Title of Invention

"PYROOLO [2,1-C][1,4]BENZO-DIAZEPINE HYBRID"

Abstract The present invention relates to a process for the preparation of pyrrolo [2,l-c][l,4]benzodiazepine hybrids . This invention relates to a process for the preparation of new pyrrolo[2,l-c][l,4]benzodiazepine hybrids as potential antitumour agents. More particularly, it provides a process for the preparation of 7-methoxy-8-[N-(l"-pyrenyl)-alkane-3'-carboxamide]-oxy(llaS)l,2,3,l latetraydro5Hpyrrolo[2,lc][l,4]benzodiazepin-5-one, with aliphatic chain length variation of these compounds and it also describes the DNA binding, anticancer (antitumour) activity.
Full Text PYRROLO[2,l-c][l,4]BENZODIAZEPINE HYBRIDS Field of invention
The present invention relates to a process for the preparation of novel pyrrolo [2.1-c][l,4]benzodiazepine hybrids useful as potential antitumour agents. This invention relates to a process for the preparation of new pyrrolo[2,l-c][l,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-tetraydro 5H-pyrrolo[2,l-c][l,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:

(Formula Removed)
Background of the invention
Pyrrolo[2,l-6'][l,4]benzodiazepine antitumour antibiotics are commonly known as anthramycin class of compounds. In the last few years, a growing interest has been show n in the development of new pyrrolo[2,l -c][ 1,4]benzodiazepines (PBDs). These antibiotics react covalently with DNA to form an N2-guanine adduct that lies within the minol groove of duplex DNA via an acid-labile aminal 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.; Kohn, K. W. and Speous, C. L. J. Mol. Biol, 1970, 57, 551.; Hurley, L. H.; Gairpla, C. and Zmijewski, M. Biochem. Biophys. Acta., 1977, 475, 521.; Kaplan, D. J. and Hurley. L. 11. 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; Kelland, L. R. and Thurston, D. E. J. Med. Clem. 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).
Recently, a noncross-linking mixed imine-amide PBD dimers have been synthesized
that have significant DNA binding ability and potent anti tumour activitiy. (Kamal, A.;
Ramesh, G., Laxman, N.; Ramulu, P.; Srinivas, O.; Neelima, K.; Kondapi, A. K.; Srinu,
Naturally occurring pyrrolo[2,l-c][l,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
metaboi:. ;nactivation
Objects if the invention
The main object of the present invention is to provide new pyrrolo[2,l-c][l,4]-
benzodiazepine hybrids useful as antitumour agents.
Another objective of the present invention is to provide a process for the preparation
of novel pyrrolo[2, l-cj| l,4]-benzodiazepine hybrids useful as antitumour agents.
Summary of the invention
Accordingly the present invention provides a process for the preparation of a novel
pyrrolo[2, l-c][l,4]benzodiazepme hybrids of formula V wherein R = H, OH and n is
FORMULA V
Accordingly the present process provides a process for preparation of pyrrolo[2,lc
|[l,4]benzodiazepine hybrids of formula V
FORMULA V
which comprises reacting pyrene amine of formula I
-[(3'-carboxy alkyl)oxy]-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-
carboxaldehyde diethyl thioacetal of formula II where R is as stated above
in the presence of isobutyl chloroformate, bases like triethyl amine, DBU in presence of
organic solvents up to retluxing for a period of 24 h isolating (2.V)-N-{4-[N-(l"-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,
reducing the above nitro compounds of formula III with SnCla^HjO in presence of organic
solvent up to a reflux temperature, isolating the (2AT)-N-{4-[N-(l"-pyrenyl)-alkane-3'-
carboxamide]-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,
reacting the above said amino compound of formula IV with known deprotecting agents manner to give novel pyrrolo[2,l-c][l,4]benzodiazepine hybrids of formula V
wherein n and R are as stated above.
Detailed description of the invention
The precursors, pyrene amine of formula I (Banik, B. K.; Becker, F. F. Bioorg. Med.
. 2001, 9, 593) and (2,S)-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. ,/. Med Chem.
1999, 42, 5131 , Reddy, B. S. P.; Damayanthi, Y.; Reddy, B. S. N.; Lown, W. J. Anti- Drug Design 2000, 75, 225) have been prepared by literature methods.
These new analogues of pyrrolo[2,l-c][l,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:
1 The ether linkage at C-8 position of DC-81 intermediates with pyrene ring moiety.
2 Refluxing the reaction mixture for 24-48 h.
3 Synthesis of C-8 linked PBD antitumour antibiotic hybrid imines.
4 Purification by column chromatography using different solvents like ethyl acetate,
hexane, dichloromethane and methanol.
Some representative compounds of forn present invention are given below
7-Methoxy-8-[N-(r'-pyrenyl)-methane-i urboxamide]-oxy-(l \aS)l,2,3,1 la
tetrahydro-5/f-pyrrolo[2,1 -c][ 1,4]benzodiazepin-5-one
7-Methoxy-8-[N-(l"-pyrenyl)-methane-r-carboxamide]-oxy-(4^)-hydroxy
1,2,3,1 la tetrahydro-5//-pyrrolo[2, l-c][l,4]benzodiazepin-5-one
5
7-Methoxy-8-[N-( 1 "-pyrenyl)-ethane-2'-carboxamide]-oxy-( 11 aS) 1,2,3,11 a
tetrahydro-5//-pyrrolo[2,1 -c][ 1,4]benzodiazepin-5-one
7-Methoxy-8-[N-( l"-pyrenyl)-ethane-2'-carboxamide]-oxy-(4/?)-hydroxy (1
1,2,3,11 a tetrahydro-5//-pyrrolo[2,1 -c][ 1,4]benzodiazepin-5-one
7-Methoxy-8-[N-( 1 "-pyrenyl)-propane-3 '-carboxamide]-oxy-( 11 aS)-1,2,3,11 a
tetra-hydro-5//-pyrrolo[2, l-c][l,4]benzodiazepin-5-one
7-Methoxy-8-[N-( I "-pyrenyl)-propane-3 '-carboxamide]-oxy-(4/?)-hydroxy (1
1,2,3,11 a-tetra-hydro-5//-pyrrolo[2, l-c][l,4]benzodiazepin-5-one
7-Methoxy-8-[N-( 1 "-pyrenyl)-butane-4'-carboxamide]-oxy-( 11 aS}-1,2,3,11 a-tetrahydro-
5//-pyrrolo[2,l-c][l,4]benzodiazepin-5-one
7-Methoxy-8-[N-( 1 "-pyrenyl)-butane-4'-carboxamide]-oxy-(4/?)-hydroxy (11 &S)-
1,2,3,11 a-tetra-hydro-5//-pyrrolo[2, l-c][l,4]benzodiazepin-5-one
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
Compound (2,S>N-[4-[(l'-carboxy methyl)oxy]-5-methoxy-2-nitrobenzoyl] pyrrolidine-
2-carboxaldehyde diethyl thioacetal of formula II (2.29 g, 5 mmol) was taken in dry
CHjCk (20 mL), TEA (707 mg, 7 mmol) was added and the mixture was cooled at 0-5 °C.
Isobutyl chloroformate (819 mg, 6 mmol) in dry CHzCb (10 mL) was added dropwise and
the mixture was kept at 0-5 °C for 15 min. A solution of 1-amino pyrene of formula I (251
mg, 5 mmol) in CHjCh was added to it at the same temperature and the solution was stirred
at room temperature for overnight. The mixture was washed with saturated NaHCCh (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 (2i1)-N-{4-[N-(l"-
pyrenyl)-methane-3'-carboxamide]-oxy~5-methoxy-2-nitro-benzoyl}pyrrolidine-2-
carboxaldehyde diethyl thioacetal of formula III as a yellow liquid.
The (2A')-N-{4-[N-(l"-pyrenyl)-methane-r-carboxamide]-oxy~5-methoxy-2-nitro
benzoy!! pyrrolidine-2-carboxaldehyde diethvl thioacetal of formula III (0.657 g, 1 mmol)
was dissolved in ethyl acetate (15 mL) and added SnCb.2H2O (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 NaHC03 solution, diluted with ethyl acetate, filtered
through celite and extracted. The combined organic phase was dried over NaiSO^ and
6
evaporated under vacuum to afford the crude (2i')-N-{4-[N-(l"-pyrenyl)-methane-rcarboxamide]-
oxy—5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl
ihioacetal of formula IV.
A solution of i2i')-N-H-[N-( l"-pyrenyi)-methane-l '-carboxamide|-oxy—5-methoxy-
2-ammobenzoyi}pyrrohdme-2-carboxaldehyde diethyl thioacetai of formula IV (627 mg, 1
mmol), HgCl2 (613 mg, 2.26 mmol) and CaCO3 (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% NaHCCh (20 mL), brine
(20 mL) and the combined organic phase is dried (Na2SC»4). The organic layer was
evaporated in vacuum and purified by column chromatography (90% CH2Cl2-MeOH) to give
compound 7-Methoxy-8-f N-( 1 "-pyreny l)-propane-3 f-carboxamide]-oxy-( 11 aS)-1,2,3,11 a
tetra-hydro-5//-pyrrolo[2,l-c][l,4]benzo-diazepin-5-one as pale yellow oil.
Example 2
Compound (4/?)-hydroxy-(21S')-N-[4-[(r-carboxy methyl)oxy]-5-methoxy-2-nitrobenzoyl]
pyrrolidine-2-carboxaldehyde diethyl thioacetai of formula II (2.37 g, 5 mmol) was
taken in dry CH2C12 (20 mL), TEA (707 mg, 7 mmol) was added and the mixture was cooled
at 0-5 °C. Isobutyl chloroformate (819 mg, 6 mmol) in dry CH2C12 (10 mL) was added
dropwise and the mixture was kept at 0-5 °C for 15 min. A solution of 1-amino pyrene
formula 1 (251 mg, 5 mmol) in CH2C12 was added to it at the same temperature and the
solution was stirred at room temperature for overnight. The mixture was washed with
saturated NaHCOa (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
(2,S)-N-{4-[N-( 1 "-pyrenyl)-methane-1 '-carboxamide]-oxy— 5-methoxy-2-nitrobenzoyl}
pyrrolidine-2-carboxaldehyde diethyl thioacetai of formula III as a yellow liquid.
The (4/0-hydroxy-(2,5>N-{4-[N-( 1 "-pyrenyl)-methane-1 '-carboxamide]-oxy-5-
methoxy-2-nitrobenzoyl} pyrrolidine-2-carboxaldehyde diethyl thioacetai of formula III
(0.673 g, 1 mmol) was dissolved in ethyl acetate (15 mL) and added SnCl2.2H20 (1.12 g, 5
mmol) was refluxed for 3 h or until the TLC indicated that reaction was completed. The
action mixture was then adjusted to pH 8 carefully with saturated NaHCO^ solution, diluted
with ethyl acetate, filtered through celite and extracted. The combined organic phase was
dried over Na2SC>4, and evaporated under vacuum to afford the crude (47?)-hydroxy-(2iS)-N-
! 4-[N-( 1 "-pyrenyl)-methane-1 '-carboxamide]-oxy~5-methoxy-2-amino-benzoyl} pyrrolidine-
A solution of(4/0-hydroxy-(2i')-N-{4-[N-( 1 "-pyrenylj-methane- i '->:arboxamide]- nethoxy-2-aminobenzoyl}pyrrolidine-2-carboxaidehyde diethyl thioacetal of formula IV
m43 nig, 1 mmol), HgCh (613 mg, 2.26 mmol) and CaCCb (246 mg, 2.46 mmol) in
starting material. The reaction mixture was diluted with EtOAc (30 mL) ana filtered through
.1 celite bed. The clear vellow organic supernatant was extracted with saturated 5% NaHCCh
t20 mL), brine (20 mL) and the combined organic phase is dried (NaiSCu). The organic layer
was evaporated in vacuum and purified by column chromatography (90% CF^Ch-MeOH) to
iiive compound 7-methoxy-8-[N-(l "-pyrenyl)-methane-r-carboxamide]-oxy-(4#)-hydroxy-
Example 3
Compound (2\)-N-[4-[(2'-carboxy ethyl)oxy]-5-methoxy-2-nitrobenzovl] pyrrolidine-
2-carboxaldehyde diethyl thioacetal of formula II (2.36 g, 5 mmol) was taken in dry CHiCh
(20 mL), TEA (707 mg, 7 mmol) was added and the mixture was cooled at 0-5 °C. Isobutyl
diiorotbrmate (819 mg, 6 mmol) in dry CILCU (10 mL) was added dropwise and the mixture
v.as kept at (">-S °C for 15 min A solution of 1-amino pyrene of formula I (251 mg, 5 mmol)
in CH2Ch 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} (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 (2,S)-N-{4-[N-(r'-pyrenyl)-
ethane-2'-carboxamide]-oxy--5-methoxy-2-nitro-benzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula III as a yellow liquid.
The (2Ar)-N-{4-[N-(r'-pyrenyl)-ethane-2'-carboxamide]-oxy—5-methoxy-2-nit.ro .
benzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula III (0.671 g, 1 mmol)
was dissolved in ethyl acetate (15 mL) and added SnCl2.2H20 (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 NaHCCh solution, diluted with ethyl acetate, filtered
through celite and extracted. The combined organic phase was dried over NaiSO^ and
evaporated under vacuum to afford the crude (2iS)-N-{4-[N-(l"-pyrenyl)-ethane-2'-
carboxamide]-oxy—5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal of formula IV.
A solution of (2.S')-N-{4-[N-(r'-pyrenyl)-ethane-2'-carboxamide]-oxy—5-methoxy-2-
aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula IV (641 mg, 1
mmol), HgCl2 (613 mg, 2.26 mmol) and CaCO3 (246 mg, 2.46 mmol) in MeCN-water
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% NaHCCh (20 mL), brine
(20 mL) and the combined organic phase is dried (l^SCM). The organic layer was
evaporated in vacuum and purified by column chromatography (90% CH^Ch-MeOH) to give
compound 7-Methoxy-8-[N-(l"-pyrenyl)-ethane-2'-carboxamide]-oxy-(l lai> 1,2,3,1 latetrahvdro-
5//-pyrrolo[2, l-f][l,4]benzodiazepm-5-one as pale yellow oil.
Example 4
Compound (4/^)-hydroxy-(2,ST)-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 CHiClz (20 mL), TEA (707 mg, 7 mmol) was added and the mixture was
cooled at 0-5 °C Isohutyl chloroformate (819 mg, 6 mmol) in dry CH2Cl2 (10 mL) was
added dropv/ise and the mixture was kept at 0-5 °C for 15 min. A solution of 1-amino pyrene
of formula I (251 mg, 5 mmol) in CH2C12 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.i (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
(4/?)-hydroxy-(2i)-N-{ 4-[N-( 1 "-pyrenyl)-ethane-2'-carboxamide]-oxy--5-methoxy-
nitrobenzoyl} pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula III as a yellow
liquid.
The(4/?)-hydroxy-(21ST)-N-{ 4-[N-( 1 "-pyrenyl)-ethane-2'-carboxamide]-oxy-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 SnCl2.2H2O (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 NaHCOs solution, diluted with ethyl
acetate, filtered through celite and extracted. The combined organic phase was dried over
Na2SC>4, and evaporated under vacuum to afford the crude (4/^)-hydroxy-(2i')-N-{4
pyrenyl)-ethane-2'-carboxamide]-oxy--5-methoxy-2-amino-benzoyl}pyrrolidme-2-
carboxaldehyde diethyl thioacetal of formula IV.
A solution of (4/^)-hydroxy-(2iS)-N-{4- -pyrenyl)-ethane-2'-carboxamide]-oxy—
methoxy-2-aminobenzoyl}pyrrolidine-2-carbo uehyde diethyl thioacetal of formula
(657 mg, 1 mmol), HgCl2 (613 mg, 2.26 mmol) and CaCO3 (246 mg, 2.46 mmol) in MeCNwater
(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 througn
a celite bed. The clear yellow organic supernatant was extracted with saturated 5% NaHCCh
(20 mL), brine (20 mL) and the combined organic phase is dried (NasSCV). The organic layer
was evaporated in vacuum and purified by column chromatography (90% ClrbCU-MeOH) to
give compound 7-Methoxy-8-[N-(l "-pyrenyl)-ethane-2'-carboxamide]-oxy-(4#)-hydroxy-
(1 13^-1,2,3,1 latetra-hydro-5//-pyrrolo[2,l-c][l,4]benzodiazepin-5-one as pale yellow oil.
Example 5
Compound (25)-N-[4-[(3'-carboxy propyl)oxy]-5-methoxy-2-nitrobenzoyl]
pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula II (2.43 g, 5 mmol) was taken in
dry CHiCh (20 mL), TEA (707 mg, 7 mmol) was added and the mixture was cooled at 0-
5°C. Isobutyl chloroformate (819 mg, 6 mmol) in dry CHjCh (10 mL) was added dropwise
and the mixture was kept at 0-5 °C for 15 min. A solution of 1-amino pyrene formula I
mg, 5 mmol) in CFfeCh was added to it at the same temperature and the solution was stirred
at room temperature for overnight. The mixture was washed with saturated NaHCCh (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%).
1H NMR (CDCh) 8 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]+.
The(21Sr)-N-{4-[N-(l"-pyrenyl)-propane-3'-carboxamide]-oxy--5-methoxy-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 SnCl2.2H2O (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 NaHCOa solution, diluted with ethyl acetate, filtered
through celite and extracted. The combined organic phase was dried over Na2SO4, and
evaporated under vacuum to afford the crude (2,5)-N-{4-[N-(l"-pyrenyl)-propane-3'-
carboxamide]-oxy-5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal of formula IV (458 mg, 70%).
'H NMR (CDC13) 6 1.10-1.40 (m, 6H), 1.50-2.30 (m, 8H), 2.40-2.80 (m, 4H), T40 (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).
A solution of (2^T)-N-{4-[N-(l"-pyrenyl)-propane-3'-carboxamide]-oxy—5-methoxy-2-
aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula IV (655 mg, 1
inmol). HgCb (613 mu, 2.26 mmol) and CaCCh (246 me, 2.46 tnmon in MeCN-water (4:1)
was stirred slowly at room temperature until TLC indicates complete loss of rarting material.
The reaction mixture was diluted with EtOAc (30 mL) and filtered throuuh a ceiite bed. The
.icar yellow organic supernatant was extracted with saturated 5% NaHCO, (20 mLA brine
(20 mL) and the combined organic phase is dried (NajSO/O. The organic layer was
evaporated in vacuum and purified by column chromatography (90% CHiCh-MeOH) to give
compound 7-Methoxy-8-[N-( 1 "-pyrenyl)-propane-3'-carboxamide]-oxy-( 11 aS)-
\ 2,3,1 latetra-hydro-5//-pyrrolo[2,l-t'][l,4]benzodiazepin-5-one as pale vellow oil of
formula V (285 mg, 54%).
'HNMR(CDC13)8 1.40-2.40 (m, I OH), 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 I'M + HI".
Example 6
Compound (4^?)-hydroxy-(2S)-N-[4-[(3'-carboxy propyl)oxy]-5-methoxy-2-
nitrobenzoyll pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula II (2.51 g, 5 mmol)
'.as taken in dry CH?CN (20 mL). TEA (707 mg, 7 mrnol) was added and the mixture was
cooled at 0-5 °C. Isobutyl chloroformate (819 mg, 6 mmol) in dry C^Ch (10 mL) was
added dropwise and the mixture was kept at 0-5 °C for 15 rnin. A solution of 1-amino pyrene
of formula 1 (251 mg, 5 mrnol) in CHjC^ 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^ (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
(4/^)-hydroxv-(21Sr)-N-{4-[N-(]"-pyrenyl)-propane-3'-carboxamide]-oxy--5-methoxy-2-
nitrobenzoyl} pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula III as a yellow
liquid.
The (4K)-hydroxy-(26T)-N-{4-[N-(l"-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 SnCl2.2H2O (1.12 g, 5
mmol) was refluxed for 3 h or until the TLC indicated that reaction was completed. The
•action mixture was then adjusted to pH 8 carefully with saturated NaHCCh solution, diluted
with ethyl acetate, filtered through ceiite and extracted. The combined organic phase was
dried over Na2SC>4, and evaporated under vacuum to afford the crude (4/?)-hydroxy-(2iS)-N-
! 4-[N-( 1 "-pyrenyl)-propane-3 '-carboxamide]-oxy—5-methoxy-2-aminobenzoyl} pyrrolidine-
2-carboxaldehyde diethyl thioacetal of formula IV.
11
A solution of (4^)-hydroxy-(26>N-{4-[N-(l"-pyrenyl)-propane-3'-carboxamide]-oxy-
-5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula IV
(o71 mg, 1 mmol), HgCl2 (613 mg, 2.26 mmol) and CaC03 (246 rng, 2.46 mmol) in MeCNwater
( 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% NaHCOi
(20 mL), brine (20 mL) and the combined organic phase is dried (Na2SC>4). The organic layer
was evaporated in vacuum and purified by column chromatography (90% CH2Cl2-MeOH) to
give compound 7-Methoxy-8-[N-(l"-pyrenyl)-propane-3'-carboxamide]-oxy-(4/?) hydroxy-
( 1 lai')-l,2,3,llatetra-hydro-5//-pyrrolo[2,l-c][l,4]benzodiazepin-5-one as pale yellow oil of
formula V
Example 7
Compound (2i*)-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 CH2C12
(20 mL), TEA (707 mg, 7 mmol) was added and the mixture was cooled at 0-5 °C. Isobutyl
chloroformate (819 mg, 6 mmol) in dry CH2C1?. (10 mL) was added dropwise and the mixture
was kept at 0-5 °C for 15 min. A solution of 1-amino pyrene of formula I (251 mg, 5 mmol)
in CH2Ch was added to it at the same temperature and the solution was stirred at room
temperature for overnight. The mixture was washed with saturated NaHCCh (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 (21S)-N-{4-[N-(l"-pyrenyl)-
butane-3'-carboxamide]-oxy-5-methoxy-2-nitrobe-nzoyl}pyrrolidine-2-carboxaldehyde
diethyl thioacetal of formula III as a yellow liquid (1.92 g, 55%).
1HNMR(CDC13)8 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 + Hf.
The nitro diethyl thioacetal (21Sr)-N-{4-[N-(l"-pyrenyl)-butane-3'-carboxamide]-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 SnCl2.2H2O (1.12 g,
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 NaHCCh solution, diluted
with ethyl acetate, filtered through celite and extracted. The combined organic phase was
dried over Na2SO4, and evaporated under vacuum to afford the crude amino diethyl thioacetal
12
(2.V)-N-{4-[N-(l"-pyrenyl)-butane-3'-carboxamide]-oxy-5-methoxy-2-aminobenzoyl)
pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula IV (482 mg, 72%).
!HNMR(CDCb)8 1.10-1.40 (m, 6H), 1.50-2.30 (m, 1 OH), 2.40-2.80 (m, 6H), 3.40 (s, 3H),
> 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),
8 30 (m, 9H), 9.10-9.25 (m, 1H).
A solution of (2.S>N-{4-[N-(l"-pyrenyl)-butane-3'-carboxamide]-oxy-5-methoxy-2-
ammobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula IV (669 mg, 1
mmol), HgCl2 (613 mg, 2.26 mmol) and CaCO3 (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% NaHCCh (20 mL), brine
(20 mL) and combined organic phase is dried (Na2SC»4). The organic layer was evaporated invacuum and purified by column chromatography (90% CHzCk-MeOH) to give compound 7-
Methoxy-8-[N-( 1 "-pyrenyl)-butne-4'-carboxamide]-oxy-( 11 aS)-1,2,3,11 a-tetra-hydro-5//-
pyrrolo[2, l-c][l,4]benzodiazepin-5-one of formula V as pale yellow oil (266 mg, 49%).
'H NMR (CDC13) 6 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 + Hf.
Example 8
Compound (4^)-hydroxy-(2i>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 CHaCli (20 mL), TEA (707 mg, 7 mmol) was added and the mixture was
cooled at 0-5 °C Isobutyl chloroformate (819 mg, 6 mmol) in dry CH2C12 (10 mL) was
added dropwise and the mixture was kept at 0-5 °C for 15 min. A solution of 1-amino pyrene
of formula 1 (251 mg, 5 mmol) in CHjCh was added to it at the same temperature and the
solution was stirred at room temperature for overnight. The mixture was washed with
saturated NaHCCh (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
(4/^)-hydroxy-(25)-N-{ 4-[N-( 1 "-pyrenyl)-butane-3'-carboxamide]-oxy-5-methoxy--2-nitrobenzoyl)
pyrrolidine-2-carboxaldehyde diethyl thi 1 of formula III as a yellow liquid.
The nitro diethyl thioacetal (4/?)-hyaroxy-(2i>N-{4-[N-(l"-pyrenyl)-butane-3'-
carboxamide]-oxy-5-methoxy-2-nitrobenzo-yl} pyrrolidine-2-carboxaldehyde diethyl
thioacetal of formula III (0.715 g, 1 mmol) was dissolved in ethyl acetate (15 mL) and added
(1.12 g, 5 mmol) was refluxed for 3 h or until the TLC indicated that reaction
13
was completed. The reaction mixture was then adjusted to pH 8 carefully with saturated
NaHCCh solution, diluted with ethyl acetate, filtered through celite and extracted. The
combined organic phase was dried over Na2SO4 and evaporated under vacuum to afford the
crude amino diethyl thioacetal (4R)-hydrox.y-(2S)-N-{4-[N-(l"-pyrenyl)-butane-3(-
carboxamide]-oxy-5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal of formula IV.
A solution of (4^)-hydroxy-(24S)-N-{4-[N-(l"-pyrenyl)-butane-3'-carboxamide]-oxy-
5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula IV
(685 mg, 1 mmol), HgCl2 (613 mg, 2.26 mmol) and CaCO3 (246 mg, 2.46 mmol) in MeCNwater
(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% NaHCCh
(20 mL), brine (20 mL) and the combined organic phase is dried The organic layer
was evaporated in vacuum and purified by column chromatography (90% CF^Cb-MeOH) to
give compound 7-Methoxy-8-[N-( 1 "-pyrenyl)-butne-4'-carboxamide]-oxy-(4#)-hydroxy-
( 1 laS)-l,2,3,1 la-tetra-hydro-5//-pyrrolo[2,l-c][l,4]benzodiazepin-5-one of formula V as
pale yellow oil.
Biological Activity:
In vitro biological activity studies were carried out at National Cancer Institute
(USA).
Cytotoxicity:
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 sulforhodamine 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 logioTGI and logioLC50 as
well as logio 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 logio TGI and logio LC50 showed similar pattern to the logio
GI50 mean graph mid points.
14
Table 1. In vitro one dose primary anticancer assay pyrene linked PBD hybrid of formula
'One dose of Ve and Vg at 10"4 molar concentration
The novel pyrrolobenzodiazepine hybrid formula Vila has shown to possess 10 nano
molar potency (at the LCso 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 LCSO values of nine cancers (average of six to nine cancer cell lines)
of compound Vila listed in Table 3
Table 2. logic GI50 logio TGI and logic LCSO mean graphs midpoints(MG_MID^ of in vitro
cytotoxicity data for the compound Ve against human tumour cell lines.
Compound LogioGISO LogioTGI LogioLCSO
Table 3. Log LCSO (concentration in mol/L causing 50% lethality) Values for





We Claim:
1 Pyrrolo[2,l-c][l,4]benzodiazepine hybrid of formula V wherein R is H , OH
and n is 1-4.
(Formula Removed)

2 A pyrrolobenzodiazepine hybrid as claimed in claim 1 has structural formula
(Formula Removed)
3 A pyrrolobenzodiazepine hybrid as claimed in claim 1 has structural formula
(Formula Removed)

4 A pyrrolobenzodiazepine hybrid as claimed in claim 1 has structural formula
(Formula Removed)


5 A pyrrolobenzodiazepine hybrid as claimed in claim 1 has stmctural formula
(Formula Removed)

6 A pyrrolobenzodiazepine hybrid as claimed in claim 1 has structural formula
(Formula Removed)

7 A pyrrolobenzodiazepine as claimed in claim 1 has structural formula
(Formula Removed)

8 A pyrrolobenzodiazepine hybrid as claimed in claim 1 has structural formula
(Formula Removed)


9. A pyrrolobenzodiazepine hybrid as claimed in claim 1 has structural formula
(Formula Removed)

10. A process for preparing a pyrrolo [2,l-c][l,4]benzodiazepine hybrid of formula V


(Formula Removed)
where R and H as defined in claim 1
Formula V which comprises reacting pyrene amine of formula I

(Formula Removed)
with (2S)-N-[4-[(3'-carboxy methyl)oxy]-5-methoxy-2-nitrobenzoyl] pyrrolidine-2-carboxaldehyde diethyl thioacetal of fonnula II where Ris as stated above 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
(Formula Removed)
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 formulaIII where n is 1-4 and R is as defined above

(Formula Removed)
reducing the nitro compounds of formula III with SnCl2.2H2O in presence of an organic solvent as here in described up to a reflux temperature, isolating the (2S)-N-{4-[N-( 1 "-pyrenyl)-alkane-3'-carboxamide]-oxy~5-methoxy-2-aminobenzoyl)pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula IV where n is 1-4 and R is as stated above, /=(Formula Removed)

reacting the amino compound of foemula IV with a deprotecting agents to obtain pyrrolo[2,l-c][l,4]benzodiazepine hybrids of formula V wherein n and R are as stated above.
11. The process as claimed in claim lOwherein the organic solvent used for the reduction
of the nitro compound of formula III comprises ethyl acetate.
12. Pyrene linked pyrrolo [2,l-c][l,4]benzodiazepine hybrids substantially as herein
described with references to the examples.

Documents:

456-DEL-2003-Abstract-(28-01-2009).pdf

456-del-2003-abstract.pdf

456-DEL-2003-Claims (25.02.2009).pdf

456-DEL-2003-Claims-(28-01-2009).pdf

456-del-2003-claims.pdf

456-del-2003-complete specification (granted).pdf

456-DEL-2003-Correspondence-Others(25.02.2009).pdf

456-DEL-2003-Correspondence-Others-(28-01-2009).pdf

456-del-2003-correspondence-others.pdf

456-del-2003-correspondence-po.pdf

456-del-2003-description (complete)-(25-02-2009).pdf

456-DEL-2003-Description (Complete)-(28-01-2009).pdf

456-del-2003-description (complete).pdf

456-del-2003-form-1.pdf

456-del-2003-form-18.pdf

456-DEL-2003-Form-2-(28-01-2009).pdf

456-del-2003-form-2.pdf

456-DEL-2003-Form-3-(28-01-2009).pdf

456-del-2003-form-3.pdf

456-DEL-2003-Petition-137-(28-01-2009).pdf

abstract.jpg


Patent Number 232387
Indian Patent Application Number 456/DEL/2003
PG Journal Number 13/2009
Publication Date 27-Mar-2009
Grant Date 16-Mar-2009
Date of Filing 27-Mar-2003
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG NEW DELHI-110 001,INDIA
Inventors:
# Inventor's Name Inventor's Address
1 OLEPU SRINIVAS CHEMICAL TECHNOLOGY, HYDERABAD-500 007 INDIA
2 AHMED KAMAL CHEMICAL TECHNOLOGY, HYDERABAD-500 007 INDIA
3 GUJJAR RAMESH CHEMICAL TECHNOLOGY, HYDERABAD-500 007 INDIA
4 PODDUTOORI RAMULU CHEMICAL TECHNOLOGY, HYDERABAD-500 007 INDIA
PCT International Classification Number C07D 487/04
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA