Indian Patents. 257917:"PYRROLO[2,1-C][1,4] BENZODIAZEPINE-NAPTHALIMIDE CONJUGATES LINKED THROUGH PIPERAZINE MOIETY AND PROCESS FOR PREPARATION THEREOF"

Title of Invention	"PYRROLO[2,1-C][1,4] BENZODIAZEPINE-NAPTHALIMIDE CONJUGATES LINKED THROUGH PIPERAZINE MOIETY AND PROCESS FOR PREPARATION THEREOF"
Abstract	The present invention relates to novel pyrrolo[2,l-c][l,4]benzodiazepine-napthalimide hybrids linked through piperazine moiety as potential antitumour agents. The present invention also relates to a process for the preparation of novel pyrrolo[2,l-c][l,4]benzodiazepine-napthalimide hybrids linked through piperazine moiety useful as potential antitumour agents.

Full Text	Field of the invention The present invention relates to novel pyrrolo[2,1-c][1,4]benzodiazepine-napthalinide hybrids linked through piperazine moiety as potential antitumour agents. The present invention also relates to a process for the preparation of novel pyrrolo[2,1-c][1,4]benzodiazepine-napthalimide hybrids linked through piperazine moiety useful as potential antitumour agents. The present invention particularly relates to the synthesis of pyrrolo[2,1-c][l,4]benzodiazepme-napthalimide hybrids linked through piperazine moiety as possible anticancer agents. The structural formula of novel pyrrolo[2,1-c][1,4]benzodiazepine-napthalimide hybrids (VIII) is as follows, wherein n1=2,3,4. (Formula Removed) Background of the invention Pyrrolo[2,1-c][1,4]benzodiazepines are a family of DNA interactive antitumour antibiotics derived from Streptomyces species. Examples of naturally occurring pyrrolo[2,1-c][1,4] benzodiazepines include anthramycin, tomaymycin, sibiromycin and DC-81. These compounds show their biological activity through covalent binding via their N10-CII imine/carbinol amine moiety to the C2- amine position of a guanine residue within the minor groove of DNA giving rise to the preference for Pu-G-Pu sequences. (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, 91, 551.; Hurley, L. H.; Gairpla, C. and Zmijewski, M. Biochem. Biophy. Acta., 1977, 475, 521., Kaplan, D. J. and Hurley, L. H. Biochemistry, 1981, 20, 7572.). The molecules have a right-handed twist, when viewed from the C-ring towards the A-ring. This enables the PBD to mirror the curvature of B-form DNA and maintain isohelical contact with the walls and floor of the minor groove. In the last few years a growing Compound Oh 18h Villa 21.9 22.7 Vlllb 25.8 26.7 Vlllc 23.4 24.2 Vllld 13.1 14.3 Vllle 20.9 21.7 D'C-81 0.3 0.7 For CT-DNA alone at pH 7.00 ± 0.01, Tm = 69.8°C ± 0.01 (mean value from 10 separate determinations), all Tm values are ± 0.1-0.2° C. For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentration = 100µ,M and ligand concentration = 20µ, M in aqueous sodium phosphate buffer [10 mM sodium phosphate + 1mM EDTA, pH 7.00± 0.01]. interest has been shown in the development of new pyrrolo[2,l-c][l,4]benzodiazepine hybnds. Many PBD conjugates have been synthesized and investigated for their anticancer activity (Thurston, D E.; Morris, S. J., Hartley, J. A. Chem. Commun. 1996, 563.; Damayanthi, Y., Reddy, B. S P ; Lown, J. W. J Org Chem 1999, 64, 290.; Kamal, A.; Reddy, B. S. N.; Reddy, G S. K.; Ramesh, G Bioorg Med Chem Lett. 2002, 12, 1933, Kamal, A.; Reddy, B. S. N.; Reddy Indian patent application No 209/DEL/2000). Recently C-8 linked PBD dimers with C2/C2 exounsaturation have been designed and synthesized (Gregson, S. J.; Howard, P W.; Hartley, J A.; Brooks, N. A.; Adam, L. J.; Jenkins, T. C.; Kelland, L. R. and Thurston, D. E., J. Med. Chem. 2001, 44, 737). Also, non cross-linking mixed imine-amide PBD dimers have been synthesized that have significant DNA binding ability and potent antitumor activity (Kamal, A.; Ramesh, G.; Laxman, N.; Ramulu, P.; Snmvas, O.; Neehma, K.; Kondapi, A. K.; Srinu, V. B.; Nagarajaram, H. M. J. Med. Chem. 2002, 45, 4679). During earlier studies in this laboratory PBDs have been linked to naphthahmides through alkane chain which have shown promising anticancer activity (Kamal, A.; Reddy, B. S. N.; Reddy, G. S. K.; Ramesh, G Bioorg Med. Chem. Lett. 2002, 12, 1933, Kamal, A.; Reddy, B. S. N.; Reddy Indian patent application No.209/DEL/2000). However, in the present invention the PBD and naphthahmide moieties have been linked through piperazine moiety with alkyl side arms, instead of simple alkane chain spacers. By incorporation of a piperazine moiety in the spacer these new hybnds not only exhibit enhanced in vitro anticancer activity but remarkable DNA binding affinity for a number of this type of hybrids as illustrated in Table 1 and 2. (Table Removed) Objects of the invention The main objective of the present invention is to provide new pyrrolo[2,l- c][l,4]benzodiazepines useful as anticancer agents. Another objective of the present invention is to provide a process for the preparation of novel pyrrolo[2,l- c][l,4]benzodiazepines useful as antitumor agents. Summary of the invention Accordingly, the present invention provides a novel pyrrolo[2,l- c][l,4benzodiazepines of formula VIII where ni = 2, 3,4, n2 = 2, 3, 4. (Formula Removed) The present invention also provides a process for the preparation of pyrrolo[2,l- c][l,4]benzodiazepmes of formula VIII shown above where n[ = 2, 3, 4, n2 = 2, 3, 4, which comprises of reacting (2S)-N-[4-hydroxy-5-methoxy-2- nitrobenzoyl]pyrrohdine-2-carboxaldehyde diethyl thioacetal of formula IV with dibromoalkanes in an aprotic water miscible organic solvent in the presence of a mild inorganic base at refluxing temperature for a period of 48h, isolating 2S-N-[4-(n-bromo alkoxy)-5-methoxy-2-nitrobenzoyl]pyrrohdine-2-carbaxaldehyde diethyl thioacetal of formula V, reacting the compound of formula V with piperazine attached naphthahmide in presence of mild inorganic bases isolating compound of formula VI, reducing it with SnCl2.2H2O in presence of organic solvent at a reflux temperature, reacting the above ammo compound of formula VII with known deprotecting agents in a conventional manner to give novel pyrrol[2,l-c][l,4]benzodiazepine of formula VIII wherein n is as stated above. Detailed description of the invention The precursor, (2S)-N-[4-hydroxy-5-methoxy-2-mtrobenzoyl]pyrrolidine-2-carboxaldehyde diethylthioacetal of formula IV (Thurston, D. E.; Murthy, V. S.; Langley, D. R.; Jones, G.; B. Synthesis, 1990, 81) has been prepared by literature methods. Some representative compounds of formula VIII of present invention are given below 1. 7-Methoxy-8-{2-[4-[2-(l,3-dioxo-benz[rfe]isoquinohn-2-yl)ethyl]piperazin-l-yl]ethyl}-oxy-(llaS)-l,2,3,l la tetrahydro-5H-pyrrolo [2,l-c][l,4]benzodiazepin-5-one 2. 7-Methoxy-8-{3-[4-[2-(l,3-dioxo-benz[Je]isoquinohn-2-yl)ethyl]piperazin-l-yl]propyl}-oxy-(llaS)-1,2,3,1 la tetrahydro-5H-pyrrolo [2,l-c][l,4]benzodiazepin-5-one 3. 7-Methoxy-8- {4-[4-[2-( 1,3-dioxo-benz[de]isoquinohn-2-yl)ethyl]piperazin-1 -yl]butyl}-oxy-(l laS)-l,2,3,lla tetrahydro-5H-pyrrolo [2,l-c][l,4]benzodiazepin-5-one 4. 7-Methoxy-8- {3-[4-[3-( 1,3-dioxo-benz[de]isoquinohn-2-yl)propyl]piperazin-1 - yl]propyl}-oxy-(l laS)-1,2,3,1 la tetrahydro-5H-pyrrolo [2,l-c][l,4]benzodiazepin-5- one 5. 7-Methoxy-8-{4-[4-[4-(l,3-dioxo-benz[de]isoquinolin-2-yl)butyl]piperazin-l- yl]butyl}-oxy-(llaS)-l,2,3,lla tetrahydro-5H-pyrrolo [2,l-c][l,4]benzodiazepin-5- one These new analogues of pyrrolo[2,l-c][l,4]benzodiazepine hybrids have shown promising anticancer activity in in selected human cancer cell lines of colon (HT-29, HCT-15), lung (A-549, HOP-62), cervix (SiHa) origin. 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-I which comprises of 1. The ether linkage at C-8 position of DC-81 intermediates with napthahmide moiety 2. Refluxing the reaction mixture for 24-48h. 3. Synthesis of C-8 linked PBD hybnds 4. Purification by column chromatography using different solvents like ethyl acetate, hexane, dichloromethane and methanol. (Formula Removed) Reagents and conditions (i) Dibromo alkanes, K2C03, acetonitnle, reflux, 12h, (n) N-Boc piperazine, K2C03, acetonitnle, reflux, 8h, (m) CF3COOH, CHCI3, rt, 12h (Formula Removed) Reagents and conditions (i) Dibromo alkanes,K2C03, acetonitrile, reflux, 24 h, (u) compound I, K2C03, acetonitnle, reflux, 12 h, (in) SnCI2 2H20, methanol, reflux, 5 h, (iv) HgCI2, CaC03, acetonitnle/H20, r t, 12 h 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 To a solution of (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula IV (800 mg, 2 mmol) in acetone were added anhydrous K2CO3 (829 mg, 6 mmol) and 1,2 dibromo ethane (940 mg, 5mmol) and the mixture was refluxed for 48h.After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under redused pressure, purification by column chromatography afforded compound V. 1H NMR (CDCI3) 1.30-1 45 (m, 6H), 1.70-2.35 (m, 4H), 2.70-2 85 (m, 4H), 3.12-3 30 (m, 2H), 3.70 (t, 2H, J=6 3 Hz), 3.95 (s, 3H), 4.40 (t, 2H, J=6Hz), 4.60-4 75 (m, 1H), 4 82 (d, 1H, J=4.3 Hz), 6 80 (s, 1H), 7.65 (s, 1H) To a solution of 2S-N-[4-(2-bromo ethoxy)-5-methoxy-2-nitrobenzoyl]pyrrohdine-2-carbaxaldehyde diethyl thioacetal of formula V (507 mg, 1 mmol), piperazine attached naphthahmide (340 mg, 1.1 mmol) in acetone was added anhydrous K2CO3 (415 mg, 3 mmol) and the mixture was refluxed for 24h. After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under reduced pressure, purification by column chromatography afforded compound VI. 1H NMR (CDCI3, 200 MHz) 1.22-1.40 (m, 6H), 1.70-2.35 (m, 4H), 2.55-2.95 (m, 16H), 3.15-3.32 (m, 2H), 3.92 (s, 3H), 4.15-4.35 (m, 4H), 4.57-4.72 (m, 1H), 4.80 (d, 1H, 7=4.3 Hz), 6.77 (s, 1H), 7.60-7.80 (m, 3H), 8.30 (t, 2H, J = 8 Hz), 8.55 (d, 2H, J = 7.6 Hz). To a solution of 2S-N-{4-[2-[4-[2-(l,3-dioxo-benz[Je]isoquinohn-2-yl)ethyl]piperazin-l-yl]ethyl]-oxy-5-methoxy-2-nitrobenzoyl}pyrrohdine-2-carbaxaldehyde diethyl thioacetal of formula VI (736 mg, 1 mmol) in methanol was added SnCl2.2H20 (1.12 gr, 5 mmol) and the mixture was refluxed until the TLC indicated the completion of reaction. The methanol was evaporated and 10% NaHCO3 solution was added. The aqueous layer was extracted with ethyl acetate, the combined organic phases were dried over Na2SO4 and evaporated under vacuum to afford the amino thioacetal (VII) and directly used in the next step. A solution of VII (706 mg, 1 mmol) HgCl2 (624 mg, 2.3 mmol) and CaC03 (250 mg, 2.5 mmol) in CH3CN-H2O (4:1) was stirred at room temperature until the TLC indicated complete consumption of the starting matenal. The reaction mixture was diluted with ethyl acetate and filtered through a celite bed. The organic layer was concentrated, dried and purified by column chromatography to give the compound VIII. 1H NMR (CDCI3, 200 MHz) 1.90-2.40 (m, 4H), 2.45-2.92 (m, 12H), 3.55-3.82 (m, 3H), 3 92 (s, 3H), 4.05-4.40 (m, 4H), 6.77 (s, 1H), 7.45 (s, 1H), 7.62 (d, \H,J= 4.39 Hz), 7.76 (t, 2H, J = 7.69 Hz), 8,20 (d, 2H, J= 8.2 Hz), 8.60 (d, 2H, J= 7.32 Hz). Example 2 To a solution of (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrohdine-2-carboxaldehyde diethyl thioacetal of formula IV (800 mg, 2 mmol) in acetone were added anhydrous K2CO3 (828 mg, 6 mmol) and 1,3 dibromo propane (1 gr, 5 mmol) and the mixture was refluxed for 48h.After completion of reaction K2C03 was removed by filtration and the solvent was evaporated under reduced pressure, purification by column chromatography afforded compound V. 1H NMR (CDCI3,200 MHz) 1 25-1 40 (m, 6H), 1.72-2.42 (m, 6H), 2.70-2.8 (m, 4H), 3.15-3.30 (m, 2H), 3.60 (t, 2H, J = 6 20 Hz), 3.95 (s, 3H), 4.20 (t, 2H, J = 4 96 Hz), 4.60-4.75 (m, 1H), 4.82 (d, 1H, .7=4.33 Hz), 6.78 (s, 1H), 7 68 (s, 1H). To a solution of 2S-N-[4-(3-bromo propoxy)-5-methoxy-2-nitrobenzoyl]pyrrohdine-2-carbaxaldehyde diethyl thioacetal of formula V (521 mg, 1 mmol), piperazine attached naphthahmide (340 mg, 1.1 mmol) in acetone was added anhydrous K2CO3 (415 mg, 3 mmol) and the mixture was refluxed for 24h.After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under reduced pressure, purification by column chromatography afforded compound VI. 1H NMR (CDCI3, 200 MHz) 1.30-1 42 (m, 6H), 1.70-2.30 (m, 6H), 2.40-2.82 (m, 16H), 3.15-3.30 (m, 2H), 3.92 (s, 3H), 4 15 (m, 2H), 4.30 (m , 2H), 4.60-4.70 (m, 1H), 4.82 (d, 1H, 7=4.25 Hz), 6 75 (s, 1H), 7.65 (s, 1H), 7 75 (t, 2H, 7=7.4 Hz), 8.2 (d, 2H, J= 8 Hz), 8.6 (d, 2H, J = 7.6 Hz). To a solution of 2S-N-{4-[3-[4-[2-(l,3-dioxo-benz[de]isoquinohn-2-yl)ethyl]piperazin-l-yl]propyl]-oxy-5-methoxy-2-nitrobenzoyl}pyrrohdine-2-carbaxaldehyde diethyl thioacetal of formula VI (750 mg, 1 mmol) .in methanol was added SnCl2.2H20 (1.12 gr, 5 mmol) and the mixture was refluxed until the TLC indicated the completion of reaction. The methanol was evaporated and 10% NaHCCh solution was added. The aqueous layer was extracted with ethyl acetate, the combined organic phases were dned over Na2S04 and evaporated under vacuum to afford the amino thioacetal (VII) and directly used in the next step. A solution of VII (720 mg, 1 mmol) HgCl2 (624 mg, 2.3 mmol) and CaC03 (250 mg, 2.5 mmol) in CH3CN-H20 (4:1) was stirred at room temperature until the TLC indicated complete consumption of the starting matenal. The reaction mixture was diluted with ethyl acetate and filtered through a celite bed. The organic layer was concentrated, dned and punfied by column chromatography to give the compound VIII. 1H NMR (CDCI3, 200 MHz) 1.60-2.16 (m, 6H), 2.25-2.80 (m, 12H), 3.50-3.82 (m, 3H), 3.95 (s, 3H), 4.05-4 40 (m, 4H), 6.80 (s, 1H), 7.45 (s, 1H), 7.62 (d, 1H, J= 3.33 Hz), 7.79 (t, 2H, J= 7.32 Hz), 8.20 (d, 2H, J= 8.05 Hz), 8.60 (d, 2H, J = 7.32 Hz). Example 3 To a solution of (2S)-N-[4-hydroxy-5-methoxy-2-mtrobenzoyl]pyrrohdine-2-carboxaldehyde diethyl thioacetal of formula (800 mg, 2 mmol) in acetone were added anhydrous K2CO3 (829 mg, 6 mmol) and 1,4 dibromo butane (1.07 gr, 5 mmol) and the mixture was refluxed for 48h.After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under redused pressure, purification by column chromatography afforded compound V 1H NMR (CDCI3,300 MHz) 1.30-1.40 (m, 6H), 1.75-2.40 (m, 8H), 2.70-2.85 (m, 4H), 3.15-3.30 (m, 2H), 3.50 (t, 2H, J = 6.25 Hz), 3.95 (s, 3H), 4.10 (m, 2H), 4.60-4.70 (m, 1H), 4.82 (d, 1H, .7=4.3 Hz), 6.75 (s, 1H), 7.62 (s, 1H). To a solution of 2S-N-[4-(4-bromo butoxy)-5-methoxy-2-mtrobenzoyl]pyrrohdine-2-carbaxaldehyde diethyl thioacetal of formula V (535 mg, 1 mmol), piperazine attached naphthalimide (340 mg, 1.1 mmol) in acetone was added anhydrous K2CO3 (415 mg, 3 mmol) and the mixture was refluxed for 24h.After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under redused pressure, purification by column chromatography afforded compound VI. 1H NMR (CDCI3, 200 MHz) 1.25-1.40 (m, 6H), 1.60-2.15 (m, 8H), 2.35-2.85 (m, 16H), 3.15-3.30 (m, 2H), 3.92 (s, 3H), 4.12 (m, 1H), 4.30 (m, 2H), 4.60-4.72 (m, 1H), 4.80 (d, 1H, .7=4.23 Hz), 6.75 (s, 1H), 7.60 (s, 1H), 7.75 (t, 2H, 7=7.45 Hz), 8.2 (d, 2H, J= 8.25 Hz), 8.56 (d, 2H, J= 7.62 Hz). To a solution of 2S-N-{4-[4-[2-(l,3-dioxo-benz[Je]isoquinohn-2-yl)ethyl]piperazin- l-yl]butyl]-oxy-5-methoxy-2-mtrobenzoyl}pyrrohdine-2-carbaxaldehyde diethyl thioacetal (764 mg, 1 mmol) of formula VI .in methanol was added SnCl2.2H20 (1.12 gr, 5 mmol) and the mixture was refluxed until the TLC indicated the completion of reaction. The methanol was evaporated and 10% NaHCC>3 solution was added. The aqueous layer was extracted with ethyl acetate, the combined organic phases were dried over Na2SO4 and evaporated under vacuum to afford the amino thioacetal (VII) and directly used in the next step. A solution of VII (734 mg, 1 mmol) HgCl2 (624 mg, 2.3 mmol) and CaC03 (250 mg, 2.5 mmol) in CH3CN-H20 (4:1) was stirred at room temperature until the TLC indicated complete consumption of the starting material. The reaction mixture was diluted with ethyl acetate and filtered through a cehte bed. The organic layer was concentrated, dried and purified by column chromatography to give the compound VIII. 1H NMR (CDCI3, 200 MHz) 1.56-2.15 (m, 8H), 2.25-2.80 (m, 16H), 3 45-3.82 (m, 3H), 3.92 (s, 3H), 4.0-4.15 (m, 2H), 4 22-4 37 (t, 2H), 6.70 (s, 1H), 7.42 (s, 1H), 7.60 (d, 1H, J = 4 25 Hz), 7.72 (t, 2H, J = 7.4 Hz), 8.16 (d, 2H, /= 8 1 Hz), 8 56 (d, 2H, J = 7.42 Hz). Example 4 To a solution of (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrohdine-2-carboxaldehyde diethyl thioacetal of formula IV (800 mg, 2 mmol) in acetone were added anhydrous K2CO3 (829 mg, 6 mmol) and 1,3 dibromo propane (1 gr, 5mmol) and the mixture was refluxed for 48h.After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under redused pressure, purification by column chromatography afforded compound V. 1H NMR (CDCI3,200 MHz) 1.25-1.40 (m, 6H), 1.72-2.42 (m, 6H), 2.70-2.8 (m, 4H), 3.15-3.30 (m, 2H), 3.60 (t, 2H, J = 6.20 Hz), 3.95 (s, 3H), 4 20 (t, 2H, J= 4.96 Hz), 4.60-4.75 (m, 1H), 4.82 (d, 1H, 7=4.33 Hz), 6.78 (s, 1H), 7 68 (s, 1H). To a solution of 2S-N-[4-(3-bromo propoxy)-5-methoxy-2-mtrobenzoyl]pyrrolidine-2-carbaxaldehyde diethyl thioacetal of formula V (521 mg, 1 mmol), piperazine attached naphthahmide (324 mg, 1 mmol) in acetone were added anhydrous K2CO3 (415 mg, 3 mmol) and the mixture was refluxed for 24h. After completion of reaction K.2CO3 was removed by filtration and the solvent was evaporated under reduced pressure, purification by column chromatography afforded compound VI. 1H NMR (CDCI3, 200 MHz) 1.25-1.42 (m, 6H), 1.70-2.40 (m, 8H), 2.60-3.30 (m, 18H), 3.92 (s, 3H), 4.05 (m, 4H), 4.70-4.80 (m, 1H), 4.82 (d, 1H, .7=4.25 Hz), 6.77 (s, 1H), 7.60 (s, 1H), 7.75 (t, 2H, 7=7.35 Hz), 8.18 (d, 2H, /= 8 Hz), 8.55 (d, 2H, J = 7.55 Hz). To a solution of 2S-N-{4-[3-[3-[4-[3-(l,3-dioxo-benz[de]isoquinohn-2-yl)propyl]piperazin-l-yl]propyl]-oxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carbaxaldehyde diethyl thioacetal of formula VI (764 mg, 1 mmol) .in methanol was added SnCl2.2H20 (1.12 gr, 5 mmol) and the mixture was refluxed until the TLC indicated the completion of reaction. The methanol was evaporated and 10% NaHCO3 solution was added. The aqueous layer was extracted with ethyl acetate, the combined organic phases were dried over Na2S04 and evaporated under vacuum to afford the ammo thioacetal (VII) and directly used in the next step. A solution of VII (734 mg, 1 mmol) HgCl2 (624 mg, 2.3 mmol) and CaC03 (250 mg, 2.5 mmol) in CH3CN-H20 (4:1) was stirred at room temperature until the TLC indicated complete consumption of the starting material. The reaction mixture was diluted with ethyl acetate and filtered through a cehte bed. The organic layer was concentrated, dried and purified by column chromatography to give the compound VIII. 1H NMR (CDC13, 200 MHz) 1.75-2.18 (m, 8H), 2.22-2.80 (m, 16H), 3.45-4 30 (m, 10H), 6.75 (s, IH), 7.45 (s, IH), 7.60 (d, IH, J- 4.2 Hz), 7.72 (t, 2H, ./= 7.4 Hz), 8.20 (d, 2H,J= 8.1 Hz), 8.58 (d, 2H, J= 7.35 Hz). Example 5 To a solution of (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrohdine-2-carboxaldehyde diethyl thioacetal of formula IV (800 mg, 2 mmol) in acetone were added anhydrous K2CO3 (829 mg, 6 mmol) and 1,4 dibromo butane (1 gr, 5 mmol) and the mixture was refluxed for 48h.After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under redused pressure, purification by column chromatography afforded compound V. 1H NMR (CDC13,300 MHz) 1.30-1.40 (m, 6H), 1.75-2.40 (m, 8H), 2.70-2.85 (m, 4H), 3.15-3.30 (m, 2H), 3.50 (t, 2H, J = 6.25 Hz), 3.95 (s, 3H), 4.10 (m, 2H), 4.60-4.70 (m, IH), 4.82 (d, IH, 7=4.3 Hz), 6.75 (s, IH), 7 62 (s, IH) To a solution of 2S-N-[4-(4-bromo butoxy)-5-methoxy-2-mtrobenzoyl]pyrrolidine-2-carbaxaldehyde diethyl thioacetal of formula V (538mg, 1 mmol), piperazine attached naphthahmide (337 mg, 1 mmol) in acetone were added anhydrous K2CO3 (415 mg, 3 mmol) and the mixture was refluxed for 24h. After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under reduced pressure, purification by column chromatography afforded compound VI. 'H NMR (CDC13,200 MHz) 1.60-2.33 (m, 12H), 2.52-3.0 (m, 16H), 3.12-3.30 (m, 2H), 3.95 (s, IH), 4.02-4.25 (m, 4H), 4.60-4.72 (m, IH), 4.80 (d, IH, .7=4.3 Hz), 6.75 (s, IH), 7.60 (s, IH), 7.75 (t, 2H, 7=7.45 Hz), 8.18 (d, 2H, J= 8.2 Hz), 8.56 (d, 2H, J = 7.6 Hz). To a solution of 2S-N-{4-[4-[4-[4-(l,3-dioxo-benz[de]isoquinohn-2-yl)butyl]piperazin-l-yl]butyl]-oxy-5-methoxy-2-nitrobenzoyl}pyrrohdine-2-carbaxaldehyde diethyl thioacetal (792 mg, 1 mmol) of formula VI .in methanol was added SnCl2.2H20 (1.12 gr, 5 mmol) and the mixture was refluxed until the TLC indicated the completion of reaction. The methanol was evaporated and 10% NaHCO3 solution was added. The aqueous layer was extracted with ethyl acetate, the combined organic phases were dried over Na2SO4 and evaporated under vacuum to afford amino thioacetal (VII) and directly used in the next step. A solution of VII (762 mg, 1 mmol) HgCl2 (624 mg, 2.3 mmol) and CaCO3 (250 mg, 2.5 mmol) m CH3CN-H2O (4:1) was stirred at room temperature until the TLC indicated complete consumption of the starting material. The reaction mixture was diluted with ethyl acetate and filtered through a cehte bed. The organic layer was concentrated, dried and purified by column chromatography to give the compound VIII. 1H NMR (CDC13, 300 MHz) 1.50-2.10 (m, 12H), 2.25-2.80 (m, 16H), 3.45-4.30 (m, 10H), 6.75 (s, 1H), 7.45 (s, 1H), 7.62 (d, 1H, J= 4.2 Hz), 7.75 (t, 2H, J = 7.3 Hz), 8.20 (d, 2H, J= 8.1 Hz), 8.56 (d, 2H,7= 7.40 Hz). Biological activity In vitro cytotoxicity against human cancer cell lines: The human cancer cell lines procured from National Cancer Institute, Fredenck, U.S.A or National Center for Cell Science; Pune, India, were used in present study. Cells were grown in tissue culture flasks in complete growth medium (RPMI-1640 medium with 2 raM glutamine, 100 ng/ml streptomycin, pH 7.4, sterilized by filtration and supplemented with 10% fetal calf serum and 100 units/ml penicillin before use) at 37°C in an atmosphere of 5% CO2 and 90% relative humidity in a carbon dioxide incubator. The cells at subconfluent stage were harvested from the flask by treatment with trypsin (0.5% in PBS containing 0.02% EDTA) for determination of cytotoxicity. Cells with viability of more than 98% as determined by trypan blue exclusion were used for assay. The cell suspension of the required cell density were prepared in complete growth medium with gentamycin (50µg/ml) for determination of cytotoxicity. A stock solutions of (2 X 10"2 M) of test material were prepared in DMSO . The stock solutions were serially diluted with complete growth medium containing 50µg/ml of gentamycin to obtain working test solutions of required concentrations. In vitro cytotoxicity against human cancer cell lines was determined (Monks, A., Scudiero, D., Skehan, P, Shoemaker R., Paull, K., Vistica, D., Hose, C, Langley, J., Cronise, P., Vaigro-Wolff, A., Gray-Goodrich, M., Campbell,H., Mayo, J and Boyd M. J. Natl. Cancer Inst.,1991,83, 757-766) using 96-well tissue culture plates. The 100 µl of cell suspension was added to each well of the 96-well tissue culture plate. The cells were incubated for 24 hours. Test matenals in complete growth medium (100µl) were added after 24 hours incubation to the wells containing cell suspension. The plates were further incubated for 48 hours (at 37°C in an atmosphere of 5% and 90% relative humidity in a carbon dioxide incubator) after addition of test material and then the cell growth was stopped by gently layering trichloroacetic acid (TCA, 50ul, 50%) on top of the medium in all the wells. The plates were incubated at 4°C for one hour to fix the cells attached to the bottom of the wells. The liquid of all the wells was gently pipetted out and discarded. The plates were washed five times with distilled water to remove TCA, growth medium low molecular weight metabolites, serum proteins etc and air-dned. Cell growth was measured by staining with sulforhodamine B dye (Skehan et al., 1990). The adsorbed dye was dissolved in Tns-Buffer (100 m 1, 0.01M, pH 10.4) and plates were gently stirred for 5 minutes on a mechanical stirrer. The optical density was recorded on ELISA reader at 540 nm. The cell growth was calculated by subtracting mean OD value of respective blank from the mean OD value of experimental set. Percent growth in presence of test material was calculated considering the growth in absence of any test material as 100% and in turn percent growth inhibition in presence of test material will be calculated. Cytotoxicity: Compounds evaluated for the primary anticancer activity. The cytotoxicity data for some representative compounds has shown in Table 1. Table 1. The percentage growth inhibition data for napthahmide-PBD hybrids Compd Cancer (Table Removed) anot tested Table 2. DNA Thermal Denaturation Studies: (Table Removed) For CT-DNA alone at pH 7.00 □ 0.01, Tm = 69.8 □C □ 0.01 (mean value from 10 separate determinations), all □ Tm values are □ 0.1 - 0.2 □C. For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentration = 100 □M and ligand concentration = 20 DM in aqueous sodium phosphate buffer [10 mM sodium phosphate + 1 mM EDTA, pH 7.00 □ 0.01]. We claim 1. A novel pyrrolo[2,1-c][1,4]benzodiazepines of formula VIII where n1 = 2, 3, 4, n2 =2,3,4. (Formula Removed) 2. 7-Methoxy-8- {2-[4-[2-( 1,3-dioxo-benz[de]isoquinolin-2'-yl)ethyl]piperazin-1 - yl]ethyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo [2,1- c][1,4]benzodiazepin-5-one as disclosed in claim 1 has structural formula shown below. (Formula Removed) 3. 7-Methoxy-8- {3-[4-[2-( 1,3-dioxo-benz[de]isoquinolin-2-yl)ethyl]piperazin-1 - yl]propyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo [2,1-c][1,4]benzodiazepin- 5-one as disclosed in claim 1 has structural formula shown below. (Formula Removed) 4. 7-Methoxy-8-{4-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)ethyi]piperazin-l- yl]butyl}-oxy-(11 aS)-1,2,3,11 a tetrahydro-5H-pyrrolo [2,1 -c][1,4]benzodiazepin-5- one as disclosed in claim 1 has structural formula shown below. (Formula Removed) 5. 7-Methoxy-8-{3-[4-[3-(1,3-dioxo-benz[de]isoquinolin-2-yl)propyl]piperazin-l- yl]propyl}-oxy-(11 aS)-1,2,3,11 a tetrahydro-5H-pyrrolo [2,1 -c][1,4]benzodiazepin- 5-one as disclosed in claim 1 has structural formula shown below. (Formula Removed) 6. 7-Methoxy-8- {4-[4-[4-( 1,3-dioxo-benz[de]isoquinolin-2-yl)butyl]piperazin-1 - yl]butyl}-oxy-(11 aS)-1,2,3,11 a tetrahydro-5H-pyrrolo [2,1 -c][1,4]benzodiazepin-5- one as disclosed in claim 1 has structural formula shown below. (Formula Removed) 7. 7-Methoxy-8- {4-[4-[2-( 1,3-dioxo-benz[de]isoquinolin-2-yl)propyl]piperazin-1 - yl]butyl}-oxy-(11 aS)-1,2,3,11 a tetrahydro-5H-pyrrolo [2,1 -c][1,4]benzodiazepin-5- one as disclosed in claim 1 has structural formula shown below. (Formula Removed) 8. 7-Methoxy-8-{3-[4-[2-(l,3-dioxo-benz[de]isoquinolin-2-yl)butyl]piperazin-l- yl]propyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo [2,1-c][1,4]benzodiazepin- 5-one as disclosed in claim 1 has structural formula shown below. (Formula Removed) 9. 7-Methoxy-8- {3-[4-[3-( 1,3-dioxo-benz[de]isoquinolin-2-yl)propyl]piperazin-1. yl]ethyl}-oxy-(11 aS)-1,2,3,11a tetrahydro-5H-pyrrolo [2,1 -c][1,4]benzodiazepin-5- one as disclosed in claim 1 has structural formula shown below. (Formula Removed) 10. A process for the preparation of pyrrolo[2,1-c][1,4]benzodiazepines of formula VIII (Formula Removed) where n1 = 2, 3, 4, n2 = 2, 3, 4, which comprises of reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula IV with dibromoalkanes in an aprotic water miscible organic solvent in the presence of a mild inorganic base at refluxing temperature for a period of 48h, isolating 2S-N-[4-(n-bromo alkoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carbaxaldehyde diethyl thioacetal of formula V, reacting the compound of formula V with piperazine attached naphthalimide in presence of mild inorganic bases isolating compound of-formula VI, reducing it with SnCI2.2H20 in presence of organic solvent at a reflux temperature, reacting the above amino compound of formula VII with a deprotecting agent to obtain the pyrrol[2,l-c][l,4]benzodiazepine of formula VIII wherein n is as stated above.

Full Text

Field of the invention
The present invention relates to novel pyrrolo[2,1-c][1,4]benzodiazepine-napthalinide hybrids linked through piperazine moiety as potential antitumour agents. The present invention also relates to a process for the preparation of novel pyrrolo[2,1-c][1,4]benzodiazepine-napthalimide hybrids linked through piperazine moiety useful as potential antitumour agents.
The present invention particularly relates to the synthesis of pyrrolo[2,1-c][l,4]benzodiazepme-napthalimide hybrids linked through piperazine moiety as possible anticancer agents. The structural formula of novel pyrrolo[2,1-c][1,4]benzodiazepine-napthalimide hybrids (VIII) is as follows, wherein n1=2,3,4.
(Formula Removed)
Background of the invention
Pyrrolo[2,1-c][1,4]benzodiazepines are a family of DNA interactive
antitumour antibiotics derived from Streptomyces species. Examples of naturally
occurring pyrrolo[2,1-c][1,4] benzodiazepines include anthramycin, tomaymycin,
sibiromycin and DC-81. These compounds show their biological activity through
covalent binding via their N10-CII imine/carbinol amine moiety to the C2-
amine position of a guanine residue within the minor groove of DNA giving rise
to the preference for Pu-G-Pu sequences. (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, 91, 551.; Hurley, L. H.; Gairpla, C. and Zmijewski, M.
Biochem. Biophy. Acta., 1977, 475, 521., Kaplan, D. J. and Hurley, L. H. Biochemistry, 1981, 20, 7572.). The molecules have a right-handed twist, when viewed from the C-ring towards the A-ring. This enables the PBD to mirror the curvature of B-form DNA and maintain isohelical contact with the walls and floor of the minor groove. In the last few years a growing

Compound Oh 18h
Villa 21.9 22.7
Vlllb 25.8 26.7
Vlllc 23.4 24.2
Vllld 13.1 14.3
Vllle 20.9 21.7
D'C-81 0.3 0.7
For CT-DNA alone at pH 7.00 ± 0.01, Tm = 69.8°C ± 0.01 (mean value from 10 separate determinations), all Tm values are ± 0.1-0.2° C. For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentration = 100µ,M and ligand concentration = 20µ, M in aqueous sodium phosphate buffer [10 mM sodium phosphate + 1mM EDTA, pH 7.00± 0.01].
interest has been shown in the development of new pyrrolo[2,l-c][l,4]benzodiazepine hybnds. Many PBD conjugates have been synthesized and investigated for their anticancer activity (Thurston, D E.; Morris, S. J., Hartley, J. A. Chem. Commun. 1996, 563.; Damayanthi, Y., Reddy, B. S P ; Lown, J. W. J Org Chem 1999, 64, 290.; Kamal, A.; Reddy, B. S. N.; Reddy, G S. K.; Ramesh, G Bioorg Med Chem Lett. 2002, 12, 1933, Kamal, A.; Reddy, B. S. N.; Reddy Indian patent application No 209/DEL/2000). Recently C-8 linked PBD dimers with C2/C2 exounsaturation have been designed and synthesized (Gregson, S. J.; Howard, P W.; Hartley, J A.; Brooks, N. A.; Adam, L. J.; Jenkins, T. C.; Kelland, L. R. and Thurston, D. E., J. Med. Chem. 2001, 44, 737). Also, non cross-linking mixed imine-amide PBD dimers have been synthesized that have significant DNA binding ability and potent antitumor activity (Kamal, A.; Ramesh, G.; Laxman, N.; Ramulu, P.; Snmvas, O.; Neehma, K.; Kondapi, A. K.; Srinu, V. B.; Nagarajaram, H. M. J. Med. Chem. 2002, 45, 4679). During earlier studies in this laboratory PBDs have been linked to naphthahmides through alkane chain which have shown promising anticancer activity (Kamal, A.; Reddy, B. S. N.; Reddy, G. S. K.; Ramesh, G Bioorg Med. Chem. Lett. 2002, 12, 1933, Kamal, A.; Reddy, B. S. N.; Reddy Indian patent application No.209/DEL/2000). However, in the present invention the PBD and naphthahmide moieties have been linked through piperazine moiety with alkyl side arms, instead of simple alkane chain spacers. By incorporation of a piperazine moiety in the spacer these new hybnds not only exhibit enhanced in vitro anticancer activity but remarkable DNA binding affinity for a number of this type of hybrids as illustrated in Table 1 and 2.
(Table Removed)
Objects of the invention
The main objective of the present invention is to provide new pyrrolo[2,l-
c][l,4]benzodiazepines useful as anticancer agents. Another objective of the present
invention is to provide a process for the preparation of novel pyrrolo[2,l-
c][l,4]benzodiazepines useful as antitumor agents.
Summary of the invention
Accordingly, the present invention provides a novel pyrrolo[2,l-
c][l,4benzodiazepines of formula VIII where ni = 2, 3,4, n2 = 2, 3, 4.
(Formula Removed)
The present invention also provides a process for the preparation of pyrrolo[2,l-
c][l,4]benzodiazepmes of formula VIII shown above where n[ = 2, 3, 4, n2 = 2, 3, 4,
which comprises of reacting (2S)-N-[4-hydroxy-5-methoxy-2-
nitrobenzoyl]pyrrohdine-2-carboxaldehyde diethyl thioacetal of formula IV with
dibromoalkanes in an aprotic water miscible organic solvent in the presence of a mild inorganic base at refluxing temperature for a period of 48h, isolating 2S-N-[4-(n-bromo alkoxy)-5-methoxy-2-nitrobenzoyl]pyrrohdine-2-carbaxaldehyde diethyl thioacetal of formula V, reacting the compound of formula V with piperazine attached naphthahmide in presence of mild inorganic bases isolating compound of formula VI, reducing it with SnCl2.2H2O in presence of organic solvent at a reflux temperature, reacting the above ammo compound of formula VII with known deprotecting agents in a conventional manner to give novel pyrrol[2,l-c][l,4]benzodiazepine of formula VIII wherein n is as stated above. Detailed description of the invention
The precursor, (2S)-N-[4-hydroxy-5-methoxy-2-mtrobenzoyl]pyrrolidine-2-carboxaldehyde diethylthioacetal of formula IV (Thurston, D. E.; Murthy, V. S.; Langley, D. R.; Jones, G.; B. Synthesis, 1990, 81) has been prepared by literature methods.
Some representative compounds of formula VIII of present invention are given below
1. 7-Methoxy-8-{2-[4-[2-(l,3-dioxo-benz[rfe]isoquinohn-2-yl)ethyl]piperazin-l-yl]ethyl}-oxy-(llaS)-l,2,3,l la tetrahydro-5H-pyrrolo [2,l-c][l,4]benzodiazepin-5-one
2. 7-Methoxy-8-{3-[4-[2-(l,3-dioxo-benz[Je]isoquinohn-2-yl)ethyl]piperazin-l-yl]propyl}-oxy-(llaS)-1,2,3,1 la tetrahydro-5H-pyrrolo [2,l-c][l,4]benzodiazepin-5-one
3. 7-Methoxy-8- {4-[4-[2-( 1,3-dioxo-benz[de]isoquinohn-2-yl)ethyl]piperazin-1 -yl]butyl}-oxy-(l laS)-l,2,3,lla tetrahydro-5H-pyrrolo [2,l-c][l,4]benzodiazepin-5-one
4. 7-Methoxy-8- {3-[4-[3-( 1,3-dioxo-benz[de]isoquinohn-2-yl)propyl]piperazin-1 -
yl]propyl}-oxy-(l laS)-1,2,3,1 la tetrahydro-5H-pyrrolo [2,l-c][l,4]benzodiazepin-5-
one
5. 7-Methoxy-8-{4-[4-[4-(l,3-dioxo-benz[de]isoquinolin-2-yl)butyl]piperazin-l-
yl]butyl}-oxy-(llaS)-l,2,3,lla tetrahydro-5H-pyrrolo [2,l-c][l,4]benzodiazepin-5-
one
These new analogues of pyrrolo[2,l-c][l,4]benzodiazepine hybrids have shown promising anticancer activity in in selected human cancer cell lines of colon (HT-29, HCT-15), lung (A-549, HOP-62), cervix (SiHa) origin. 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-I which comprises of
1. The ether linkage at C-8 position of DC-81 intermediates with napthahmide
moiety
2. Refluxing the reaction mixture for 24-48h.
3. Synthesis of C-8 linked PBD hybnds
4. Purification by column chromatography using different solvents like ethyl acetate,
hexane, dichloromethane and methanol.
(Formula Removed)

Reagents and conditions
(i) Dibromo alkanes, K2C03, acetonitnle, reflux, 12h, (n) N-Boc piperazine, K2C03, acetonitnle, reflux, 8h, (m) CF3COOH, CHCI3, rt, 12h (Formula Removed)
Reagents and conditions
(i) Dibromo alkanes,K2C03, acetonitrile, reflux, 24 h, (u) compound I, K2C03, acetonitnle, reflux, 12 h, (in) SnCI2 2H20, methanol, reflux, 5 h, (iv) HgCI2, CaC03, acetonitnle/H20, r t, 12 h
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
To a solution of (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula IV (800 mg, 2 mmol) in acetone were added anhydrous K2CO3 (829 mg, 6 mmol) and 1,2 dibromo ethane (940 mg, 5mmol) and the mixture was refluxed for 48h.After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under redused pressure, purification by column chromatography afforded compound V.
1H NMR (CDCI3) 1.30-1 45 (m, 6H), 1.70-2.35 (m, 4H), 2.70-2 85 (m, 4H), 3.12-3 30 (m, 2H), 3.70 (t, 2H, J=6 3 Hz), 3.95 (s, 3H), 4.40 (t, 2H, J=6Hz), 4.60-4 75 (m, 1H), 4 82 (d, 1H, J=4.3 Hz), 6 80 (s, 1H), 7.65 (s, 1H)
To a solution of 2S-N-[4-(2-bromo ethoxy)-5-methoxy-2-nitrobenzoyl]pyrrohdine-2-carbaxaldehyde diethyl thioacetal of formula V (507 mg, 1 mmol), piperazine attached naphthahmide (340 mg, 1.1 mmol) in acetone was added anhydrous K2CO3 (415 mg, 3 mmol) and the mixture was refluxed for 24h. After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under reduced pressure, purification by column chromatography afforded compound VI. 1H NMR (CDCI3, 200 MHz) 1.22-1.40 (m, 6H), 1.70-2.35 (m, 4H), 2.55-2.95 (m, 16H), 3.15-3.32 (m, 2H), 3.92 (s, 3H), 4.15-4.35 (m, 4H), 4.57-4.72 (m, 1H), 4.80 (d, 1H, 7=4.3 Hz), 6.77 (s, 1H), 7.60-7.80 (m, 3H), 8.30 (t, 2H, J = 8 Hz), 8.55 (d, 2H, J = 7.6 Hz).
To a solution of 2S-N-{4-[2-[4-[2-(l,3-dioxo-benz[Je]isoquinohn-2-yl)ethyl]piperazin-l-yl]ethyl]-oxy-5-methoxy-2-nitrobenzoyl}pyrrohdine-2-carbaxaldehyde diethyl thioacetal of formula VI (736 mg, 1 mmol) in methanol was added SnCl2.2H20 (1.12 gr, 5 mmol) and the mixture was refluxed until the TLC indicated the completion of reaction. The methanol was evaporated and 10% NaHCO3 solution was added. The aqueous layer was extracted with ethyl acetate, the combined organic phases were dried over Na2SO4 and evaporated under vacuum to afford the amino thioacetal (VII) and directly used in the next step.
A solution of VII (706 mg, 1 mmol) HgCl2 (624 mg, 2.3 mmol) and CaC03 (250 mg, 2.5 mmol) in CH3CN-H2O (4:1) was stirred at room temperature until the TLC indicated complete consumption of the starting matenal. The reaction mixture was diluted with ethyl acetate and filtered through a celite bed. The organic layer was concentrated, dried and purified by column chromatography to give the compound VIII.
1H NMR (CDCI3, 200 MHz) 1.90-2.40 (m, 4H), 2.45-2.92 (m, 12H), 3.55-3.82 (m, 3H), 3 92 (s, 3H), 4.05-4.40 (m, 4H), 6.77 (s, 1H), 7.45 (s, 1H), 7.62 (d, \H,J= 4.39 Hz), 7.76 (t, 2H, J = 7.69 Hz), 8,20 (d, 2H, J= 8.2 Hz), 8.60 (d, 2H, J= 7.32 Hz). Example 2
To a solution of (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrohdine-2-carboxaldehyde diethyl thioacetal of formula IV (800 mg, 2 mmol) in acetone were added anhydrous K2CO3 (828 mg, 6 mmol) and 1,3 dibromo propane (1 gr, 5 mmol)
and the mixture was refluxed for 48h.After completion of reaction K2C03 was removed by filtration and the solvent was evaporated under reduced pressure, purification by column chromatography afforded compound V. 1H NMR (CDCI3,200 MHz) 1 25-1 40 (m, 6H), 1.72-2.42 (m, 6H), 2.70-2.8 (m, 4H), 3.15-3.30 (m, 2H), 3.60 (t, 2H, J = 6 20 Hz), 3.95 (s, 3H), 4.20 (t, 2H, J = 4 96 Hz), 4.60-4.75 (m, 1H), 4.82 (d, 1H, .7=4.33 Hz), 6.78 (s, 1H), 7 68 (s, 1H).
To a solution of 2S-N-[4-(3-bromo propoxy)-5-methoxy-2-nitrobenzoyl]pyrrohdine-2-carbaxaldehyde diethyl thioacetal of formula V (521 mg, 1 mmol), piperazine attached naphthahmide (340 mg, 1.1 mmol) in acetone was added anhydrous K2CO3 (415 mg, 3 mmol) and the mixture was refluxed for 24h.After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under reduced pressure, purification by column chromatography afforded compound VI.
1H NMR (CDCI3, 200 MHz) 1.30-1 42 (m, 6H), 1.70-2.30 (m, 6H), 2.40-2.82 (m, 16H), 3.15-3.30 (m, 2H), 3.92 (s, 3H), 4 15 (m, 2H), 4.30 (m , 2H), 4.60-4.70 (m, 1H), 4.82 (d, 1H, 7=4.25 Hz), 6 75 (s, 1H), 7.65 (s, 1H), 7 75 (t, 2H, 7=7.4 Hz), 8.2 (d, 2H, J= 8 Hz), 8.6 (d, 2H, J = 7.6 Hz).
To a solution of 2S-N-{4-[3-[4-[2-(l,3-dioxo-benz[de]isoquinohn-2-yl)ethyl]piperazin-l-yl]propyl]-oxy-5-methoxy-2-nitrobenzoyl}pyrrohdine-2-carbaxaldehyde diethyl thioacetal of formula VI (750 mg, 1 mmol) .in methanol was added SnCl2.2H20 (1.12 gr, 5 mmol) and the mixture was refluxed until the TLC indicated the completion of reaction. The methanol was evaporated and 10% NaHCCh solution was added. The aqueous layer was extracted with ethyl acetate, the combined organic phases were dned over Na2S04 and evaporated under vacuum to afford the amino thioacetal (VII) and directly used in the next step.
A solution of VII (720 mg, 1 mmol) HgCl2 (624 mg, 2.3 mmol) and CaC03 (250 mg, 2.5 mmol) in CH3CN-H20 (4:1) was stirred at room temperature until the TLC indicated complete consumption of the starting matenal. The reaction mixture was diluted with ethyl acetate and filtered through a celite bed. The organic layer was concentrated, dned and punfied by column chromatography to give the compound VIII.
1H NMR (CDCI3, 200 MHz) 1.60-2.16 (m, 6H), 2.25-2.80 (m, 12H), 3.50-3.82 (m, 3H), 3.95 (s, 3H), 4.05-4 40 (m, 4H), 6.80 (s, 1H), 7.45 (s, 1H), 7.62 (d, 1H, J= 3.33 Hz), 7.79 (t, 2H, J= 7.32 Hz), 8.20 (d, 2H, J= 8.05 Hz), 8.60 (d, 2H, J = 7.32 Hz).
Example 3
To a solution of (2S)-N-[4-hydroxy-5-methoxy-2-mtrobenzoyl]pyrrohdine-2-carboxaldehyde diethyl thioacetal of formula (800 mg, 2 mmol) in acetone were added anhydrous K2CO3 (829 mg, 6 mmol) and 1,4 dibromo butane (1.07 gr, 5 mmol) and the mixture was refluxed for 48h.After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under redused pressure, purification by column chromatography afforded compound V 1H NMR (CDCI3,300 MHz) 1.30-1.40 (m, 6H), 1.75-2.40 (m, 8H), 2.70-2.85 (m, 4H), 3.15-3.30 (m, 2H), 3.50 (t, 2H, J = 6.25 Hz), 3.95 (s, 3H), 4.10 (m, 2H), 4.60-4.70 (m, 1H), 4.82 (d, 1H, .7=4.3 Hz), 6.75 (s, 1H), 7.62 (s, 1H).
To a solution of 2S-N-[4-(4-bromo butoxy)-5-methoxy-2-mtrobenzoyl]pyrrohdine-2-carbaxaldehyde diethyl thioacetal of formula V (535 mg, 1 mmol), piperazine attached naphthalimide (340 mg, 1.1 mmol) in acetone was added anhydrous K2CO3 (415 mg, 3 mmol) and the mixture was refluxed for 24h.After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under redused pressure, purification by column chromatography afforded compound VI. 1H NMR (CDCI3, 200 MHz) 1.25-1.40 (m, 6H), 1.60-2.15 (m, 8H), 2.35-2.85 (m, 16H), 3.15-3.30 (m, 2H), 3.92 (s, 3H), 4.12 (m, 1H), 4.30 (m, 2H), 4.60-4.72 (m, 1H), 4.80 (d, 1H, .7=4.23 Hz), 6.75 (s, 1H), 7.60 (s, 1H), 7.75 (t, 2H, 7=7.45 Hz), 8.2 (d, 2H, J= 8.25 Hz), 8.56 (d, 2H, J= 7.62 Hz).
To a solution of 2S-N-{4-[4-[2-(l,3-dioxo-benz[Je]isoquinohn-2-yl)ethyl]piperazin-
l-yl]butyl]-oxy-5-methoxy-2-mtrobenzoyl}pyrrohdine-2-carbaxaldehyde diethyl
thioacetal (764 mg, 1 mmol) of formula VI .in methanol was added SnCl2.2H20 (1.12 gr, 5 mmol) and the mixture was refluxed until the TLC indicated the completion of reaction. The methanol was evaporated and 10% NaHCC>3 solution was added. The aqueous layer was extracted with ethyl acetate, the combined organic phases were dried over Na2SO4 and evaporated under vacuum to afford the amino thioacetal (VII) and directly used in the next step.
A solution of VII (734 mg, 1 mmol) HgCl2 (624 mg, 2.3 mmol) and CaC03 (250 mg, 2.5 mmol) in CH3CN-H20 (4:1) was stirred at room temperature until the TLC indicated complete consumption of the starting material. The reaction mixture was diluted with ethyl acetate and filtered through a cehte bed. The organic layer was
concentrated, dried and purified by column chromatography to give the compound VIII.
1H NMR (CDCI3, 200 MHz) 1.56-2.15 (m, 8H), 2.25-2.80 (m, 16H), 3 45-3.82 (m, 3H), 3.92 (s, 3H), 4.0-4.15 (m, 2H), 4 22-4 37 (t, 2H), 6.70 (s, 1H), 7.42 (s, 1H), 7.60 (d, 1H, J = 4 25 Hz), 7.72 (t, 2H, J = 7.4 Hz), 8.16 (d, 2H, /= 8 1 Hz), 8 56 (d, 2H, J = 7.42 Hz). Example 4
To a solution of (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrohdine-2-carboxaldehyde diethyl thioacetal of formula IV (800 mg, 2 mmol) in acetone were added anhydrous K2CO3 (829 mg, 6 mmol) and 1,3 dibromo propane (1 gr, 5mmol) and the mixture was refluxed for 48h.After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under redused pressure, purification by column chromatography afforded compound V. 1H NMR (CDCI3,200 MHz) 1.25-1.40 (m, 6H), 1.72-2.42 (m, 6H), 2.70-2.8 (m, 4H), 3.15-3.30 (m, 2H), 3.60 (t, 2H, J = 6.20 Hz), 3.95 (s, 3H), 4 20 (t, 2H, J= 4.96 Hz), 4.60-4.75 (m, 1H), 4.82 (d, 1H, 7=4.33 Hz), 6.78 (s, 1H), 7 68 (s, 1H). To a solution of 2S-N-[4-(3-bromo propoxy)-5-methoxy-2-mtrobenzoyl]pyrrolidine-2-carbaxaldehyde diethyl thioacetal of formula V (521 mg, 1 mmol), piperazine attached naphthahmide (324 mg, 1 mmol) in acetone were added anhydrous K2CO3 (415 mg, 3 mmol) and the mixture was refluxed for 24h. After completion of reaction K.2CO3 was removed by filtration and the solvent was evaporated under reduced pressure, purification by column chromatography afforded compound VI. 1H NMR (CDCI3, 200 MHz) 1.25-1.42 (m, 6H), 1.70-2.40 (m, 8H), 2.60-3.30 (m, 18H), 3.92 (s, 3H), 4.05 (m, 4H), 4.70-4.80 (m, 1H), 4.82 (d, 1H, .7=4.25 Hz), 6.77 (s, 1H), 7.60 (s, 1H), 7.75 (t, 2H, 7=7.35 Hz), 8.18 (d, 2H, /= 8 Hz), 8.55 (d, 2H, J = 7.55 Hz).
To a solution of 2S-N-{4-[3-[3-[4-[3-(l,3-dioxo-benz[de]isoquinohn-2-yl)propyl]piperazin-l-yl]propyl]-oxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carbaxaldehyde diethyl thioacetal of formula VI (764 mg, 1 mmol) .in methanol was added SnCl2.2H20 (1.12 gr, 5 mmol) and the mixture was refluxed until the TLC indicated the completion of reaction. The methanol was evaporated and 10% NaHCO3 solution was added. The aqueous layer was extracted with ethyl acetate, the combined organic phases were dried over Na2S04 and evaporated under vacuum to afford the ammo thioacetal (VII) and directly used in the next step.
A solution of VII (734 mg, 1 mmol) HgCl2 (624 mg, 2.3 mmol) and CaC03 (250 mg, 2.5 mmol) in CH3CN-H20 (4:1) was stirred at room temperature until the TLC indicated complete consumption of the starting material. The reaction mixture was diluted with ethyl acetate and filtered through a cehte bed. The organic layer was concentrated, dried and purified by column chromatography to give the compound VIII.
1H NMR (CDC13, 200 MHz) 1.75-2.18 (m, 8H), 2.22-2.80 (m, 16H), 3.45-4 30 (m, 10H), 6.75 (s, IH), 7.45 (s, IH), 7.60 (d, IH, J- 4.2 Hz), 7.72 (t, 2H, ./= 7.4 Hz), 8.20 (d, 2H,J= 8.1 Hz), 8.58 (d, 2H, J= 7.35 Hz). Example 5
To a solution of (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrohdine-2-carboxaldehyde diethyl thioacetal of formula IV (800 mg, 2 mmol) in acetone were added anhydrous K2CO3 (829 mg, 6 mmol) and 1,4 dibromo butane (1 gr, 5 mmol) and the mixture was refluxed for 48h.After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under redused pressure, purification by column chromatography afforded compound V. 1H NMR (CDC13,300 MHz) 1.30-1.40 (m, 6H), 1.75-2.40 (m, 8H), 2.70-2.85 (m, 4H), 3.15-3.30 (m, 2H), 3.50 (t, 2H, J = 6.25 Hz), 3.95 (s, 3H), 4.10 (m, 2H), 4.60-4.70 (m, IH), 4.82 (d, IH, 7=4.3 Hz), 6.75 (s, IH), 7 62 (s, IH)
To a solution of 2S-N-[4-(4-bromo butoxy)-5-methoxy-2-mtrobenzoyl]pyrrolidine-2-carbaxaldehyde diethyl thioacetal of formula V (538mg, 1 mmol), piperazine attached naphthahmide (337 mg, 1 mmol) in acetone were added anhydrous K2CO3 (415 mg, 3 mmol) and the mixture was refluxed for 24h. After completion of reaction K2CO3 was removed by filtration and the solvent was evaporated under reduced pressure, purification by column chromatography afforded compound VI. 'H NMR (CDC13,200 MHz) 1.60-2.33 (m, 12H), 2.52-3.0 (m, 16H), 3.12-3.30 (m, 2H), 3.95 (s, IH), 4.02-4.25 (m, 4H), 4.60-4.72 (m, IH), 4.80 (d, IH, .7=4.3 Hz), 6.75 (s, IH), 7.60 (s, IH), 7.75 (t, 2H, 7=7.45 Hz), 8.18 (d, 2H, J= 8.2 Hz), 8.56 (d, 2H, J = 7.6 Hz).
To a solution of 2S-N-{4-[4-[4-[4-(l,3-dioxo-benz[de]isoquinohn-2-yl)butyl]piperazin-l-yl]butyl]-oxy-5-methoxy-2-nitrobenzoyl}pyrrohdine-2-carbaxaldehyde diethyl thioacetal (792 mg, 1 mmol) of formula VI .in methanol was added SnCl2.2H20 (1.12 gr, 5 mmol) and the mixture was refluxed until the TLC indicated the completion of reaction. The methanol was evaporated and 10% NaHCO3
solution was added. The aqueous layer was extracted with ethyl acetate, the combined
organic phases were dried over Na2SO4 and evaporated under vacuum to afford amino
thioacetal (VII) and directly used in the next step.
A solution of VII (762 mg, 1 mmol) HgCl2 (624 mg, 2.3 mmol) and CaCO3 (250 mg,
2.5 mmol) m CH3CN-H2O (4:1) was stirred at room temperature until the TLC
indicated complete consumption of the starting material. The reaction mixture was
diluted with ethyl acetate and filtered through a cehte bed. The organic layer was
concentrated, dried and purified by column chromatography to give the compound
VIII.
1H NMR (CDC13, 300 MHz) 1.50-2.10 (m, 12H), 2.25-2.80 (m, 16H), 3.45-4.30 (m,
10H), 6.75 (s, 1H), 7.45 (s, 1H), 7.62 (d, 1H, J= 4.2 Hz), 7.75 (t, 2H, J = 7.3 Hz),
8.20 (d, 2H, J= 8.1 Hz), 8.56 (d, 2H,7= 7.40 Hz).
Biological activity
In vitro cytotoxicity against human cancer cell lines: The human cancer cell lines procured from National Cancer Institute, Fredenck, U.S.A or National Center for Cell Science; Pune, India, were used in present study. Cells were grown in tissue culture flasks in complete growth medium (RPMI-1640 medium with 2 raM glutamine, 100 ng/ml streptomycin, pH 7.4, sterilized by filtration and supplemented with 10% fetal calf serum and 100 units/ml penicillin before use) at 37°C in an atmosphere of 5% CO2 and 90% relative humidity in a carbon dioxide incubator. The cells at subconfluent stage were harvested from the flask by treatment with trypsin (0.5% in PBS containing 0.02% EDTA) for determination of cytotoxicity. Cells with viability of more than 98% as determined by trypan blue exclusion were used for assay. The cell suspension of the required cell density were prepared in complete growth medium with gentamycin (50µg/ml) for determination of cytotoxicity. A stock solutions of (2 X 10"2 M) of test material were prepared in DMSO . The stock solutions were serially diluted with complete growth medium containing 50µg/ml of gentamycin to obtain working test solutions of required concentrations. In vitro cytotoxicity against human cancer cell lines was determined (Monks, A., Scudiero, D., Skehan, P, Shoemaker R., Paull, K., Vistica, D., Hose, C, Langley, J., Cronise, P., Vaigro-Wolff, A., Gray-Goodrich, M., Campbell,H., Mayo, J and Boyd M. J. Natl. Cancer Inst.,1991,83, 757-766) using 96-well tissue culture plates. The 100 µl of cell suspension was added to each well of the 96-well tissue culture plate. The cells were incubated for 24 hours. Test matenals in complete growth medium
(100µl) were added after 24 hours incubation to the wells containing cell suspension. The plates were further incubated for 48 hours (at 37°C in an atmosphere of 5% and 90% relative humidity in a carbon dioxide incubator) after addition of test material and then the cell growth was stopped by gently layering trichloroacetic acid (TCA, 50ul, 50%) on top of the medium in all the wells. The plates were incubated at 4°C for one hour to fix the cells attached to the bottom of the wells. The liquid of all the wells was gently pipetted out and discarded. The plates were washed five times with distilled water to remove TCA, growth medium low molecular weight metabolites, serum proteins etc and air-dned. Cell growth was measured by staining with sulforhodamine B dye (Skehan et al., 1990). The adsorbed dye was dissolved in Tns-Buffer (100 m 1, 0.01M, pH 10.4) and plates were gently stirred for 5 minutes on a mechanical stirrer. The optical density was recorded on ELISA reader at 540 nm. The cell growth was calculated by subtracting mean OD value of respective blank from the mean OD value of experimental set. Percent growth in presence of test material was calculated considering the growth in absence of any test material as 100% and in turn percent growth inhibition in presence of test material will be calculated. Cytotoxicity:
Compounds evaluated for the primary anticancer activity. The cytotoxicity data for
some representative compounds has shown in Table 1.
Table 1. The percentage growth inhibition data for napthahmide-PBD hybrids
Compd Cancer
(Table Removed)
anot tested Table 2. DNA Thermal Denaturation Studies:
(Table Removed)
For CT-DNA alone at pH 7.00 □ 0.01, Tm = 69.8 □C □ 0.01 (mean value from 10 separate determinations), all □ Tm values are □ 0.1 - 0.2 □C. For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentration = 100 □M and ligand concentration = 20 DM in aqueous sodium phosphate buffer [10 mM sodium phosphate + 1 mM EDTA, pH 7.00 □ 0.01].

We claim
1. A novel pyrrolo[2,1-c][1,4]benzodiazepines of formula VIII where n1 = 2, 3, 4,
n2 =2,3,4.
(Formula Removed)
2. 7-Methoxy-8- {2-[4-[2-( 1,3-dioxo-benz[de]isoquinolin-2'-yl)ethyl]piperazin-1 -
yl]ethyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo [2,1-
c][1,4]benzodiazepin-5-one as disclosed in claim 1 has structural formula
shown below.
(Formula Removed)
3. 7-Methoxy-8- {3-[4-[2-( 1,3-dioxo-benz[de]isoquinolin-2-yl)ethyl]piperazin-1 -
yl]propyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo [2,1-c][1,4]benzodiazepin-
5-one as disclosed in claim 1 has structural formula shown below.
(Formula Removed)
4. 7-Methoxy-8-{4-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-yl)ethyi]piperazin-l-
yl]butyl}-oxy-(11 aS)-1,2,3,11 a tetrahydro-5H-pyrrolo [2,1 -c][1,4]benzodiazepin-5-
one as disclosed in claim 1 has structural formula shown below.
(Formula Removed)
5. 7-Methoxy-8-{3-[4-[3-(1,3-dioxo-benz[de]isoquinolin-2-yl)propyl]piperazin-l-
yl]propyl}-oxy-(11 aS)-1,2,3,11 a tetrahydro-5H-pyrrolo [2,1 -c][1,4]benzodiazepin-
5-one as disclosed in claim 1 has structural formula shown below.
(Formula Removed)
6. 7-Methoxy-8- {4-[4-[4-( 1,3-dioxo-benz[de]isoquinolin-2-yl)butyl]piperazin-1 -
yl]butyl}-oxy-(11 aS)-1,2,3,11 a tetrahydro-5H-pyrrolo [2,1 -c][1,4]benzodiazepin-5-
one as disclosed in claim 1 has structural formula shown below.
(Formula Removed)
7. 7-Methoxy-8- {4-[4-[2-( 1,3-dioxo-benz[de]isoquinolin-2-yl)propyl]piperazin-1 -
yl]butyl}-oxy-(11 aS)-1,2,3,11 a tetrahydro-5H-pyrrolo [2,1 -c][1,4]benzodiazepin-5-
one as disclosed in claim 1 has structural formula shown below.
(Formula Removed)
8. 7-Methoxy-8-{3-[4-[2-(l,3-dioxo-benz[de]isoquinolin-2-yl)butyl]piperazin-l-
yl]propyl}-oxy-(11aS)-1,2,3,11a tetrahydro-5H-pyrrolo [2,1-c][1,4]benzodiazepin-
5-one as disclosed in claim 1 has structural formula shown below.

(Formula Removed)
9. 7-Methoxy-8- {3-[4-[3-( 1,3-dioxo-benz[de]isoquinolin-2-yl)propyl]piperazin-1.
yl]ethyl}-oxy-(11 aS)-1,2,3,11a tetrahydro-5H-pyrrolo [2,1 -c][1,4]benzodiazepin-5-
one as disclosed in claim 1 has structural formula shown below.
(Formula Removed)
10. A process for the preparation of pyrrolo[2,1-c][1,4]benzodiazepines of formula
VIII
(Formula Removed)
where n1 = 2, 3, 4, n2 = 2, 3, 4, which comprises of reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula IV with dibromoalkanes in an aprotic water miscible organic solvent in the presence of a mild inorganic base at refluxing temperature for a period of 48h, isolating 2S-N-[4-(n-bromo alkoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carbaxaldehyde diethyl thioacetal of formula V, reacting the compound of formula V with piperazine attached naphthalimide in presence of mild inorganic bases isolating compound of-formula VI, reducing it with SnCI2.2H20 in presence of organic solvent at a reflux temperature, reacting the above amino compound of formula VII with a deprotecting agent to obtain the pyrrol[2,l-c][l,4]benzodiazepine of formula VIII wherein n is as stated above.

Documents:

1218-del-2004-abstract.pdf

1218-DEL-2004-Claims-(09-05-2012).pdf

1218-del-2004-claims.pdf

1218-DEL-2004-Correspondence Others-(09-05-2012).pdf

1218-DEL-2004-Correspondence Others-(23-08-2011).pdf

1218-del-2004-correspondence-others.pdf

1218-DEL-2004-Description (Complete)-(09-05-2012).pdf

1218-del-2004-description (complete).pdf

1218-del-2004-drawings.pdf

1218-del-2004-form-1.pdf

1218-del-2004-form-18.pdf

1218-del-2004-form-2.pdf

1218-DEL-2004-Form-3-(23-08-2011).pdf

1218-del-2004-form-3.pdf

1218-del-2004-form-5.pdf

1218-DEL-2004-Petition-137-(23-08-2011).pdf

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Patent Number

257917

Indian Patent Application Number

1218/DEL/2004

PG Journal Number

47/2013

Publication Date

22-Nov-2013

Grant Date

19-Nov-2013

Date of Filing

30-Jun-2004

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	AHMED KAMAL	INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY (IICT), HYDERABAD, ANDHRAPRADESH, INDIA.
2	RONDLA RAMU	INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY (IICT), HYDERABAD, ANDHRAPRADESH, INDIA.
3	GOLLAPALLI BHASKER	INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY (IICT), HYDERABAD, ANDHRAPRADESH, INDIA.

PCT International Classification Number

C07D 243/12

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA