U.S. patent application number 10/396129 was filed with the patent office on 2004-09-30 for pyrene-linked pyrrolo[2,1--c][1,4]benzodiazepine hybrids useful as anti-cancer agents.
This patent application is currently assigned to COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH. Invention is credited to Gujjar, Ramesh, Kamal, Ahmed, Olepu, Srinivas, Poddutoori, Ramulu.
Application Number | 20040192677 10/396129 |
Document ID | / |
Family ID | 32988738 |
Filed Date | 2004-09-30 |
United States Patent
Application |
20040192677 |
Kind Code |
A1 |
Kamal, Ahmed ; et
al. |
September 30, 2004 |
PYRENE-LINKED PYRROLO[2,1--C][1,4]BENZODIAZEPINE HYBRIDS USEFUL AS
ANTI-CANCER AGENTS
Abstract
The present invention relates to a process for the preparation
of novel pyrrolo [2,1 -c][1,4]benzodiazepine hybrids useful as
antitumour agents. This invention also relates to a process for the
preparation of new pyrrolo[2,1-c][1,4]benzodiazepine hybrids as
potential antitumour agents. More particularly, it provides a
process for the preparation of
7-methoxy-8-[N-(1"-pyrenyl)-alkane-3'-carboxamide]-oxy-(11aS)-1,2,3,11a-t-
etraydro 5H-pyrrolo[2,1 -c][1,4]benzodiazepin-5-one, with aliphatic
chain length variation of these compounds and it also describes the
DNA binding, aticancer (antitumour) activity. The structral formula
of this novel pyrrolo[2,1-c][1,4]benzodiazepine is given below:
1
Inventors: |
Kamal, Ahmed; (Hyderabad,
IN) ; Gujjar, Ramesh; (Hyderabad, IN) ;
Poddutoori, Ramulu; (Hyderabad, IN) ; Olepu,
Srinivas; (Hyderabad, IN) |
Correspondence
Address: |
LADAS & PARRY
26 WEST 61ST STREET
NEW YORK
NY
10023
US
|
Assignee: |
COUNCIL OF SCIENTIFIC AND
INDUSTRIAL RESEARCH
|
Family ID: |
32988738 |
Appl. No.: |
10/396129 |
Filed: |
March 25, 2003 |
Current U.S.
Class: |
514/220 ;
540/496 |
Current CPC
Class: |
A61K 31/551 20130101;
C07D 487/04 20130101 |
Class at
Publication: |
514/220 ;
540/496 |
International
Class: |
A61K 031/551; C07D
487/04 |
Claims
The process for the preparation of new
pyrrolo[2,1-c][1,4]benzodiazepine hybrids is disclosed and claimed
in our U.S. patent application Ser. No. 10/396,103:
1. A compound of formula V wherein R is H, OH and n is 1-4
11FORMULA V.
2. A compound as claimed in claim 1 of the structural formula
12
3. A compound as claimed in claim 1 of the structural formula
13
4. A compound as claimed in claim 1 of the structural formula
14
5. A compound as claimed in claim 1 of the structural formula
15
6. A compound as claimed in claim 1 of the structural formula
16
7. A compound as claimed in claim 1 of the structural formula
17
8. A compound as claimed in claim 1 of the structural formula
18
9. A compound as claimed in claim 1 of the structural formula
19
10. A process for preparing a compound of formula V 20wheren is
1-4andR is H or, OH FORMULA Vwhich comprises reacting pyrene amine
of formula I 21with (2S)-N-{4-[(3'-carboxy
alkyl)oxy]-5-methoxy-2-nitrobenzoyl}pyrroli- dine-2-carboxyaldehyde
diethyl thioacetal of formula II where R is as defined above;
22isolating (2S)-N-{4-[N-(1"-pyrenyl)-alkane-3'-carboxami-
de]-oxy--5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal III where n is 1-4 and R is as defined above,
23reducing the nitro compound of formula III with
SnCl.sub.2.2H.sub.2O in the presence of an organic solvent up to a
reflux temperature, isolating the
(2S)-N-{4-[N-(1"-pyrenyl)-alkane-3'-carboxamide]-oxy--5-methoxy-2-aminobe-
nzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula IV
where n is 1-4 and R is as defined above, 24and reacting the amino
compound of formula IV with a deprotecting agent to obtain pyrrolo
[2,1-c][1,4]benzodiazepine hybrids of formula V wherein n and R are
as stated above.
11. A process as claimed in claim 10 wherein the reaction between
the compound of formula I and the compound of formula II is carried
out in the presence of isobutyl chloroformate and in the presence
of a base selected from the group consisting of triethyl amine and
DBU; and in the presence of an organic solvent selected from the
group consisting of ethyl acetate, hexane and dichloromethane.
12. The process as claimed in claim 10 wherein the organic solvent
used for the reduction of the nitro compound of formula III
comprises ethyl acetate.
13. A method for the treatment of cancer comprising administering
to a subject suffering therefrom, a therapeutically effective
amount of a compound of formula V wherein R is H, or OH and n is
1-4 wherein the cancer to be treated is leukemia, non-small-cell
lung, colon, CNS, melanoma, ovarian, prostate, or breast cancer
25wheren is 1-4andR is H or, OH FORMULA V.
14. (Cancelled).
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a process for the
preparation of novel pyrrolo [2,1-c][1,4]benzodiazepine hybrids
useful as potential antitmour agents. This invention also relates
to a process for the preparation of new
pyrrolo[2,1-c][1,4]benzodiazepine hybrids as potential antitumour
agents. More particularly, it provides a process for the
preparation of
7-methoxy-8-[N-(1"-pyrenyl)-alkane-3'-carboxamide]-oxy-(11-
aS)-1,2,3,11a-tetraydro 5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one,
with aliphatic chain length variation of these compounds and it
also describes the DNA binding, anticancer (antitumour) activity.
The structural formula of this novel
pyrrolo[2,1-c][1,4]benzodiazepine is given below: 2
BACKGROUND OF THE INVENTION
[0002] Pyrrolo[2,1-c][1,4]benzodiazepine antitumour antibiotics are
commonly known as anthramycin class of compounds. In the last few
years, a growing interest has been shown in the development of new
pyrrolo[2,1-c][1,4]benzodiazepines (PBDs). These antibiotics react
covalently with DNA to form an N2-guanine adduct that lies within
the minor groove of duplex DNA via an acid-labile animal bond to
the electrophilic imine at the N10-C11 position (Kunimoto, S.;
Masuda T.; Kanbayashi, N.; Hamada, M.; Naganawa, H.; Miyamoto, M.;
Takeuchi, T.; and Unezawa, H. J. Antibiot., 1980, 33, 665.; Kobn,
K. W. and Speous, C. L. J. Mol. Biol., 1970, 51, 551.; Hurley, L.
H.; Gairpla, C. and Zmijewski, M. Biochem. Biophys. Acta., 1977,
475, 521.; Kaplan, D. J. and Hurley, L. H. Biochmestry, 1981, 20,
7572). The molecules have a right-handed twist, which allows them
to follow the curvature of the minor groove of B-form
double-stranded DNA spanning three base pairs. Recently, PBD dimers
have been developed that comprises two C2-exo-methylene substituted
DC-81 subunits tethered through their C-8 position via an inert
propanedioxy linker (Gregson, S. J.; Howard, P. W.; Hartely, J. A.;
Brooks, N. A; Adams, L. J.; Jenkins, T. C.; Keland, L. R. and
Thurston, D. E. J. Med. Chem. 2001, 44, 737). A recent development
has been the linking of two PBD units through their C-8 positions
to give bisfunctional alkylating agents capable of cross-linking
DNA (Thurston, D. E.; Bose, D. S.; Thomson, A. S.; Howard, P. W.;
Leoni, A.; Croker, S. J.; Jenkins, T. C.; Neidle, S. and Hurley, L.
H. J. Org. Chem., 1996, 61, 8141).
[0003] Recently, a noncross-lining mixed imine-amide PBD dimers
have been synthesized that have significant DNA binding ability and
potent anti tumour activity. (Kamal, A.; Ramesh, G., Laxman, N.;
Ramulu, P.; Srinivas, O.; Neelima, K.; Kondapi, A. K. Srinu, V. B.;
Nagarajaram, H. M. J. Med. Chem. 2002, 45, 4679). 3
[0004] Naturally occurring pyrrolo[2,1-c][1,4]benzodiazepines
belong to a group of antitumour antibiotics derived from
Streptomyces species. Recently, there is much impetus for the PBD
systems as they can recognize and bind to specific sequence of DNA.
Examples of naturally occurring PBD's include anthramycin, DC-81,
tomaymycin sibiromycin and neothramycin. However, the clinical
efficacy for these antibiotics is hindered by several limitations,
such as poor water solubility, cardiotoxicity, development of drug
resistance and metabolic inactivation.
Objects if the Invention
[0005] The main object of the present invention is to provide new
pyrrolo[2,1-c][1,4]-benzodiazepine hybrids useful as antitumour
agents.
[0006] Another objective of the present invention is to provide a
process for the preparation of novel
pyrrolo[2,1-c][1,4]-benzodiazepine hybrids useful as antitumour
agents.
Summary of the Invention
[0007] Accordingly the present invention provides a process for the
preparation of a novel pyrrolo[2,1-c][1,4]benzodiazepine hybrids of
formula V wherein R.dbd.H, OH and n is 1-4 4
[0008] Accordingly the present process provides a process for
preparation of pyrrolo[2,1-c][1,4]benzodiazepine hybrids of formula
V 5
[0009] which comprises reacting pyrene amine of formula I 6
[0010] with (2S)-N-{4-[(3'-carboxy
alkyl)oxy]-5-methoxy-2-nitrobenzoyl}pyr- rolidine-2-carboxaldehyde
diethyl thioacetal of formula II where R is as stated above 7
[0011] in the presence of isobutyl chloroformate, bases like
triethyl amine, DBU in presence of organic solvents up to refluxing
for a period of 24 h isolating
(2S)-N-{4-[N-(1"-pyrenyl)-alkane-3'-carboxamide]-oxy--5-
-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal III where n is 1-4 and R is as stated above by
conventional methods, 8
[0012] reducing the above nitro compounds of formula III with
SnCl.sub.2.2H.sub.2O in presence of organic solvent up to a reflux
temperature, isolating the
(2S)-N-{4-[N-(1"-pyrenyl)-alkane-3'-carboxamid-
e]-oxy--5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula IV where n is 1-4 and R is as stated
above by known methods, 9
[0013] reacting the above said amino compound of formula IV with
known deprotecting agents in a conventional manner to give novel
pyrrolo[2,1-c][1,4]benzodiazepine hybrids of formula V wherein n
and R are as stated above.
Detailed Description of the Invention
[0014] The precursors, pyrene amine of formula I (Banik, B. K.;
Becker, F. F. Bioorg. Med. Chem. 2001, 9, 593) and
(2S)-N-{4-[(3'-carboxy
alkyl)oxy]-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula II (Baraldi, P. G.; Balboni, G.;
Cacciari, B.; Guiotto, A.; Manfredini, S.; Romagnoli, R.; Spalluto,
G.; Thurston, D. E.; Howard, P. W.; Bianchi, N.; Rutigiiano, C.;
Mischiati, C. and Gambari, R. J. Med Chem. 1999, 42, 5131.; Reddy,
B. S. P.; Damayanthi, Y.; Reddy, B. S. N.; Lown, W. J. Anti-Cancer
Drug Design 2000, 15, 225) have been prepared by literature
methods.
[0015] These new analogues of pyrrolo[2,1-c][1,4]benzodiazepine
hybrids linked at C-8 position have shown promising DNA binding
activity and efficient anticancer activity in various cell lines.
The molecules synthesized are of immense biological significance
with potential sequence selective DNA-binding property. This
resulted in design and synthesis of new congeners as illustrated in
Scheme-1, which comprise;
[0016] 1. The ether linkage at C-8 position of DC-81 intermediates
with pyrene ring moiety.
[0017] 2. Refluxing the reaction mixture for 24-48 h.
[0018] 3. Synthesis of C-8 linked PBD antitumour antibiotic hybrid
imines.
[0019] 4. Purification by column chromatography using different
solvents like ethyl acetate, hexane, dichloromethane and methanol.
10
[0020] Some representative compounds of formula V present invention
are given below
[0021] 1.
7-Methoxy-8-[N-(1"-pyrenyl)-methane-1'-carboxamide]-oxy-(11aS)1,-
2,3,11a tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one
[0022] 2.
7-Methoxy-8-[N-(1"-pyrenyl)-methane-1'-carboxamide]-oxy-(4R)-hyd-
roxy (11aS) 1,2,3,11a
tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one
[0023] 3.
7-Methoxy-8-[N(1"-pyrenyl)-ethane-2'-carboxamide]-oxy-(11aS)1,2,-
3,11a tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one
[0024] 4.
7-Methoxy-8-[N-(1"-pyrenyl)-ethane-2'-carboxamide]-oxy-(4R)-hydr-
oxy (11aS) 1,2,3,11a
tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one
[0025] 5.
7-Methoxy-8-[N-(1"-pyrenyl)-propan-3'-carboxamide]-oxy-(11aS)-1,-
2,3,11a tetra-hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one
[0026] 6.
7-Methoxy-8-[N-(1"-pyrenyl)-propane-3'-carboxamide]-oxy-(4R)-hyd-
roxy
(11aS)-1,2,3,11a-tetra-hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-on-
e
[0027] 7.
7-Methoxy-8-[N-(1"-pyrenyl)-butane4'-carboxamide]-oxy-(11aS)-1,2-
,3,11a-tetra-hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one
[0028] 8.
7-Methoxy-8-[N-(1"-pyrenyl)-butane-4'-carboxamide]-oxy-(4R)-hydr-
oxy
(11aS)-1,2,3,11a-tetra-hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one
[0029] The process for the preparation of new
pyrrolo[2,1-c][1,4]benzodiaz- epine hydrids is disclosed and
claimed in our copending copatent application no.______
[0030] The following examples are given by way of illustration and
therefore should not be construed to the present limit of the scope
of invention.
Example 1
[0031] Compound (2S)-N-[4-[(1'-carboxy
methyl)oxy]-5-methoxy-2-nitrobenzoy- l]pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula II (2.29 g, 5 mmol) was taken in dry
CH.sub.2Cl.sub.2 (20 mL), TEA (707 mg, 7 mmol) was added and the
mixture was cooled at 0-5.degree. C. Isobutyl chloroformate (819
mg, 6 mmol) in dry CH.sub.2Cl.sub.2 (10 mL) was added dropwise and
the mixture was kept at 0-5.degree. C. for 15 min. A solution of
1-amino pyrene of formula I (251 mg, 5 mmol) in CH.sub.2Cl.sub.2
was added to it at the same temperature and the solution was
stirred at room temperature for overnight. The mixture was washed
with saturated NaHCO.sub.3 (50 mL), brine, dried and solvent was
evaporated. The crude material was chromatographed over silica gel
using ethyl acetate/hexane (8:2) solvent to give compound
(2S)-N-{4-[N-(1"-pyrenyl)-methane-3'-carboxamide]-oxy--5-
-methoxy-2-nitro-benzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal of formula III as a yellow liquid.
[0032] The
(2S)-N-{4-[N-(1"-pyrenyl)-methane-1'-carboxamide]-oxy--5-methox-
y-2-nitro benzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal
of formula III (0.657 g, 1 mmol) was dissolved in ethyl acetate (15
mL) and added SnCl.sub.2.2H.sub.2O (1.12 g, 5 mmol) was refluxed
for 3 h or until the TLC indicated that reaction was completed. The
reaction mixture was then adjusted to pH 8 carefully with saturated
NaHCO.sub.3 solution, diluted with ethyl acetate, filtered through
celite and extracted. The combined organic phase was dried over
Na.sub.2SO.sub.4, and evaporated under vacuum to afford the crude
(2S)-N-{4-[N-(1"-pyrenyl)-methane-1'-car-
boxamide]-oxy--5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula IV.
[0033] A solution of
(2S)-N-{4-[N-(1"-pyrenyl)-methane-1'-caboxamide]-oxy--
-5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal of formula IV (627 mg, 1 mmol), HgCl.sub.2 (613 mg, 2.26
mmol) and CaCO.sub.3 (246 mg, 2.46 mmol) in MeCN-water (4:1) was
stirred slowly at room temperature until TLC indicates complete
loss of starting material. The reaction mixture was diluted with
EtOAc (30 mL) and filtered through a celite bed. The clear yellow
organic supernatant was extracted with saturated 5% NaHCO.sub.3 (20
mL), brine (20 mL) and the combined organic phase is dried
(Na.sub.2SO.sub.4). The organic layer was evaporated in vacuum and
purified by column chromatography (90% CH.sub.2Cl.sub.2--MeOH) to
give compound
7-Methoxy-8-[N-(1"-pyrenyl)-propane-3'-carboxamide]-oxy--
(11aS)-1,2,3,11a
tetra-hydro-5H-pyrrolo[2,1-c][1,4]benzo-diazepin-5-one as pale
yellow oil.
Example 2
[0034] Compound (4R)-hydroxy-(2S)-N-[4-[(1'-carboxy
methyl)oxy]-5-methoxy-2-nitro-benzoyl]pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula II (2.37 g, 5 mmol) was taken in dry
CH.sub.2Cl.sub.2 (20 mL), TEA (707 mg, 7 mmol) was added and the
mixture was cooled at 0-5.degree. C. Isobutyl chloroformate (819
mg, 6 mmol) in dry CH.sub.2Cl.sub.2 (10 mL) was added dropwise and
the mixture was kept at 0-5.degree. C. for 15 min. A solution of
1-amino pyrene formula I (251 mg, 5 mmol) in CH.sub.2Cl.sub.2 was
added to it at the same temperature and the solution was stirred at
room temperature for overnight. The mixture was washed with
saturated NaHCO.sub.3 (50 mL), brine, dried and solvent was
evaporated. The crude material was chromatographed over silica gel
using ethyl acetate/hexane (8:2) solvent to give compound
(2S)-N-{4-[N-(1"-pyrenyl)-methane-1'-carboxamide]-oxy--5-methoxy-2-nitrob-
enzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula
III as a yellow liquid.
[0035] The
(4R)-hydroxy-(2S)-N-{4-[N-(1"-pyrenyl)-methane-1'-carboxamide]--
oxy--5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal of formula III (0.673 g, 1 mmol) was dissolved in ethyl
acetate (15 mL) and added SnCl.sub.2.2H.sub.2O (1.12 g, 5 mmol) was
refluxed for 3 h or until the TLC indicated that reaction was
completed. The reaction mixture was then adjusted to pH 8 carefully
with saturated NaHCO.sub.3 solution, diluted with ethyl acetate,
filtered through celite and extracted. The combined organic phase
was dried over Na.sub.2SO.sub.4, and evaporated under vacuum to
afford the crude
(4R)-hydroxy-(2S)-N-{4-[N-(1"-pyrenyl)-methane-1'-carboxamide]-oxy--5-met-
hoxy-2-amino-benzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal of formula IV.
[0036] A solution of
(4R)-hydroxy-(2S)-N-{4-[N-(1"-pyrenyl)-methane-1'-car-
boxamide]-oxy--5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula IV (643 mg, 1 mmol), HgCl.sub.2 (613
mg, 2.26 mmol) and CaCO.sub.3 (246 mg, 2.46 mmol) in MeCN-water
(4:1) was stirred slowly at room temperature until TLC indicates
complete loss of starting material. The reaction mixture was
diluted with EtOAc (30 mL) and filtered through a celite bed. The
clear yellow organic supernatant was extracted with saturated 5%
NaHCO.sub.3 (20 mL), brine (20 mL) and the combined organic phase
is dried (Na.sub.2SO.sub.4). The organic layer was evaporated in
vacuum and purified by column chromatography (90%
CH.sub.2Cl.sub.2--MeOH) to give compound
7-methoxy-8-[N-(1"-pyrenyl)-meth-
ane-1'-carboxamide]-oxy-(4R)-hydroxy-(11aS)-1,2,3,11atetra-hydro-5H-pyrrol-
o[2,1-c][1,4]benzodiazepin-5-one as pale yellow oil.
Example 3
[0037] Compound (2S)-N-[4-[(2'-carboxy
ethyl)oxy]-5-methoxy-2-nitrobenzoyl- ]pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula II (2.36 g, 5 mmol) was taken in dry
CH.sub.2Cl.sub.2 (20 mL), TEA (707 mg, 7 mmol) was added and the
mixture was cooled at 0-5.degree. C., Isobutyl chloroformate (819
mg, 6 mmol) in dry CH.sub.2Cl.sub.2 (10 mL) was added dropwise and
the mixture was kept at 0-5.degree. C. for 15 min. A solution of
1-amino pyrene of formula I (251 mg, 5 mmol) in CH.sub.2Cl.sub.2
was added to it at the same temperature and the solution was
stirred at room temperature for overnight. The mixture was washed
with saturated NaHCO.sub.3 (50 mL), brine, dried and solvent was
evaporated. The crude material was chromatographed over silica gel
using ethyl acetate/hexane (8:2) solvent to give compound
(2S)-N-{4[-(1"-pyrenyl)-ethane-2'-carboxamide-oxy--5-methoxy2-nitro-benzo-
yl}pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula III
as a yellow liquid.
[0038] The
(2S)-N-{4-[N-(1"-pyrenyl)-ethane-2'-carboxamide]-oxy--5-methoxy-
-2-nitro benzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal of
formula III (0.671 g, 1 mmol) was dissolved in ethyl acetate (15
mL) and added SnCl.sub.2.2H.sub.2O (1.12 g, 5 mmol) was refluxed
for 3 h or until the TLC indicated that reaction was completed. The
reaction mixture was then adjusted to pH 8 carefully with saturated
NaHCO.sub.3 solution, diluted with ethyl acetate, filtered through
celite and extracted. The combined organic phase was dried over
Na.sub.2SO.sub.4, and evaporated under vacuum to afford the crude
(28)-N-{4-[N-(1"-pyrenyl)-ethane-2'-carb-
oxamide]-oxy--5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula IV.
[0039] A solution of
(2S)-N-{4-[N-(1"-pyrenyl)-ethane-2'-carboxamide]-oxy--
-5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal of formula IV (641 mg, 1 mmol), HgCl.sub.2 (613 mg, 2.26
mmol) and CaCO.sub.3 (246 mg, 2.46 mmol) in MeCN-water (4:1) was
stirred slowly at room temperature until TLC indicates complete
loss of starting material. The reaction mixture was diluted with
EtOAc (30 mL) and filtered through a celite bed. The clear yellow
organic supernatant was extracted with saturated 5% NaHCo.sub.3 (20
mL), brine (20 mL) and the combined organic phase is dried
(Na.sub.2SO.sub.4). The organic layer was evaporated in vacuum and
purified by column chromatography (90% CH.sub.2Cl.sub.2--MeOH) to
give compound
7-Methoxy-8-[N-(1"-pyrenyl)-ethane-2'-carboxamide]-oxy-(-
11aS)-1,2,3,11atetra-hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one
as pale yellow oil.
Example 4
[0040] Compound (4R)-hydroxy-(2S)-N-[4-[(2'-carboxy
ethyl)oxy]-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula II (2.44 g, 5 mmol) was taken in dry
CHCl.sub.2 (20 mL), TEA (707 mg, 7 mmol) was added and the mixture
was cooled at 0-5.degree. C. Isobutyl chloroformate (819 mg, 6
mmol) in dry CH.sub.2Cl.sub.2 (10 mL) was added dropwise and the
mixture was kept at 0-5.degree. C. for 15 min. A solution of
1-amino pyrene of formula I (251 mg, 5 mmol) in CH.sub.2Cl.sub.2
was added to it at the same temperature and the solution was
stirred at room temperature for overnight. The mixture was washed
with saturated NaHCO.sub.3 (50 mL), brine, dried and solvent was
evaporated. The crude material was chromatographed over silica gel
using ethyl acetate/hexane (8:2) solvent to give compound
(4R)-hydroxy-(2S)-N-{4-[N-(1"-pyrenyl)-ethane-2'-carboxamide]-oxy--5-meth-
oxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal
of formula III as a yellow liquid.
[0041] The
(4R)-hydroxy-(2S)-N-{4-[N-(1"-pyrenyl)-ethane-2'-carboxamide]-o-
xy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal of formula III (0.687 g, 1 mmol) was dissolved in ethyl
acetate (15 mL) and added SnCl.sub.2.2H.sub.2O (1.12 g, 5 mmol) was
refluxed for 3 h or until the TLC indicated that reaction was
completed. The reaction mixture was then adjusted to pH 8 carefully
with saturated NaHCO.sub.3 solution, diluted with ethyl acetate,
filtered through celite and extracted. The combined organic phase
was dried over Na.sub.2SO.sub.4, and evaporated under vacuum to
afford the crude
(4R)hydroxy-(2S)-N-{4-[-(1"-pyrenyl)-ethane-2'-carboxamide]-oxy-5-methoxy-
-2-amino-benzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal of
formula IV.
[0042] A solution of
(4R)-hydroxy-(2S)-N-{4-[N-(1"-pyrenyl)-ethane-2'-carb-
oxamide-oxy--5-methoxy-2aminobenzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula IV (657 mg, 1 mmol), HgCl.sub.2 (613
mg, 2.26 mmol) and CaCO.sub.3 (246 mg, 2.46 mmol) in MeCN-water
(4:1) was stirred slowly at room temperature until, TLC indicates
complete loss of starting material. The reaction mixture was
diluted with EtOAc (30 mL) and filtered through a celite bed. The
clear yellow organic supernatant was extracted with saturated 5%
NaHCO.sub.3 (20 mL), brine (20 mL) and the combined organic phase
is dried (Na.sub.2SO.sub.4), The organic layer was evaporated in
vacuum and purified by column chromatography (90%
CH.sub.2Cl.sub.2--MeOH) to give compound
7-Methoxy-8-[N-(1"-pyrenyl)-etha-
ne-2'-carboxamide]-oxy-(4R)-hydroxy-(11aS)-1,2,3,11atetra-hydro-5H-pyrrolo-
[2,1-c][1,4]benzodiazepin-5-one as pale yellow oil.
Example 5
[0043] Compound (2S)-N-[4-[(3'-carboxy
propyl)oxy]-5-methoxy-2-nitrobenzoy- l]pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula II (2.43 g, 5 mmol) was taken in dry
CH.sub.2Cl.sub.2 (20 mL), TEA (707 mg, 7 mmol) was added and the
mixture was cooled at 0-5.degree. C. Isobutyl chloroformate (819
mug, 6 mmol) in dry CH.sub.2Cl.sub.2 (10 mL) was added dropwise and
the mixture was kept at 0-5.degree. C. for 15 min. A solution of
1-amino pyrene formula I (251 mg, 5 mmol) in CH.sub.2Cl.sub.2 was
added to it at the same temperature and the solution was stirred at
room temperature for overnight. The mixture was washed with
saturated NaHCO.sub.3 (50 mL), brine, dried and solvent was
evaporated. The crude material was chromatographed over silica gel
using ethyl acetate/hexane (8:2) solvent to give compound
(2S)-N-{4-N-(1"-pyrenyl)-propane-3'-carboxamide]-oxy--5--
methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal of formula III as a yellow liquid (1.92 g, 56%).
[0044] .sup.1H NMR (CDCl.sub.3) .delta. 1.10-1.40 (m, 6H),
1.40-2.40 (m, 6H), 2.50-2.90 (m, 4H), 3.10-3.25 (m, 2H), 3.60 (s,
3H), 4.0-4.20 (m, 2H), 4.55-4.85 (m, 2H), 6.70 (s, 1H), 7.62 (s,
1H), 7.70-8.40 (m, 9H), 8.60-8.90 (m, 1H); MS (FAB) 686
[M+H].sup.+.
[0045] The
(2S)-N-{4-[N-(1"-pyrenyl)-propane-3'-carboxamide]-oxy--5-methox-
y-2-nitro benzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal
of formula III (0.685 g, 1 mmol) was dissolved in ethyl acetate (15
mL) and added SnCl.sub.2.2H.sub.2O (1.12 g, 5 mmol) was refluxed
for 3 h or until the TLC indicated that reaction was completed. The
reaction mixture was then adjusted to pH 8 carefully with saturated
NaHCO.sub.3 solution, diluted with ethyl acetate, filtered through
celite and extracted. The combined organic phase was dried over
Na.sub.2SO.sub.4, and evaporated under vacuum to afford the crude
(2S)-N-{4-[N-(1"-pyrenyl)-propane-3'-car-
boxamide]-oxy-5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula IV (458 mg, 70%).
[0046] .sup.1H NMR (CDCl.sub.3) .delta. 1.10-1.40 (m, 6H),
1.50-2.30 (m, 8H), 2.40-2.80 (m, 4H), 3.40 (s, 3H), 3.45-3.60 (m,
2H), 4.05-4.15 (m, 2H), 4.50-4.70 (m, 2H), 6.25 (s, 1H), 6.70 (s,
1H), 7.65-8.30 (m, 9H), 9.10-9.25 (m, 1H).
[0047] A solution of
(2S)-N-{4-[N-(1"-pyrenyl)-propane-3'-carboxamide]-oxy-
--5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal of formula IV (655 mg, 1 mmol), HgCl.sub.2 (613 m, 2.26
mmol) and CaCO.sub.3 (246 mg, 2.46 mmol) in MeCN-water (4:1) was
stirred slowly at room temperature until TLC indicates complete
loss of starting material. The reaction misfire was diluted with
EtOAc (30 mL) and filtered through a celite bed. The clear yellow
organic supernatant was extracted with saturated 5% NaHCO.sub.3 (20
mL), brine (20 mL) and the combined organic phase is dried
Na.sub.2SO.sub.4). The organic layer was evaporated in vacuum and
purified by column chromatography (90% CH.sub.2Cl.sub.2--MeOH) to
give compound
7-Methoxy-8-[N-(1"-pyrenyl)-propane-3'-carboxamide]-oxy--
(11aS)-1,2,3,11atetra-hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one
as pale yellow oil of formula V (285 mg, 54%).
[0048] .sup.1H NMR (CDCl.sub.3) .delta. 1.40-2.40 (m, 10H),
2.60-2.90 (m, 2H), 3.40-4.05 (m, 4H), 4.10-4.40 (m, 2H), 6.85 (s,
1H), 7.40 (s, 1H), 7.65 (d, 1H), 7.75-8.20 (m, 8H), 8.20-8.40 (m,
1H), 9.0-9.10 (m, 1H); MS (FAB) 530 [M+H].sup.+.
Example 6
[0049] Compound (4R)-hydroxy-(2S)-N-[4-[(3'-carboxy
propyl)oxy]-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula II (2.51 g, 5 mmol) was taken in dry
CH.sub.2Cl.sub.2 (20 mL), TEA (707 mg, 7 mmol) was added and the
mixture was cooled at 0-5.degree. C. Isobutyl chloroformate (819
mg, 6 mmol) in dry CH.sub.2Cl.sub.2 (10 mL) was added dropwise and
the mixture was kept at 0-5.degree. C. for 15 min. A solution of
1-amino pyrene of formula I (251 mg, 5 mmol) in CH.sub.2Cl.sub.2
was added to it at the same temperature and the solution was
stirred at room temperature for overnight. The mixture was washed
with saturated NaHCO.sub.3 (50 mL), brine, dried and solvent was
evaporated. The crude material was chromatographed over silica gel
using ethyl acetate/hexane (8:2) solvent to give compound (4R
hydroxy-(2S)-N-{4-[N-(1"-pyrenyl)-propane-3'-carboxa-
mide]-oxy--5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula III as a yellow liquid.
[0050] The
(4R)-hydroxy-(2S)-N-{4-[N-(1"-pyrenyl)-propane-3'-carboxamide]--
oxy--5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal of formula III (0.701 g, 1 mmol) was dissolved in ethyl
acetate (15 mL) and added SnCl.sub.2.2H.sub.2O (1.12 g, 5 mmol) was
refluxed for 3 h or until the TLC indicated that reaction was
completed. The reaction mixture was then adjusted to pH 8 carefully
with saturated NaHCO.sub.3 solution, diluted with ethyl acetate,
filtered through celite and extracted. The combined organic phase
was dried over Na.sub.2SO.sub.4, and evaporated under vacuum to
afford the crude
(4R)-hydroxy-(2S)-N-{4-[N-(1"-pyrenyl)-propane-3'-carboxamide-oxy--5-meth-
oxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal
of formula IV.
[0051] A solution of
(4R)-hydroxy-(2S)-N-{4-[N-(1"-pyrenyl)-propane-3'-car-
boxamide]-oxy--5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula IV (671 mg, 1 mmol), HgCl.sub.2 (613
mg, 2.26 mmol) and CaCO.sub.3 (246 mg, 2.46 mmol) in MeCN-water
(4:1) was stirred slowly at room temperature until TLC indicates
complete loss of starting material. The reaction mixture was
diluted with EtOAc (30 mL) and filtered through a celite bed. The
clear yellow organic supernatant was extracted with saturated 5%
NaHCO.sub.3 (20 mL), brine (20 mL) and the combined organic phase
is dried (Na.sub.2SO.sub.4). The organic layer was evaporated in
vacuum and purified by column chromatography (90%
CH.sub.2Cl.sub.2--MeOH) to give compound
7-Methoxy-8-[N-(1"-pyrenyl)-prop- ane-3'-carboxamide]-oxy-(4R)
hydroxy-(11aS)-1,2,3,11atetra-hydro-5H-pyrrol-
o[2,1-c][1,4]benzodiazepin-5-one as pale yellow oil of formula
V.
Example 7
[0052] Compound (2S)-N-{4-[(3'-carboxy
butyl)oxy]-5-methoxy-2-nitrobenzoyl- }pyrrolidine-2-car-boxaldehyde
diethyl thioacetal of formula II (2.50 g, 5 mmol) was taken in dry
CH.sub.2Cl.sub.2 (20 mL), TEA (707 mg, 7 mmol) was added and the
mixture was cooled at 0-5.degree. C. Isobutyl chloroformate (819
mg, 6 mmol) in dry CH.sub.2Cl.sub.2 (10 mL) was added dropwise and
the mixture was kept at 0-5.degree. C. for 15 min. A solution of
1-amino pyrene of formula I (251 mg, 5 mmol) in CH.sub.2Cl.sub.2
was added to it at the same temperature and the solution was
stirred at room temperature for overnight. The mixture was washed
with saturated NaHCO.sub.3 (50 mL), brine, dried and solvent was
evaporated. The crude material was chromatographed over silica gel
using ethyl acetate/hexane (8:2) solvent to give compound
(2S)-N-{4-[N-(1"-pyrenyl)-butane-3'-carboxamide]-oxy-5-m-
ethoxy-2-nitrobe-nzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal of formula III as a yellow liquid (1.92 g, 55%).
[0053] .sup.1H NMR (CDCl.sub.3) .delta. 1.10-1.40 (m, 6H),
1.40-2.40 (m, 8H), 2.50-2.90 (m, 4H), 3.10-3.25 (m, 2H), 3.60 (s,
3H), 4.0-4.20 (m, 2H), 4.55-4.85 (m, 2H), 6,70 (s, 1H), 7.62 (s,
1H), 7.70-8.40 (m, 9H), 8.60-8.90 (m, 1H); MS (FAB) 700
[M+H].sup.+.
[0054] The nitro diethyl thioacetal
(2S)-N-{4-[N-(1"-pyrenyl)-butane-3'-ca-
rboxamide]-oxy-5-methoxy-2-nitrobenzo-yl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula III (0.699 g, 1 mmol) was dissolved
in ethyl acetate (15 mL) and added SnCl.sub.2.2H.sub.2O (1.12 g, 5
mmol) was refluxed for 3 h or until the TLC indicated that reaction
was completed. The reaction mixture was then adjusted to pH 8
carefully with saturated NaHCO.sub.3 solution, diluted with ethyl
acetate, filtered through celite and extracted. The combined
organic phase was dried over Na.sub.2SO.sub.4, and evaporated under
vacuum to afford the crude amino diethyl thioacetal
(2S)-N-{4-[N-(1"pyrenyl)-butane-3'-carboxamide]-oxy-5--
methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal of formula IV (482 mg, 72%).
[0055] .sup.1H NMR (CDCl.sub.3) .delta. 1.10-1.40 (m, 6H),
1.50-2.30 (m, 10H), 2.40-2.80 (m, 6H), 3.40 (s, 3H), 3.45-3.60 (m,
2H), 4.05-4.15 (m, 2H), 4.50-4.70 (m 2H), 6.25 (s, 1H), 6.70 (s,
1H), 7.65-8.30 (m, 9H), 9.10-9.25 (m, 1H).
[0056] A solution of
(2S)-N-{4-[N-(1"-pyrenyl)-butane-3'-carboxamide]-oxy--
5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal of formula IV (669 mg, 1 mmol), HgCl.sub.2 (613 mg, 2.26
mmol) and CaCO.sub.3 (246 mg, 2.46 mmol) in MeCN-water (4:1) was
stirred slowly at room temperature until TLC indicates complete
loss of starting material. Reaction mixture was diluted with EtOAc
(30 mL) and filtered through a celite bed. The clear yellow organic
supernatant was extracted with saturated 5% NaHCO.sub.3 (20 mL),
brine (20 mL) and combined organic phase is dried
(Na.sub.2SO.sub.4). The organic layer was evaporated in vacuum and
purified by column chromatography (90% CH.sub.2Cl.sub.2--MeOH) to
give compound
7-Methoxy-8-[N-(1"-pyrenyl)-butane-4'-carboxamide]-oxy-(-
11aS)-1,2,3,11a-tetra-hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one
of formula V as pale yellow oil (266 mg, 49%)
[0057] .sup.1H NMR(CDCl.sub.3) .delta. 1.40-2.40 (m, 12H),
2.60-2.90 (m, 2H), 3.40-4.05 (m, 4H), 4.10-4.40 (m, 2H), 6.85 (s,
1H), 7.40 (s, 1H), 7.65 (d, 1H), 7.75-8.20 (m, 8H), 8.20-8.40 (m,
1H), 9.0-9.10 (m, 1H); MS (FAB) 544 [M+H].sup.+.
Example 8
[0058] Compound (4R)-hydroxy-(2S)-N-{4-[(3'-carboxy
butyl)oxy]-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-car-boxaldehyde
diethyl thioacetal of formula II (2.58 g, 5 mmol) was taken in dry
CH.sub.2Cl.sub.2 (20 mL), TEA (707 mg, 7 mmol) was added and the
mixture was cooled at 0-5.degree. C. Isobutyl chloroformate (819
mg, 6 mmol) in dry CH.sub.2Cl.sub.2 (10 mL) was added dropwise and
the mixture was kept at 0-5.degree. C. for 15 min. A solution of
1-amino pyrene of formula I (251 mg, 5 mmol) in CH.sub.2Cl.sub.2
was added to it at the same temperature and the solution was
stirred at room temperature for overnight. The mixture was washed
with saturated NaHCO.sub.3 (50 mL), brine, dried and solvent was
evaporated. The crude material was chromatographed over silica gel
using ethyl acetate/hexane (8:2) solvent to give compound
(4R)-hydroxy-(2S)-N-{4-[N-(1"-pyrenyl)-butane-3'-carboxa-
mide]-oxy-5-methoxy-2-nitrobe-nzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula III as a yellow liquid.
[0059] The nitro diethyl thioacetal
(4R)-hydroxy-(2S)-N-{4-[N-(1"-pyrenyl)-
-butane-3'-carboxamide]-oxy-5-methoxy-2-nitrobenzo-yl}pyrrolidine-2-carbox-
aldehyde diethyl thioacetal of formula III (0.715 g, 1 mmol) was
dissolved in ethyl acetate (15 mL) and added SnCl.sub.2.2H.sub.2O
(1.12 g, 5 mmol) was refluxed for 3 h or until the TLC indicated
that reaction was completed. The reaction mixture was then adjusted
to pH 8 carefully with saturated NaHCO.sub.3 solution, diluted with
ethyl acetate, filtered through celite and extracted. The combined
organic phase was dried over Na.sub.2SO.sub.4, and evaporated under
vacuum to afford the crude amino diethyl thioacetal
((4R)-hydroxy-(2S)-N-{4-[N-(1"-pyrenyl)-butane-3'-carb-
oxamide]-oxy-5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula IV.
[0060] A solution of
(4R)-hydroxy-(2)-N-{4-[N-(1"-pyrenyl)-butane-3'-carbo-
xamide]-oxy-5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula IV (685 mg, 1 mmol), HgCl.sub.2 (613
mg, 2.26 mmol) and CaCO.sub.3 (246 mg, 2.46 mmol) in MeCN-water
(4:1) was stirred slowly at room temperature until TLC indicates
complete loss of starting material. The reaction mixture Was
diluted with EtOAc (30 mL) and filtered through a celite bed. The
clear yellow organic supernatant was extracted with saturated 5%
NaHCO.sub.3 (20 mL), brine (20 mL) and the combined organic phase
is dried (Na.sub.2SO.sub.4). The organic layer was evaporated in
vacuum and purified by column chromatography (90%
CH.sub.2Cl.sub.2--MeOH) to give compound
7-Methoxy-8-[N-(1"-pyrenyl)-buta-
ne-4'-carboxamide]-oxy-(4R)-hydroxy-(11aS)-1,2,3,11a-tetra-hydro-5H-pyrrol-
o[2,1-c][1,4]benzodiazepin-5-one of formula V as pale yellow
oil.
[0061] Biological Activity:
[0062] In vitro biological activity studies were carried out at
National Cancer Institute (USA).
[0063] Cytotoxicity:
[0064] Compounds Ve and Vg were evaluated the primary anti-cancer
activity (Table 1) and. further Ve have been evaluated in vitro
against sixty human tumour cells derived from nine cancer types
(leukemia, non-small-cell lung, colon, CNS, melanoma, ovarian,
prostate, and breast cancer). For each compound, dose response
curves for each cell line were measured at a minimum of five
concentrations at 10 fold dilutions. A protocol of 48 h continuous
drug exposure was used and a sulforbodamine B (SRB) protein assay
was used to estimate cell viability or growth. The concentration
causing 50% cell growth inhibition (GI50), total cell growth
inhibition (TGI 0% growth) and 50% cell death (LC50, -50% growth)
compared with the control was calculated. The mean graph midpoint
values of log.sub.10TGI and log.sub.10LC50 as well as log.sub.10
GI50 for Ve are listed in Table 2. As demonstrated by mean graph
pattern, compound Ve exhibits an interesting profile of activity
and selectivity for various cell lines. The mean graph mid point of
log.sub.10 TGI and log.sub.10 LC50 showed similar pattern to the
log.sub.10 GI50 mean graph mid points.
1TABLE 1 In vitro one dose primary anticancer assay.sup.a pyrene
linked PBD hybrid of formula Ve and Vg Growth percentages (Lung)
(Breast) (CNS) PBD hybrids NCI-H460 MCF7 SF-268 Ve 11 31 70 Vg 106
72 131 .sup.aOne dose of Ve and Vg at 10.sup.-4 molar
concentration
[0065] The novel pyrrolobenzodiazepine hybrid formula VIIa has
shown to possess 10 nano molar potency (at the LC.sub.50 level)
against one non-small cell lung cancer (NCI-H226) and one colon
cancer (HCC-2998). and 0.1 micro molar potency against leukemia
cancer (SR), melanoma cancer (M14), renal cancer (A498) and CNS
cancer (SF-539) and also have 10 micro molar potency against two
CNS cancer cell lines (SF539, SNB75) and one prostate cancer
(DU-145). The LC50 values of nine cancers (average of six to nine
cancer cell lines) of compound VIIa listed in Table 3
[0066] Table 2. log.sub.10 GI50 log.sub.10 TGI and log.sub.10LC50
mean graphs midpoints(MG_MID) of in vitro cytotoxicity data for the
compound Ve against human tumour cell lines.
2 Compound Log.sub.10 GI50 Log.sub.10 TGI Log.sub.10 LC50 Ve -7.75
-6.89 -4.74
[0067]
3TABLE 3 Log LC50 (concentration in mol/L causing 50% lethality)
Values for Compounds Ve Compound Cancer Ve Leukemia -4.65
Non-small-cell lung -4.67 Colon -5.00 CNS -5.23 Melanoma -5.62
Ovarian >-4.00 Renal -5.05 Prostate -5.30 Breast >-4.00 Each
cancer type represents the average of six to nine different cancer
cell lines.
* * * * *