 starting from the corresponding PEG compound.
With Regard to P.sub.6
##STR00052##
[0100] can be prepared according to the schemes below: Case where
ALK=CH.sub.2CH.sub.2
##STR00053##
Case where ALK.noteq.CH.sub.2CH.sub.2
##STR00054##
Stage (i):
[0101] activation of the alcohol in the mesylate form; the reaction
is carried out in an anhydrous polar aprotic solvent, such as DCM,
by treatment with mesyl chloride in the presence of a base, such as
TEA.
Stage (ii):
[0102] mesylate/halogen exchange; the reaction is carried out at
reflux of a polar aprotic solvent, such as acetone, with a sodium
halide, such as sodium iodide.
Stage (iii):
[0103] deprotection using a solution of hydrochloric acid (for
example a solution in dioxane) or of trifluoroacetic acid.
Stage (iv):
[0104] activation of the acid; the reaction is carried out at
ambient temperature in a polar aprotic solvent, such as DCM, by
treatment with NHS in the presence of a coupling agent, such as
DCC.
Stage (v):
[0105] elongation of the PEG chain; the reaction is carried out in
an anhydrous polar aprotic solvent, such as THF or DMF, by
treatment of a halogenated ester with the alkoxide of a PEG diol
monoprotected in the tetrahydropyran (THP) ether form. The
preparation of this type of monoprotected PEG diol is well
described in the literature; see, for example, Richard A. et al.,
Chem. Eur. J., 2005, 11, 7315-7321 or Sakellariou E. G. et al.,
Tetrahedron, 2003, 59, 9083-9090.
Stage (vi):
[0106] deprotection using a 0.1N hydrochloric acid solution (for
example a solution in dioxane or ethanol).
[0107] A person skilled in the art can draw his inspiration from
the operating conditions of the examples described below.
Process for the Preparation of the Conjugate
[0108] The conjugate is obtained by the process consisting in:
(i) bringing into contact and allowing to react an optionally
buffered aqueous solution of the binding agent and a solution of a
compound of formula (I); (ii) and then optionally separating the
conjugate formed in stage (i) from the compound of formula (I)
and/or from the binding agent not having reacted and/or from the
aggregates which might be formed.
[0109] According to an alternative form, the conjugate which has
formed in stage (i) is separated in stage (ii) from the unreacted
binding agent and from the aggregates possibly present in the
solution. According to another alternative form, the conjugate of
stage (i) is separated in stage (ii) only from the unreacted
compound of formula (I) and from the aggregates which might be
formed and the binding agent which might not have reacted is left
in the solution.
[0110] The compound of formula (I) preferably comprises an
activated functional group C(.dbd.O)Z.sub.bR.sub.b, which is
reactive with regard to the RCG2 groups, in particular with regard
to the amino groups present on the antibodies. A relatively
unreactive or insufficiently reactive chemical group can be easily
converted into a more reactive group using one or more chemical
reactions known to a person skilled in the art; for example
##STR00055##
or else
##STR00056##
[0111] An example of a process applicable in the case of an
antibody and of a compound of formula (I) is that given in Example
1.
[0112] The aqueous solution of the binding agent can be buffered
using, for example, buffers such as, for example, potassium
phosphate or N-(2-hydroxyethyl)piperazine-N'-2-ethanesulphonic acid
(HEPES buffer). The buffer depends on the nature of the binding
agent. The compound of formula (I) is dissolved in a polar organic
solvent, for example DMSO or DMA.
[0113] The reaction takes place at a temperature generally of
between 20 and 40.degree. C. The duration of the reaction can vary
between 1 and 24 h. The reaction between the binding agent and
compound of formula (I) can be monitored by SEC with a
refractometric and/or ultraviolet detector, in order to determine
therefrom the state of progression. If the degree of grafting is
inadequate, the reaction can be allowed to take place for a longer
time and/or further compound of formula (I) can be added. Reference
may be made to the general method given in the Examples part for
more details on specific conditions which can be used for the
conjugation.
[0114] A person skilled in the art has available various
chromatographic techniques for the separation of stage (ii): the
conjugate can be purified, for example, by steric exclusion
chromatography (SEC), by adsorption chromatography (such as
ion-exchange chromatography, IEC), by hydrophobic interaction
chromatography (HIC), by affinity chromatography, by chromatography
on mixed supports, such as ceramic hydroxyapatite, or by HPLC.
Purification by dialysis or diafiltration can also be used.
[0115] The term "aggregates" is understood to mean the combinations
which can be formed between two or more binding agents, the binding
agents having or not having been modified by conjugation. The
aggregates are capable of being formed under the influence of a
large number of parameters, such as a high concentration of binding
agent in the solution, the pH of the solution, high shear forces,
the number of dimers grafted and their hydrophobic nature, or the
temperature (see the references cited in the introduction to J.
Membrane Sci., 2008, 318, 311-316), the influence of some of them
sometimes not being precisely clarified. In the case of proteins or
antibodies, reference may be made to AAPS Journal, "Protein
Aggregation and Bioprocessing", 2006, 8(3), E572-E579. The content
of aggregates can be determined using known techniques, such as SEC
(see, in this regard, Analytical Biochemistry, 1993, 212(2),
469-480).
[0116] After stage (i) or (ii), the solution of the conjugate can
be subjected to a stage (iii) of ultrafiltration and/or of
diafiltration. The conjugate in aqueous solution is thus obtained
on conclusion of these stages.
Antibody
[0117] The antibody (see, in this regard, Janeway et al.,
"Immunobiology", 5th edition, 2001, Garland Publishing, New York)
can be chosen from those described in particular in Applications WO
04043344, WO 08010101, WO 08047242 and WO 05009369 (anti-CA6). The
antibody can in particular be monoclonal, polyclonal or
multispecific. An antibody fragment may also be concerned. A
murine, human, humanized or chimeric antibody may also be
concerned.
Conjugate
[0118] A conjugate generally comprises of the order of 1 to 10
pyrrolo[1,4]benzodiazepine dimer(s) attached to the binding agent
(this is the degree of grafting or drug-to-antibody ratio (DAR)).
This number varies according to the nature of the binding agent and
of the dimer and also the operating conditions used for the
conjugation (for example, the number of equivalents of dimer with
respect to the binding agent, the reaction time, the nature of the
solvent and the nature of the possible cosolvent). Bringing the
binding agent and the dimer into contact results in a mixture
comprising: several conjugates differing individually from one
another by different DARs; optionally the unreacted binding agent
(in the case of an incomplete reaction); and possibly aggregates.
The DAR which is determined on the final solution, for example by
UV spectroscopy, thus corresponds to a mean DAR.
[0119] In the case where the binding agent is an antibody, UV
spectroscopy can be a method used to determine the DAR. This method
draws its inspiration from that presented in Antony S. Dimitrov
(Ed.), LLC, 2009, "Therapeutic Antibodies and Protocols", vol. 525,
445, Springer Science. It consists in measuring the absorbance of a
solution of conjugate after the separation stage (ii) at two
wavelengths, denoted LO1 and LO2. Use is made of the following
molar extinction coefficients of the naked antibody and of the
pyrrolo[1,4]benzodiazepine dimer measured prior to the
conjugation.
[0120] The absorbances of the solution of conjugate at LO1 and LO2
(A.sub.LO1 and A.sub.LO2) are measured either on the corresponding
peak of the SEC spectrum (this makes it possible to calculate a
"DAR(SEC)") or by using a conventional UV spectrophotometer (this
makes it possible to calculate a "DAR(UV)"). The absorbances can be
expressed in the form:
A.sub.LO1=(c.sub.D.times.e.sub.D LO1)+(c.sub.A.times.e.sub.A
LO1)
A.sub.LO2=(c.sub.D.times.e.sub.D LO2)+(c.sub.A.times.e.sub.A
LO2)
equations for which: [0121] c.sub.D and c.sub.A respectively denote
the concentrations in the solution of the part of the conjugate
relating to the pyrrolo[1,4]benzodiazepine dimer and the part of
the conjugate relating to the antibody; [0122] e.sub.D LO1 and
e.sub.D LO2 respectively denote the molar absorption coefficients
of the pyrrolo[1,4]benzodiazepine dimer before conjugation at the
two wavelengths LO1 and LO2; [0123] e.sub.A LO1 and e.sub.A LO2
respectively denote the molar absorption coefficients of the naked
antibody at the two wavelengths LO1 and LO2.
[0124] The term "naked antibody" is understood to mean the antibody
to which no pyrrolo[1,4]benzodiazepine dimer is attached, that is
to say the antibody before the conjugation stage.
[0125] The resolution of these two equations results in:
c.sub.D=[(e.sub.A LO1.times.A.sub.LO2)-(e.sub.A
LO2.times.A.sub.LO1)]/[(e.sub.D LO2.times.e.sub.A LO1)-(e.sub.A
LO2.times.e.sub.D LO1)]
c.sub.A=[A.sub.LO1-(c.sub.D.times.e.sub.D LO1)]/e.sub.A LO1
[0126] The mean DAR then corresponds to c.sub.D/c.sub.A. In the
case of the pyrrolo[1,4]benzodiazepine dimers, the two wavelengths
LO1=280 nm and LO2=320 nm are considered. The mean DAR measured on
the SEC spectrum is preferably between 1 and 10, preferably between
1.5 and 7.
[0127] The conjugate can be used as anticancer agent. Due to the
presence of the binding agent, the conjugate is rendered highly
selective with regard to the tumour cells rather than the healthy
cells. This makes it possible to direct the compound of formula (I)
which has an anticancer activity to the close surroundings of these
or directly inside these (in this regard, see the following
publications, which describe the use of conjugates of monoclonal
antibodies in the treatment of cancers: "Antibody-drug conjugates
for cancer therapy", Carter P. J. et al., Cancer J., 2008, 14,
154-169; "Targeted cancer therapy: conferring specificity to
cytotoxic drugs", Chari R., Acc. Chem. Res., 2008, 41, 98-107). It
is possible to treat solid or non-solid cancers.
[0128] The conjugate is formulated in the form of a buffered
aqueous solution at a concentration generally of between 1 and 10
mg/ml. This solution can be injected in the infusion form as is or
else can be rediluted to form an infusion solution.
EXAMPLES
[0129] The chemical shifts (delta in ppm) are expressed in ppm.
Method A: High Pressure Liquid Chromatography Mass Spectrometry
(LCMS)
[0130] The spectra were obtained on a Waters UPLC-SQD device in
positive and/or negative electrospray mode (ES+/-). Chromatographic
conditions: column: ACQUITY BEH C18 1.7 .mu.m-2.1.times.50 mm;
solvents: A: H.sub.2O (0.1% formic acid), B: CH.sub.3CN (0.1%
formic acid); column temperature: 50.degree. C.; flow rate: 1
ml/min; gradient (2 min): from 5 to 50% of B in 0.8 min; 1.2 min:
100% of B; 1.85 min: 100% of B; 1.95 min: 5% of B.
Method B: High Pressure Liquid Chromatography Mass Spectrometry
(LCMS)
[0131] The spectra were obtained on a Waters ZQ device in positive
and/or negative electrospray mode (ES+/-). Chromatographic
conditions: column: XBridge C18 2.5 .mu.m 3.times.50 mm; solvents:
A: H.sub.2O (0.1% formic acid), B: CH.sub.3CN (0.1% formic acid);
column temperature: 70.degree. C.; flow rate: 0.9 ml/min; gradient
(7 min): from 5 to 100% of B in 5.3 min; 5.5 min: 100% of B; 6:3
min: 5% of B.
Method C: Mass Spectrometry (MS)
[0132] The spectra were recorded using chemical ionization
(reactant gas: ammonia) on a WATERS GCTof device (direct
introduction without LC).
Method D: High Pressure Liquid Chromatography Mass Spectrometry
(LCMS)
[0133] The spectra were obtained on a Waters UPLC-SQD device in
positive and/or negative electrospray mode (ES+/-). Chromatographic
conditions: column: ACQUITY BEH C18 1.7 .mu.m 2.1.times.50 mm;
solvents: A: H.sub.2O (0.1% formic acid), B: CH.sub.3CN (0.1%
formic acid); column temperature: 70.degree. C.; flow rate: 1
ml/min; gradient (2 min): from 5 to 50% of B in 1 min; 1.3 min:
100% of B; 1.45 min: 100% of B; 1.75 min: 5% of B.
Method E: High Pressure Liquid Chromatography Mass Spectrometry
(LCMS)
[0134] The spectra were obtained on a Waters UPLC-SQD device in
electrospray ionization mode in positive and/or negative mode
(ES+/-). Chromatographic conditions: column: ACQUITY BEH C18 1.7
.mu.m 2.1.times.50 mm; solvents: A: H.sub.2O (0.1% formic acid), B:
CH.sub.3CN (0.1% formic acid); column temperature: 70.degree. C.;
flow rate: 1 ml/min; gradient (4 min): from 5 to 100% of B in 3.15
min; 3.75 min: 5% of B.
Method F: High Pressure Liquid Chromatography Mass Spectrometry
(LCMS)
[0135] The spectra were obtained on a Waters UPLC-SQD device in
electrospray ionization mode in positive and/or negative mode
(ES+/-). Chromatographic conditions: column: ACQUITY BEH C18, 1.7
.mu.m 2.1.times.30 mm; solvents: A: H.sub.2O (0.1% formic acid), B:
CH.sub.3CN (0.1% formic acid); column temperature: 45.degree. C.;
flow rate: 0.6 ml/min; gradient (2 min): from 5 to 50% of B in 1
min; 1.3 min: 100% of B; 1.45 min: 100% of B; 1.75 min: 5% of
B.
Method G: Deglycosylation and Mass Spectrometry (HRMS) of a
Conjugate
[0136] Deglycosylation is a technique for enzymatic digestion using
glycosidase. It is carried out starting from 500 .mu.l of
conjugate+100 .mu.l of Tris HCl 50 mM buffer+10 .mu.l of
glycanase-F enzyme (100 units of lyophilized enzyme/100 .mu.l of
water). The mixture is vortexed and maintained overnight at
37.degree. C. The deglycosylated sample is then ready to be
analysed by HRMS. As the case may be, the HRMS analysis of the
sample can also be carried out without prior deglycosylation. In
both cases, the mass spectra were obtained on a Waters Q-Tof-2
device in positive electrospray mode (ES+). Chromatographic
conditions: column: 4 .mu.m BioSuite 250 URH SEC 4.6.times.300 mm
(Waters); solvents: A: 25 mM ammonium formate+1% of formic acid: B:
CH.sub.3CN; column temperature 30.degree. C.: flow rate 0.4 ml/min;
isocratic 70% A+30% B (15 min).
[0137] All the intermediate compounds described in this patent
application are claimed for their use in the preparation of a
compound of formula (I). More particularly, for each example, all
the intermediate compounds described are claimed for their use in
the preparation of the respective compound of formula (I).
Example 1
1.1. Preparation of the Conjugate
[0138] A conjugate is prepared by reacting hu2H11 (also known as
hu53 2H11 on page 15 of WO 2008010101; it is an antibody comprising
a VH having the amino acid sequence SED ID No. 24) and
N-hydroxysuccinimidyl
3-(2-{2-[2-(2-{3-[3-(2-([2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3-
,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridi-
n-4-yloxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)-2,5-dioxopyrrol-
idin-1-yl]propanoylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoate.
[0139] 516 .mu.g of N-hydroxysuccinimidyl
3-(2-{2-[2-(2-{3-[3-(2-{[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3-
,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridi-
n-4-yloxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)-2,5-dioxopyrrol-
idin-1-yl]propanoylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoate in
solution in 540 .mu.l of DMA are added to 8.19 mg of hu2H11 in 2.16
ml of an aqueous buffer with a 0.05 M concentration of
N-(2-hydroxyethyl)piperazine-N'-2-ethanesulphonic acid (HEPES), a
0.05 M concentration of NaCl and a 2 mM concentration of
ethylenediaminetetraacetic acid (EDTA) of pH=8. After stirring at
AT for 3 h, the mixture is filtered through Millex.RTM.-SV 0.45
.mu.M (PVDF Durapore Millipore) and purified on a Superdex.TM. 200
prep grade column (Hiload.TM. 26/60 GE column) preequilibrated in a
saline phosphate buffer brought to pH=6.5 by addition of HCl. The
fractions of interest are combined and concentrated on Amicon
Ultra-15 (Ultracel 50 k Millipore) and then filtered through
Sephadex G-25 (NAP-5 and NAP-10 GE columns) preequilibrated in an
aqueous buffer with a 10 mM concentration of histidine comprising
10% of sucrose and 5% of NMP.
[0140] The conjugate obtained (2.5 ml) is quantitatively determined
by spectrophotometry using the extinction coefficients of
4-{2-[methyl(2-methyl-2-mercaptopropyl)amino]ethoxy}-2,6-bis[(S)-2-eth-(E-
)-ylidene-7-methoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-
-one-8-yloxymethyl]pyridine (e.sub.319 nm=8848 M.sup.-1 cm.sup.-1
and e.sub.280 nm=8634 M.sup.-1 cm.sup.-) and of hu2H11 (e.sub.280
nm=208 380 M.sup.-1 cm.sup.-1): a mean of 3.8 tomaymycin dimers per
antibody molecule at the concentration of 1.52 mg/ml was
determined.
1.2. N-hydroxysuccinimidyl
3-(2-{2-[2-(2-{3-[3-(2-{[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3-
,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridi-
n-4-yloxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)-2,5-dioxopyrrol-
idin-1-yl]propanoylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoate
##STR00057##
[0142] A solution of 9.82 mg of
4-{2-[methyl(2-methyl-2-mercaptopropyl)amino]ethoxy)-2,6-bis[(S)-2-eth-(E-
)-ylidene-7-methoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-
-one-8-yloxymethyl]pyridine in 50 .mu.l of DMA and a solution of
6.8 mg of N-hydroxysuccinimidyl
3-{2-[2-(2-{2-[3-(2,5-dioxo-2,5-dihydropyrrol-1-yl)propanoylamino]ethoxy}-
ethoxy)ethoxy]ethoxy}propanoate in 50 .mu.l of DMA are added to 3.3
mg of diisopropylethylamine supported on resin (3.72 mmol/g). The
mixture obtained is stirred at AT for 24 h and then filtered
through silica (Interchrom Puriflash Silica 15/35U 2G) using a
gradient from 0 to 10% of methanol in DCM. The fractions comprising
the desired product are combined, concentrated under RP and then
purified by flash chromatography on silica (Interchrom Puriflash
Silica 15/35U 2G) using a gradient from 0 to 10% of MeOH in DCM.
The fractions comprising the desired product are combined and
concentrated under RP. 1.16 mg of N-hydroxysuccinimidyl
3-(2-{2-[2-(2-{3-[3-(2-{[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3-
,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridi-
n-4-yloxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)-2,5-dioxopyrrol-
idin-1-yl]propanoylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoate are
thus obtained. LC/MS (E): rt=1.38 min; [M+H].sup.+: m/z 1322.
1.3.
4-{2-[methyl(2-methyl-2-mercaptopropyl)amino]ethoxy)-2,6-bis[(S)-2-et-
h-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazep-
in-5-one-8-yloxymethyl]pyridine
##STR00058##
[0144] A solution of 40 mg of tris(2-carboxyethyl)phosphine
hydrochloride and of 36.6 mg of NaHCO.sub.3 in 680 .mu.l of water
is added to 40 mg of
4-{2-[methyl(2-methyl-2-methyldisulphanylpropyl)amino]ethoxy}-2,6-bis[(S)-
-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzod-
iazepin-5-one-8-yloxymethyl]pyridine in solution in 2.1 ml of
methanol and 930 .mu.l of DMF. The mixture is stirred at AT for 45
min, then concentrated under RP and purified by flash
chromatography on silica (Merck SuperVarioFlash 15 g column, Si60
15-40 .mu.m), using a gradient from 0 to 10% of MeOH in a
DCM/acetonitrile 9:1 mixture. The fractions comprising the desired
product are combined and concentrated under RP. 21 mg of
4-{2-[methyl(2-methyl-2-mercaptopropyl)amino]ethoxy}-2,6-bis[(S)-2--
eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiaz-
epin-5-one-8-yloxymethyl]pyridine are thus obtained. LC/MS (E):
rt=1.28 min; [M+H].sup.+: m/z 809; [M+H.sub.2O+H].sup.+: m/z
827.
1.4.
4-{2-[methyl(2-methyl-2-(methyldisulphanyl)propyl)amino]ethoxy)-2,6-b-
is[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]-
benzodiazepin-5-one-8-yloxymethyl]pyridine
##STR00059##
[0146] 19.6 .mu.l of methanesulphonyl chloride are added to a
solution, cooled to -25.degree. C., of 22 mg of
4-{2-[methyl(2-methyl-2-(methyldisulphanyl)propyl)amino]ethoxy}-2,6-bis(h-
ydroxymethyl)pyridine and of 53 .mu.l of TEA in 0.5 ml of DCM.
After stirring for 30 min, the mixture is hydrolysed and the
organic phase is washed with water, then dried over MgSO.sub.4 and
concentrated under RP. The residue thus obtained (22 mg) is added
to a solution of 20 mg of tomaymycin in 0.7 ml of DMF, and also 30
mg of K.sub.2CO.sub.3 and 12 mg of KI. The mixture is stirred at
30.degree. C. for 2 h and then hydrolysed with 4 ml of water. The
resulting precipitate is washed with water, dried under vacuum,
then dissolved in DCM, concentrated under RP and purified by flash
chromatography on silica (Merck SuperVarioFlash 15 g column, Si60
15-40 .mu.m), using a gradient from 0 to 5% of MeOH in DCM. The
fractions comprising the desired product are combined and
concentrated under RP. 8 mg of
4-{2-[methyl(2-methyl-2-(methyldisulphanyl)propyl)amino]ethoxy}-2,6-bis[(-
S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benz-
odiazepin-5-one-8-yloxymethyl]pyridine are thus obtained. .sup.1H
NMR (500 MHz, d-chloroform): 1.28 (s, 6H); 1.76 (d, J=6.4 Hz, 6H);
2.39 (s, 3H); 2.43 (s, 3H); 2.60 (s, 2H); 2.91 (s, 2H); 2.97 (s,
4H); 3.91 (d, J=4.2 Hz, 2H); 4.00 (s, 6H); 4.09 (s, 2H); 4.27 (s,
4H); 5.27 (s, 4H); 5.61 (q, J=6.4 Hz, 2H); 6.85 (s, 2H); 7.00 (s,
2H); 7.56 (s, 2H); 7.65 (d, J=4.2 Hz, 2H). LC/MS (A): rt=0.74 min;
[M+H].sup.+: m/z 855; [M-H].sup.-: m/z 853.
1.5.
4-{2-[methyl(2-methyl-2-(methyldisulphanyl)propyl)amino]ethoxy}-2,6-b-
is(hydroxymethyl)pyridine
##STR00060##
[0148] 365 .mu.l of formic acid are added to a suspension, cooled
to 0.degree. C., of 322 mg of
4-[2-(2-methyl-2-(methyldisulphanyl)propylamino)ethoxy]-2,6-bis(hydroxyme-
thyl)pyridine in 262 .mu.l of formaldehyde. The mixture is heated
at 100.degree. C. for 1% hours. After returning to ambient
temperature, the mixture is hydrolysed and then a 5N aqueous sodium
hydroxide solution is added until a pH=12 is obtained. The aqueous
phase is extracted 3.times. with AcOEt and the combined organic
phases are dried over MgSO.sub.4 and concentrated under RP. 310 mg
of
4-{2-[methyl(2-methyl-2-(methyldisulphanyl)propyl)amino]ethoxy)-2,6-bis(h-
ydroxymethyl)pyridine are thus obtained.
[0149] .sup.1H NMR (300 MHz, d.sub.6-DMSO): 1.26 (s, 6H); 2.39 (s,
3H); 2.40 (s, 3H); 2.60 (s, 2H); 2.88 (t, J=5.7 Hz, 2H); 4.13 (t,
J=5.7 Hz, 2H); 4.45 (d, J=6.0 Hz, 4H); 5.31 (t, J=6.0 Hz, 2H); 6.85
(s, 2H). LC/MS (A): rt=0.22 min; [M+H].sup.+: m/z 333;
[M+HCO2H--H].sup.-: m/z 377.
1.6.
4-[2-(2-methyl-2-(methyldisulphanyl)propylamino)ethoxy]-2,6-bis(hydro-
xymethyl)pyridine
##STR00061##
[0151] 270 .mu.l of 2-(methyldithio)isobutyraldehyde and 730 .mu.l
of titanium isopropoxide are added to a suspension of 390 mg of
4-[2-aminoethoxy]-2,6-bis(hydroxymethyl)pyridine (prepared after
deprotection of the Boc group from
4-(2-(tert-butoxycarbonylamino)ethoxy)-2,6-bis(hydroxymethyl)pyridine
described on page 101 of WO 07085930) in 2 ml of THF. After 20 min,
an additional 270 .mu.l of 2-(methyldithio)isobutyraldehyde and an
additional 730 .mu.l of titanium isopropoxide are added and the
mixture is stirred at AT for 2 h. 6 ml of ethanol are then added to
the mixture, the mixture is stirred at AT for 20 min and then 124
mg of sodium cyanoborohydride are added to the mixture. After
stirring for 45 min, an additional 124 mg of sodium
cyanoborohydride are added and, after stirring for 1 h, the mixture
is concentrated under RP and the residue is diluted in AcOEt and
water. The resulting precipitate is filtered off and dissolved in a
1M aqueous HCl solution. The aqueous phase obtained is brought to
basic pH with a 5M aqueous sodium hydroxide solution and extracted
three times with DCM, and the combined organic phases are
concentrated under RP. 322 mg of
4-[2-(2-methyl-2-(methyldisulphanyl)propylamino)ethoxy]-2,6-bis(hydroxyme-
thyl)pyridine are thus obtained.
[0152] .sup.1H NMR (400 MHz, d.sub.6-DMSO): 1.26 (s, 6H); 1.81
(broad m, 1H); 2.39 (s, 3H); 2.67 (broad s, 2H); 2.94 (broad t,
J=5.7 Hz, 2H); 4.11 (t, J=5.7 Hz, 2H); 4.45 (d, J=5.5 Hz, 4H); 5.32
(t, J=5.5 Hz, 2H); 6.85 (s, 2H). LC/MS (A): rt=0.24 min;
[M+H].sup.+: m/z 347.
Example 2
2.1. Preparation of the Conjugate
[0153] A conjugate is prepared as for Ex. 1 by reacting hu2H11 and
N-hydroxysuccinimidyl
3-{2-[2-(2-{2-[2-(2-([2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11-
a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridin-4-
-yloxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)acetylamino]ethoxy}-
ethoxy)ethoxy]ethoxy}propanoate. The conjugate obtained is
quantitatively determined by spectrophotometry using the extinction
coefficients of
4-{2-(methyl(2-methyl-2-mercaptopropyl)amino]ethoxy}-2,6-bis[(S)-2-eth-(E-
)-ylidene-7-methoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepine--
5-one-8-yloxymethyl]pyridine (e.sub.319 nm=8848 M.sup.-1 cm.sup.-1,
e.sub.280 nm=8634 M.sup.-1 cm.sup.-1) and of hu2H11 (e.sub.280
nm=208 380 M.sup.-1 cm.sup.-1): a mean of 5.6 tomaymycin dimers per
antibody molecule was determined.
2.1. N-hydroxysuccinimidyl
3-{2-[2-(2-{2-[2-{[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-t-
etrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridin-4-yl-
oxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)acetylamino]ethoxy}eth-
oxy)ethoxy]ethoxy}propanoate
##STR00062##
[0155] 5.5 mg of N,N'-disuccinimidyl carbonate and 15 .mu.l of
DIPEA are added to 12 mg of
3-{2-[2-(2-{2-[2-(2-{[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11-
a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridin-4-
-yloxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)acetylamino]ethoxy}-
ethoxy)ethoxy]ethoxy}propanoic acid in solution in 1 ml of THF and
1 ml of DCM. After 3 h at AT, 4 ml of DCM are added and the
resulting organic phase is washed twice with water, dried over
MgSO.sub.4 and concentrated under RP and the residue is purified by
flash chromatography on silica (Interchrom Puriflash Silica 15/35U
2G) using a gradient from 3 to 8% of methanol in DCM. 8 mg of
N-hydroxysuccinimidyl
3-{2-[2-(2-{2-[2-(2-{[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11-
a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridin-4-
-yloxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)acetylamino]ethoxy}-
ethoxy)ethoxy]ethoxy}propanoate are thus obtained.
[0156] LC/MS (E): rt=1.36 min; [M+2H.sub.2O+Na].sup.+: m/z 1270;
[M+H.sub.2O+Na].sup.+: m/z 1252; [M+H.sub.2O+H].sup.+: m/z 1229;
[M+H].sup.-: m/z 1211.
2.2.
3-{2-[2-(2-{2-[2-(2-.omicron.[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-meth-
oxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymeth-
yl]pyridin-4-yloxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)acetyla-
mino]ethoxy}ethoxy)-ethoxy]ethoxy}propanoic acid
##STR00063##
[0158] 17.5 .mu.l of an aqueous lithium hydroxide solution and 100
.mu.l of water are added to 18 mg of methyl
3-{2-[2-(2-{2-[2-(2-{[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11-
a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridin-4-
-yloxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)acetylamino]ethoxy}-
ethoxy)ethoxy]ethoxy}propanoate in solution in 270 .mu.l of THF.
After 2 h, the mixture is diluted in DCM and a phosphate buffer is
added to pH=3. The resulting aqueous phase is extracted 3.times.
with DCM, the combined organic phases are dried over MgSO.sub.4 and
concentrated under RP and the residue is purified by flash
chromatography on silica (Interchrom Puriflash Silica 15/35U 2G)
using a gradient from 3 to 15% of methanol in DCM. 11.5 mg of
3-{2-[2-(2-{2-[2-(2-{[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11-
a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridin-4-
-yloxy)ethyl](methyl)aminol-1,1-dimethylethylsulphanyl)acetylamino]ethoxy}-
-ethoxy)ethoxy]ethoxy}propanoic acid are thus obtained. LC/MS (D):
rt=0.88 min; [M+H.sub.2O+Na].sup.+: m/z 1154; [M+H.sub.2O+H].sup.+:
m/z 1133; [M+H].sup.+: m/z 1115.
2.3. Methyl
3-{2-[2-(2-{2-[2-(2-{[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11-
a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridin-4-
-yloxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)acetylamino]ethoxy}-
ethoxy)ethoxy]ethoxy}propanoate
[0159] Prepared according to Ex. 1 from methyl
3-{2-[2-(2-{2-[2-(2-{[2-(2,6-bis(hydroxymethyl)pyridin-4-yloxy)ethyl]meth-
ylamino}-1,1-dimethylethylsulphanyl)acetylamino]ethoxy}ethoxy)ethoxy]ethox-
y}-propanoate:
##STR00064##
[0160] LC/MS (D): rt=0.93 min; [M+H.sub.2O+H].sup.+: m/z 1146;
[M+H].sup.+: m/z 1128.
2.4. Methyl
3-{2-[2-(2-{2-[2-(2-{[2-(2,6-bis(hydroxymethyl)pyridin-4-yloxy)ethyl](met-
hyl)-amino-1,1-dimethylethylsulphanyl)acetylamino]ethoxy}ethoxy)ethoxy]eth-
oxy}propanoate
##STR00065##
[0162] 73.7 mg of methyl
3-[2-(2-{2-[2-(2-iodoacetylamino)ethoxy]ethoxy}ethoxy)ethoxy]propanoate
in solution in 1 ml of DMF and 39 .mu.l of DIPEA are added to 45 mg
of
4-{2-[(2-mercapto-2-methylpropyl)(methyl)amino]ethoxy}-2,6-bis(hydroxymet-
hyl)pyridine in solution in 1 ml of DMF. After 24 h at AT, the
mixture is concentrated under RP and purified by flash
chromatography on silica (Analogix Super Flash SiO.sub.2 SF25-8 g),
using a gradient from 0 to 10% of methanol in DCM. 53 mg of methyl
3-{2-[2-(2-{2-[2-(2-{[2-(2,6-bis(hydroxymethyl)pyridin-4-yloxy)ethyl](met-
hyl)amino}-1,1-dimethylethylsulphanyl)acetylamino]ethoxy}ethoxy)ethoxy]eth-
oxy}propanoate are thus obtained.
[0163] .sup.1H NMR (400 MHz, d.sub.6-DMSO): 1.21 (s, 6H); 2.40 (s,
3H); 2.50 to 2.56 (m, 4H); 2.87 (t, J=5.8 Hz, 2H); 3.15 to 3.23 (m,
4H); 3.40 (t, J=5.8 Hz, 2H); 3.47 to 3.52 (m, 12H); 3.59 (s, 3H);
3.62 (t, J=6.2 Hz, 2H); 4.13 (t, J=5.8 Hz, 2H); 4.45 (d, J=5.8 Hz,
4H); 5.31 (t, J=5.8 Hz, 2H); 6.85 (s, 2H); 7.98 (t, J=5.8 Hz, 1H).
LC/MS (A): rt=0.35 min; [M+H].sup.+: m/z 620; [M+2H]2.sup.+: m/z
310.5 (base peak); [M-H+HCO.sub.2H].sup.-: m/z 664.
2.5. Methyl
3-[2-(2-{2-[2-(2-iodoacetylamino)ethoxy]ethoxy}ethoxy)ethoxy]propanoate
##STR00066##
[0165] 117.4 mg of N-hydroxysuccinimidyl iodoacetate in solution in
3 ml of DCM are added to 100 mg of
3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propanoic acid.
After 2 h at AT, 330 .mu.l of MeOH are added and the mixture is
cooled to 0.degree. C. 360 .mu.l of a 2M solution of
trimethylsilyldiazomethane in hexane are added. After 1 h, the
mixture is neutralized by addition of 50 .mu.l of acetic acid and
then a saturated aqueous NaHCO.sub.3 solution is added until pH=8
is obtained. The organic phase is dried over MgSO.sub.4 and
concentrated under RP, and the residue is purified by flash
chromatography on silica (Interchrom Puriflash Silica 15/35U 10G)
using a gradient from 0 to 10% of methanol in DCM. 132 mg of methyl
3-[2-(2-{2-[2-(2-iodoacetylamino)ethoxy]ethoxy}ethoxy)ethoxy]propanoate
are thus obtained.
[0166] .sup.1H NMR (400 MHz, d.sub.6-DMSO): 2.54 (t, J=6.4 Hz, 2H);
3.20 (q, J=5.8 Hz, 2H); 3.41 (t, J=5.8 Hz, 2H); 3.48 to 3.53 (m,
12H); 3.60 (s, 3H); 3.63 (t, J=6.4 Hz, 2H); 3.65 (s, 2H); 8.27
(broad t, J=5.8 Hz, 1H). LC/MS (A): rt=0.54 min; [M+H].sup.+: m/z
448; [M+HCO.sub.2H--H].sup.-: m/z 492.
2.6.
4-{2-[(2-mercapto-2-methylpropyl)(methyl)amino]ethoxy}-2,6-bis(hydrox-
ymethyl)pyridine
##STR00067##
[0168] A solution of 198 mg of tris(2-carboxyethyl)phosphine
hydrochloride in 730 .mu.l of water is added to 80 mg of
4-{2-[methyl(2-methyl-2-(methyldisulphanyl)propyl)amino]ethoxy}-2,6-bis(h-
ydroxymethyl)pyridine in solution in 1.95 ml of methanol. After 2 h
at AT, the mixture is concentrated under RP and the residue is
taken up in 10 ml of water. The aqueous phase is brought to pH=8 by
addition of an aqueous sodium hydroxide solution and then extracted
2.times. with AcOEt. The combined organic phases are washed with a
saturated aqueous NaCl solution and concentrated under RP. 68 mg of
4-{2-[(2-mercapto-2-methylpropyl)(methyl)amino]ethoxy}-2,6-bis(hydroxymet-
hyl)pyridine are obtained.
[0169] .sup.1H NMR (400 MHz, d.sub.6-DMSO): 1.28 (s, 6H); 2.43 (s,
3H); 2.54 (s, 2H); 2.62 (s, 1H); 2.91 (t, J=5.7 Hz, 2H); 4.15 (t,
J=5.7 Hz, 2H); 4.45 (d, J=5.8 Hz, 4H); 5.30 (t, J=5.8 Hz, 2H); 6.85
(s, 2H). LC/MS (A): rt=0.11 min; [M+H].sup.+: m/z 301.
Example 3
3.1. Preparation of the Conjugate
[0170] A conjugate is prepared as for Ex. 1 by reacting hu2H11 and
N-hydroxysuccinimidyl
3-(2-{2-[2-(2-{2-[1-(3,5-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-te-
trahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]phenyl-4-yl)--
1-methylethylsulphanyl]acetylamino}ethoxy)ethoxy]-ethoxy}ethoxy)propanoate-
.
[0171] The conjugate obtained is quantitatively determined by
spectrophotometry using the extinction coefficients of
1-(1-methyl-1-(methyldisulphanyl)ethyl)-3,5-bis[(S)-2-eth-(E)-ylidene-7-m-
ethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxym-
ethyl]benzene (e.sub.319 nm=8460 M.sup.-1 cm.sup.-1 and e.sub.280
nm=10 531 M.sup.-1 cm.sup.-1) and of hu2H11 (e.sub.280 nm=208 380
M.sup.-1 cm.sup.-1): a mean of 4.2 tomaymycin derivatives per
antibody molecule was determined.
3.2. N-hydroxysuccinimidyl
3-(2-{2-[2-(2-{2-[1-(3,5-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-te-
trahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]phenyl-4-yl)--
1-methylethylsulphanyl]acetylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoate
[0172] Prepared as for Ex. 2 from
3-(2-{2-[2-(2-{2-[1-(3,5-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-te-
trahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]phenyl-4-yl)--
1-methylethylsulphanyl]acetylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoic
acid:
##STR00068##
[0173] LC/MS (F): rt=1.27 min; [M+H].sup.+: m/z 1123.
3.3.
3-(2-{2-[2-(2-{2-[1-(3,5-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11-
a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]phenyl-4-
-yl)-1-methylethylsulphanyl]acetylamino}ethoxy)ethoxy]ethoxy}ethoxy)propan-
oic acid
[0174] Prepared as for Ex. 2 from methyl
3-(2-{2-[2-(2-{2-[1-(3,5-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-te-
trahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]phenyl-4-yl)--
1-methylethylsulphanyl]acetylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoate:
##STR00069##
[0175] LC/MS (F): rt=1.19 min; [M+H].sup.+: m/z 1026.
3.4. Methyl
3-(2-{2-[2-(2-{2-[1-(3,5-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-te-
trahydro-pyrrolo[2,1-c]1,4]benzodiazepin-5-one-8-yloxymethyl]phenyl-4-yl)--
1-methylethylsulphanyl]acetylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoate
[0176] Prepared as for Ex. 2 from methyl
3-(2-{2-[2-(2-{2-[1-(3,5-bis(hydroxymethyl)phenyl)-1-methylethylsulphanyl-
]acetylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoate:
##STR00070##
[0177] LC/MS (F): rt=1.28 min; [M+H].sup.+: m/z 1040.
3.5. Methyl
3-(2-{2-[2-(2-{2-[1-(3,5-bis(hydroxymethyl)phenyl)-1-methylethylsulphanyl-
]-acetylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoate
[0178] Prepared as for Ex. 2 from
3,5-bis(hydroxymethyl)-1-(1-mercapto-1-methylethyl)benzene:
##STR00071##
[0179] .sup.1H NMR (400 MHz, d.sub.6-DMSO): 1.64 (s, 6H); 2.53 (t,
J=6.2 Hz, 2H); 2.93 (s, 2H); 3.15 (q,
[0180] J=6.0 Hz, 2H); 3.37 (t, J=6.0 Hz, 2H); 3.49 (m, 12H); 3.59
(s, 3H); 3.62 (t, J=6.2 Hz, 2H); 4.49 (d, J=5.6 Hz, 4H); 5.13 (t,
J=5.6 Hz, 2H); 7.14 (s, 1H); 7.32 (s, 2H); 7.84 (t, J=6.0 Hz, 1H).
LC/MS (B): rt=2.98 min; [M+H].sup.+: m/z 532; base peak: m/z 354;
[M-H+HCO.sub.2H].sup.-: m/z 576.
3.8. 3,5-bis(hydroxymethyl)-1-(1-mercapto-1-methylethyl)benzene
##STR00072##
[0182] A solution of 43 mg of
5-hydroxymethyl-1-(1-mercapto-1-methylethyl)-3-[(tert-butyl)dimethylsilan-
yloxymethyl]benzene in 1 ml of an acetic acid/THF/water (3/1/1)
mixture is stirred at AT for 4.5 h and concentrated under RP, and
then the residue is taken up in 3 ml of water. The pH of the
aqueous phase is brought to 7 by addition of a 10% aqueous
Na.sub.2CO.sub.3 solution and then the aqueous phase is extracted
with AcOEt. The organic phase is dried over MgSO.sub.4 and
concentrated under RP. 18 mg of
3,5-bis(hydroxymethyl)-1-(1-mercapto-1-methylethyl)benzene are thus
obtained.
[0183] .sup.1H NMR (400 MHz, d.sub.6-DMSO): 1.76 (s, 6H); 3.19 (s,
1H); 4.48 (d, J=5.9 Hz, 4H); 5.14 (t, J=5.9 Hz, 2H); 7.13 (s, 1H);
7.36 (s, 2H). MS (C): CI: [M+NH.sub.4].sup.+: m/z 230.
3.9.
5-Hydroxymethyl-1-(1-mercapto-1-methylethyl)-3-[(tert-butyl)dimethyls-
ilanyloxymethyl]benzene
##STR00073##
[0185] 114 .mu.l of hydrazine hydrate are added to 336 mg of
1-{3-[(tert-butyl)dimethylsilanyloxymethyl]-5-(hydroxymethyl)phenyl}-1-me-
thylethyl thioacetate in solution in 3.7 ml of acetonitrile. After
3 h at AT, the mixture is concentrated under RP and the residue is
purified by flash chromatography on silica (Biotage 25+M) using a
gradient from 0 to 55% AcOEt in heptane. 230 mg of
5-hydroxymethyl-1-(1-mercapto-1-methylethyl)-3-[(tert-butyl)dimethylsilan-
yloxymethyl]benzene are thus obtained.
[0186] .sup.1H NMR (400 MHz, d.sub.6-DMSO): 0.08 (s, 6H); 0.92 (s,
9H); 1.76 (s, 6H); 3.19 (s, 1H); 4.48 (d, J=5.7 Hz, 2H); 4.71 (s,
2H); 5.14 (t, J=5.7 Hz, 1H); 7.10 (s, 1H); 7.33 to 7.42 (m, 2H). MS
(C): CI: [M+NH.sub.4].sup.+: m/z 344.
3.10.
1-[3-[(tert-butyl)dimethylsilanyloxymethyl]-5-(hydroxymethyl)phenyl]-
-1-methylethyl thioacetate
##STR00074##
[0188] 404 .mu.l of thioacetic acid and 376 mg of zinc iodide are
added to 1 g of
1-(1-hydroxy-1-methylethyl)-3,5-bis[(tert-butyl)dimethylsilanyloxy-
methyl]benzene in solution in 4.7 ml of 1,2-dichloroethane. The
mixture is heated at 50.degree. C. for 40 min. After returning to
AT, the salts are removed by filtration, the organic phase is
concentrated under RP and the residue is purified by flash
chromatography on silica (Biotage 40+M) using a gradient from 0 to
30% AcOEt in heptane. 248 mg of
1-[3-[(tert-butyl)dimethylsilanyloxymethyl]-5-(hydroxymethyl)phenyl]-1-me-
thylethyl thioacetate are thus obtained.
[0189] .sup.1H NMR (400 MHz, d.sub.6-DMSO): 0.08 (s, 6H); 0.91 (s,
9H); 1.79 (s, 6H); 2.17 (s, 3H); 4.48 (d, J=5.6 Hz, 2H); 4.70 (s,
2H); 5.16 (t, J=5.6 Hz, 1H); 7.11 (s, 1H); 7.33 (s, 1H); 7.35 (s,
1H). LC/MS (A): rt=1.23 min; [M+Na].sup.+: m/z 391.
3.11.
1-(1-Hydroxy-1-methylethyl)-3,5-bis[(tert-butyl)dimethylsilanyloxyme-
thyl]benzene
##STR00075##
[0191] 10.5 ml of n-BuLi as a 1.6M solution in hexane are added
dropwise to 4.32 g of
1-bromo-3,5-bis[(tert-butyl)dimethylsilanyloxymethyl]benzene (G. T.
Crisp and P. D. Turner, Tetrahedron, 2000, 56 (42), 8335) in
solution in 150 ml of THF cooled to -71.degree. C. After 1 h 30,
4.27 ml of acetone are added dropwise. The mixture is allowed to
return to AT and then hydrolysed with a saturated aqueous
NH.sub.4CI solution. The aqueous phase is extracted with 100 ml of
AcOEt, the combined organic phases are dried over MgSO.sub.4 and
concentrated under RP, and the residue is purified by flash
chromatography on silica (Biotage 65+M) using a gradient from 0 to
20% AcOEt in heptane. 2.48 g of
1-(1-hydroxy-1-methylethyl)-3,5-bis[(tert-butyl)dimethylsilanyloxymethyl]-
benzene are thus obtained.
[0192] .sup.1H NMR (400 MHz, d-chloroform): 0.11 (s, 12H); 0.96 (s,
18H); 1.59 (s, 6H); 1.69 (s, 1H); 4.76 (s, 4H); 7.21 (s, 1H); 7.33
(s, 2H). LC/MS (B): rt=6.43 min; [M+Na].sup.+: m/z 447; base peak:
m/z 407.
Example 4
4.1. Preparation of the Conjugate
[0193] A conjugate is prepared as for Ex. 1 by reacting hu2H11 and
N-hydroxysuccinimidyl
3-{2-[2-(2-{2-[2-(4-{4-[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,-
11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridin-
-4-yloxy)ethyl]piperazin-1-yl}-1,1-dimethyl-4-oxobutylsulphanyl)acetylamin-
o]ethoxy}ethoxy)ethoxy]ethoxy}propanoate. The conjugate obtained is
quantitatively determined by spectrophotometry using the extinction
coefficients e.sub.320 nm=7876 M.sup.-1 cm.sup.-1 and e.sub.280
nm=4334 M.sup.-1 cm.sup.-1 for the tomaymycin dimer and e.sub.280
nm=208 380 M.sup.-1 cm.sup.-1 for hu2H11: a mean of 3.13 tomaymycin
dimers per antibody molecule was determined.
4.2. N-hydroxysuccinimidyl
3-{2-[2-(2-[2-{2-(4-(4-[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,-
11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]-pyridi-
n-4-yloxy)ethyl]piperazin-1-yl}-1,1-dimethyl-4-oxobutylsulphanyl)acetylami-
no]-ethoxy}ethoxy)ethoxy]ethoxy}propanoate
[0194] Prepared according to Example 2 from
3-{2-[2-(2-{2-[2-(4-{4-[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,-
11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridin-
-4-yloxy)ethyl]piperazin-1-yl}-1,1-dimethyl-4-oxobutylsulphanyl)acetylamin-
o]ethoxy}ethoxy)-ethoxy]ethoxy}propanoic acid
##STR00076##
[0195] LC/MS (B): rt=3.27 min; [M+H].sup.+: m/z 1309;
[M+2H].sup.2+: m/z 655.
[0196] 4.3.
3-{2-[2-(2-{2-[2-(4-{4-[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,-
11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridi-
n-4-yloxy)ethyl]piperazin-1-yl}-1,1-dimethyl-4-oxobutylsulphanyl)acetylami-
no]ethoxy}ethoxy)ethoxy]ethoxy}propanoic acid
[0197] Prepared according to Example 2 from methyl
3-{2-[2-(2-{2-[2-(4-{4-[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,-
11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridin-
-4-yloxy)ethyl]piperazin-1-yl}-1,1-dimethyl-4-oxobutylsulphanyl)acetylamin-
o]ethoxy}ethoxy)ethoxy]ethoxy}propanoate
##STR00077##
[0198] LC/MS (B): rt=3.16 min; [M+H].sup.+: m/z 1212;
[M+2H].sup.2+: m/z 606.
4.4. Methyl
3-{2-[2-(2-{2-[2-(4-{4-[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,-
11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridin-
-4-yloxy)ethyl]-piperazin-1-yl}-1,1-dimethyl-4-oxobutylsulphanyl)acetylami-
no]ethoxy}ethoxy)ethoxy]-ethoxy}propanoate
[0199] Prepared according to Example 2 from methyl
3-{2-[2-(2-{2-[2-(4-{4-[2-(2,6-bis(hydroxymethyl)pyridin-4-yloxy)ethyl]pi-
perazin-1-yl}-1,1-dimethyl-4-oxobutylsulphanyl)-acetylamino]ethoxy}ethoxy)-
ethoxy]ethoxy}propanoate
##STR00078##
[0200] .sup.1H NMR (500 MHz, d.sub.6-DMSO): 1.22 (s, 6H); 1.69 (m,
8H); 2.37 (m, 2H); 2.40 (m, 2H); 2.48 (m, 2H); 2.53 (t, J=6.1 Hz,
2H); 2.72 (m, 2H); 2.92 (m, 2H); 3.04 (m, 2H); 3.11 (s, 2H); 3.19
(q, J=5.4 Hz, 2H); 3.39 (t, J=5.4 Hz, 2H); 3.42 (m, 4H); 3.49 (m,
12H); 3.59 (s, 3H); 3.62 (t, J=6.1 Hz, 2H); 3.86 (s, 6H); 3.88 (m,
2H); 4.10 (m, 4H); 4.22 (m, 2H); 5.17 (d, J=13.0 Hz, 2H); 5.22 (d,
J=13.0 Hz, 2H); 5.55 (m, 2H); 6.94 (s, 2H); 7.09 (s, 2H); 7.38 (s,
2H); 7.77 (d, J=3.9 Hz, 2H); 8.01 (t, J=5.4 Hz, 1H). LC/MS (B):
rt=3.30 min; [M+H].sup.+: m/z 1225; [M+2H].sup.2+: m/z 613 (base
peak).
4.5. Methyl
3-{2-[2-(2-{2-[2-(4-{4-[2-(2,6-bis(hydroxymethyl)pyridin-4-yloxy)ethyl]pi-
perazin-1-yl}-1,1-dimethyl-4-oxobutylsulphanyl)acetylamino]ethoxy}ethoxy)e-
thoxy]ethoxy}-propanoate
[0201] Prepared according to Example 2 from
4-(2-[4-(4-mercapto-4-methylpentanoyl)piperazin-1-yl]ethoxy)-2,6-bis(hydr-
oxymethyl)pyridine
##STR00079##
[0202] .sup.1H NMR (500 MHz, d.sub.6-DMSO): all the signals are
broad with 1.22 (s, 6H); 1.71 (m, 2H); 2.20 to 2.58 (partially
masked m, 8H); 2.75 (m, 2H); 3.12 (m, 2H); 3.19 (m, 2H); 3.47 to
3.53 (m, 18H); 3.60 (m, 5H); 4.18 (m, 2H); 4.46 (m, 4H); 5.31 (m,
2H); 6.86 (s, 2H); 8.00 (m, 1H). LC/MS (B): rt=2.20 min;
[M+H].sup.+: m/z 717; [M-H+HCO.sub.2H].sup.-: m/z 761.
4.6.
4-(2-[4-(4-mercapto-4-methylpentanoyl)piperazin-1-yl]ethoxy)-2,6-bis(-
hydroxymethyl)pyridine
[0203] Prepared according to Example 2 from
4-(2-[4-(4-methyl-4-(methyldisulphanyl)pentanoyl)piperazin-1-yl]ethoxy)-2-
,6-bis(hydroxymethyl)pyridine
##STR00080##
[0204] .sup.1H NMR (400 MHz, d.sub.6-DMSO): 1.32 (s, 6H); 1.76 (m,
2H); 2.36 to 2.53 (partially masked m, 6H); 2.66 (s, 1H); 2.75 (t,
J=5.5 Hz, 2H); 3.40 to 3.52 (m, 4H); 4.18 (t, J=5.5 Hz, 2H); 4.46
(s, 4H); 5.30 (very broad m, 2H); 6.86 (s, 2H). LC/MS (B): rt=0.76
min; [M+H].sup.+: m/z 398; [M-H].sup.-: m/z 396.
4.7.
4-(2-[4-(4-methyl-4-(methyldisulphanyl)pentanoyl)piperazin-1-yl]ethox-
y)-2,6-bis(hydroxymethyl)pyridine
##STR00081##
[0206] 344 .mu.L of TEA and then, after stirring for 10 min, 748 mg
of 4-methyl-4-(methyldisulphanyl)pentanoic acid, 417 .mu.l of
diisopropylcarbodiimide and 69 mg of 1-hydroxybenzotriazole hydrate
are added to 600 mg of
4-[2-(piperazin-1-yl)ethoxy]-2,6-bis(hydroxymethyl)pyridine in
solution in 12 ml of DMF. After 15 h at AT, the mixture is
concentrated under RP, 15 ml of water are added and extraction is
carried out 2.times. with AcOEt. The combined organic phases are
dried over MgSO.sub.4 and concentrated under RP, and the residue is
purified by flash chromatography on silica (Analogix Super Flash
SiO.sub.2 SF25-80 g) using a gradient from 2 to 10% of MeOH in DCM.
390 mg of
4-(2-[4-(4-methyl-4-(methyldisulphanyl)pentanoyl)piperazin-1-yl]ethoxy)-2-
,6-bis(hydroxymethyl)pyridine are thus obtained.
[0207] .sup.1H NMR (400 MHz, d.sub.6-DMSO): 1.26 (s, 6H); 1.79 (m,
2H); 2.36 (m, 2H); 2.39 (s, 3H); 2.40 to 253 (partially masked m,
4H); 2.75 (t, J=5.6 Hz, 2H); 3.45 (m, 4H); 4.18 (t, J=5.6 Hz, 2H);
4.46 (d, J=5.9 Hz, 4H); 5.30 (t, J=5.9 Hz, 2H); 6.86 (s, 2H). LC/MS
(A): rt=0.42 min; [M+H].sup.+: m/z 444; [M-H+HCO.sub.2H].sup.-: m/z
488.
4.8.
4-[2-(Piperazin-1-yl)ethoxy]-2,6-bis(hydroxymethyl)pyridine
##STR00082##
[0209] 19 ml of a 4M solution of HCl in dioxane are added to 2.3 g
of
4-(2-(4-(tert-butoxycarbonyl)piperazin-1-yl)ethoxy)-2,6-bis(hydroxymethyl-
)pyridine in solution in 33 ml of dioxane. After 12 h at AT, the
resulting precipitate is recovered by filtration on a sintered
glass funnel, taken up in MeOH and then concentrated under RP, and
the residue is diluted in 40 ml of an MeOH/water 1/1 mixture. The
resulting solution is deposited on Mega BE-SCX, 25 GM 150 ML
(Varian). After washing the phase with MeOH, the product of
interest is eluted with a 2N solution of ammonia in methanol. The
MeOH/NH.sub.3 phase is concentrated under RP. 1.6 g of
4-[2-(piperazin-1-yl)ethoxy]-2,6-bis(hydroxymethyl)pyridine are
thus obtained.
[0210] .sup.1H NMR (400 MHz, d.sub.6-DMSO): 2.40 (m, 4H); 2.68 (m,
6H); 4.15 (t, J=5.7 Hz, 2H); 4.44 (m, 4H);
[0211] 5.29 (m, 2H); 6.85 (s, 2H). LC/MS (A): rt=0.10 min;
[M+H].sup.+: m/z 268; [M+2H]2.sup.+: m/z 134.5; base peak: m/z
113.
4.9.
4-(2-(4-(tert-butoxycarbonyl)piperazin-1-yl)ethoxy)-2,6-bis(hydroxyme-
thyl)pyridine
##STR00083##
[0213] 779 mg of sodium borohydride and 2.29 g of CaCl.sub.2 are
added to 3.1 g of diethyl
4-[2-(4-(tert-butoxycarbonyl)piperazin-1-yl)ethoxy]pyridine-2,6-dicarboxy-
late in solution in 105 ml of EtOH. After 3 h at AT, the mixture is
hydrolysed and concentrated under RP. Water is added to the residue
obtained and the resulting aqueous phase is extracted 4.times. with
AcOEt. The combined organic phases are washed with a saturated
aqueous NaCl solution and then concentrated under RP. 2.4 g of
4-(2-(4-(tert-butoxycarbonyl)piperazin-1-yl)ethoxy)-2,6-bis(hydroxymethyl-
)pyridine are thus obtained.
[0214] .sup.1H NMR (400 MHz, d.sub.6-DMSO): 1.39 (s, 9H); 2.43 (m,
4H); 2.73 (t, J=5.6 Hz, 2H); 3.30 (partially masked m, 4H); 4.17
(t, J=5.6 Hz, 2H); 4.45 (d, J=5.9 Hz, 4H); 5.30 (t, J=5.9 Hz, 2H);
6.86 (s, 2H). LC/MS (A): rt=0.24 min; [M+H].sup.+: m/z 368.
4.10. Diethyl
4-[2-(4-(tert-butoxycarbonyl)piperazin-1-yl)ethoxy]pyridine-2,6-dicarboxy-
late
##STR00084##
[0216] 4.5 ml of TEA and then 2 ml of methanesulphonyl chloride are
added to a solution, cooled to 0.degree. C., of 5 g of
1-(tert-butoxycarbonyl)-4-(2-hydroxyethyl)piperazine in 102 ml of
DCM. After 1 h, the mixture is brought back to AT. After stirring
for an additional 1 h, the mixture is hydrolysed and the organic
phase is washed 2.times. with water, then dried over MgSO.sub.4 and
concentrated under RP. 140 ml of DMF are added to the residue thus
obtained (6.7 g) and the mixture is brought to 60.degree. C. 190 mg
of the diethyl ester of chelidamic acid (Scrimin P., Tecilla P.,
Tonellato U. and Vendrame T. J., Org. Chem., 1989, 54, 5988) and
549 mg of K.sub.2CO.sub.3 are then added and the mixture is heated
at 80.degree. C. for 20 h. After concentrating under RP, the
mixture is hydrolysed and extracted 3.times. with AcOEt, the
combined organic phases are washed with a saturated NaCl solution
and concentrated under RP, and the residue is purified by flash
chromatography on silica (Analogix Super Flash SiO.sub.2 SF25-150
g) using a gradient from 60 to 85% of AcOEt in heptane. 3.1 g of
diethyl
4-[2-(4-(tert-butoxycarbonyl)piperazin-1-yl)ethoxy]pyridine-2,6-dicarboxy-
late are thus obtained.
[0217] .sup.1H NMR (400 MHz, d.sub.6-DMSO): 1.34 (t, J=7.2 Hz, 6H);
1.39 (s, 9H); 2.44 (m, 4H); 2.76 (t, J=5.7 Hz, 2H); 3.30 (partially
masked m, 4H); 4.34 (t, J=5.7 Hz, 2H); 4.38 (q, J=7.1 Hz, 4H); 7.74
(s, 2H). LC/MS (B): rt=2.81 min; [M+H].sup.+: m/z 452;
[MH+HCO.sub.2H].sup.-: m/z 496.
Example 5
5.1.
4-{2-[4-(2-Methyl-2-(methyldisulphanyl)propyl)piperazin-1-yl]ethoxy}--
2,6-bis(hydroxymethyl)pyridine
[0218] Prepared according to Example 1 from
4-[2-(piperazin-1-yl)ethoxy]-2,6-bis(hydroxymethyl)-pyridine.
##STR00085##
[0219] .sup.1H NMR (400 MHz, d.sub.6-DMSO): 1.25 (s, 6H); 2.39 (s,
3H); 2.40 (s, 2H); 2.44 to 2.57 (partially masked m, 8H); 2.69 (t,
J=5.7 Hz, 2H); 4.14 (t, J=5.7 Hz, 2H); 4.45 (d, J=5.7 Hz, 4H); 5.29
(t, J=5.7 Hz, 2H); 6.85 (s, 2H). LC/MS (B): rt=0.51 min;
[M+H].sup.+: m/z 402.
Example 6
6.1. Preparation of the Conjugate
[0220] A conjugate is prepared as for Ex. 1 by reacting hu2H11 and
N-hydroxysuccinimidyl
3-{2-[2-(2-{2-[2-(2-{[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11-
a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridin-4-
-yloxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)-acetyl(methyl)amin-
o]ethoxy}ethoxy)ethoxy]ethoxy}propanoate. The conjugate obtained is
quantitatively determined by spectrophotometry using the extinction
coefficients e.sub.319 nm=7789 M.sup.-1 cm.sup.-1 and e.sub.280
nm=4362 M.sup.-1 cm.sup.-1 for the tomaymycin dimer and e.sub.280
nm=208 380 M.sup.-1 cm.sup.-1 for hu2H11: a mean of 2.90 tomaymycin
dimers per antibody of molecule was determined.
6.2. N-hydroxysuccinimidyl
3-{2-[2-(2-{2-[2-(2-{[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11-
a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridin-4-
-yloxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)acetyl(methyl)amino-
]ethoxy}ethoxy)ethoxy]ethoxy}propanoate
[0221] Prepared according to Example 2 from
3-{2-[2-(2-{2-[2-(2-{[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11-
a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridin-4-
-yloxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)acetyl(methyl)amino-
]ethoxy}ethoxy)-ethoxy]ethoxy}propanoic acid:
##STR00086##
[0222] LC/MS (F): rt=1.02 min; [M+H].sup.+: m/z 1225;
[M+H.sub.2O+H].sup.+: m/z 1243
6.3.
3-{2-[2-(2-{2-[2-(2-{[2-(2,6-bis[(S)-2-Eth-(E)-ylidene-7-methoxy-1,2,-
3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyri-
din-4-yloxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)acetyl(methyl)-
amino]ethoxy}ethoxy)ethoxy]ethoxy}propanoic acid
[0223] Prepared according to Ex. 2 from methyl
3-{2-[2-(2-{2-[2-(2-{[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11-
a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridin-4-
-yloxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)acetyl(methyl)amino-
]ethoxy}ethoxy)-ethoxy]ethoxy}propanoate:
##STR00087##
[0224] LC/MS (F): rt=0.97 min; [M+H].sup.+: m/z 1129
[0225] 6.4. Methyl
3-{2-[2-(2-{2-[2-(2-{[2-(2,6-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11-
a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one-8-yloxymethyl]pyridin-4-
-yloxy)ethyl](methyl)amino}-1,1-dimethylethylsulphanyl)acetyl(methyl)amino-
]ethoxy}-ethoxy)ethoxy]ethoxy}propanoate
[0226] Prepared according to Ex. 2 from methyl
3-{2-[2-(2-{2-[2-(2-{[2-(2,6-bis(hydroxymethyl)pyridin-4-yloxy)ethyl](met-
hyl)amino}-1,1-dimethylethylsulphanyl)acetyl(methyl)amino]ethoxy}ethoxy)-e-
thoxy]ethoxy}propanoate:
##STR00088##
[0227] .sup.1H NMR (500 MHz, d.sub.6-DMSO): the absorptions are
broad with 1.20 (s, 6H); 1.58 to 1.73 (m, 6H); 2.38 (s, 3H); 2.52
to 3.07 (m, 13H); 3.36 to 3.63 (m, 23H); 3.67 to 3.91 (m, 8H); 4.06
to 4.26 (m, 6H); 5.02 to 5.26 (m, 4H); 5.33 to 5.60 (m, 2H); 6.39
to 7.42 (m, 6H); 7.76 (m, 2H). LC/MS (A): rt=0.77 min; [M+H].sup.+:
m/z 1142; [M+2H].sup.2+: m/z 571.5 (base peak).
6.5. Methyl
3-{2-[2-(2-{2-[2-(2-{[2-(2,6-bis(hydroxymethyl)pyridin-4-yloxy)ethyl](met-
hyl)-amino}-1,1-dimethylethylsulphanyl)acetyl(methyl)amino]ethoxy}ethoxy)e-
thoxy]ethoxy}-propanoate
[0228] Prepared according to Ex. 2 from methyl
3-{2-[2-(2-{2-[(2-iodoacetyl)(methyl)amino]ethoxy}-ethoxy)ethoxy]ethoxy}p-
ropanoate:
##STR00089##
[0229] .sup.1H NMR (400 MHz, d.sub.6-DMSO): 50/50 resolution of
rotamers with: 1.22 (s, 3H); 1.24 (s, 3H); 2.41 (s, 3H); 2.53 (t,
J=6.2 Hz, 2H); 2.56 (m, 2H); 2.80 (s, 1H); 2.88 (t, J=5.9 Hz, 2H);
3.05 (s, 2H); 3.35 to 3.58 (m, 18H); 3.59 (s, 3H); 3.62 (t, J=6.2
Hz, 2H); 4.13 (t, J=5.9 Hz, 2H); 4.45 (d, J=5.9 Hz, 4H); 5.29 (t,
J=5.9 Hz, 2H); 6.85 (s, 2H). LC/MS (B): rt=2.18 min; [M+H].sup.+:
m/z 634; [M-H+HCO.sub.2H].sup.-: m/z 678.
6.6. Methyl
3-{2-[2-(2-{2-[(2-iodoacetyl)(methyl)amino]ethoxy}ethoxy)ethoxy]ethoxy}-p-
ropanoate
##STR00090##
[0231] 117.4 mg of N-hydroxysuccinimidyl iodoacetate in solution in
6.5 ml of DCM are added to 215 mg of methyl
3-(2-{2-[2-(2-(methylamino)ethoxy)ethoxy]ethoxy}ethoxy)propanoate.
After 2 h at AT, the mixture is concentrated under RP and the
residue is purified by flash chromatography on silica (Analogix
Super Flash SiO.sub.2 SF25-24 g) using a gradient from 0 to 6% of
MeOH in DCM. 210 mg of methyl
3-{2-[2-(2-{2-[(2-iodoacetyl)(methyl)amino]ethoxy}ethoxy)ethoxy-
]ethoxy}-propanoate are thus obtained. .sup.1H NMR (400 MHz,
d.sub.6-DMSO): 55/45 resolution of rotamers with 2.54 (t, J=6.2 Hz,
2H); 2.83 (s, 1.35H); 3.02 (s, 1.65H); 3.36 to 3.55 (m, 16H); 3.60
(s, 3H); 3.63 (t, J=6.2 Hz, 2H); 3.83 (s, 1.1H); 3.88 (s, 0.9H).
LC/MS (A): rt=0.62 min; [M+H].sup.+: m/z 462.
6.7. Methyl
3-(2-{2-[2-(2-(methylamino)ethoxy)ethoxy]ethoxy}ethoxy)propanoate
##STR00091##
[0233] 3 ml of a 4M solution of HCl in dioxane are added to 390 mg
of methyl
3-[2-(2-{2-[2-((tert-butoxycarbonyl)(methyl)amino}ethoxy]ethoxy}et-
hoxy)ethoxy]propanoate in solution in 5.3 ml of dioxane. After 12 h
at AT, the mixture is concentrated under RP and the residue is
dissolved in a minimum of methanol and deposited on Mega BE-SCX, 10
GM 60 ML (Varian). After washing the phase with MeOH, the product
of interest is eluted with a 2N solution of ammonia in methanol.
The methanol/NH.sub.3 phase is concentrated under RP. 270 mg of
methyl
3-(2-{2-[2-(2-(methylamino)ethoxy)ethoxy]ethoxy}ethoxy)propanoate
are thus obtained. .sup.1H NMR (400 MHz, d.sub.5-DMSO): 2.28 (s,
3H); 2.54 (t, J=6.2 Hz, 2H); 2.59 (t, J=5.9 Hz, 2H); 3.44 (t, J=5.9
Hz, 2H); 3.47 to 3.54 (m, 12H); 3.60 (s, 3H); 3.63 (t, J=6.2 Hz,
2H). LC/MS (A) (ELSD): rt=0.30 min; [M+H].sup.+: m/z 294.
6.8. Methyl
3-[2-(2-{2-[2-((tert-butoxycarbonyl)(methyl)amino)ethoxy]ethoxy}ethoxy)-e-
thoxy]propanoate
##STR00092##
[0235] 2 ml of a 2M solution of trimethylsilyldiazomethane in
hexane are added to a solution, cooled to 0.degree. C., of 500 mg
of
3-[2-(2-{2-[2-((tert-butoxycarbonyl)(methyl)amino)ethoxy]ethoxy}ethoxy)-e-
thoxy]propanoic acid in 4.8 ml of MeOH. After 2 h, the mixture is
neutralized by addition of 120 .mu.l of acetic acid and then
concentrated under RP, and the residue is purified by flash
chromatography on silica (Analogix Super Flash SiO.sub.2 SF25-40 g)
using a gradient from 0 to 5% of methanol in DCM. 400 mg of methyl
3-[2-(2-{2-[2-((tert-butoxycarbonyl)(methyl)amino)ethoxy]ethoxy}ethoxy)et-
hoxy]propanoate are thus obtained.
[0236] .sup.1H NMR (400 MHz, d.sub.6-DMSO): 1.38 (s, 9H); 2.54 (t,
J=6.2 Hz, 2H); 2.80 (broad s, 3H); 3.29 (partially masked t, J=5.9
Hz, 2H); 3.45 to 3.55 (m, 14H); 3.59 (s, 3H); 3.63 (t, J=6.2 Hz,
2H). LC/MS (A): rt=0.84 min; [M+Na].sup.+: m/z 416 (base peak);
LC/MS (A): rt=0.84 min; [M+Na].sup.+: m/z 416 (base peak).
6.9.
3-[2-(2-{2-[2-((tert-Butoxycarbonyl)(methyl)amino)ethoxy]ethoxy}ethox-
y)ethoxy]-propanoic acid
##STR00093##
[0238] 85.4 mg of NaH are added portionwise to a solution, cooled
to 0.degree. C., of 520 mg of
3-[2-(2-{2-[2-((tert-butoxycarbonyl)amino)ethoxy]ethoxy}ethoxy)ethoxy]pro-
panoic acid in 14 ml of THF. After stirring for 5 min, 150 .mu.l of
iodomethane are added. The mixture is then stirred at AT for 2 h
and then an additional 150 .mu.l of iodomethane are added. After 12
h at AT, the mixture is hydrolysed and then brought to acidic pH by
addition of aqueous acetic acid at 0.degree. C. The aqueous phase
is extracted 3.times. with AcOEt, the combined organic phases are
dried over MgSO.sub.4 and concentrated under RP, and the crude
product obtained is reacted again according to the same protocol
with 85 mg of NaH and 176 .mu.l of iodomethane for an additional 2
h. 500 mg of
3-[2-(2-{2-[2-((tert-butoxycarbonyl)(methyl)amino)ethoxy]ethoxy}ethoxy)et-
hoxy]propanoic acid are thus obtained.
[0239] LC/MS (F): rt=1.01 min; [M+H].sup.+: m/z 380.
Evaluation of the Inhibition of the Proliferation of the
MDA-MB-231, MDA-A1 and HCT116 Cell Lines by the Compounds of
Formula IA with Z.sub.bR.sub.b=--OMe and Y.dbd.Y'=--OMe
[0240] The MDA-MB-231, MDA-A1 or HCT116 cells in their exponential
growth phase are trypsinized and resuspended in their respective
culture medium (DMEM/F12 Gibco #21331, 10% FCS Gibco #10500-056, 2
nM glutamine Gibco #25030 for the MDA cells; DMEM Gibco #11960, 10%
FCS Gibco #10500-056, 2 mM glutamine Gibco #25030 for the HCT116
cells). The cell suspension is seeded in Cytostar 96-well culture
plates (GE Healthcare Europe, #RPNQ0163) in the complete culture
medium containing serum at a density of 5000 cells/well
(MDA-MB-231, MDA-A1, HCT116). After incubation for 4 hours,
successive dilutions of the tomaymycin dimers are added to the
wells in triplicate for each concentration. The cells are cultured
for 3 days at 37.degree. C. under an atmosphere containing 5%
CO.sub.2 in the presence of the cytotoxic agents. On the 4.sup.th
day, 10 .mu.l of a .sup.14C-thymidine solution (0.1 .mu.Ci/well,
Perkin Elmer #NEC56825000) are added to each well. The
incorporation of .sup.14C-thymidine is measured 96 hours after the
start of the experiment using a MicroBeta radioactivity counter
(Perkin Elmer). The data are expressed in the form of a % of
survival by determining the ratio between the count obtained with
the cells treated with the cytotoxic agent and the count obtained
with the cells of the control wells (treated with the culture
medium alone).
TABLE-US-00002 TABLE II IC.sub.50 [nM] Inhibition of the
proliferation (.sup.14C-thymidine pulse at 96 h) MDA- MDA-
Structure of the compound of formula (IA) HCT116 A1 MB231
##STR00094## 0.030 1.991 0.009 ##STR00095## 0.120 2.260 0.070
##STR00096## 0.127 >10 0.084 ##STR00097## 1.250 >10 0.450
[0241] It is found that the test compounds for which
Z.sub.bR.sub.b=--OMe have a powerful anticancer activity; this
allows it to be supposed that the similar compounds characterized
by another Z.sub.bR.sub.b group are liable to exhibit an identical
activity.
Evaluation of the Inhibition of the Proliferation of the MDA-MB-231
Cell Lines by the hu2H11-Cytotoxic Agent Conjugates
[0242] MDA-MB-231 cells in their exponential growth phase are
trypsinized and resuspended in their culture medium (DMEM/F12 Gibco
#21331, 10% FCS Gibco #10500-056, 2 nM glutamine Gibco #25030). The
cell suspension is seeded in Cytostar 96-well culture plates (GE
Healthcare Europe, #RPNQ0163) in the complete culture medium
containing serum at a density of 5000 cells/well. After incubation
for 4 hours, successive dilutions of the antibody-cytotoxic agent
immunoconjugates are added to the wells at concentrations
decreasing from 10.sup.-7 to 10.sup.-12 M (in triplicate for each
concentration). The cells are cultured at 37.degree. C. in an
atmosphere containing 5% CO.sub.2 in the presence of the
antibody-cytotoxic agent immunoconjugates for 3 days. On the
4.sup.th day, 10 .mu.l of a .sup.14C-thymidine solution (0.1
.mu.Ci/well, Perkin Elmer #NEC56825000) are added to each well. The
incorporation of .sup.14C-thymidine is measured 96 hours after the
start of the experiment with a MicroBeta radioactivity counter
(Perkin Elmer). The data are expressed in the form of a % of
survival by determining the ratio between the count obtained with
the cells treated with the immunoconjugate and the count obtained
with the cells of the control wells (treated with the culture
medium alone). In certain experiments*, the naked antibody hu2H11
was added to the wells at a concentration of 1 .mu.M at the start
of the experiment and the inhibition of the proliferation was
measured as described above.
TABLE-US-00003 TABLE III IC.sub.50 [pM] Mean IC.sub.50 Inhibition
of the proliferation (.sup.14C-thymidine pulse at 96 h) Mean
(+naked IC.sub.50 Structure IC.sub.50 hu2H11*) ratio ##STR00098##
31 540 17 ##STR00099## 910 5095 6 ##STR00100## 54 3981 74
##STR00101## 1255 9125 7 ##STR00102## 68.5 1119 16
Monomer Stability of the Noncleavable Tomaymycin Dimer
Conjugates
[0243] The noncleavable hu2H11 conjugates prepared with tomaymycin
dimers described in Examples 3 and 5 of International Application
WO 09016516 have a tendency to experience a decrease in their
monomer purity over time after storing for several months at
3-5.degree. C. Specifically, these conjugates, formulated in an
aqueous buffer at pH 6.5 with a histidine concentration of 10 mM
containing 10% of sucrose and 5% of NMP, exhibiting a mean of 3 to
3.5 tomaymycin dimers per antibody molecule, with an initial
monomer purity of the order of 97.5%, can decline in monomer purity
by 6 to 15% in 6 to 8 months. This phenomenon was not observed with
the conjugate of Example 3 which, with a mean of 3.5 tomaymycin
dimers per antibody molecule, retains a monomer purity of greater
than 99% in 4 months under the same storage conditions, thus
suggesting a better stability of this particular conjugate. This is
a general objective desired in the field of conjugates to be able
to have available a conjugate which retains a monomer purity over
time.
Evaluation of the Antitumour Activity of hu2H11-Cytotoxic Agent
Conjugates on Female SCID Mice Carrying an Advanced Human Breast
Adenocarcinoma, MDA-MB-231
[0244] Two conjugates of the same antibody hu2H11, denoted C1
(prepared with the tomaymycin dimer described in Example 5 of
International Application WO 09016516) and C2 (conjugate of Example
3), were evaluated at 4 dose levels on measurable mammary tumours
MDA-MB-231 implanted s.c. on female SCID mice. The control groups
were not treated. The doses of the two conjugates are given in
.mu.g of tomaymycin dimer/kg. They were administered at 80, 40, 20
and 10 .mu.g/kg by injection of intravenous (IV) bolus type on day
13 for C1 and on day 24 for C2.
[0245] As regards the evaluation of the antitumour activity of the
conjugates, the animals were weighed daily and the tumours were
measured twice weekly using a calliper rule. The antitumour
activity was evaluated at the Maximum Tolerated Dose (MTD). A dose
producing a loss in weight of 20% at nadir or 10% (or more)
mortality linked to the conjugate is regarded as being toxic. The
body weight of the animals includes the weight of the tumours. The
weight of the tumours is calculated according to the formula:
weight (mg)=[length (mm).times.width (mm).sup.2]/2. The efficacy
parameters are the .DELTA.T/.DELTA.C, the mean percentage of
regression, the partial and complete regressions (PR and CR) and
the number of tumour-free mice at the end of the study (TFM).
[0246] The change in the volume of the tumours for each treated
mouse (T) and control mouse (C) is calculated for each tumour by
subtracting the volume of the tumour on the day of the start of the
study from the volume of the tumour on the day of observation
specified. The mean AT is calculated for the treated group and the
mean AC is calculated for the control group. The ratio
.DELTA.T/.DELTA.C is then calculated and expressed as a percentage:
.DELTA.T/.DELTA.C=(delta T/delta C).times.100.
[0247] A dose is regarded as being therapeutically active for a
.DELTA.T/.DELTA.C of less than 40% and very active for a
.DELTA.T/.DELTA.C of less than 10%. When the .DELTA.T/.DELTA.C is
less than 0, the dose is regarded as highly active and the
percentage of regression is then calculated (according to Plowman
J., Dykes D. J., Hollingshead M., Simpson-Herren L. and Alley M.
C., Human tumor xenograft models in NCI drug development: Feibig H.
H. BA, editor. Basel: Karger.; 1999, pp. 101-125).
[0248] The percentage of tumour regression is defined as being the
percentage of decrease in the tumour volume in the treated group on
the day of observation specified in comparison with its volume on
the first day of treatment. The percentage of tumour regression is
calculated at a specific time t and for each animal. The mean
percentage of regression is then calculated for the group.
Percentage of regression ( at time t ) = volume t 0 - volume t
volume t 0 .times. 100 ##EQU00001##
[0249] Partial regression (PR): The regressions are defined as
being partial when the decrease in the tumour volume reaches 50% of
the volume of the tumour at the beginning of treatment.
[0250] Complete regression (CR): The complete regression is
obtained when the volume of the tumour=0 mm.sup.3 (a CR is regarded
as being present when the volume of the tumour cannot be
measured).
[0251] TFM: The tumour-free mice are defined as being mice not
exhibiting a detectable tumour at the end of the study (beyond 100
days of treatment).
TABLE-US-00004 TABLE IV Evaluation of the antitumour activity of
the conjugates C1 and C2 on female SCID mice carrying an advanced
human breast adenocarcinoma, MDA-MB-231 Mean Adminis- Dose in loss
in Mean tration .mu.g/kg of weight Mean percentage route/Dose in
tomaymycin Day of in % at .DELTA.T/.DELTA.C of re- Biological
Conju- ml/kg by dimer (mg adminis- nadir in % gression Regressions
TFM interpre- gate injection prot/kg) tration Mortality (day) (day)
(day) Partial Complete (day 120) tation C1 IV 80 (3.76) 13 5/5 --
-- -- -- -- -- Toxic DAR 10 ml/kg 40 (1.88) 0/5 -9.6 (20) 7 (28) --
3/5 0/5 0/5 MTD, very 2.9 active 20 (0.94) 0/5 -3.5 (30) 13 (28) --
0/5 0/5 0/5 Active 10 (0.47) 0/5 -1.1 (27) 23 (28) -- 0/5 0/5 0/5
Active C2 IV 80 (2.98) 24 4/6 -- -- -- -- -- -- Toxic DAR 10 ml/kg
40 (1.49) 0/6 -5.0 (31) <0 (46) 96.2 (46) 6/6 1/6 1/6 MTD,
highly 3.5 active 20 (0.75) 0/6 -4.4 (31) <0 (46) 66.4 (46) 4/6
0/6 0/6 Highly active 10 (0.37) 0/6 -4.0 (31) 14 (46) -- 0/6 0/6
0/6 Active Abbreviations: MTD = Maximum Tolerated Dose, TFM =
Tumour-Free Mice at the end of the study
[0252] According to a single-dose administration scheme, the
highest dose tested for these two conjugates in this study (80
.mu.g/kg) proved to be toxic, resulting in loss in weight and
mortality.
[0253] At the MTD (40 .mu.g/kg), C1 is very active and results in a
loss in weight of 9.6% at nadir, a .DELTA.T/.DELTA.C of 7% and 3
PRs for 5 SCID mice. An antitumour activity was also observed at
the lower doses of 20 and 10 .mu.g/kg without resulting in PR.
[0254] At the MTD (40 .mu.g/kg), C2 is highly active and results in
a loss in weight of 5% at nadir and a tumour regression of 96.2%
with 6 PRs for 6 mice, including 1 TFM. It is also very active at
the lower dose of 20 .mu.g/kg with a tumour regression of 66.4% and
4 PRs for 6 mice. An antitumour activity was also observed at the
lowest dose of 10 .mu.g/kg without, however, resulting in
regression or PR.
[0255] In conclusion, it is found that C2 exhibits a strong
anticancer activity and showed a better activity than C1 with a
tumour regression, PR and TFM at the MTD which are not observed
with C1 at the same doses.
* * * * *