AU1500901A - Fused azepinone cyclin dependent kinase inhibitors - Google Patents

Fused azepinone cyclin dependent kinase inhibitors Download PDF

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AU1500901A
AU1500901A AU15009/01A AU1500901A AU1500901A AU 1500901 A AU1500901 A AU 1500901A AU 15009/01 A AU15009/01 A AU 15009/01A AU 1500901 A AU1500901 A AU 1500901A AU 1500901 A AU1500901 A AU 1500901A
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Australia
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group
benzazepin
aliphatic
hydrogen
indolo
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AU15009/01A
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AU780528B2 (en
Inventor
Rick Gussio
Ravi K. Jalluri
Conrad Kunick
Laurent Meijer
Edward A. Sausville
Daniel W. Zaharevitz
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Centre National de la Recherche Scientifique CNRS
US Department of Health and Human Services
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Centre National de la Recherche Scientifique CNRS
US Department of Health and Human Services
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Priority claimed from PCT/US1999/013579 external-priority patent/WO1999065910A1/en
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Priority to AU15009/01A priority Critical patent/AU780528B2/en
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Assigned to KUNICK, CONRAD, CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE, UNITED STATES OF AMERICA, REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES, THE reassignment KUNICK, CONRAD Alteration of Name(s) of Applicant(s) under S113 Assignors: KUNICK, CONRAD, MEIJER, LAURENT, UNITED STATES OF AMERICA, REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES, THE
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Description

AUSTRALIA
Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES CONRAD KUNICK LAURENT MEIJER Invention Title: FUSED AZEPINONE CYCLIN DEPENDENT KINASE INHIBITORS The following statement is a full description of this invention, including the best method of performing it known to me/us: FUSED AZEPINONE CYCLIN DEPENDENT KINASE INHIBITORS This application is both a divisional of Australian patent application 45714/99 and an application for patent of addition to Australian patent application 45714/99. This application principally concerns improvements in, or modifications of, a main invention described in 45714/99.
FIELD
This invention concerns fused azepinone cyclin dependent kinase inhibitors, compositions comprising these compounds, and methods for administering such compounds for diseases of cellular proliferation and/or abnormal protein phosphorylation.
BACKGROUND
A major advance in the understanding of the control of the cell cycle has been the discovery of a family of enzymes called cyclin dependent kinases (cdk).
Structurally they consist of a catalytic subunit and a regulatory subunit. The catalytic subunit is similar to the catalytic region in a number of serine/threonine o kinases and at least eight distinct subunits have been described (cdkl(=cdc2)-cdk8).
20 The regulatory subunit is necessary for activity and a number of proteins in this family have been described (cyclin A-cyclin Most cyclins can interact with more than one cdk and each known cyclin-cdk pair seems to have a distinct role in i: regulating the cell cycle. These activities are regulated not only through transcriptional and translational control of the subunits, but also through phosphorylation and dephosphorylation of the subunits. In addition, negative regulatory proteins have been discovered (p15, p 1 6
N
K4 p21 cipl and p27 Kipl which bind to the cyclin-cdk complex and inhibit activity. Structural understanding of the cdks and their regulation has been advanced by the solution of crystal structures for cdk2, cyclin A, cdk2-cyclin A, and cdk2-cyclin A-p 2 7 ki p l [Russo et al., "Crystal Structure of the p 2 7 Kipl Cyclin-Dependent Kinase-4 Bound to the Cyclin-A-Cdk2 Complex," Nature, 382(6589):325-331 (1996).] \\mbfilc\hon S\suzannep\eep\SccM47 14-9. I Divisional SPECIdoc 16/01/01 -2- It is clear then that the cdks are important in the control of the cell cycle. As a result, it appears that alterations in cdk expression, function or regulation are associated with diseases of cellular proliferation. Alterations that would increase cdk activity (overexpression of the catalytic and/or positive regulatory subunit, or underexpression or deletion of negative regulatory proteins) have been reported in many cancers. The most common observation has been the deletion of the p16 (also called MTS 1, CDKN2, p1 6 NK) gene. This gene codes for a protein that inhibits the activity of cdk4 and cdk6. This loss of inhibitory activity has been observed in a wide variety of primary human tumors and human tumor-derived cell lines, including lung, breast, brain, bone, skin, bladder, kidney, ovary, liver, colon, pancreas and leukemias. Overexpression of cdkl in ovarian carcinoma and overexpression of cyclin D in non-small cell lung cancer also has been observed.
Clinical studies have shown that alterations in cdk pathways have prognostic significance. Deletion of the p16 gene has shown to be associated with poor prognosis in B cell lymphomas Garcia-Sanz et al., "Deletions and Rearrangement of Cyclin-Dependent Kinase 4 Inhibitor Gene p16 are Associated with Poor Prognosis in B cell Non-Hodgkin's Lymphomas," Leukemia, 11(11): 1915- (1997)], and pediatric acute lymphoblastic leukemia U.R. Kees et al.
"Homozygous Deletion of the pl6/MTS1 Gene in Pediatric Acute Lymphoblastic Leukemia is Associated with Unfavorable Clinical Outcome," Blood, 89(11):4161-6 (1997)]. High expression of cyclin D1 has also been shown to predict early relapse in pediatric ALL. Kees et al., "Deletions of the p16 Gene in Pediatric Leukemia and Corresponding-Cell Lines," Oncogene, 12(10):2235-9 (1996).] High expression of cdkl predicts disease recurrence in prostate adenocarcinoma. [B.V.
25 Kallakury et al., "The Prognostic Significance ofp 3 4 cdc 2 and Cyclin Dl Protein Expression in Prostate Adenocarcinoma," Cancer, 80(4):753-63 (1997). Loss of p21 expression resulted in a significantly higher risk of recurrence following surgery "for gastric carcinoma. M. Ogawa et al., "Loss of p21WAF l
/CIP
l Expression Correlates with Disease Progression in Gastric Carcinoma," Br. J. Cancer, 75(11):1617-20 (1997).] Higher p27 expression has correlated with longer survival times in breast C. Catzavelos et al., "Decreased Levels of the Cell-Cycle Inhibitor of p27'
IP
Protein: Prognostic Implications in Primary Breast Cancer," Nat. Med., 3(2):227-30 \\mlbfilc\honcSzancet\Keep\Speci 4514-99.1 Divisional SPECIdoc 16101/01 -3- (1997)], and non-small cell lung cancer Esposito, "Prognostic Role of the Cyclin-Dependent Kinase Inhibitor p27 in Non-Small Lung Cancer," Cancer Res., 57(16):3381-5 (1997)].
Atherosclerosis is another disease associated with excessive cellular proliferation. An important signal for proliferation of vascular smooth muscle cells is increased expression of cdk2 and associated regulatory subunits, cyclin E and cyclin A. C. Ihling, et al., "Topographical Association Between the Cyclin- Dependent Kinases Inhibitor P21, p 5 3 Accumulation, and Cellular Proliferation in Human Atherosclerotic Tissue," Arterioscler. Thromb. Vasc. Biol., 17(10):2218-24 (1997).] This is consistent with the observation that high levels ofhomocysteine, known to cause occlusive arterial disease, causes increases in aortic cdk activity. [B.
Lubec et al., "Homocysteine Increases Cyclin-dependent Kinase in Aortic Rat Tissue," Circulation, 94(10):2620-5 (1996).] The involvement of cdk2 also is consistent with the report that an antisense cdk2 oligonucleotide can prevent graft coronary arteriosclerosis. Suzuki et al., "Prevention of Graft Coronary Arteriosclerosis by Antisense cdk2 Kinase Oligonucleotide," Nat. Med., 3(8):900-3 (1997).] Other diseases in which there is evidence that inhibitors of cdks may be of therapeutic use include mesangial proliferative glomerulonephritis Pippin et al., "Direct in vivo Inhibition of the Nuclear Cell Cycle Cascade in Experimental Mesangial Proliferative Glomerulonephritis with Roscovitine, a Novel Cyclin- Dependent Kinase Antagonist," J. Clin. Invest., 1900(9):2512-20 (1997)], infection with human cytomegalo-virus Bresnahan et al., "Inhibition of Cellular cdk2 Activity Blocks Human Cytomegalovirus Replication," Virology, 231(2):239-47 i 25 (1997)], and malaria Graeser et al., "Plasmodium Falciparum Protein Kinase and the Malarial Nuclear Division Cycles," Mol. Biochem. Parasitol., 82(1):37-49 (1996)]. Abnormal phosphorylation of tau protein is a characteristic of Alzheimer's disease. Recent reports have shown that this phosphorylation is carried out, at least in part, by brain cdk5 A. Sengupta et al., "Potentiation of GSK-3-Catalyzed Alzheimer-like Phosphorylation of Human tau by cdk5," Mol.Cell. Biochem., 167(1- 2):99-105 (1997)]. Inhibitors of cdk5 should be useful in the treatment of the disease.
\\melbfllc\homeS\suz nct\Keep\Spcci\457 4-99.1 Divisional SPECIdoc 16101/01 The potential role of cdk inhibitors in therapy of numerous diseases has led to efforts to find small molecules that inhibit all or some of the cdks. Several small molecules have been discovered that inhibit cdks specifically. These include the purine analogs, olomoucine, roscovitine, and CVT-313; the flavonoid, flavopiridol; and butyrolactone I. Other potent inhibitors of cdks are known, including staurosporine, UCN-01, and suramin, but these compounds also are potent inhibitors of other protein kinases. Crystal structures for several specific cdk inhibitors in complex with cdk2 have been determined, including olomoucine, roscovitine, and flavopiridol. One specific inhibitor ofcdks has reached clinical trials, flavopiridol Sadlacek et al., "Flavopiridol (186-8275, NSC-649890), a New Kinase Inhibitor for Tumor Therapy," International Journal of Oncology, 9:1143 (1996)], which has shown antitumor activity in Phase I trials in a number of tumor types and is progressing to Phase II trials. All other known cdk inhibitors have been shown to inhibit the growth of tumor cells in culture, although none are as potent as flavopiridol. No in vivo anti-tumor studies have been reported for these compounds, although there is an anecdotal report of a response to olomoucine in a spontaneous dog melanoma.
Several cdk inhibitors have shown activity in models for other diseases.
Animal studies have shown that CVT-313 is an effective inhibitor of neointimal proliferation in a rat restenosis model. Brooks et al., "CVT-313, a Specific and S'Potent Inhibitor of CDK2 that Prevents Neointimal Proliferation," J. Biol. Chem., 272(46):29207-11 (1997).] Roscovitine has been reported to improve renal function *ooi* •in a rat model of glomerulonephritis, and to be an inhibitor of human cytomegalovirus replication in culture. Roscovitine also inhibits DNA synthesis in 25 plasmodiumfalciparum, the malarial parasite. R. Graser et al., supra.
7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one is a known compound.
This compound originally was synthesized by Dr. Conrad Kunick, then at the Pharmazeutisches Institut der Universitit Bonn. C. Kunick, "Synthese von 7,12dihydro-indolo[3,2-d][1]benzazepin-6(5H)-onen und 6,11-dihydroindolo[3',2':2,3]azepino[4,5-b]indol-5(4H)-on," Arch. Pharm. (Weinheim), 325:297- 299 (1992).
\\melb-fles\honcS\suaznnec\Keep\SpccM 7 514-99.1 Divisional SPECIdoc 16/01/01 From the above discussion it is clear that known cyclin-dependent kinase inhibitors are useful for treating diseases of cellular proliferation and/or abnormal protein phosphorylation. But, there still is a need for new potent, and preferably selective, cdk inhibitors.
SUMMARY
The invention of 45714/99 provides a new class ofcyclin dependent kinase inhibitors that also have shown antiproliferative activity in human tumor cell line assays. These compounds typically satisfy the formula
R
2
A
R
3 wherein A is oxygen or sulfur bonded to the ring by a single or a double bond, Y and Z are conjugated rings, the Y ring has at least one carbon atom with a substituent RI selected from the group consisting of alkoxy, amino, acyl, aliphatic substituents, particularly alkyl, alkenyl and alkinyl substituents, and even more 15 particularly lower aliphatic substituents, cyano, nitro, carboxyl, halogen, hydrogen, hydroxyl and imino, R 2 is selected from the group consisting of hydrogen, benzyl, •lower alkyl, and lower alkyl ester, the Z ring has a substituent R 3 selected from the group consisting of hydrogen, lower aliphatic substituents, particularly lower alkyl substituents, or cyclic alkyl. "Lower" as used herein typically refers to compounds 20 or substituents having 10 or fewer carbon atoms in a chain, and includes all position, geometric and stereoisomers of such substituents or compounds. Furthermore, if R 1 and R 2 are hydrogen and the Z ring comprises a five membered ring fused to a six membered ring then the six membered ring includes a substituent other than bromine. A is most typically a double bond, and the majority of compounds made to date have A double bonded to oxygen.
The compounds of the present invention further satisfy the formula \\rnIb filcs\ho S\suzannet\Keep\Spcc 7 I 4-99.1 Divisional SPECI.A& 16/01/01
RIO
wherein A is oxygen or sulfur coupled to the ring by a single or double bond; R 2 is selected from the group consisting of hydrogen, aryl, and lower aliphatic substituents; R 4
-R
7 are independently selected from the group consisting of alkoxy, amino including aliphatic amines such as aminoalkyl, acyl, aliphatic, aliphatic alcohols, aliphatic nitriles, cyano, nitro, carboxyl, halogen, hydrogen, hydroxyl, imino, a, 3 unsaturated ketones and epoxy, including aliphatic epoxys such as epoxyalkyl; R 8 -R 1 are independently selected from the group consisting of aliphatic, aliphatic alcohols, alkoxy, acyl, cyano, nitro, epoxy, haloalkyl groups, halogen, hydrogen, hydroxyl sulfoxide, sulfone and aminosulfonyl; R 12 is selected 10 from the group consisting of aliphatic, aliphatic alcohols, carboxylic acids and hydrogen, and wherein: at least one of R 4
R
7 is aminoalkyl or epoxy, and/or at least one of R 8 RII is sulfoxide, sulfone or aminosulfonyl.
As used herein, the term "epoxy" should be construed as including within its scope aliphatic epoxy such as epoxyalkyl. Particular examples of compounds satisfying these formulas have: R 2 selected from the group consisting of H, -CH 2
COOCH
3
CH
3 and -CH 2 Ph, preferably hydrogen; R4-R 7 independently selected from the group consisting of alkoxy, amino including aliphatic amines such as aminoalkyl, ~acyl, alkyl, alkenyl, alkinyl, cyano, nitro, carboxyl, halogen, hydrogen, hydroxyl and imino, lower aliphatic alcohols, lower aliphatic nitriles, and a, P unsaturated ketones and epoxy, including aliphatic epoxy such as epoxyalkyl, cyano groups, and nitro groups, particularly -OH, -C(=NH)NH 2
-CO
2 H, -Br and -OCH 3 Rs-RI 1 independently selected from the group consisting of alcohols, alkoxy, acyl, alkyl, alkenyl, alkinyl, cyano, nitro, epoxy, haloalkyl, halogen, hydrogen, hydroxyl and lower alkyl, particularly halogens, -OH, -CH 2 0H, -CH 2
CHOCH
2 \\mclbfilcs\hom$\suzannet\Kcep\Speci45714-99.1 Divisional SPECI.doc 16/01/01 -7-
CH
2
CH
2
CHOCH
2
-CF
3 -OCH3 sulfoxide, sulfone and aminosulfonyl; and R 12 selected from the group consisting of alcohols, carboxylic acids, hydrogen and lower alkyl groups, particularly -CH 2 CH20H, -CH3 and -CH 2
CH
3 The present invention also provides compositions comprising effective amounts of a compound, or compounds, satisfying the formulas above. Such compositions may further comprise inert carriers, excipients, diagnostics, direct compression binders, buffers, stabilizers, fillers, disintegrants, flavors, colors, lubricants, other active ingredients, other materials conventionally used in the formulation of pharmaceutical compositions, and mixtures thereof.
A method for inhibiting the proliferation of living cells in a subject, such as hyperproliferative cells and/or neoplastic cells, or for treating a neoplasm in a subject also is provided. The method first comprises providing a compound or compounds, or a composition comprising the compound or compounds, as described above. An effective amount of the compound(s) or composition(s) is then administered to a subject to inhibit the proliferation of living cells. Administering the compound(s) or composition(s) generally comprises administering topically, orally, intramuscularly, intranasally, subcutaneously, intraperitoneally, intralesionally or intravenously. The currently preferred administration method is ego* intravenous. The effective amount should be as high as the subject can tolerate, but O typically is from about 0.1 gram to about 3.0 grams of a compound, and from about mg/kg of subject/dose to about 400 mg/kg of subject/dose, preferably from about mg/kg of subject/dose to about 50 mg/kg of subject/dose.
S
BRIEF DESCRIPTION OF THE DRAWINGS Figures 1 to 32 correspond exactly to Figures 1 to 32 of parent application 45714/99. If difficulty is experienced in reading these figures, reference should be made to the figures of 45714/99, which are by virtue of this reference intended to be expressly incorporated into the disclosure of the present application. It is noted that these figures were deemed to be acceptable by the Receiving Office at which the parent application was originally filed.
H!uznng\Kcp\SpccM5114-99.1 DivLiioIa SPECIAMENDED 20.4.2001doc 20/04/0 7a FIG. 1 provides mean plots of data obtained by in vitro human tumor cell line screens for 9-bromo-7, 12-dihydro-2,3-dimethoxy-indolo[3,2-d] ]benzazepin- FIG. 2 provides mean plots of data obtained by in vitro human tumor cell line screens for 7,12-dihydro-2,3-dimethoxy-9-trifluoromethyl-indolo[3,2d] [1]benzazepin-6(5H)-one.
FIG. 3 provides mean plots of data obtained by in vitro human tumor cell line screens for 9-bromo-7, 12-dihydro-indolo [1]benzazepin-6(5H)-one.
0 *000% H~suzanneci\e~p\SpecM714-99.1 Divsion SPECIAMENDED 20.4.2001.doc 20/04MI FIG. 4 provides mean plots of data obtained by in vitro human tumor cell line screens for 7,12-dihydro-9-trifluoromethyl-indolo[3,2-d][1 one.
FIG. 5 provides mean plots of data obtained by in vitro human tumor cell line screens for 9-chloro-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one.
FIG. 6 provides mean plots of data obtained by in vitro human tumor cell line screens for 8-bromo-6,11-dihydro-thieno[3',2':2,3]azepino[4,5-b]indol-5(4H)one.
FIG. 7 provides mean plots of data obtained by in vitro human tumor cell line screens for 9-methoxy-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one.
FIG. 8 provides mean plots of data obtained by in vitro human tumor cell line screens for 9-bromo-7,12-dihydro-indolo[3,2-d][1 FIG. 9 provides mean plots of data obtained by in vitro human tumor cell line screens for 9-bromo-7,12-dihydro-12-(2-hydroxyethyl)-indolo[3,2d][1]benzazepin-6(5H)-one.
FIG. 10 provides mean plots of data obtained by in vitro human tumor cell line screens for 6-methylthio-7,12-dihydro-indolo[3,2-d][1]benzazepine.
FIG. 11 provides mean plots of data obtained by in vitro human tumor cell line screens for 2,3-dimethoxy-9-nitro-7,12-dihydro-indolo[3,2-d][1]benzazepin- 20 FIG. 12 provides mean plots of data obtained by in vitro human tumor cell line screens for 9-cyano-7,12-dihydro-indolo[3,2-d][1 FIG. 13 provides mean plots of data obtained by in vitro human tumor cell line screens for 2,3-dimethoxy-9-cyano-7,12-dihydro-indolo[3,2-d][1]benzazepin- 25 FIG. 14 provides mean plots of data obtained by in vitro human tumor cell line screens for 9-nitro-7,12-dihydro-indolo[3,2-d][1 FIG. 15 provides mean plots of data obtained by in vitro human tumor cell line screens for 3-(6-oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[3,2d] [1 ]benzazepin-2-yl)-propionitrile FIG. 16 provides mean plots of data obtained by in vitro human tumor cell line screens for 2-bromo-9-nitro-7,12-dihydro-indolo[3,2-d][1 \\melbfie\hon, \sxuzannc\Kcp\Spcci\45714-99.1 Divisioal SPECIdoc 16/01/01 one.
FIG. 17 provides mean plots of data obtained by in vitro human tumor cell line screens for 3-(6-oxo-9-trifluoromethyl-5 12-tetrahydro-indolo[3,2d] [1I ]benzazepin-2-yl)-acrylonitrile.
FIG. 18 provides mean plots of data obtained by in vitro human tumor cell line screens for 2-(3-hydroxy- 1 -propinyl), 9-trifluoromethyl-7, 12-dihydroindolo[3,2-d] I1]benzazepin-6(5TH-one.
FIG. 19 provides mean plots of data obtained by in vitro human tumor cell line screens for 2-iodo-9-bromo-7, 12-dihydro-indolo[3 [1 one.
FIG. 20 provides mean plots of data obtained by in vitro human tumor cell line screens for 2-(3-oxo- 1 -butenyl), 9-trifluoromethyl-7, 12-tetrahydro-indolo[3 ,2d] [1I ]benzazepin-6(511)-one.
FIG. 21 provides mean plots of data obtained by in vitro human tumor cell line screens for 8-chloro-6, 11 -dihydro-thieno[3',2 t :2,3]azepino[4,5-b]indol-5(4H)one.
OV. FIG. 22 provides mean plots of data obtained by in vitro human tumor cell line screens for 2-iodo,9-triflouromethyl-7, 12-dihydro-indolo[3,2-d] [1]benzazepin- 6(511)-one.
FIG. 23 provides mean plots of data obtained by in vitro human tumor cell ~line screens for 7,1 2-dihydro-pyrido[3',2' :4,5 ]pyrrolo[3 [1]benzazepin-6(5H)one.
FIG. 24 provides mean plots of data obtained by in vitro human tumor cell 0:line screens for I11 -methyl-7, 12-dihydro-indolo [3 [1I 0'25 FIG. 25 provides mean plots of data obtained by in vitro human tumor cell line screens for 1-hydroxycyclohexyl)-ethinyl], 9-trifluoromethyl-7, 12dihydro-indolo[3,2-d] I ]benzazepin-6(511)-one.
FIG. 26 provides mean plots of data obtained by in vitro human tumor cell line screens for 2-cyano-7, 12-dihydro-indolo[3 [1 FIG. 27 provides mean plots of data obtained by in vitro human tumor cell line screens for 2-iodo-7, 12-dihydro-indolo [3 [1]benzazepin-6(511)-one- \\FneIbfies\hoS\suzanne\Kep\Speci'\4Sl 14-99.1 Divisional SPECIdoc 16/01/01 10 FIG. 28 provides mean plots of data obtained by in vitro human tumor cell line screens for 11 -ethyl-7,12-dihydro-indolo[3,2-d][1 FIG. 29 provides mean plots of data obtained by in vitro human tumor cell line screens for 8-methyl-6,11-dihydro-thieno[3',2':2,3]azepino[4,5-b]indol-5(4H)one.
FIG. 30 provides mean plots of data obtained by in vitro human tumor cell line screens for 3-(6-Oxo-9-trifluoromethyl-5,6,7,12-tetrahydro-indolo[3,2-d][1]benzazepin-2-yl)-acrylic acid, methyl ester.
FIG. 31 provides graphs illustrating in vitro time course assays for 9-nitro- 7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one.
FIG. 32 provides graphs illustrating in vitro time course assays for 9-nitro- 7,12-dihydro-indolo[3,2-d] [1 FIG. 33 provides mean plots of data obtained by in vitro human tumor cell line screens for 2-(2,3-Epoxypropyl)-9-trifluoromethyl-7, 12-dihydroindolo[3,2d][1]benzazepin-6(5H)-one.
FIG. 34 provides mean plots of data obtained by in vitro human tumor cell line screens for 2-(Epoxyethyl)-9-trifluoromethyl-7,12-dihydroindolo[3,2d][1]benzazepin-6(5H)-one.
FIG. 35 provides mean plots of data obtained by in vitro human tumor cell line screens for 9-Bromo-2-(2,3-epoxypropyl)-7,12-dihydroindolo[3,2- [1 FIG 36. provides mean plots of data obtained by in vitro human tumor cell line screens for 2-(2-oxopropyl)-9-trifluoromethyl-7,12-dihydroindolo[3,2d] [1 DETAILED DESCRIPTION In the following detailed description references to "the present invention" *refer to the main invention that is the subject ofAU 45714/99 which overlaps with the invention of the present divisional application for a patent of addition. This text should therefore be read in conjunction with the claims which specify the subject \\clbilc\homcS'suantKKep\Spc\457 4-99.1 Divisional SPECIldoc 16/01/01 -11 matter of the present application. This text is retained in the present specification for completeness.
The compounds of the present invention are specific cdk inhibitors, and also have significant biological activity in human tumor cell line assays. The following paragraphs describe how to make the compounds of the present invention, pharmaceutical compositions comprising such compounds, and methods for administering such compounds for treating diseases of cellular proliferation and/or abnormal protein phosphorylation and/or infectious diseases, such as cancer, atherosclerosis, Alzheimer's disease, and malaria.
L COMPOUNDS A. General Molecular Formula Compounds of the present invention generally satisfy Formula I below.
R
3 1 Formula 1 With reference to Formula 1, A is a oxygen or sulfur coupled to the B ring by a I single or double bond, typically a double bond. If A is a single bond, then it also includes a hydrogen or lower aliphatic group, particularly a lower alkyl group bonded thereto. Y and Z are conjugated rings or conjugated heterocyclic rings, and generally are independently 5- or 6-membered rings. "Conjugated" means that the rings include at least one double bond separated by a single bond from a double bond or heteroatom having a lone pair of electrons, such as O, S or N, or two or more double bonds separated by single bonds. Unless specified otherwise, the terms "ring" and "rings" include rings having just carbon atoms form the ring structure, as well as rings that include heteroatoms, heterocycles, that form the ring structure. Working examples of compounds made according to the present \\melb files\hom\suzannet\Kp\Speci\45714-99. I Divisional SPEC.doc 16/01/01 12invention have included benzene rings and thiophenes as the Y ring, and the Z ring has included pyrolidines and pyridines.
Y and Z also can comprise fused ring systems. For example, as described in more detail below, one example of a class of compounds satisfying Formula 1 have a Z ring comprising a five-membered ring fused to a six-membered ring.
Additional examples of ring structures are provided below in Table 1.
Y and Z also can include substituents other than hydrogen coupled thereto.
Examples of compounds satisfying Formula 1 include a Y ring having at least one carbon atom with a substituent Ri coupled thereto. R 1 typically is selected from the group consisting of acyl, aliphatic substituents, particularly alkyl, alkenyl and alkinyl substituents, cyano, nitro, alkoxy amino NR 2 including primary, secondary and tertiary amines), carboxyl (-CO 2 halogen, hydrogen, hydroxyl and imino [nitrogen-containing organic groups having a carbon-tonitrogen double bond]. Particular compounds of the present invention have included R 1 groups selected from the group consisting of H, -OH, -C(=NH)-NH 2
CO
2 H, Br and -OCH 3
R
2 typically is selected from the group consisting of hydrogen, aryl, lower aliphatic, particularly lower alkyl, alkyl aryl, benzyl, and lower alkyl ester.
Examples of particular groups attached to the amide nitrogen of Formula 1 include H, -CH 2
COOCH
3
-CH
3 and -CH 2 Ph. Compounds satisfying Formula 1 include a Z ring having at least one carbon atom with a substituent R 3 coupled thereto. R 3 generally is selected from the group consisting of hydrogen, lower alkyl or cyclic alkyl, cyclohexyl. Moreover, if RI and R 2 are hydrogen and the Z ring comprises a five membered ring fused to a six membered ring then the six membered ring includes a substituent other than bromine.
While Formula 1 is generic to compounds of the present invention, most of H-\suzneocKp\SpeclM5714-99.1 Divisioaal SPEC1AMENDED 20A.2001.oc 20/04,01 13the compounds of the invention further satisfy Formula 2.
R, RI A RlO Formula 2 With reference to Formula 2, A is oxygen or sulfur coupled to the B ring by a single bond or a double bond, generally a double bond. R 2 is selected from the group consisting of hydrogen, acyl, aliphatic substituents, particularly alkyl, alkenyl and alkinyl substituents, even more particularly lower alkyl substituents, cyano, nitro, aryl, and lower alkyl ester, preferably hydrogen. R 4
-R
7 are independently selected from the group consisting of alkoxy, amino, carboxyl, halogen, hydrogen, hydroxyl, imino, aliphatic alcohols, particularly lower alkyl alcohols, aliphatic nitriles, particularly lower aliphatic nitriles, and a, P unsaturated ketones. Particular examples of R 4
-R
7 without limitation, include -OH, -C(=NH)NH 2
-CO
2 H, cyanoethyl, 3-hydroxy-1-propinyl, 3-oxo-1-butenyl, 2-(1-hydroxycyclohexyl)ethinyl, halogens, particularly -Br, and -OCH 3
R
8 -Rll are independently selected from the group consisting of aliphatic alcohols, particularly lower alkyl alcohols, alkoxides, acyl substituents, aliphatic substituents, particularly alkyl, alkenyl and alkinyl substituents, even more particularly lower alkyl substituents, cyano, nitro, epoxides, a,3-unsaturated carbonyl-bearing groups, ethers, haloaliphatic substituents, such as haloalkyl fluoromethyl, difluoromethyl, trifluoromethyl), halogen, hydrogen, and hydroxyl. Particular examples of R 8 -Rl include, without limitation, -CN, Br, Cl, and F, -OH, -CH 2 OH, -CH 2
CHOCH
2 (propylene oxide),
-CH
2
CH
2
CHOCH
2 (butylene oxide), -CF 3 and -OCH 3
R
1 2 generally is selected from the group consisting of lower aliphatic, particularly lower alkyl, lower aliphatic alcohols, particularly lower alkyl alcohols, H.imuzanmgcKecp\SpccM5714-99.1 Divisional SPECIAMENDED 20A.2001.doc 20/04/01 ICF_ X 13a carboxylic acids, and hydrogen. Particular examples of R 12 include, without limitation, -CH 2
CH
2 OH, -CH 3 and -CH 2
CH
3 Examples of compounds satisfying both Formulas 1 and 2 can be selected, without limitation, from the group consisting of 7, 12-dihydro-indolo[3,2d] [1]benzazepin-6(5H)-one, 2-bromo-7, 12-dihydro-indolo[3,2-d] [1]benzazepin- 9- bromo-7, 12-dihydro-indo lo benzazepin-6(5H)- one, 9-chloro- 7,1 2-dihydro-indolo[3,2-d] [1]benzazepin-6(5H)-one, 1 l-chloro-7, 12-dihydroindolo [1]benzazepin-6(5H)-one, 1O-bromo-7, 12-dihydro-indolo[3,2- H~uzanncgKep\SpecM571499.1 Divisionaa SPECIAN4ENDED 20A.2001 doc 20/04M1I 14 d] [1 ]benzazepin-6(5H)-one, 8-bromo-6, 11 -dihydro-thieno[3',2' :2,3 azepino[4,5 b]indol-5(4H)-one, 9-bromo-7, 12-dihydro-4-methoxy-indolo[3 [1]benzazepin- 9-bromo-7, 12-dihydro-4-hydroxy-indolo[3 [1]benzazepin-6(5H)one, 7,1 2-dihydro-4-methoxy-indolo [3 [1]benzazepin-6(5H)-one, 9-bromo-7, 12dihydro-2,3-dimethoxy-indolo[3 [1]benzazepin-6(5H)-one, 9-bromo-7, 12dihydro-2,3-dihydroxy-indolo[3 [1]benzazepin-6(5H)-one, 7,1 2-dihydro-2,3dimethoxy-indolo[3 [1]benzazepin-6(5H)-one, 7,1 2-dihydro-9-trifluormethylindolo[3 [1]benzazepin-6(5H)-one, 7,1 2-dihydro-2,3-dimethoxy-9trifluoromethyl-indolo[3 [1]benzazepin-6(5H)-one, 2-bromo-7, 12-dihydro-9trifluoromethyl-indolo[3 [1]benzazepin-6(5H)-one, 9-bromo-7, 12-dihydroindolo [3 [1]benzazepin-6(5H)-thione, 9-bromo-5, 12-bis-(t-butyloxycarbonyl)- 7,1 2-dihydro-indolo[3 [1]benzazepin-6(5H)-one, 9-bromo- 12-(tbutyloxycarbonyl)-7, 12-dihydro-indolo[3 [1]benzazepin-6(5H)-one, 9-bromo- 5,7-bis-(t-butyloxycarbonyl)-7, 12-dihydro-indolo[3 [1]benzazepin-6(5H)-one, 9-bromo-5 12-tri-(t-butyloxycarbonyl)-7, 12-dihydro-indolo [3 [1 ]benzazepin- 9-bromo-7, 12-dihydro-5-methyloxycarbonylmethyl-indolo[3 ,2d] [1 benzazepin-6(5H)-one, 9-bromo-7, 12-dihydro- 12-methyloxycarbonylmethylindolo[3 [1]benzazepin-6(5H)-one, 9-bromo-7, 12-dihydro- 12-(2-hydroxyethyl)indolo[3 [1]benzazepin-6(5H)-one, 2,9-dibromo-7, 12-dihydro-indolo[3 ,2d] [1 ]benzazepin-6(5H)-one, 8,1 0-dichloro-7, 12-dihydro-indolo[3 ,2- [1]benzazepin-6(5H)-one, 9-cyano-7, 12-dihydro-indolo[3 ,2-dj [1]benzazepin- 6(H-oe *-rm-,2dhdo5mty-noo32d[~ezzpn65)oe 9-bromo-7, 1 2-dihydro-5-methyl-indolo[3,2-d] []benzazepin-6(5H)-one, 9 5-el9bromo-7,12-dihydro-5-methyl-indolo[3 ,2-d][1]benzazepin-6(5H)-one, 9--2 ethyl- 7,1I2-dihydro-indolo [3 1 ]benzazepin-6(5H)-one, 9-bromo-7, 12-d-ihydro- 1 2-(2-propenyl)-indolo[3 [1]benzazepin-6(5H)-one, 7,1 2-dihydro-9-methylindolo[3,2-d] [1]benzazepin-6(5H)-one, 7,1 2-dihydro-9-methoxy-indolo[3,2d] [1 ]benzazepin-6(5H)-one, 9-fluoro-7, 12-dihydro-1I2-(2-propenyl)-indolo[3 ,2d] [1 ]benzazepin-6(5H)-one, 11 -bromo-7, 12-dihydro-indolo[3 [1]benzazepin- 6(5H)-one, 9-bromo-7, 12-dihydro-2-(methyliminoamnine)-indolo[3 ,2d] [1 ]benzazepin-6(5H)-one, 9-bromo-7,1I2-dihydro-2-(carboxylic acid)-indolo[3 ,2d] [1]benzazepin-6(5H)-one, 9-bromo-7, 12-dihydro- 1 -hydroxy-indolo[3 ,2- \\melb-files\honx$\suzanct\Kecp\SpcM57 14-99.1 Divisional SPECIdoc 16/0 1/01 15 d] [1]benzazepin-6(5H)-one, 9-bromo-7, 12-dihydro- 1-hydroxymethyl-indolo[3 ,2d] [1]benzazepin-6(5H)-one, 7,1 2-dihydro-4-hydroxy-indolo[3 [1]benzazepinand 7,1 2-dihydro-2,3-dihydroxy-indolo [1]benzazepin-6(5H)-one, 2,3-dimethoxy-9-nitro-7, 12-dihydro-indolo[3 [1]benzazepin-6(511)-one, 9cyano-7, 12-dihydro-indolo[3,2-d] [1]benzazepin-6(511)-one, 2,3-dimethoxy-9-cyano- 7,1 2-dihydro-indolo[3 [1]benzazepin-6(511)-one, 9-nitro-7, 12-dihydroindolo[3,2-d] [1]benzazepin-6(5TH-one, 2-bromo-9-nitro-7, 12-dihydro-indolo[3 ,2d] [1]benzazepin-6(5H)-one, 2-(3-hydroxy-l1-propinyl), 9-trifluoromethyl-7, 12dihydro-indolo[3 [1]benzazepin-6(5H)-one, 2-iodo-9-bromo-7, 12-dihydroindolo[3 [1]benzazepin-6(5H)-one, 2-(3 -oxo-l1-butenyl), 9-trifluoromethyl-7, 12tetrahydro-indolo[3 [1 benzazepin-6(5R)-one, 2-iodo,9-trifluoromethyl-7, 12dihydro-indolo[3 [1]benzazepin-6(5TH-one, 7,1 2-dihydropyrido[3',2' :4,5]pyrrolo[3,2-d] [1I ]benzazepin-6(5H)-one, 11I -methyl-7, 12-dihydroindolo[3 [1]-benzazepin-6(5H)-one, 1-hydroxycyclohexyl)-ethinyl], 9- ~*15 trifluoromethyl-7, 12-dihydro-indolo[3 [1 ]benzazepin-6(5H)-one, 2-cyano-7, 12dihydro-indolo[3 [1]benzazepin-6(511)-one, 2-iodo-7, 12-dihydro-indolo[3 ,2d] [1]benzazepin-6(5H)-one, 11 -ethyl-7, 12-dihydro-indolo[3 [1]-benzazepin- 6(511)-one, 8-bromo-6, 1-dihydro-thieno[3 ',2':2,3]azepino[4,5-b]indol-5(4H)-one, 8chloro-6, 11 -dihydro-thieno[3',2' :2,3]azepino[4,5-b]mndol-5(4H)-one, 8-methyl-6,1 11dihydro-thieno[3',2':2,3] azepino [4,5-b]indol-5(4H)-one, 3-(6-oxo-9-trifluoromethyl- 5,6,7,1 2-tetrahydro-indolo[3 [1]benzazepin-2-yl)-propionitrile, 3-(6-oxo-9tr *looehl5671-erhdoidl 3,-][I]ezzpn2y)arlntie 3-6o9tri fluoromethyl-5 ,6,7,12-tetrahydro-indolo [3 1 benzazepin-2-yl)-aroitle acrylic acid, methyl ester.
Additional examples of compounds of the present invention are provided below as Formulas \\rmelb_flcs\hon S\s~Wnct\Kcp\SpcMS71I4-99. I Divisional SPECIDoC 16/01/01 -16-
H
Formula 3
H
Formula 4 oo* o Formula B. Synthesis of Compounds There are several synthetic approaches to the basic ring system of Formula 1.
The first known example of 7,12-dihydroindolo[3,2-d] [1]benzazepin to be described was the lactam shown below in Scheme 1, which was synthesized as an intermediate in a sequence towards the iboga alkaloid selenium dehydrogenation products. This lactam was obtained via an eleven step procedure, the last step being the lactam ring closure initiated by cleavage of the \\mclb filcs\homn$\suzannct\Keep\Speci\45714-99.1 Divisional SPECI.doc 16/01/01 17tosyl protecting group in the lactam precursor shown in Scheme 1.
Ts H 1 0 NH N HBr/AcOH Scheme 1 One method for synthesizing 7,12-dihydroindolo[3,2-d][1]benzazepin was reported by Kunick as shown below in Scheme 2.
H
H I
H
R
Scheme 2 Kunick's method employs a Fischer indolization. An analog synthesis was published shortly after by the group of A.P. Kozikowski, who prepared the unsubstituted lactam as a putative ligand of the mitochondrial DBI (diazepam binding inhibitor) receptor complex.
10 The annelated azepinones are key intermediates in the synthesis of [b,d]-fused azepinones. Several strategies towards the structures have been reported. A general route to [1 ]benzazepin-2,5(3H, 4H)-diones was described by Witte and Boekelheide Witte and V. Boekelheide, "Stereoselective Syntheses of Isoquinuclidones," J Org. Chem., 32:2849-2853 (1972), which is incorporated herein by reference], illustrated below as Scheme 3.
\\mlb-filcs\honS\sazannt\Keep\Spcc'4S7 4-99.1 Divisional SPECI doc 16/01/01 18n
R--
'~~COEt
NR
H oY Cr0 3
IN
CO
2 Et H ,9 MeOH
H,
NaH 4 Scheme 3 Kunick has reported an alternative general strategy in having fewer steps and good 5 overall yields.
\cbfiles\hofleSsuzalPt\Keep\Speci4S7 14-99.1 Divisional SPECIdoc 16/01/01 -19- R NIO COEt NH COEt COEt H O H O N N R DMSO/HO R HO OCO Scheme 4 On the basis of the ring system of Formula 2, the synthesis of compounds according to the present invention may be achieved by the following strategies: Introducing substituents in Ring A, and formation of heterocyclic rings. Substituents can be coupled to position 2 via electrophilic aromatic substitution at several stages in the synthetic pathway. For example, halogens and nitro substituents can be introduced. Other substituents can be introduced by nucleophilic exchange of the halogen or via diazonium intermediates, derived from the nitro compound. Carbon chains may be introduced at the 2 position by •palladium catalyzed reactions employing halogen substituted derivatives. For 10 compounds having one or more substituents in positions 1, 3, and/or 4, or a hetero atom in ring A, the synthetic route starts with an appropriate amino-substituted aromatic carboxylic acid. An example of this strategy is provided below as Scheme \\lbfilcs\homc$\sant\Kcep\Speci\45714-99.1 Divisional SPECIdoc 16/01/01 20 OMe EtOH, HC1 CI CO 2 Et
H
N COEt
KH
0 COEt
H
N, 1 H S0 4 M~r, Scheme \\rnxlb tilcs\hofl1$\suzaflfld'KcqFSpecCM\57I4-99.1 Divisional SPECIdoc 16/0 1/01 -21- Modifications at positions 5 and 6. A nitrogen substitution at position is achieved by reaction with an alkyl halide in the presence of a base. Sulfur can be exchanged for the oxygen by reaction with phosphorus sulfide. This thiolactam serves as starting material for compounds annelated at the 5,6 site, or the synthesis ofmethylthioimidates by reaction with iodomethane.
Introducing substituents into the D-Ring. Substituents intended at position 9 or 11, or appropriate precursor groups, are included in the phenylhydrazine (Scheme Substitutions at position 10 are included via electrophilic aromatic substitution. Halogen substituents, e.g. the bromo substituent in the 9, 10 or 11 position, may be exchanged for other substituents, e.g. the cyano group, via a Rosenmund-von Braun reaction. The bromo substituent furthermore may serve as a reaction center for palladium catalyzed coupling reactions, e.g.
Heck-reactions. Methyl substituents at positions 9 and 11 may be oxidized to carboxyl groups, which can be transformed by well known methods to other 15 functional groups, e.g. alcohols, esters, ketones, aldehydes, etc.
Introduction of substituents in position 12. The nitrogen in position 12 can be alkylated selectively with alkyl halides using appropriate bases. Aminoalkyl chains can be placed at position 12 by a Mannich reaction.
S* Substitution of the indole part C,D for other ring systems. The aryl-(CO)-CH 2 motif of the intermediate shown in the Schemes above provides the structural basis for the annelation of various heterocyclic ring systems. For example, the following heterocycles have been annelated at this site utilizing the electrophilicity of the keto function and the C,H-acidity of the CH 2 group in the vicinal position: pyrazoles, pyrimidines, pyranes, pyridines, quinolines, and quinoxalines. Alternatively, the lactam ring closure strategy can be used for the preparation of the derivatives in which the C,D-system is substituted for another system.
Additional information concerning general synthetic approaches are provided below in Examples 1-5. Synthesis of particular compounds are described in Examples 6-30.
\\melbfies\ho cS\s nnet\Kecp\Spcc\457I14-99. 1 Divisional SPECldoc 16/01/01 -22- II. COMPOSITIONS The compounds described herein can be formulated into compositions for administration to humans and animals subjects) to, for example, inhibit the proliferation of living cells, particularly hyperproliferative or neoplastic cells. Such compositions include "effective amounts" of the compounds described above, and may further comprise inert carriers, excipients, diagnostics, direct compression binders, buffers, stabilizers, fillers, disintegrants, flavors, colors, lubricants, other active ingredients, other materials conventionally used in the formulation of pharmaceutical compositions, and mixtures thereof.
The method of the present invention comprises administering to humans or animals "effective amounts" of a compound, a mixture of compounds, or compositions comprising "effective amounts" of a compound or mixture of compounds. Persons of ordinary skill in the art will realize that an "effective amount" varies. It currently is believed that "administering an effective amount" comprises administering to subjects a total amount of compound per treatment of from about 0.3 gram to about 3 grams, preferably from about 0.5 gram to about 1 gram, of a compound or compounds, or compositions comprising the compound(s), according to the present invention. Moreover, it typically is desirable to provide as large a dose as possible to a subject, depending upon the ability of the subject receiving the compound, or compositions comprising the compound(s), to tolerate the dose. Effective amounts also can be stated with reference to amounts of a material per unit mass of the subject receiving the compounds. It currently is believed that such dosage for-the present invention should be from about of subject/dose to about 400mg/Kg of subject/dose.
Tests performed using nearly sixty different cancer cell lines (see Examples below) indicate that the cytotoxic profile of the compounds of the present invention share certain similarities with other agents that are useful as antineoplastics. Thus, it would be within the purview of persons skilled in the art of preparing pharmaceutical formulations to add such compounds to pharmaceutical inert carriers, excipients, etc. suitable for administration to a subject, in a manner similar to that used for preparing such formulations of known antineoplastics. Compounds \\eIblles\ho=S\srznt\Keep\.Spcci45714-99.1 Divisional SPECIdoc 16101/01 -23of the present invention can be administered to subjects using dosage protocols that are substantially similar to protocols used with other antineoplastics.
The compounds or compositions can be administered by any number of methods including, but not limited to, topically, orally, intramuscularly, intranasally, subcutaneously, intraperitoneally, intralesionally or intravenously. Currently, oral and intravenous administration are believed to be the preferred methods for administering the compounds and compositions.
III. BIOLOGICAL RESULTS Compounds of the present invention also have been subjected to various biological analyses to determine their biological activity. The biological analyses include cdk inhibition assays and the drug screening procedure employed by the National Cancer Institute for the screening of drugs having possible anticancer utility.
A. Enzyme Inhibition Assays The enzyme inhibition activity of compounds of the present invention have been assayed by Dr. Larent Meijer of CNRS, Roscoff, France, using his cdk inhibition assay. The assay is described by V. Rialet's and L. Meijer's "A Screening Test for Antimitotic Compounds Using the Universal M Phase-specific Protein Kinase, p34cdc 2 /Cyclin bcdc 13 Affinity-Immobilized on pl3sucl-Coated Microtitration Plates," Anticancer Res., 11(4):1581-90 (1991), which is incorporated herein by reference. The results of these assays are provided below in Table 1.
Where only substitutions are stated in Table 1, such substitutions refer to substituents on core compound 7,12-dihydro-indolo [3,2-d][1]benzazapine one (or thione). Otherwise complete IUPAC names are provided.
The following IC 50 values of known compounds are provided for purposes of comparison: flavopiridol 0.4 gM; olomoucine 7.0 gm; roscovitine 4.2 gM; CVT-313 4.2 uM; and butyrolactone I 0.6 M.
\\melbfilcs\homS\sszanwt\Kcep Spe457 14-99.1 Divisional SPECIdoc 16/01/01 -24
S
S. 0
S
On.
eon
S
Sn.
0@ 5 0
S
a
S
S
P.S.
S
S
a .55.
05 S S
S
Substitution IIC 5 o(ItM) cdc2/cyclin B 9-cyano 0.024 0.044 2,3-dimethoxy, 9-nitro 0.024 9-nitro 0.035 2,3-dimethoxy, 9-cyano 0.044 3-(6-oxo-9-trifluoromethyl- 0.047 5,6,7, 12-tetrahydro-indolo[3,2d] [1I ]benzazepin-2-yl)propionitrile 2-Br, 9-nitro 0.053 2,3-dimethoxy-9-Br 0.500 0.200 2-Br,9-trifluoromethyl 0.240 3-(6-oxo-9-trifluoromethyl- 5,6,7,12-tetrahydro-indolo[3,2- 0.270 d] [1I ]benzazepin-2-yl)acrylonitrile 2,3-dimethoxy;-9- 0.280 0.430 trifluoromethyl 2-Br,9-Br 0.300 10.100 2-(3-hydroxy- 1-propinyl), 0.300 trifluoromethyl 2-1,9-Br 0.320 2-(3-oxo-lI-butenyl), 9- 0.320 \\welb-flles\homS\szannc\Kccp\Spci\457 14-99.1 Divisional SPECIdoc 16/01/01 a Substitution IC 5 0(jAM) cdc2/cyclin B trifluoromethyl 9-Br 0.400 0.850 9-trifluoromethyl 0.400 0.600 8-trifluoromethyl-6,1 1- 0.500 dihydrothieno[3',2':2,3]azepino[4,5b] indol-5 (4H)-one 8-bromo-6,1 I1-dihydro- 0.600 thieno[3',2':2,3]azepino,[4,5- (4H)-one.
2-1,9-trifluoromethyl 0.700 9-OCH 3 0.900 2.100 10O-Br 1.300 2.700 11-Br 1.300 1.400 11I-Cl 1.400 2.900 12-CH 2 COOMe 1.400 9-F 1.600 1.300 9-CH 3 2.000 6.300 7,12-dihydro- 2.200 pyrido[3',2':4,5]pyrrolo[3,2d] [1I 6 9-Br 2.300 8.000 8,1 0-dichioro 2.500 1 2-CH 2
CH
2 OH 3.0001 lb-fls\homcS\suzannet\Kcep\SpecMN57I 4-99.1 Divisional SPECIdoc 16/0 1/01 26 Substitution IC50(iAM) cdc2/cyclin B 2,3-dihydroxy,9-Br 3.000 8.000 11 -methyl 3.000 -hydroxycyclohexyl)- 3.200 ethinyl], 9-trifluoromethyl 2-Br 3.300 5.000 2-cyano, 3.300 2-1 3.700 11 -ethyl 3.800 8-methyl-6, 11 -dihydro- 4.000 thieno[3',2':2,3]azepino[4,5b]indol-5(4H)-one 2,3-dimethoxy 4.300 5.400 3-(6-Oxo-9-trifluoromethyl- 4.300 5,6,7, 12-tetrahydro-indolo[3,2d] [1I ]-benzazepin-2-yl)-acrylic acid, methyl ester 12-CH 3 ,9-Br 6.200 5-CH 2 COOMe,9-Br 6.400 5.300 None 7.000 10.100 5-CH 3 ,9-Br 20.000 130.000 I 2-ethyl,9-Br 23.000 2 -Ph 35.000 270.000 4-OH,9-Br 40.000 1000.000 6-thiomethyl,9-Br 43.000 160.000 \mcb files/hon $\suzannes\Keep\Speci*\45714.99.1 Divisional SPECIdoc 16/01 /01 -27- TABLE 1 Table 1 clearly shows that compounds provided as being representative of the present invention are potent inhibitors of cdc2/cyclin B. Compounds of the present invention typically have IC5', of less than about 50 jiM, preferably less than about 10 jiM, even more preferably less than about 5 jiM, and particularly preferred compounds have IC 5 of less than about I jiM. The first 12 compounds listed in Table 1 have IC 5 lower than most known compounds, and in the range of about 0.3 jiM or less. The 9-cyano compound currently is the best known compound in terms of its inhibitory effects for cdc2/cyclin B.
Preferred compounds of the present invention having IC 5 of less than about 10 jiM can be selected from the group consisting of 9-cyano-7,12-dihydro-indolo[3,2d] [1 ]benzazepin-6(5H)-one, 9-bromo-7, 12-dihydro-2,3-dimethoxy-indolo[3 ,2d] [1 ]benzazepin-6(5H)-one, 2-bromo-7, 12-dihydro-9-trifluoromethyl-indolo[3 ,2d] [1]benzazepin-6(5H)-one, 7,1 2-dihydro-2,3-dimethoxy-9-trifluoromethylindolo[3,2-d] [1]benzazepin-6(5H)-one, 2,9-dibromo-7, 12-dihydro-indolo[3 ,2d] [1]benzazepin-6(5H)-one, 7,1 2-dihydro-9-trifluormethyl-indolo[3 ,2d] [1 ]benzazepin-6(5H)-one, 9-chloro-7, 12-dihydro-indolo[3 [1]benzazepin- 8-bromo-6, 1-dihydro-thieno ]azepino[4,5-b]indol-5(4H)-one, 7,1 2-dihydro-9-methoxy-indolo[3,2-d] [1]benzazepin-6(5H)-one, 1 O-bromo-7, 12dihydro-indolo[3 [1]benzazepin-6(5H)-one, 11 -bromo-7, 12-dihydro-indolo[3 ,2d] [1]benzazepin-6(5H)-one, 11 -chloro-7, 12-dihydro-indolo[3 [1]benzazepin- 9-fluoro-7, 12-dihydro-indolo [3 [1]benzazepin-6(5H)-one, 9-methyl- 7,1 2-dihydro-indolo [3 [1]benzazepin-6(5H)-one, 9-bromo-7, 12-dihydroindolo[3 [1]benzazepin-6(5H)-thione, 8,1 0-dichloro-7, 12-dihydro-indolo[3 ,2d]1bnaein65)oe *.rm-,2dhdo1-2-yrxehl-noo32 o [1 ]benzazepin-6(5H)-one, 9-bromo-7, 12-dihydro- 12-(2hydroxyt)-indolo[3 2d] [1 ]benzazepin-6(5H)-one, 2-bromo-7, 12-dihydro-indolo[3 [1]benzazepin- 7,1 2-dihydro-2,3 -dimethoxy-indolo[3 [1 benzazepin-6(5H)-one, 9bromo-7, 12-dihydro-1I2-methyl-indolo[3 [1]benzazepin-6(5H)-one, 9-bromo- 7,1 2-dihydro-5-methyloxycarbonylmethyl-indolo[3 [1]benzazepin-6(5H)-one, 7,1 2-dihydro-indolo[3 [1]benzazepin-6(5H)-one, 7,1 2-dihydrol-fils~hoS~suannc\Kcc\Spe i \45799. I Divisional SPECIADc 16/01/01 -28pyrido [3 ',2':4,5]pyrrolo[3,2-d] [1]benzazepin-6(5H)-one, 11 -methyl-7, 12-dihydroindolo[3 [1]-benzazepin-6(5H)-one, 2- 1-hydroxycyclohexyl)-ethinyl], 9trifluoromethly-7, 12-dihydro-indolo [3 [1]benzazepin-6(511)-one, 2-cyano-7, 12dihydro-indolo [3 [1]benzazepin-6(5H)-one, 2-iodo-7, 12-dihydro-indolo[3 ,2d] [1]benzazepin-6(5H)-one, 11 -ethyl-7, 12-dihydro-indolo [3 [1]-benzazepin- 6(511)-one, 8-methyl-6, 11 -dihydro-thieno[3 azepino[4,5-b]indol-5(4H)-one, 2-CH=CH-COOCH 3 9-trifluoromethyl-7, 12-dihydro-indolo[3,2-d] I1 ]-benzazepin- 6(511)-one, 9-nitro-7, 12-dihydro-2,3 -dimethoxy-indolo[3 [1]benzazepin-6(5R)one, -nitro-7, 12-dihydro-indolo[3 [1]benzazepin-6(511)-one, -cyano-7, 12dihydro-2,3-dimethoxy-indolo[3 [1 benzazepin-6(511)-one, 9-trifluoromethyl- 7,1 2-dihydro-2-CH 2
CH
2 -CN-indolo [1]benzazepin-6(511)-one, 2-bromo-9nitro-7, 12-dihydro-indolo[3 [1]benzazepin-6(5H)-one, 9-cyano-7, 12-dihydro- 2,3dimethoxy-indolo[3 [1]benzazepin-6(511)-one, 9-trifluoromethyl-7, 12dihydro-2-CH=CH-CN-indolo[3 [1]benzazepin-6(5T)-one, 9-trifluoromethyl- 7,1 2-dihydro-2-(3-hydroxy-l1-propinyl)-indolo[3 [1]benzazepin-6(5H)-one, 2iodo-9-bromo-7, 12-dihydro-indolo[3 [1]benzazepin-6(511)-one, 9trifluoromethyl-7, 12-dihydro-2-(3 -oxo- 1 -butenyl)-indolo[3 [1I ]benzazepin- 6(511)-one, 8-bromo-6, 1-dihydro-thieno[3',2' :2,3]azepino indol-5(4H)-one, 8- 0:chloro-6, 1-dihydro-thieno [3',2':2,3]azepino[4,5-b]indol-5(4H)-one, 2-iodo-9trifluoromethyl-7, 12-dihydro-indolo[3,2-d] [1]benzazepin-6(511)-one, 9-OCH 3 12dihydro-indolo[3 [1]benzazepin-6(511)-one, I 2-CH 2 COOMe-7, 12-dihydroindolo[3 [1]benzazepin-6(511)-one, and 6=S-9-bromo-7, 12-dihydro-indolo[3 ,2o o. [1]benzazepin-6(511)-one.-.
Particularly preferred compounds for cdk inhibition have IC 5 of less than about 1 1 4M. These compounds include 9-cyano-7,12-dihydro-indolo[3,2- 0 d] [1 ]benzazepin-6(5H)-one, 9-bromo-7, 12-dihydro-2,3 -dimethoxy-indolo[3 ,2- 0: d][1 ]benzazepin-6(5H)-one, 2-bromo-7, 12-dihydro-9-trifluoromethyl-indolo[3 ,2d] [1]benzazepin-6(5H)-one, 7,1 2-dihydro-2,3-dimethoxy-9-trifluoromethylindolo[3 [1]benzazepin-6(5H)-one, 2,9-dibromo-7, 12-dihydro-indolo[3 ,2d] [1]benzazepin-6(5H)-one, 7,1 2-dihydro-9-trifluoromethyl-indolo[3,2d] [1]benzazepin-6(5H)-one, 9-chloro-7, 12-dihydro-indolo[3 [1]benzazepin- 8-bromo-6, 11 -dihydro-thieno[3',2' :2,3 ]azepino[4,5-b]indol-5(4H)-one, \\mIb fies\hon\szannet\Kep\SpciU45714-99.1 Divisional SPECIdoc 16/01/01 -29- 7,1 2-dihydro-9-methoxy-indolo[3,2-d] [1]benzazepin-6(5H)-one, (green compounds need LUPAC names) 2,3-dimethoxy-9-nitro-7, 12-dihydro-indolo[3 ,2d] [1]benzazepin-6(511)-one, 2,3 -dimethoxy, 9-cyano-7, 12-dihydro-indolo[3 ,2d] [1]benzazepin-6(511)-one, 2-CH 2
CH
2 -CN,9-trifluoromethyl-7, 12-dihydroindolo[3,2-d] 111]benzazepin-6(5H)-one, 2-bromo-9-nitro-7, 12-dihydro-indolo[3,2d] [1]benzazepin-6(5H)-one, 2-CH=CH-CN, 9-trifluoromethyl-7, 12-dihydroindolo[3 [1]benzazepin-6(51f)-one, 2-(3 -hydroxy-l1-propinyl), 9trifluoromethyl-7, 12-dihydro-indolol3 [1]benzazepin-6(5TH-one, 2-iodo-9bromo-7, 12-dihydro-indolo[3,2-d] [1]benzazepin-6(511)-one, 2-(3 -oxo-l1-butenyl), 9trifluoromethyl-7, 12-tetrahydro-indolo[3 [1]benzazepin-6(5H)-one, 8-chioro- 6,1 1-dihydro-thieno[3',2':2,3]azepino[4,5-b]indol-5(4H)-one, 2-iodo,9trifluoromethyl-7, 12-dihydro-indolo[3 [1]benzazepin-6(5H)-one, 9-nitro-7, 12dihydro-indolo[3 [1]benzazepin-6(5H)-one, 9-bromo-7, 12-dihydro- 2,3dimethoxy-indolo[3,2-d] [1]benzazepin-6(511)-one, 9-bromo-7, 12-dihydroindolo[3 1 ]benzazepin-6(5H)-one, 8-bromo-6, 11 -dihydrothieno[3',2' :2,3]azepino[4,5-b]indol-5 (4H)-one and 9-OCH 3 12-dihydroindolo[3,2-d] 1 B. In Vitro Human Tumor Cell Line Assay The human tumor cell line screening procedure uses a diverse, diseaseoriented panel consisting of approximately 60 different human tumor cell lines organized into disease-specific subpanels. The compounds of the present invention were tested over a range of concentrations for cytotoxic or growth-inhibitory effects against cell lines comprising the panel. The eight subpanels represented diverse histologies (leukemias, melanomas, and tumors of the lung, colon, kidney, breast, ovary, and brain).
Compounds of the present invention were tested over a period of several days. During this period the cells were continuously exposed to various concentrations of the compounds tested. The tests produced individual doseresponses, one for each cell line one for each example), and the data are disclosed in dose-response curves. The data provided by these dose response curves are summarized using a mean-graph format illustrated by the FIGS. 1-30.
\\nbfies\hoeS\szant\Keep\Speci\457 14-99.1 Divisional SPECIdoc 16/01/01 To produce data for the mean-graph format, a compound concentration that produced a target level response was calculated for each cell line. Three different response parameters were evaluated. The first response parameter was the growth inhibition ("G1 5 0 G1 50 is the concentration of compounds made according to the present invention that results in an apparent 50% decrease in the number of tumor cells relative to control tumor cells (not exposed to the compounds of the present invention) at the end of the incubation period.
The second response parameter was the total growth inhibition TGI is the concentration at which the number of tumor cells remaining at the end of the incubation period substantially equal the number of tumor cells existing at the start of the incubation period.
The third response parameter was the lethal concentration ("LC 5
LC
5 0 is the concentration of compounds made according to the present invention that caused an apparent 50% reduction in the number of tumor cells relative to the number of tumor cells present at the start of the incubation period.
•In a typical GI5 0 mean graph left graph of FIG. the relative position of the vertical reference line along the horizontal concentration axis was obtained by averaging the negative logloGI50 values for all the cell lines tested against the compound. Horizontal bars were then plotted for the individual negative logloGISo values of each cell line relative to the vertical reference line. The G1 50 graph thus provides a characteristic fingerprint for the compound, displaying the individual cell lines that are proportionately more sensitive than average (bars extending to the right of the reference line) or proportionately less sensitive than average (bars extending to the left of the reference line). The length of a bar is proportional to the difference between the log 0
GI
5 0 value obtained with the particular cell line and the -mean (represented by the vertical reference line).
Similar mean graphs are shown in FIG. 2 for the TGI (middle graph) and
LC
5 0 (right-hand graph) response parameters.
FIGS. 1-30 provide mean graph formats for particular compounds representative of the present invention. These FIGS. clearly show that compounds satisfying Formulas 1 and 2 above are useful for inhibiting the growth of human \\metbfiles\holS\suannct\Kec\pSpccMA574-99. Divisional SPECIdoc 16/01/01 9 9 9 *99* 9 9999.9 9 -31 tumor cells. Certain results from the human tumor cell assays are summarized below in Table 2.
In Vitro Antitumor Activity on Cell Lines of the NCI Cancel Cell Screen by Compounds of the Present Invention: G1 5 0[RM] Substitution Averag SR HCT-116 RXF- .1 393___ 2,3-dimethoxy,9-Br 7 4 2 2 2-Br,9-trifluoromethyl >100 >100 >100 >100 2,3-dimethoxy,9-trifluoromethyl 4 2 2 2 2-Br,9-Br >100 >100 >100 >100 9-Br (Parent Compound) 43 4 2 Roscovitine 18 7 8 8 9-trifluoromethyl 72 31 4 83 8-bromo-6,11I-dihydro- >89 28 >100 >100 thieno[3',2':2,3]azepino[4,5-b]indol- 5(4H)-one 12-CH 2 COOMe 66 >100 >100 >100 6 =S,9-Br 3 2 8,10-dichloro >91 >100 >100 >100 12-CH 2
CH
2 OH 21 17 16 19 2,3dihydroxy,9-Br >100 >100 >100 >100 2,3-dimethoxy 32 52 26 19 12-CH 3 ,9-Br 34 3 7 18 2 COOMe,9-Br 26 19 20 9 Olomoucine 51 1 27 138 1 \\nmlb-flcs~homeS\saznetUlCeepSpccM'\4574-99.I Divisional SPECIdoc 16/01/01 o. .o .00.
S.*00 *00 -32- Substitution Averag SR HCT-1 16 RXFe 393 3 ,9-Br 26 0.3 20 12-ethyl,9-Br 51 5 34 22 2 -Ph 36 8 20 4-OH,9-Br 39 50 27 6-thiomethyl,9-Br 3 0.4 2 2 12-COO-tBu,9-Br 6 3 3 4 12-allyl,9-Br 65 40 >100 51 5,7-di-COO-tBu,9-Br >100 >100 >100 >100 5,7,12-tri-COO-tBu,9-Br 37 >100 8 39 4-methoxy,9-Br 20 33 10 4-methoxy 79 >100 >100 >100 5,12-di-COO-C-(CH 3 3 ,9-Br 44 14 87 2,3-dimethoxy,9-nitro 1.2 0.20 0.30 0.76 9-cyano 89 3 21 >100 2,3-dimethoxy,9-cyano 5.4 0.43 0.48 0.89 9-nitro 0.37 0.02 0.07 0.27 3-(6-oxo-9-trifluoromethyl-5,6,7, 12- 2.2 0.05 0.79 1.9 tetrahydro-indolo[3 ,2d] [1I ]benzazepin-2-yl)-propionitrile 2-Br, 9-nitro 87 24 >100 >100 3-(6-oxo-9-trifluoromethyl-5,6,7, 12- 83 >100 >100 tetrahydro-indolo[3 ,2d] [1I ]benzazepin-2-yl)-acrylonitrile \\melb-flcs\homcS\suzanct\c p\SpcMi\457499.1 Divisional SPECIdoc 16101/01 33 Substitution Averag SR HCT-116 RXFe 393 2-(3-hydroxy-1-propinyl), 9- 14 0.33 2.8 8.1 trifluoromethyl 2-1,9-Br 55 >100 47 2-(3-oxo-1-butenyl), 9- 2.7 0.5 1.3 2.9 trifluoromethyl 8-chloro-6,1 I1-dihydro- 85 12 >100 >100 thieno[3',2':2,3]azepino[4,5-b]indol- 5(4H)-one 2-I,9-trifluoromethyl 85 >100 >100 >100 7,12-dihydro- >100 >100 >100 >100 pyrido[3',2':4,5]pyrrolo[3 ,2d] 1 ]benzazepin-6(5H) -one.
11I-methyl 33 11 17 13 2-[2-(l1 -hydroxycyclohexyl)-ethinyl], 2.5 2.6 2.3 2.2 9-trifluoromethyl 2-cyano 96 >100 >100 >100 2-1 25 20 24 .11 -ethyl 58 7.0 74 24 8 -methyl-6,1 I1-dihydro- 38 15 23 thieno[3',2':2,3]azepino[4,5-b]indol- 5(4H)-one 3-(6-Oxo-9-trifluoromethyl-5,6,7,12- 96 tetrahydro-indolo[3,2-d] [1I]benzazepin-2-yl)-acrylic acid, methyl ester TABLE 2 \\melbfiles\homcS\suzannt\Kecp\SpecMS47 14-99.1 Divsional SPECIIoc 16/01/01 -34- Table 2 illustrates that compounds satisfying Formulas 1 and 2 not only are potent cyclin dependent kinase inhibitors, but also are potent inhibitors of the growth of human tumor cells.
Additional biological activity information is available for 9-bromo-7,12dihydro-indolo[3,2-d][l]benzazepin-6(5H)-one. For example, it also has been tested in the human tumor cell line screen and showed modest differential activity.
This compound had an overall GI 50 of 43 tM and was most active in several colon lines (HCT-116 and KM12), along with a CNS line (SF-539). The compound also was able to totally inhibit the growth of two cell lines, HCT-116 and SF-539, both at a concentration of 28 gM.
Exponentially growing SUDHL-4 cells were exposed to 9-bromo-7,12dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one for 48 hours. The IC 50 for cell proliferation for this compound is about 3 pM. Cell cycle analysis of these cells revealed a prominent G2/M arrest, which is consistent with inhibition of cdkl.
Preliminary toxicity studies of 9-bromo-7,12-dihydro-indolo[3,2d][1]benzazepin-6(5H)-one demonstrate that the maximum tolerated single dose to be about 400 mg/Kg.
Hollow fiber analyses also have been performed using 9-bromo-7,12dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one. These studies demonstrated that this compound showed substantial growth inhibition in the hollow fiber assay, i.e., 56% inhibition at 150 mg/Kg in the i.p. implanted fibers, 98% inhibition at 150 mg/Kg in the s.c. implanted fibers. See Example 31 below for the procedure used for the in vivo tests. Data is reported as %T/C according to the protocol of Example 31 for each of the 2 compound doses against each of the cell lines with separate values calculated for the intraperitoneal and subcutaneous samples, with a target value for %T/C being about 50 or less.
IV. EXAMPLES The following examples are provided to illustrate certain particular features of the present invention. These examples should not be construed to limit the invention to the particular features exemplified.
\\mlbfilcs\home$\suzanct\Keep\Spcc\45714-99. Divisional SPECIdoc 16/01/01 Example 1 This example describes a general procedure for synthesizing compounds satisfying general Formula 2. A slurry of an appropriate 1H-[l]benzazepine- 2,5(3H,4H)-dione (6 mmol) in glacial acetic acid (10 mL) is placed in a roundbottomed flask equipped with oil bath, thermometer, reflux condenser and magnetic stirrer. After addition of the appropriate phenyl hydrazine (7 mmol) the mixture is heated to 70 0 C with stirring for one hour. After cooling to room temperature concentrated sulfuric acid (0.5 mL) is added and the mixture is stirred at 70 0 C for the indicated reaction time. After cooling to room temperature, the mixture is poured into a 10% aqueous sodium acetate solution (50 mL). A precipitate is formed, which is filtered off with suction and purified by recrystallization or column chromatography.
Example 2 15 This example describes a second general procedure for synthesizing compounds satisfying general Formula 2. A slurry of an appropriate 1H- []benzazepine-2,5(3H,4H)-dione (1 mmol) in glacial acetic acid (2 mL) is placed in a round-bottomed flask equipped with oil bath, thermometer, reflux condenser and magnetic stirrer. A suspension of sodium acetate (123 mg, 1.5 mmol) and the appropriate substituted phenylhydrazine (1.5 mmol) or the appropriate phenyl hydrazine hydrochloride (1.5 mmol) in glacial acetic acid(5 mL) is added dropwise with stirring. After stirring at 70 0 C for 1 hour the mixture is cooled to room temperature. Concentrated sulfuric acid (0.1 mL) is added and the mixture is stirred at 70 0 C for 1 hour. After cooling to room temperature, the mixture is poured into a 5% aqueous sodium acetate solution (15 mL). A precipitate forms, which is filtered off with suction and purified by recrystallization from the given solvent.
Example 3 This example describes a third general procedure for synthesizing compounds satisfying general Formula 2. To a suspension of 1H-[1]benzazepine- 2,5(3H,4H)-dione (528 mg, 3 mmol) in glacial acetic acid (5 mL) is added a suspension of the appropriate substituted phenyl hydrazine (3.5 mmol) and sodium \\melb_filcs\home$\suzannct\Kccp\Spcci\45714-99.1 Divisional SPECI.doc 16/01/01 -36acetate (287 mg, 3.5 mmol) in glacial acetic acid dropwise and with stirring. The mixture is stirred for 1 hour at 70 0 C and then cooled to room temperature.
Concentrated sulfuric acid (0.25 mL) is added and the mixture is stirred for 1 hour at 70 0 C. The mixture is allowed to cool to room temperature and then poured into 5% aqueous sodium acetate solution (50 mL). A precipitate forms, which is filtered off with suction and then purified Example 4 This example describes a fourth general procedure for synthesizing compounds satisfying general Formula 2 involving the synthesis of phenols by cleavage ofmethoxy compounds. Boron tribromide (1002 mg, 4 mmol) was added to a solution of the appropriate methoxy compound (1 mmol) in dichloromethane mL). The mixture was stirred by means of a magnetic stirrer, and the reaction was monitored by thin layer chromatography (silica gel, eluent acetone/toluene 1:1).
When the spot caused by the starting methoxy compound was no longer detectable, water (10 mL) was added and the mixture was stirred for 1 hour. A solid formed, which was filtered off with suction, washed with water and recrystallized for purification.
o Example This example describes a fifth general procedure for synthesizing compounds satisfying general Formula 2 involving the reactions with alkyl halides. Powdered potassium hydroxide (56 mg, 1 mmol) was added with stirring and cooling by an ice bath to a solution of 9-bromo-7,12-dihydro-indolo[3,2-d] [1 ]benzazepin-6(51)-one (327 mg, 1 mmol) in dry acetone (120 mL). After stirring the mixture for 1 hour at 0C, the appropriate alkyl halide (10 mmol) was added and stirring was continued S* for 3 days at room temperature. After addition of water (120 mL) a solid was formed, which was filtered off and recrystallized from ethanol/toluene.
Example 6 This example describes the synthesis of 9-cyano-7,12-dihydro-indolo[3,2d][1]benzazapin-6(5H)-one.
\\TcIlbfilc\hmcS\suzannct\KccpSpeci\45714-99.1 Divisional SPEC] doc 16/01/01 -37- H O 4
N
6 A B 7 2 HN 2 11 9-bromo-7,12-dihydro-indolo[3,2-d] [1 ]benzazepin-6(5H)-one was first synthesized as a precursor for the synthesis of the 9-cyano derivative using the general procedure described above in Example 2. The synthesis was performed using 1H- [1]benzazepine-2,5(3H,4H)-dione (1.05 g) and 4-bromo-phenylhydrazine (1.31 g), reaction time 1 hour. Pale yellow crystals in a 58% yield were obtained having: a mp. 330 0 C (1,4-dioxane); ir (KBr): 3220 1640 cm- 1 'H-nmr (DMSO-d 6 300 MHz): 6 (ppm) 3.50 2H, CH 2 7.21-7.30 3H), 7.34-7.41 ooo* 2H), 7.74 (bd, 1H, 7.5 Hz) 7.89 1H, 1.5 Hz), 10.05 1H, lactam NH), 11.75 1H, indole NH); 'C-nmr (DMSO-d 6 75 MHz): 6 (ppm) 31.3, 107.0, 111.4, 113.1, 120.1, 122.0, 122.1, 123.4, 124.2, 126.7, 128.1, 128.1, 133.8, 135.4, S 135.8, 171.1; C 16 HjjBrN 2 0 (327.2); Calcd. C 58.7, H 3.39, N 8.6; Found C 58.3, H 3.35, N 8.4.
A mixture of 9-bromo-7,12-dihydro-indolo[3,2-d] [1]benzazepin-6(5H)-one (327 mg, 1 mmol) and copper(I) cyanide (180 mg, 2 mmol) in dimethyl formamide (15 mL) was refluxed for 12 hours. After cooling to room temperature water mL) was added. The precipitate was filtered off with suction, washed with water, and then suspended in a mixture of water (25 mL) and 1,2-diaminoethane (40 mL).
After stirring for 15 minutes, the solid was filtered off with suction, washed twice with 10% aqueous sodium cyanide solution and recrystallized twice from ethanol/toluene to yield 42% colorless crystals, m. p. >330 0 C; ir (KBr): 3350, 3180 2200 1670 cm-' 'H-nmr (DMSO-d 6 400 MHz): 6 (ppm) 3.59 2H, CH 2 7.27-7.32 2H), 7.43 (dt, 1H, 1.0/7.6 Hz), 7.51 (dd, 1H, \\rnlb-fle\homS\suzannet\Kcp\Speci\45714-99.1 Divisional SPECIdoc 16101/01 -38- 1.3/8.4 Hz), 7.59 I H, 8.1 Hz), 7.76 (dd, I1H, 1.0/7.6 Hz), 8.32 I 10. 16 (s, 1H, lactam-NH), 12.19 1H, indole-NH); 3 C-nMr (DMSO-d 6 100.6 MHz): 6 (ppm) 31.2, 101.2, 108.2, 112.5, 120.5, 121.8, 122.3, 123.7, 123.9, 124.6, 126.3, 127.0, 128.7, 134.9, 135.8, 138.9, 171.3; C 17
HIIN
3 0 (273.31); Calcd. C 74.71, H 4.06, N 15.38; Found C 74.45, H 4.26, N 14.75.
Example 7 This example describes the synthesis of 7, 12-dihydro-9-trifiuoromethylindolo[3, 2-d][1]benzazepin-6(SH)-one.
H 0
N
Al B
C
HN
*15
\D
7
CF,
The syteiSa efre codn otegnrlpoeuedsrbdi Thean sthoesiswaspeformedracin(6g, 5 thegeerl proeureadecibnyied n of cream-colored crystals from ethanol, m. p. 330'C; ir (KBr): 3200(NH), 1650 go** cm-' 1 H-nnir (DMSO-d 6 400 MHz): 6 (ppm) 3.61 2H, CH 2 7.27-7.32 (in, 2H), 7.40-7.47 (in, 2H), 7.62 1H, 8.6 Hz), 7.78 (dd, 1H, 1.5/7.6 Hz), 8.13 (s, 0.15(s, H, lcta-NH), 12.06 I1H, indole-N}{); 3 C-nmr (DMSO-d 6 100.6 MHz): 6 (ppm) =31.2, 108.4, 112.0, 115.9 JC,F =4.1 Hz), 118.3 (q, JC,F=3.2 Hz), 119.9 JC,F= 34 Hz), 122.1, 122.3, 123.7, 125.8, 127.0, 128.6, 134.7, 135.7, 138.7, 171.4 (one signal missing due to peak overlapping); C 17
H
11
IFN
2 0 (316.3 Calcd. C 64.5 5, H 3.5 1, N 8.86; Found C 64.46, H 3.60, N 8.97.
\\nilbfilesqonc\suzannetpKeep\SpecM'\57 14-99.1 Divisional SPECIdoc 16/01/01 39- Example 8 This example describes the synthesis of 7,12-dihydro-9-methyl-indolo[3,2d][IJbenzazepin-6(SH)-one.
H 0
N
Al B
C
HN
CF,
The synthesis was performed according to the general procedure described in Example 3 above using 4-methylphenylhydrazine hydrochloride (555 mg, mmol). The reaction yield was 59% cream-colored crystals after recrystallization from ethanol, m. p. >3300 C; ir (KBr): 3220 1640 cm-' 'H-nnir (DMSO4d 6 400 Mffz): 6 (ppm) 2.41 3H, CHA) 3.46 2H, CHA) 7. 00 (dd, 1H1, 1.0/8.1 Hz), 7.22-7.38 (in, 4H), 7.43 lH), 7.73 1H, 6.9 Hz), 10.05 1H, ~lactam-NH), 11.42 1H, indole-NH); 'Cnr(DMSO-d 6 100.6 MLHz): 6 (ppm) =21.1, 31.5, 107.0, 111.1, 117.4, 122.2, 122.9, 123.5, 123.7, 126.7, 127.6, 127.7, 132.4, 135.3, 135.8, 171.5 (one signal missing due to peak overlapping); C 17 H1 14
N
2 0 (262.31); Calcd. C 77.84, H 5.38, N 10.68; Found C 77.47, H 5.39, N 10.57.
\\rrilb-fles\homcS\suzmalct\KctVSpemi4 7 l 499 Divisional SPECIdoc 16101/01 Example 9 This example describes the synthesis of 7, 12-dihydro-9-methoxy-indolo[3,2d][J]benzazepin-6(SH)-one.
H 0
N
C
HN
D,,,.OCH,
The synthesis was performed according to the general procedure described above in Example 3 using 4-methoxyphenyihydrazine hydrochloride (611 mg, mimol). Purification by column chromatography (6 cm column of silica gel 100-200 mesh, eluent dichioromethane) yields 48% cream-colored powder, m. p. 330'C (shrinking starting at 290'C); ir (KBr): 3200 1640 cm-' 'Hnnir (DMSO-d 6 400 Miflz): 5 (ppm) 3.49 2H, CH 2 3.80 3H, OCHA) 6.81 (dd, 1H, 2.0/8.6 Hz), 7.17 1H, 2.5 Hz), 7.22-7.28 (in, 2H), 7.30-7.38 (in, 2H), 7.72 (dd, 1H, 7.6 Hz), 10.04 1H, lactam-NH), 11.38 1H, indole-NH); 1 3 c- 60060: 20 nmr: not recorded; CI 7 H1 4
N
2 0 2 (278.3 Calcd. C 73.37, H 5.07, N 10.07; Found C 72.32, H 5.39, N 9.75.
e .0.
\\mbies\hocl\szmnct\Keep\SpciMS7 14-99.1 Divsional SPECIdoc 16/01/01 -41 Example This example describes the synthesis of 9-fluoro-7,12-dihydro-indolof3,2d][1Jbenzazepin-6(SH) -one.
H 0
N
AlB
C
HN
\D F The synthesis was performed according to the general procedure described above in Example 4 using 4-fluorophenyihydrazine hydrochloride (569 mg, mmol). Purification by column chromatography (6 cm. column of silica gel 100-200 mesh, eluent dichloromethane) yields 52% cream-colored powder, m. p. 330'C (shrinking starting at 180'C); ir (KBr): 3220 1635 cm-' 'Hnmr (DMSO-d 6 400 M]Hz): 6 (ppm) 3.50 2H, CH 2 7.00 1H, 2.5/9.2/9.2 Hz), 7.23-7.3 1 (in, 2H), 7.35-7.44 (in, 2H), 7.48 (dd, IH, 2.5/9.7 Hz), 7.73 (dd, 1H, 1.5/7.6 Hz), 10.08 1H, lactam-N-H), 11.67 1H, indole-NH); C1 6 H, IFN 2 0 (266.27); Calcd. C 72. 17, H 4.16, N 10.52; Found C 72.03, H 4.23, N 10.47.
Example I11 This example describe's the synthesis of 9-bromo- 7, 12-dihydro-2 ,3dimethoxy-indolo[3, 2-d][1]benzazepin-6(SH) -one.
300 \\imIb-fics\homcszannct\Kcep\SpcM\57 4-99.1 Divisional SPECIdoC W601/01 42 The preparation was performed according to the general procedure described above in Example 2 from 7,8-dimethoxy- lH-[ I]benzazepin-2,5(3H,41f)-dione (235 mg, Immol) and 4-bromophenyihydrazine hydrochloride (336 mg, 1.5 mmol), yielding 55% red-brown crystals from ethanol/toluene, m.p. 330'C, ir (KBr): 3340, 3210 1660 cm-1 'H-nmr (DMSO-d 6 400 MHz): 6 (ppm) 3.45 2H, CHA) 3.80 3H, OCHA) 3.87 3H, OCHA) 6.87 I1H), 7.23 -7.27 (in, 2H), 7.39 1H, 8.6 Hz), 7.86 1H, 2.0 Hz), 9.83 1H, lactam-NH), 11.70 (s, 1H, indole-NH); 3 C-nMr (DMSO-d 6 100.6 MHz): 3 (ppm) 31.3, 55.5, 55.7, 105.6, 106.0, 109.4, 111.5, 113.0, 114.4, 120.0, 124.0, 128.3, 129.4, 134.4, 135.7, 145.3, 148.9, 170.7; C 18
HI
5 BrN 2
O
3 (387.24); Caled. C 55.83 H 3.90 N 7.24 Br 20.63; Found C 55.80 H 3.95 N 7.25 Br 20.34.
Example 12 This example describes the synthesis of 2-bromo-7,12-dihydro-indolo[3,2d][1]benzazepin-6(5H)-one.
H 0 The synthesis was performed corresponding to the general procedure :.25 described above in Example 2, using 7-bromo-1H-[1]benzazepine-2,5(3H,4TH- 0 dione (1.52 g) and phenyl hydrazine (0.69 mL) with a reaction time of about 1 hour Off-white crystals were obtained in a 63% yield, mp. 330'C (ethanol); ir (KBr): 3260 1650 cm- 1 'H-nmr (DMSO-d 6 300 MHz): 6 (ppm) 3.53 (s, 2H, CHA) 7.08 (ddd, I1H, J 1/7/8 Hz), 7.19 (ddd, IlH, 1/7/8 Hz), 7.20 I1H, Hz), 7.43 (ddd, 1H, 0.5/1/8 Hz), 7.52 (dd, 1H, 2.5/8.5 Hz), 7.66 (ddd, 1H, 0.5/1/8Hz) 7.92 1H, 2.5 Hz), 10.16 IH, lactam NH), 11.63 1H, indole NH); 3 C-nmr (DMSO-d 6 75 MHz): 6 (ppm) 31.5, 108.3, 111.3, 115.4, 117.9, \\nielb-fils\hoffieS~zannctJc,\SpcMS5714-99.1 Divisional SPECIdOC 16/01/01 -43 119.1, 122.4, 124.0, 124.6, 126.2, 128.7, 130.2, 130.8, 134.4, 137.4, 171.0;
C
16 HI 1 BrN 2 O (327.2); Calcd. C 58.7, H 3.39, N 8.6; Found C 58.6, H 3.28, N 8.7.
Example 13 This example describes the synthesis of 7, 12-dihydro-2, 3-dimethoxy-9trifluormethyl-indolo[3, 2-d][IJbenzazepin-6(SH)-one.
15 Th syntesis ws peormdcrepnigt h eea rcdr dion (23mg 1h syntesi wand 4trfmuorehyphnigthydrgnera 264gcedureol yielding 38% pale yellow crystals, m. p. 330"C; ir (KBr): 3240 1635 cm-1 'H-nmr (DMSO-d 6 400 M~Hz): (ppm) 3.55 2H, CH 2 .81 3H1,
OCH
3 ),3.88 3H, OCH 3 ),6.90 1H1), 7.31 1H1), 7.43 (dd, 1H1, 1.0/8.6 Hz), 1H, 8.6 Hz), 8.08 1H), 9.87 1H, lactam-NH), 11.96 III, indole- NH); 3 C-nMr (DMSO-d 6 100.6 MHz): 6 (ppm) =31.2, 55.5, 55.7, 106.1, 106.8, 106.8, 109.5, 111.8, 114.3, 115.4 JC, F 4.4 Hz), 117.9 Jc, F 3.7 Hz), 120.0, 125.9, 129.6, 135.2, 138.5, 145.3, 149.1, 170.7; CjqHj 5
F
3
N
2 0 3 (376.35); Calcd. C ::25 60.64, H 4.02, N 7.45; Found C 60.70, H 4.07, N 7.46.
\\nIbfilcs\oe\szanet\KeepSpciMS7 14-99.1 Divisional SPECIdoc 16/01/01 -44- Example 14 This example describes the synthesis of 2-bromo-7,12-dihydro-9trifluormethyl-indolo[3, 2-d]]benzazepin-6(SH)-one.
0 The~ sBtei a copihdacrigt h eea rcdr decrbe aov i Eamle2 sig -bom-1-[]bnzzein2,(3, 11-don mg 1 mol an 4Brifurehlhnlyrzn 24m,15mo) 5 iedng51 clrlsscrsal fomehaoltoune m p 30 0 r(Kr) Hz,81(,g),I1.2 (sl aHd 4-rfurehlactamnyh),z 125 1 5 indolN); 3 (HM63Oc-1(=,) 10.6Hz): 31.,40~) 109.2,12.2,11.66. (,J2H, 4.Hz) 1. 24.1d, 124, 12.3 z,12.5872.131.12H,13.25(, 134.8, 134.9, 138.8, 171.2; C 17
H
10 BrF 3
N
2 O (395.19); Calcd. C 51.67, H 2.55, N 7.09, Br 20.22; Found C 51.62, H 2.58, N 7.04, Br 20.12.
\\mcibjiles\horn$s'uane\Kcep\SpecMSA714-99.1 Divisional spECIdoc 1 601/01 Example This example describes the synthesis of 9-bromo- 7, 12-dihydro-Smet hyloxycarbonylmet hyl-indolo[3, 2-dJ[J]benzazepin-6(SH)-one.
0
CH,O
N
Br A solution of 9-bromo-7, 12-dihydro-indolo[3 [1]benzazepin-6(5TH-one (327 mg, 1 mmol) in THF (35 mL) was refluxed with sodium hydride (24 mg, 1 mmol, 60% suspension in white oil) for 1.5 hours. Bromoacetic acid ethyl ester (153 mg, 1 mmol) was added and refluxing was continued for 5 hours. After :cooling to room temperature, water (50 mL) was added. The mixture was extracted three times with dichloromethane (20 mL, respectively). The combined organic layers were dried with sodium sulfate and evaporated to furnish a residue, which was recrystallized from ethanol to yield 46% colorless crystals, m. p. 240'C, ir (KBr): 3340 1750, 1655 cm'1 'H-nmr (DMSO-d 6 400 MHz): (ppm) 3. 10 (br s, 1 H. azepine-CH), 3.63 3H, COOCHA) 3.94 (br s, 1 H, azepine-CH), 4.43 (br s, 2H1, Cff 2 COOCHA) 7.29 (dd, I1H5 1.8/8.4 Hz), 7.40-7.43 ::25 (in, 2H), 7.46-7.52 (in, 2H1), 7.72-7.74 (in, 1H), 7.93 1H, 1.5 Hz), 11.94 IIH, NH); 1 3 C-nMr (DMSO-d 6 100.6 MHz): 6 (ppm) =30.9, 51.8, 52.2, 108.7, 111.7, 113.5, 120.5, 123.9, 124.6, 125.1, 125.3, 127.2, 127.8, 128.6, 133.8, 135.9, 139.4, 169.7, 170.4; CjqH 15 BrN 2 0 3 (399.25); Calcd. C 57.16, H 3.79, N 7.02, Br 20.01; Found C 57.07, H 3.90, N 6.99, Br 20.02.
\\mlbfiles\homcS\suzaflnct'Keep\Spci\45714.99.1 Divsional SPECIdoc 16/01/01 -46- Example 16 This example describes the synthesis of 9-bromo-7,12-dihydro-12-(2hydroxyethyl)-indolo[3,2-d][1]benzazepin-6(5H)-one.
H 0
N
Al B
C
HO
D
Br A solution of 9-bromo-12-methyloxycarbonylmethyl-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (399 mg, 1 mmol) in THF (80 mL) was added by means of a dropping funnel to a stirred suspension of lithium alumino 15 hydride (19 mg, 0.5 mmol) in THF (10 mL). After the addition was complete, the 0* 0 mixture was refluxed for 2 hours. An additional portion of lithium alumino hydride (19 mg, 0.5 mmol) was added and refluxing was continued for 1 hour. After cooling to room temperature, water was cautiously added until the hydrogen *00000 evolution was finished. (Caution: the evolved hydrogen is flammable and may ignite!) A precipitate of aluminium hydroxide was formed, which was redissolved 0 by dropwise addition of 25% sulfuric acid. The solution was extracted twice with dichloromethane (20 mL, respectively). The combined organic layers were dried with sodium sulfate and evaporated. The residue was recrystallized from ethanol to yield 48% colorless crystals, m.p. 267 0 C; ir (KBr): 3420, 3340 3260 (NH), 1650 cm' 'H-nmr (DMSO-d 6 MHz): 6 (ppm) 3.05(br s, 1H, azepin-CH), 3.35 (br s, 1H, azepine-CH, overlapping the H 2 0-signal), 3.68-3.75 (br m, 2H, CH 2 4.32-4.35 2H, O-CH 2 5.02 1H, 5.3 Hz, OH), 7.29-7.35 3H), 7.42- 7.46 1H), 7.59 1H, 8.6 Hz), 7.94 1H, 2.0 Hz), 7.97 1H, 7.5Hz), 10.01 1H, NH); 3 C-nmr (DMSO-d 6 100.6 MHz): 6 (ppm) 31.1, 46.6, 59.5, 109.8, 112.1, 113.0, 120.3, 121.7, 122.9, 123.5, 124.4, 127.2, 128.4, 128.8, 135.2, 136.7, 137.2, 172.5; C 1 8
H
1 5 BrN 2 0 2 (371.24); Calcd. C 58.24, H 4.07, N 7.55, Br 21.52; Found C 58.17, H 4.35, N 7.47, Br 21.68.
\\nlbfilc\hon$m\snnct\Keep\Spcci\471 4-99.1 Divisioml SPECIA.&c 16/01/01 -47 Example 17 This example describes the synthesis of 9-bromo- 7, 12-dihydro-12-methylindolo[3, 2-d]]benzazepin-6(SH)-one.
H 0
N
CH-N
Br The synthesis was performed according to the general procedure described above in Example 5 using iodomethane (1420 mg, 10 mmol) to furnish 28% of 15 yellowish crystals m. p. 313*C from ethanol/toluene; ir (KBr): 3170 1665 cm'1 'H-nnir (DMSO-d 6 400 IvtIlz): e5 (ppm) 3.08-3.99 (very broad signal, 2H, CH 2 overlapping the H 2 0-signal) 3.84 3H, CHA) 7.30-7.33 (in, 2H), 7.36 (dd, 1H, 1.6/6.9 Hz), 7.43-7.47 (in, 1H), 7.54 1H, 7.1 Hz), 7.74 (dd, 1H, 1.1/6.4 Hz), 7.95 1H, 1.5 Hz), 10.06 LH, NH); 3 C-nmr (DMSO-d 6 100.6 Mffz): 6 (ppm) =31.2, 31.8, 109.3, 112.1, 112.3, 120.4, 121.4, 122.8, 123.4, 124.6, 126.8, 128.5, 128.8, 135.1, 136.6, 137.4, 172.3; C 17
H
13 BrN 2 O (341.21); Calcd. C 59.84, H 3.84, N 8.2 1, Br 23.42; Found C 59.63, H 3.9 1, N 8. 10, Br 23.09.
\\nmtbfils\hoeS\sUZafll\KmCp\Spcci\ 45714-99.1 Divisional SPECIdoc 16/01/01 -48- Example 18 This example describes the synthesis of 9-bromo-7,12-dihydro-indolo[3,2d][1]benzazepine-6(5H)-thione.
H S
N
HN
Br A solution of 9-bromo-7,12-dihydroindolo[3,2-d]-1 (327 mg, 1 mmol) in THF (30mL) was stirred under nitrogen at 50 0 C. Phosphorus pentasulfide (250 mg, 1,12mmol) and sodium hydrogencarbonate (370 mg, 4.4 mmol) were added successively. After refluxing for 3 hours under nitrogen, the 15 mixture is allowed to cool to room temperature and then poured onto crushed ice (50 The mixture was then stirred until the ice is molten, and the precipitate which formed was filtered off with suction, washed with water and recrystallized from ethanol/toluene yielding 67% pale yellow crystals m. p. 3300, ir (KBr): 3430, 3140 cm 1 'H-nmr (DMSO-d 6 400 MHz): 6 (ppm) 3.91 2H, 20 CH 2 7.30 (dd, 1H, 1.5/8.6 Hz), 7.39-7.45 4H), 7.79 1H, 7.1 Hz), 7.86 (d, 1H, 1.5 Hz), 11.92 1H, NH), 12.07(s, 1H, NH); 13 C-nmr (DMSO-d 6 100.6 MHz): 8 (ppm) 39.6, 109.3, 111.8, 113.5, 120.3, 123.1, 123.7, 124.8, 125.5, 126.9, 127.8, 128.1, 133.3, 136.2, 136.2, 200.2; C 16
H
11 BrN 2 S (343.24); Calcd. C 55.99, H 3.23, N 8.16, Br 23.28, S 9.34; Found C 55.81, H 3.28, N 8.00, Br 22.42, S 25 9.51.
\\lbfics\hoS\snnc\KcepSpeci\457 4-99.1 Divisiona SPECIdoc 16/01101 -49- Example 19 This example describes the synthesis of 8-bromo-6,11-dihydrothieno[3',2':2,3]azepino[4, 5-b]indol-5(4H)-one.
H 0
N
S
HN
10 Br A suspension of 4-bromophenylhydrazine hydrochloride (391 mg; 1.75 mmol) and sodium acetate (144 mg, 1.75 mmol) in glacial acetic acid (2.5 mL) was added dropwise to a suspension of 4H-thieno[3,2-b]azepin-5,8(6H,7H)-dione (264 15 mg, 1.5 mmol) in glacial acetic acid (2.5 mL). After stirring for 1 hour at 70 0 C the mixture was allowed to cool to room temperature. Concentrated sulfuric acid (0.125 mL) was added and the mixture is stirred for 1.5 hours at 70 0 C. After cooling to room temperature, the mixture is poured into a 25% aqueous sodium acetate solution (25 mL). A precipitate was formed, which was filtered with suction 20 and recrystallized from ethanol/toluene to yield 26% of metallic shimmering *crystals, mp 280 0 C (decomposition above 288 0 ir (KBr): 3400 1650 cm- 'H-nmr (DMSO-d 6 400 MHz): 6 (ppm) 3.54 2H, CH 2 6.94 1H, 5 Hz), 7.24 (dd, 1H, 2.0/8.6 Hz), 7.34 1H, 8.6 Hz), 7.62 1H, 5 Hz), 7.86 (d, 1H, 1.5 Hz), 10.37 1H, lactam-NH), 11.73 1H, indole-NH); 3 C-nmr (DMSO- 25 d 6 100.6 MHz): 6 (ppm) 32.0, 104.5, 111.9, 113.2, 115.7, 120.0, 123.1, 124.2, 125.4, 128.3, 130.8, 135.1, 136.3, 168.4; C 1 4
H
9 BrN 2 0S (333.21); Calcd. C 50.46, H 2.72, N 8.41, S 9.62; Found C 50.39, H 2.90, N 8.32, S 9.53.
\\mclb_ ilcs\home$\suzannct\Kcep\Spcci\45714-99.1 Divisional SPECI.doc 16/01/01 Example This example describes the synthesis of 9-nitro-7,12-dihydro-indolo[3,2d] [1]benzazepin-6(5H)-one.
H 0
N
HN
NO
2 1H-[ ]benzazepine-2,5(3H,4H)-dione (175 mg, 1 mmol), 4-nitro-phenylhydrazine hydrochloride (284 mg, 1.5 mmol), and sodium acetate (123 mg, 1.5 mmol) were stirred in glacial acetic acid (10 mL) for 1 hour at 70 0 C. After addition of concentrated sulfuric acid (0.1 mL) stirring was continued at 70 0 C. After 1 hour and 2 hours of stirring further portions of concentrated sulfuric acid (0.1 mL, respectively) were added. After a total reaction time of 4 hours the mixture was cooled to room temperature and poured into a 5% aqueous sodium acetate solution mL). The precipitate was filtered off with suction and crystallized from ethanol/toluene to yield 33% yellow crystals, mp. 330 0 C; ir (KBr): 3380 (NH), 1660 'H-nmr (DMSO-d 6 400 MHz): 5 (ppm) 3.65 2H, CH 2 7.29- 7.34 2H), 7.43-7.47 1H), 7.60 1H, 9.2 Hz), 7.77-7.79 1H), 8.08 (dd, 1H, 8.6/2.0 Hz), 8.74 1H, 2.0 Hz), 10.22 1H, lactam NH), 12.39 1H, indole NH); "C-nmr (DMSO-d 6 125.8 MHz): 6 (ppm)= 31.2, 109.6, 111.8, 115.3, 117.3, 121.7, 122.3, 123.7, 125.8, 127.0, 129.0, 135.9, 136.1, 140.3, 140.8, 171.2;
C
6
HIIN
3 0 3 (293.29); Calcd. C 65.5, H 3.8, N 14.3; Found C 65.5, H 3.9, N 14.5.
In vitro time course assays were performed with this compound in order to determine the concentration and duration of drug application necessary to inhibit cell proliferation. Four cell lines (CoLo-205, HL-60 TB, A-498, UO-31) were cultured in media containing the compound for incremental times, then were switched to compound-free media for the rest of the observation time. The results (illustrated in FIG. 31 and 32) indicate that this particular compound causes total inhibition of growth (as measured by G 5 Io, TGI and LC 5 o) at 2-5 p.M and that short exposures were sufficient for maximum inhibition. Cells exposed to the compound for 48 Kep\.Speci\45714-99.1 Divisional SPECI.doc 16/01/01 -51hours required a lower concentration of 1-3 tM to achieve total growth inhibition.
These data are effective in determining a dosing schedule that achieves maximum cell growth inhibition, a plasma or blood level concentration of 2-5 tM compound when exposed for 45 minutes once per day.
Initial experiments to determine if these exposures are achievable in animals were done in Sprague Dawley rats. A single i.v. dose of this compound dissolved in DMSO was given to two animals, one at 5 mg/kg and one at 10 mg/kg. Plasma concentrations of the parent compound were determined by HPLC assay and are listed in the table. At 5 mg/kg the half life in the plasma was 46 minutes, clearance was 16 mL/min/kg, and concentrations in excess of 2 IM were maintained for over two hours. At 10 mg/kg the half life in plasma was 66 minutes, the clearance was 14 mL/min/kg and concentrations in excess of 2/zM were maintained for over three hours.
Time (min) Plasma Concentration (pM) Plasma Concentration (AM) 10 mg/kg dose 5 mg/kg dose 3 54.0 22.9 6 32.3 17.8 9 24.6 14.8 12 21.2 12.9 18.9 12.2 17.5 10.4 30 15.6 8.7 60 14.2 6.4 90 9.9 4.9 120 7.5 2.6 180 3.1 0.9 0 0 a
C
eg..: *s o ec 0
C
S
a.
U
S
\\nlbflcs\onSsuannetuPep\Speci\45714-99.1 Divisional SPECIdoc 16/01/01 -52- Example 21 This example describes the synthesis of 2-bromo-9-nitro-7,12-dihydroindolo[3, 2-d]/I]benzazepin-6(H)-one.
H 0
N
Br HN /0 7-bromo- 1H-[l1]benzazepine-2,5(3H,4TH-dione (254 mg, 1 mmol), 4-nitrophenyihydrazin hydrochloride (284 mg, 1.5 mmol), arnd sodium acetate (123 mg, mmol) were stirred in glacial acetic acid (10 mL) for 1 hour at 70'C. After cooling to room temperature, the mixture was poured into 5% aqueous sodium acetate solution (20 mL). The precipitate was filtered off with suction, washed with water, and crystallized from ethanol to furnish 52% yellow crystals of precursor, 7-bromo- S-(4-nitro-phenylhydrazono)-4, 5-dihydro-] H-fl] benzazepin-2 (3H)-onemp. 300'C ir (KBr): 3220 1670 'H-nmr (DMSO-d 6 400 MHz): 5 (ppm) =2.56-2.59 and 3.02-3.06 (in, AA'XX', 4H, CH 2
-CH
2 6.99 1H, 8.1 Hz), 7.33 2H, 9.2 Hz), 7.56 (dd, 1H, 8.7/2.6 Hz), 7.75 1H, 2.0 Hz), 8.16 2H, 9.6 Hz), 9.87 I1H, NH), 10. 19 1 H, NH); 1 3 C-nmr (DMSO-d 6 100.6 MHz): 8 (ppm)= 29.7, 30.5, 112.4, 116.2, 124.0, 125.8, 131.6, 132.1, 132.3, 136.8, 139.1, 147. 1, 150.8, 172.7; C 16
H
13 BrN 4
O
3 (389.22); Calcd. C 49.4, H 3.4, N 14.4, Br 20.5; Found C 49. 1, H3.4, N 14. 1, Br 2.2.
7-bromo-5-(4-nitro-phenylhydrazono)-4, 5-dihydro- 1H-[ 1 ]benzazepin-2(3H)one (389 mg, 1 mmol) was refluxed in diphenyl ether (20 mL) for 2 hours under nitrogen. After cooling to room temperature, hexane (50 mL) was added. The precipitate was filtered off with suction, washed with hexane and crystallized from ethanol/toluene to furnish 35% yellow-brown crystals, mp. 330'C; ir (KBr): 3310 1670 'H-nmr (DMSO-d 6 400 MHz): 6 (ppm) 3.69 2H, CHA) 7.23 1H, 8.6 Hz), 7.59-7.64 (in, 2H), 7.96 IH, 2.0 Hz), 8.09 (dd, 1H, 9.1/2.0 Hz), 8.77 1H, 1.5 Hz), 10.32 1H, lactam NH), 12.46 1H, indole NH); 1 3
C_
\\nmIb-filcs~homvieSsuzant\Keep\SpecdWS4714-99.l Divisional SPECIADc 16101/01 53 nmr (DMSO-d 6 100.6 MHz): 8 (ppm) 31.2, 110.5, 111.9, 115.5 8, 115.62, 117.7, 123.6, 124.3, 125.6, 129.1, 131.4, 134.6, 135.1, 140.4, 140.9, 171.0; C 16
H
10 BrN 3
O
3 (372.19); Calcd. C 51.6, H 2.7, N 11.2, Br 21.5; Found C 51.5, H 3.0, N 10.8, Br 21.3.
Example 22 This example describes the synthesis of 2,3-dimethoxy-9-nitro-7,12-dihydro indolo[3, 2-d][J]benzazepin-6(SH)-one.
H 0
H
3 00 N
H
3
CO-
HN /0 7,8-dimethoxy-1H-[1]benzazepine-2,5(3H,4TH-dione (235 mg, 1 mmol), 4-nitrophenyihydrazine hydrochloride (569 mg, 3 mmol) and sodium acetate (246 mg, 3 rnmol) were stirred in glacial acetic acid (10 mL) for 1 hour at 70'C. After cooling to room temperature, the mixture was poured into 5% aqueous sodium acetate solution (20 mL). The precipitate was filtered off with suction, washed with water, and crystallized from ethanol to furnish 60% yellow crystals of precursor 7,8dimethoxy-S-(4-nitro-phenylzydrazono)-4, S-dihydro-.JH-[J]benzazepin-2(3H)-one, mp. 286'C ir (KBr): 3260/3180 1680 'H-nmr (DMSO-d 6 400 MHz): 8 (ppm) 2.53-2.56 and 2.99-3.03 (in, AA'XX,, 4H, CH 2
-CH
2 3.77 3H,
OCH
3 ),3.81 3H, OCH 3 ),6.65 1H), 7.20 1H), 7.32 2H, 9.2 Hz), 8.13 (d, 2H, 9.2 Hz), 9.53 1H, NH), 10.06 1H, NH); 1 3 C-nmr (DMSO-d 6 100.6 MHz): 8 (ppm) 29.9, 30.7, 55.5, 55.9, 105.9, 112.1, 122.0, 125.8, 131.1, 138.6, 145.3, 148.6, 149.8, 151.1, 172.8 (one signal missing due to peak overlapping); C I H I N 4 0 5 (3 70.3 Calcd. C 5 8.4, H 4.9, N 15. 1; Found C 5 7.8, H 4.9, N 14.8.
7,8-dimethoxy-5 -(4-nitro-phenylhydrazono)-4,5 -dihydro- 1 H- 1 ]benzazepin- 2(3TH-one (370 mg, 1 mmol) was refluxed in diphenyl ether (20 mL) for 2 hours under nitrogen. After cooling to room temperature, hexane (50 mL) was added. The precipitate was filtered off with suction, washed with hexane and crystallized from \\mielb-files\honmcS\sznepJ~eq,\SpeciM57 14-99.1 DMvSional SPECIdoc 16/01/01 -54ethanol/toluene, yielding 63% yellow-brown crystals, mp. 330 0 C; ir (KBr): 3340 1660 'H-nmr (DMSO-d 6 400 MHz): 8 (ppm) 3.58 2H, CH 2 3.81 3H, OCH 3 3.88 3H, OCH 3 6.90 1H), 7.31 1H), 7.59 1H, 9.2 Hz), 8.05 (dd, 1H, 8.9/2.3 Hz), 8.69 1H, 2.0 Hz), 9.94 1H, lactam NH), 12.32 1H, indole NH); 13C-nmr (DMSO-d 6 100.6 MHz): 6 (ppm) 31.2, 55.5, 55.8, 106.0, 108.0, 109.3, 111.5, 113.8, 114.8, 116.9, 125.9, 129.9, 136.6, 140.1, 140.7, 145.3, 149.3, 170.5; C 18
H
15
N
3 0 5 (353.35); Calcd. C 61.2, H 4.3, N 11.9; Found C 60.9, H 4.4, N 11.8.
Example 23 This example describes the synthesis of 2-iodo-9-trifluoromethyl-7,12dihydro-indolo[3,2-d] H 0
N
HN
CF
3 iA solution of methyl succinyl chloride (3011 mg, 20 mmol) in toluene mL) was added dropwise by means of a dropping funnel to a mixture of iodobenzoic acid methyl ester (4156 mg, 15 mmol), pyridine (1.7 mL), and toluene mL) with stirring and cooling. The resulting suspension was refluxed for 3 hours. After cooling to room temperature, water (15 mL) was added. The organic layer was separated and washed successively with 10% hydrochloric acid (5 mL) 20 and 5% aqueous sodium carbonate solution (5 mL). Subsequently, the organic layer was dried over sodium sulfate and evaporated. The residue was crystallized from ethanol to yield 72% colorless crystals of precursor 5-iodo-2-[(4-methoxy-1,4dioxobutyl)amino]-benzoic acid methyl ester, mp. 133°C; ir (KBr): 3250 (NH), 1730/1720/1680 'H-nmr (DMSO-d 6 400 MHz): 8 (ppm) 2.59-2.69 (m, AA'BB', 4H, CH 2
-CH
2 3.60 3H, OCH 3 3.85 3H, OCH 3 7.91 (dd, 1H, 8.7/2.0 Hz), 7.99 1H, 8.7 Hz), 8.14 1H, 2.0 Hz), 10.53 1H, NH); 1 3 C-nmr (DMSO-d6, 100.6 MHz): 5 (ppm) 28.3, 31.4, 51.3, 52.5, 86.3, 120.1, 123.2, \\nmlbfilcs\home$\sannet\Xep\Speci\45714-99.1 Divisional SPECIdoc 16/01/01 138.3, 138.8, 141.9, 166.1, 170.1, 172.5; C 1 3
HI
4 IN0 5 (391.17); Calcd. C 39.9, H 3.6, N 3.6, I 32.4; Found C 39.9, H 3.7, N 3.6, I 32.4.
Potassium hydride (4 g, 100 mmol) was washed three times with toluene mL) under nitrogen, then suspended in toluene (20 mL). A solution of 5-iodo-2-[(4methoxy-l,4-dioxobutyl)amino]-benzoic acid methyl ester (7823 mg, 20 mmol) in N,N-dimethylformamide (9 mL) and toluene (80 mL) was added dropwise by means of a dropping funnel to the potassium hydride suspension with cooling and stirring under a nitrogen atmosphere. After the evolution of hydrogen had ceased, the mixture was warmed to 80 0 C and stirred continuously for 3 hours. After cooling to room temperature, glacial acetic acid (6 mL) and then water (60 mL) were cautiously added dropwise with stirring. The resulting suspension was stirred in an ice bath for 15 minutes. A precipitate formed, which was filtered off with suction and washed with water and hexanes. Crystallization from ethanol/toluene yields 41 colorless crystals of precursor 5-hydroxy-7-iodo-2-oxo-2,3-dihydro-lH- 15 [1]benzazepine-4-carbonic acid methyl ester, mp. 271°C; ir (KBr): 3150 (NH), 1680/1670 'H-nmr (DMSO-d 6 400 MHz): 8 (ppm) 2.94 2H, CH 2 3.84 3H, OCH 3 6.99 1H, 8.1 Hz), 7.85 (dd, 1H, 8.6/2.0 Hz), 8.04 1H, Hz), 10.42 1H, NH), 12.34 (br. s, 1H, OH); 3 C-nmr (DMSO-d 6 100.6 MHz): 6 (ppm) 30.6, 52.5, 87.2, 96.4, 123.7, 126.7, 136.1, 137.6, 140.1, 164.4, 170.5, 20 171.7; C 12
H
10 IN0 4 (349.12); Calcd. C 40.1, H 2.8, N 3.9, 34.4; Found C 40.1, H 3.0, N 4.0, 34.1.
Water (0.5 mL) was added to a solution of 5-hydroxy-7-iodo-2-oxo-2,3dihydro-1H-[1]benzazepine-4-carboxylic acid methyl ester (349 mg, 1 mmol) in dimethyl sulfoxide (10 mL). The mixture was stirred under nitrogen at 150 0
C.
25 Water (0.5 mL) was added after 1 hour and 2 hours of stirring, respectively. After a total reaction time of 3 hours the mixture was cooled to room temperature and poured into water (20 mL). A solid precipitated from the solution upon standing (12 hours at 6 0 C) and was filtered off with suction and crystallized from ethanol to yield 78% colorless crystals of precursor 7-iodo-lH-[1]benzazepine-2,5(3H,4H)-dione, mp. 217 0 C; ir (KBr): 3180 1650 'H-nmr (DMSO-d 6 400 MHz): 8 (ppm) 2.66-2.69 und 2.89-2.92 AA'XX', 4H, CH 2
-CH
2 6.97 1H, 8.6 Hz), 7.86 (dd, 1H, 8.6/2.0 Hz), 8.05 1H, 2.0 Hz), 10.17 1H, NH); 3 C-nmr (DMSO- \\nilbfiles\horS\suzannct\Kep\Spci\45714-99 I Divisional SPECI.doc 16/01/01 -56d 6 100.6 MHz): 6 (ppm) 28.9, 37.7, 86.9, 123.9, 128.3, 138.2, 138.9, 142.0, 173.3, 197.3; Co 0
H
8 1N0 2 (301.08); Calcd. C 39.9, H 2.7, N 4.7, 42.2; Found C 39.9, H 2.7, N 4.6, I 42.3.
Next, a mixture of 7-iodo-1H-[1]benzazepine-2,5(3H,4H)-dione (301 mg, 1 mmol), 4-trifluoromethyl-phenylhydrazine (264 mg, 1.5 mmol) and glacial acetic acid (10 mL) was stirred at 70 0 C for 1 hour. Concentrated sulfuric acid (0.1 mL) was added and stirring continued for 1 hour. The mixture was then cooled to room temperature and poured into 5% aqueous sodium acetate solution (20 mL). The precipitate was filtered off with suction, washed with water and crystallized from ethanol to yield 50% yellow crystals, mp. 330 0 C; ir (KBr): 3320 1645 'H-nmr (DMSO-d 6 400 MHz): 5 (ppm) 3.63 2H, CH 2 7.07 1H, 8.6 Hz), 7.47 (dd, 1H, 8.6/1.5 Hz), 7.61 1H, 8.6 Hz), 7.73 (dd, 1H, 8.6/2.0 Hz), 8.10 1H, 2.0 Hz), 8.16 1H), 10.24 1H, lactam NH), 12.14 1H, indole NH); 1 3 C-nmr (DMSO-d 6 100.6 MHz): 5 (ppm) =31.2, 87.7, 109.1, 112.2, 116.1 J,F 4.1 Hz), 116.2, 118.6 Jc,F= 3.1 Hz), 120.2 Jc,F= 32 Hz), 124.3, e* 125.4 JC,F 272 Hz), 125.6, 133.1, 134.9, 135.4, 136.8, 138.8, 171.2;
C
17
H
10
F
3 1N 2 0 (442.19); Calcd. C 46.2, H 2.3, N 6.3, I 28.7; Found C 46.0, H 2.4, N 6.3,128.7.
\\mcb fics\hon S\sun t\KccpSpeci\4S714-99.1 Divisional SPECIAoc 16/01/01 57 Example 24 This example describes the synthesis of 3-(6-oxo-9-trifluoromethyl-S,6, 7,12tetrahydro-indolo[3, 2-d][J]benzazepin-2-yl)-acrylonitrile.
C3 2-iodo-9-trifluoromethyl-7, 12-dihydro-indolo[3 [1]benzazepin-6(511)one (442 mg, 1 mmol), acrylonitrile (531 mg, 10 mmol), triethylamine (121 mg, 1.2 mmol), palladium(II)-acetate (45 mg, 0.2 mmol), and triphenyiphosphine (52 mg, 0.2 mmol) were stirred in N,N-dimethylformnamide (30 mL) under nitrogen at 150'C for about 8 hours, until the starting material 2-iodo-9-trifluoromethyl-7,12-dihydroindolo[3,2-d][1I]benzazepin-6(511)-one was no longer detectable by thin layer chromatography (silica gel, toluene/acetone The reaction mixture was then filtered hot and the resulting solution evaporated. The residue was crystallized from :ethanol (ethanol/toluene may also be used). Crystallization from ethanol yielded 0 43% yellow crystals, mp. 330'C; ir (KBr): 3310/3200 2220 1670 15 'H-nmr (DMSO-d 6 400 Mffz): 8 (ppm) 3.67 2H, CH 2 ),6.48 I1H, 16.8 Hz), 7.31 1H, 8.7 Hz), 7.49 (dd, 11H, 8.7/1.0 Hz), 7.64 lH, 8.6 Hz), 7.71 1H1, 16.8 Hz and dd, IH, 8.6/2.0 Hz; overlapping signals), 8.05 1H, 1.5 Hz), 8.17 IH), 10.41 1H, lactam 12.11 1H, indole NH); 1 3 C-nmir(DMSOd 6 100.6MNffz): 8 (ppm) =31.3, 96.1, 108.6, 112.1, 116.1 (q,C&F 4.1 Hz), 118.6 JC,F 3.0 Hz), 118.8, 120.2 JC,F 3 2Hz), 122.2, 122.5, 125.4 &CF 271 Hz), 125.8, 126.9, 127.6, 129.1, 133.9, 137.7, 138.8, 149.5, 171.2; C 20 Hj 2
F
3
N
3 0 (367.35); Calcd. C 65.4, H 3.3, N 11.4; Found C 65.0, H 3.3, N 11.2.
\\mlb-files\homS\suzanet\Kew,\Speci\457 14.99.1i Divisional SPECIdoc 16/01/01 -58- Example This example describes the synthesis of 3-(6-oxo-9-trifluoromethyl-S,6, 7,12tetrahydro-indolo[3, 2-d][lJbenzazepin-2-yl)-propionitrile.
A0itr f3(-x--rfurmty- 67 -erhdoidl 32 acd Th mixture was then x9trcefiviewt ihoromethy5,712trane (pinorons of 20 mL, respectively). The combined organic layers were dried by means of sodium sulfate and evaporated. Crystallization of the residue from ethanol affords 5 1% light ***yellow crystals, mp. 286*C; ir (KBr): 3340 2240 1660 'Hnm (DMSO-d 6 400 MHz): 6 (ppm) 2.88-2.99 (in, AA'BB', 4H, CH 2
-CH
2 .61 (s, 2H, CHA) 7.23 1H, 8.1 Hz), 7.36 (dd, 1H, 8.4/1.8 Hz), 7.47 (dd, 1H, 8.6/1.6 Hz), 7.63 I1H, 8.6 Hz), 7.69 1 H, 1. 6 Hz), 8.14 I1H), 10. 14 I1H, lactamn NH), 12.06 1H, indole NH); 1 3 C-nmr (DMSO-d 6 100.6 MHz): 8 (ppm) 18.0, 30.0, 31.3, 108.4, 112.1, 115.9 &CF 4.1 Hz), 118.3 (q,Jc&F 3.1 Hz), 120.0 &CF= 32 Hz), 120.2, 122.1, 122.4, 125.5 JC,F =270 Hz), 125.8, 126.8, 128.7, 134.2, 134.4, 134.6, 13 8.7, 171.2; C 2 oH 14
F
3
N
3 0 (369.36); Calcd. C 65.0, H 3.8, N 11.4; Found C64.5, H4.0, N 11.3.
\\mlb-filcs\homcS\suannet\Kcep\Spci\45714-99. I Divisional SPECIdoc 16/01/01 -59- Example 26 This example describes the synthesis of 2-(3-oxo-J-butenyl)-9trifluoromethyl- 7, 12-dihydro-indolo[3, 2-d][IJbenzazepin-6(SH)-one.
2-iodo-9-trifluoromethyl-7, 12-dihydro-indolo[3 [1]benzazepin-6(5H)one (442 mg, I mmol), methyl vinyl ketone (701 mg, 10 mmol), triethylamine (121 mg, 1.2 mmol), palladium(ID)-acetate (45 mg, 0.2 mimol), and triphenyiphosphine (52 mg, 0.2 mmol) were stirred in N,N-dimethylformnamide (30 mL) under nitrogen at 150'C for about 4 hours, until the starting material 2-iodo-9-trifluoromethyl-7,12dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one was no longer detectable by thin layer chromatography (silica gel, toluene/acetone 1: The reaction mixture was then filtered hot and the resulting solution evaporated. The residue was crystallized :from ethanol (ethanol/toluene may also be used), yielding 48% yellow crystals, mp.
330'C; ir (KBr): 3270 1670/1655 'H-nmr (DMSO-d 6 400 MHz): 8 (ppm) 2.3 7 3H1, CHA) 3.68 2H, CHA) 6.90 I1H, 16.3 Hz), 7.31 1 H, 8.1 Hz), 7.49 1H, 8.6 Hz), 7.64 1H, 8.6 Hz), 7.69 1H, 16.3 Hz), 7.75 (dd, 1H, 8.1/1.5 Hz), 8.14 I1H, 1. 5 Hz), 8.17 I1H), 10. 38 1 H, lactamn NH), 12.12 (s, I1H, indole NH); 1' 3 C-nMr (DMSO-d 6 100.6 Mffz): 8 (ppm) 27.2, 31.3, 108.4, 112.1, 116.1 JC,F =4.1 Hz), 118.5 JC,F 2.0 Hz), 120.1 JC,F 32 Hz), :20 122.2, 122.6, 125.4 JC,F 272 Hz), 125.8, 126.9, 127.0, 128.1, 128.6, 129.7, 134.1, 137.3, 142.2, 171.1, 197.9; C 2 1
H
15
F
3
N
2 0 2 (384.37); Calcd. C 65.6, H 3.9, N 7.3; Found C 65. 1, H 4.2, N 7.4.
\\flbfls\honieSnnet\Kcp\SpewMSA714.99.I Divisional SPECIdoc 16/01/01 60 Example 27 This example describes the synthesis of 2-(3-hydroxy-J-propinyl)-9trifluoromethyl- 2-dihydro-indolo[3, 2-d][J]benzazepin-6(SH)-one.
2-iodo-9-trifluoromethyl-7, 12-dihydro-indolo[3,2-d] [1]benzazepin-6(5THone (221 mg, 0.5 mmol), propargyl alcohol (112 mg, 2 mmol), bis-(triphenylphosphine)-palladium(II)-dichloride (7 mg, 0.01 mmol), and copper(I) iodide (3.8 mg, 0.02 mmol) were stirred in triethylamine (10 mL) under nitrogen at 50"C for about 5 hours, until the starting material 2-iodo-9-trifluoromethyl-7,12-dihydroindolo[3,2-d][]benzazepin-6(5H)-one was no longer detectable by thin layer chromatography (silica gel, toluene/acetone 1: Acetone (25 mL) was then added.
Subsequently, the mixture was filtered and the resulting solution evaporated. The residue was crystallized from ethanol to yield 59% yellow crystals, mp. 330'C; ir (KBr): 3500-3100 (OH, NH), 1660 1 H-nnir (DMS0-l 6 400 MHz): 8 (jppm) 3.66 2H, CHA) 4.35 2H, 6.1 Hz, CHA) 5.39 1H, 6.1 Hz, OH), 7.26 (d, 1H, 8.2 Hz), 7.45-7.48 (in, 2H), 7.61 1H, 8.1 Hz), 7.87 lH, 1.5 Hz), 8.16 (s, 1H), 10.33 1H, lactam NH), 12.15 1H, indole Ni); 1 3 C-nmr (DMSO-d 6 100.6 MHz): 5 (ppm) =31.3, 49.4, 82.9, 89.8, 108.6, 112.2, 116.1 JC,F =4.1 Hz), 118.5 JC,F 3.1 Hz), 120.1 JC,F 32 Hz), 122.1, 122.5, 125.4 JC,F= 271 Hz), 125.8, 129.8, 131.1, 133.7, 135.6, 138.8, 171.2; C 2 0
HI
3
F
3
N
2 0 2 (370.34); Calcd. C 64.9, H 3.5, N 7.6; Found C 64.5, H 3.8, N 7.3.
\\nrilbliies\homSuznnet\Keep\SpectM571 4-99.1 DivSional SPECI dC 16/0 1/01 -61- Example 28 This example describes the synthesis of -hydroxycyclohexyl)-ethinyl]- 9-trifluoromethyl- 2-dihydro-indolo[3, 2-d][J]-benzazepin-6(5H) -one.
H 3 2-iodo-9-trifluoromethyl-7, 12-dihydro-indolo[3 [1]benzazepin-6(5H)one (221 mg, 0.5 mmol), 1-ethinyl-l-cyclohexanol (248 mg, 2 mmol), bis-(triphenylphosphine)-palladium(II)-dichloride (7 mg, 0.01 mmol), and copper(I) iodide (3.8 mg, 0.02 minol) were stirred in triethylamine (10 mL) under nitrogen at for about ihour, until the starting material 2-iodo-9-trifluoromethyl-7,12-dihydroindolo[3,2-d][1I]benzazepin-6(5TH-one was no longer detectable by thin layer chromatography (silica gel, toluene/acetone 1: Acetone (25 mL) was then added.
Subsequently, the mixture was filtered and the resulting solution is evaporated. The residue was crystallized from ethanol to yield 55% colorless crystals, mp. 330'C; ir (KBr): 3540 3280/3180 1670 'H-nmr (DMSO-d 6 400 MHz): 8 (ppm) 1.23-1.28 (in, 1H), 1.51-1.60 (in, 5H), 1.66-1.68 (in, 2H), 1.87-1.90 (in, 2H), 3.64 2H, CHA) 5.47 I1H, OH), 7.25 IRH, 8.6 Hz), 7.43 (dd, I1H, 8.4/1.8 Hz), 7.47 (dd, 1H, 8.7/1.0 Hz), 7.61 1H, 8.6 Hz), 7.83 1H, 2.0 Hz), 8.15 (s, 1H), 10.31 1H, lactam 12.16 1H, indole NH); 1 3 C-nmr (DMSO-d 6 100.6 MHz): 8 (ppm) 22.8, 24.9, 31.3, 39.7, 67.0, 82.0, 94.8, 108.6, 112.1, 116.1 JC,F =4.1 Hz), 117.8, 118.5 JC,F 3.1 Hz), 120.1 JC,F =31 Hz), 122.2, 122.5, 125.4 JC,F =272Hz), 125.7, 129.7, 131.1, 133.7, 135.4, 138.8, 171.2;
C
25
H
21
F
3
N
2 0 2 (438.46); Calcd. C 68.5, H 4.8, N 6.4; Found C 68.1, H 5.0, N 6.3.
\tnlb-filcs\home$\szannct\Kecp\Spci\45714.99.1 Divisional SPECIdoc 16/01/01 62 Example 29 This example describes the synthesis of 3-(6-oxo-9-trifluoromethyl-5, 6,7,12tetrahydro-indolo[3, 2-d][J]benzazepin-2-yl)-acrylic acid methyl ester.
H3CO 2-iodo-9-trifluoromethyl-7, 12-dihydro-indolo[3 one (442 mg, 1 mmol), acrylic acid methyl ester (861 mg, 10 mmol), triethylamine (121 mg, 1.2 mmol), palladium(II)-acetate (45 mg, 0.2 mmol), and triphenylphosphine (52 mg, 0.2 mmol) were stirred in N,N-dimethylformamide (30 mL) under nitrogen at 150'C for about 14 hours, until the starting material 2-iodo-9- 10 trifluoromethyl-7, 12-dihydro-indolo[3 [1]benzazepin-6(5N)-one was no longer detectable by thin layer chromatography (silica gel, toluene/acetone 1: The reaction mixture was then filtered hot and the resulting solution evaporated. The :residue was crystallized from ethanol/toluene (ethanol may also be used) yielding beige crystals mp. 330'C; ir (KBr): 3240 1690/1635 'H-nmr (DMSO-d 6 400 Mffz): 8 (ppm) 3.68 2H1, CHA) 3.76 3H, OCHA) 6.72 (d, 1H, 16.3 Hz), 7.30 1H1, 8.6 Hz), 7.48 (dd, IH, 8.6/1.0 Hz), 7.64 1H, 8.6 Hz), 7.72 1H1, 16.3 Hz), 7.77 (dd, 1H, 8.6/1.5 Hz), 8.14 1H, 1.5 Hz), 8.17 1H, arom. 10. 38 I H, lactam NH), 12. 10 1 H, indole 3 C-nMr (DMSO-d 6 100.6 MHz): 6 (ppm) =31.3,51.4,108.4,112.1,116.1 (q,JC,F =3.6Hz), 117.4, 118.5 JC,F =3.6 Hz), 120.1 JC,F =3 3 Hz), 122.1, 122.5, 125.4 JC,F =272 Hz), 125.8, 127.0, 128.7, 129.3, 134.1, 137.3, 138.7, 143.5, 166.8, 171.8;
C
2 1
H
1
F
3
N
2 0 3 (400.37); Calcd. C 63.0, H 3.8, N 7.0; Found C 62.6, H 3.9, N \\mlbfies\home$Ssuznct\Kcep\SpccMS47 14-991 Divisional SPEC~doc 16/01/01 -63- Example This example describes the synthesis of 2,3-dimethoxy-6-oxo-5,6,7,12tetrahydro-indolo[3,2-d][1]benzazepine-9-carbonitrile.
H 0
H
3 CO N
H
3
CO,
HN
CN
9-bromo-2,3-dimethoxy-7,12-dihydro-indolo[3,2-d][1 (387 mg, 1 mmol) and copper(I) cyanide (179 mg, 2 mmol) were refluxed for 2 hours in N-methyl-2-pyrrolidone (10 mL). After cooling to room temperature, water mL) was added and the mixture was stirred for 15 minutes. The precipitate was filtered off with suction and subsequently stirred for 15 minutes in a mixture of water (10 mL) and ethylene diamine (2.5 mL). The precipitate was then filtered off with suction, washed with a 10% solution of sodium cyanide in water and crystallized from ethanol/toluene to furnish 40% of colorless crystals, mp. 330 0
C;
ir (KBr): 3300/3200 2220 1660 'H-nmr (DMSO-d 6 400 MHz): 8 (ppm) 3.53 2H, CH 2 3.80 3H, OCH 3 3.87 3H, OCH 3 6.89 1H), 7.29 1H), 7.49 (dd, 1H, 8.6/1.5 Hz), 7.58 1H, 8.2 Hz), 8.27 1H), 9.89 (s, 1H, lactam NH), 12.10 1H, indole NH); "C-nmr (DMSO-d 6 100.6 MHz): 8 (ppm) 31.1, 55.5, 55.7, 101.0, 106.0, 106.6, 109.4, 112.3, 113.9, 120.6, 123.4, 124.3, 126.4, 129.7, 135.4, 138.7, 145.3, 149.2, 170.7; C 1 9
H
15
N
3 0 3 (333.36); Calcd.
C 68.5, H 4.5, N 12.6; Found C 68.0, H 4.6, N 12.0.
*9 Example 31 This example describes the method used for hollow fiber in vivo testing of mice. Human cancer cells were cultivated in polyvinylidene fluoride (PVDF) hollow fibers. A sample of each cancer cell line was implanted intraperitoneally and subcutaneously in mice. Each mouse received plural fibers, several both intraperitoneally and subcutaneously, representing distinct cancer cell lines. Mice were treated with 9-bromo-7,12-dihydro-indolo[3,2-d][1 \\mclbfilcs\hom S\suannet\Icp\SpeccM\414-99.1 Divisional SPECIdoc 16/01/01 -64made according to the present invention at each of two test doses intraperitoneally using a QD X 4 treatment schedule. Vehicle controls consisted of mice receiving the compound diluent only. The fiber cultures were collected on the day following the last treatment. Anticancer effects were assessed by determining viable cell mass for each of the cell line using a formazan dye (MTT) conversion assay. From this, the %T/C was calculated using the average optical density of the compound-treated samples divided by the average optical density of the vehicle controls. In addition, the net increase in cell mass was determined for each sample as samples of fiber cultures were assessed for viable cell mass on the day of implantation into mice.
This allowed cytostatic and cytocidal capacities of these compounds to be assessed.
Example 32 This example describes a method for treating humans with the compounds of the present invention. Compounds satisfying Formulas 1 and/or 2 are obtained.
15 These compounds are then administered orally or intravenously to humans at a dose of from about 30 mg/kg of subject/dose up to about 400 mg/Kg of subject/dose, but preferably between about 30 mg/Kg of subject/dose to about 50 mg/kg of subject/dose, or to provide a total amount of compound or compounds to the subject per treatment of from about 0.1 gram to about 3 grams. Alternatively, compositions 20 comprising one or more compounds satisfying Formula 1 or 2, and at least one material selected from the group consisting of inert carriers, excipients, diagnostics, direct compression binders, buffers, stabilizers, fillers, disintegrants, flavors, colors, lubricants, other active ingredients, other materials conventionally used in the formulation of pharmaceutical compositions, or mixtures thereof, are administered 25 orally or intravenously to humans. The compositions are administered to provide a total amount of compound or compounds to the subject of from about 30 mg/kg of subject/dose up to about 400 mg/Kg of subject/dose, but preferably between about mg/Kg of subject/dose to about 50 mg/kg of subject/dose, or to provide a total amount of compound or compounds to the subject per treatment of from about 0.1 gram to about 3 grams.
\\mclbfiles\homeS\sanct\Kep\Spci\ 4S 14-99.1 Divisional SPECIdoc 16/01/01 65 Example XlI This example describes the synthesis of 2-(2,3-Epoxypropyl)-9-trifluoromethyl-7,12dihydroindolo[3,2-dl[ 1
-CF
3 a a To a suspension of 2-(2-propenyl)-9-trifluoromethyl-7, 12-dihydroindolof 3,2- (90 mg, 0.25 mmol) and K 2 C0 3 (100 mg) in MeOH (10 ml) was added 1 ml of MeCN. H 2 0 2 (35 5 ml) was added drop-wise at rt over a period of 3 h. Ice water (50 ml) was added and a while solid precipitated, which was filtered off with suction, washed with water, and purified by column chromatography (ethyl acetate: petrol ether 50:50) to recover 43 mg of 2-(2-propenyl)-9-trifluoromethyl-7,12dihydroindolo[3,2-d][lI]benzazepin-6(5H)-one (48 and 42 mg of 2-(2,3- Epoxypropyl)-9-trifluoromethyl-7,1I2-dihydroindolo[3 [1 mp 295-296 'C (ethanol); IR 3400, 1660, 1500, 1300, 1260, 1100 cm- I; 'H NMR (400 MHz) 2.56-2.63 (in, 1 2.77-2.80 (in, 1 2.87 J= 5.6 Hz, 2 3.20-3.22 (in, 1 3.60 2 7.22 J 8.3 Hz, 1 7.33 J 8.1 Hz, 1 7.46 J 8.6 Hz, I 7.62 J 8.1 HZ, 1 7.67 1 8.14 1 10. 12 1 12.26 1 "C NMR (100 MHz) 31.2, 37.5, 46.1, 51.8, 108.4, 112.0, 115.8 J 2 Hz), 118.0 J 31 Hz), 118.2 J 2 Hz), 122.1, 122.3, 125.5 J 270 Hz, CFA) 125.8, 127.2, 129.2, 132.9, 134.2, 134.6, 138.7, 171.3; Anal. (C 20 H I 5
N
2 0 2
F
3 372.342): calcd. C, 64.52; H 4.06; N, 7.52; found C 64.25; H, 4.34; N, 7.49.
IC
50 CDK I/cyclin B (value for Table 1) 0.9 llnrelb_film\bomc1\s~.znc\Kecp\Spcmi\5714-99.1 DivisienaI SPECI.d&l 16/0 1101 66 Example X2 This example describes the synthesis of 2-(Epoxyethyl)-9-trifluoromethyl-7, 12dihydroindolo[3,2-d][ 0 To a suspension of 2-ethenyl-9-trifluoromethyl-7,1I2-dihydroindolo[3 [1 ]benzazepin- 6(51-)-one (0.25 mmol) and K 2 C0 3 (100 mg) in MeOH (10 ml) was added 1 ml of MeCN. H 2 0 2 (35 5 mld) was added drop-wise at rt over a period of 3 h. Ice water ml) was added and a while solid precipitated, which was filtered off with suction, washed with water, and purified by column chromatography (ethyl acetate: petrol ether 50:50) to recover 2-(Epoxyethyl)-9-trifluoromethyl-7,1I2-dihydroindolo[3 [1I ]benzazepin- 6(511)-one (3 1 mp 309-3 10 'C (darkening at 280 0 IR 3 300, 1640, 1300, 1260, 1100 cm- I; 'H NMR (400 MHz) 2.97 (dd, J 5.1/2.5 Hz, 1 3.19 J 5.0 Hz, 1 H), 3.62 2 4.00-4.02 (m I 7.26 J 8. 1 Hz, I 7.3 6 (dd, J 1 =8.6 Hz, J 2 1. Hz, 1 7.47 (dd, J, 8.6 Hz, J 2 1.5 Hz, I 7.62 J 8.1 Hz, I 7.68 J Hz, 1 8.20 1 10.21 1 12.12 1 1 3 C NMR (125 MHz) 31.2, 50.2, 51.2, 108.5, 112.1, 115.9 J 5 Hz), 118.4 J 2 Hz), 120.0 J 30 Hz), *122.1, 12.4 124.1, 125.5i J 273 Hz), 125.8, 126.1, 133.0, 134.4, 135.5, 138.7, 171.3; Anal. (C 19 Hj 3
N
2 0 2
F
3 358.3 19): calcd. C, 63.69; H 3.66; N, 7.82; found C 62.93; H, 3.75; N, 7.57.
CDK I/cyclin B (value for Table 1) 0.3 \rlb~filc,\hoekS~zanflC\Kccp\SpcCMS7I4-99.1 Ojiisioml SPECIdoc 16/101 C I -67- Example X3 This example describes the synthesis of 9-Bromo-2-epoxyethyl-7,12-dihydroindolo[3,2d] NH 0 0
HN
Br To a suspension of 9-bromo-2-ethenyl-7,12-dihydroindolo[3,2-d][1 one (80 mg, 0.22 mmol) and K 2 CO3 (40 mg) in ethanol (20 ml) was added 2 ml of MeCN. H 2 0 2 (35 5 ml) was added drop-wise at rt over a period of 3 h. Ice water ml) was added and a pale yellow solid precipitated, which was filtered off with suction, washed with water, and purified by flash chromatography (ethyl acetate: petrol ether 50:50) recovering 40 mg of 9-bromo-2-ethenyl -7,12-dihydroindolo[3,2-d][ 1 ]benzazepin- 6(5H)-one and 32 mg of 9-Bromo-2-epoxyethyl-7,12-dihydroindolo[3,2t oo d][l]benzazepin-6(5H)-one mp 320-321 "C (darkening at 260 IR 3300, 1640, 1300, 1260, 830 cm'; 'H NMR (400 MHz) 2.95 (dd, J 5.6/2.5 Hz, 1 3.17 J 4.6 Hz, 1 3.49, 3.53 (AB, JAB 14.7 Hz, 2 3.99-4.01 1 7.24 J 8.6 Hz, 1 7.28 (dd, JI 8.6 Hz, J 2 2.0 Hz, 1 7.33 (dd, Jl 8.6 Hz, J2 2.0 Hz, 1 H), 7.40 J 8.6 Hz, 1 7.64 J 2.0 Hz, 1 7.91 J 2.0 Hz, 1 10.15 1 11.84 1 3 C NMR (100 MHz) 31.3, 50.2, 51.2, 107.3, 111.6, 113.3, 120.4, 122.3, 124.1, 124.6, 125.9, 128.2, 133.0, 133.7, 135.4, 136.0, 171.3; HRMS (Ci 8
H
3 BrN 2 02, 368.0160) found 368.0168 kktmlbfil"\hormcS'\suw M\Kee P\S p5714- IDivisional SPECIdoc 16/01/01 -68- Example X4 This example describes the synthesis of 9-Bromo-2-(2,3-epoxypropyl-7,12dihydroindolo[3,2-d] [1I ]benzazepin-6(5H) -one.
.00.
a* 0
C@
*see* 06 0 *ode To a suspension of 9-bromo-2-(2-propenyl)-7,1I2-dihydroindolo [1 I]benzazepinand K 2 C0 3 (40 mg) in ethanol (20 ml) was added 2 ml of MeCN. H 2 0 2 (35 ml) was added drop-wise at rt over a period of 3 h. Ice water (50 ml) was added and a pale yellow solid precipitated, which was filtered off with suction, washed with water, and purified by flash chromatography (ethyl acetate: petrol ether 50:50) recovering 9bromo-2-(2-propenyl)-7,1I2-dihydroindolo[3 I benzazepin-6(5H)-one and 9- Bromo-2-(2,3-epoxypropyl -7,1 2-dihydroindolo[3 ,2-dj [1 ]benzazepin-6(5H)-one mp 308-3 10 'C (darkening at 260 0 JR 3200, 1640, 1400, 1300, 12200, 830 cm'1; H NMR (400 MHz) 2.61 (dd, J 4.6/2.5 Hz, 1 2.78 J 4.1 Hz, 1 2.86 (d,J 5.6 Hz, 2 3.17-3.21 (m 1 3.49 7.19 J 8.1 Hz, 1 7.27 (dd, J, 8.6 Hz, J 2 2.0 Hz, 1 7.30 (dd, J I 8.1 Hz, J2 2.0 Hz, 1 7.40 J 8.6 Hz, 1 H), 7.63 1 7.90 1 10.07 1 11.80 1 3 C NMR (100 MHz) 31.3, 37.5, 46.1, 51.8, 107.1, 111.6, 113.3, 120.3, 122.2, 122.3, 124.4, 127.1, 128.0, 129.0, 132.9, 133.9, 134.1, 135.9, 171.3; Anal. (C 18 H1 3 BrN 2
O
2 383.245): calcd. C, 59.55; H, 3.95; N, 7.31 found C, 59.03; H 4.44; N, 7.21 HRMS calcd. 382.0317 found 382.0322.
\\mbfikisoenc\an\ecpSpci\471 4-99.1 Divisional SPECIdoc 1 6/01/01 -69- Example This example describes the synthesis of 2-(2-oxopropyl)-9-trifluoromethyl-7,12dihydroindolo[3,2-d] [1
O
NH
Me H C F 3 To a solution of 2 -(2-propenyl)-9-trifluoromethyl-7,12-dihydroindolo[3,2d][l]benzazepin-6(5H)-one (108 mg, 0.3 mmol) in DMF (6 ml) and H20 (1 ml) was added PdCl2 (6 mg, 0.03 mmol) and CuC12 (48 mg, 0.36 mmol). After stirring the mixture overnight at room temperature it was poured into 30 ml of water and extracted with ethyl acetate (50 ml X The organic layers were combined, washed with water i 10 ml X 3) and dried over NaSO4. Evaporation of the solution yielded a residue, which was purified by flash chromatography (ethyl acetate: petrol ether 70:30) to yield 78 mg of 2 -(2-oxopropyl)-9-trifluoromethyl-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)one mp 295-297 OC (ethanol); IR 3310, 1710, 1650, 1310, 800 cm-; 'H NMR S (400 MHz) 2.20 (s 3 3.58 2 3.78 2 7.18-7.30 2 7.46 J 8.6 Hz, 1 7.57 1 7.61 J= 8.1 Hz, 1 8.13 1 10.14 1 12.07 1 NMR (100 MHz) 29.5, 31.2, 49.9, 108.4, 112.1, 115.8, J 3 Hz), 118.3 (q 3 Hz), 120.0 J 37 Hz), 122.0, 122.3, 125.5 J 270 Hz), 125.8, 127.8, 130.0, 130.3, 134.3, 134.5, 138.7, 171.3, 205.9; Anal. (C 2 0
H
1 5
N
2 0 2
F
3 372.346): calcd. C, 64.51; H 4.06; N, 7.52; found C 64.30; H, 4.23; N, 7.50 ICso CDKl/cyclin B (value for Table 1) 0.2 \\nmlbfile\hoeS\suzanlf\KcSPecMS\457I4-99. Divisinl SPECIdoC 16/01101 r The present invention has been described with reference to preferred embodiments. Other embodiments of the invention will be apparent to those skilled in the art from a consideration of this specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.
e \\mlbfllcs\home$\suzannct\CKcp\Spec457 14-99.1 Divisiol SPECIdoc 16/01/01

Claims (34)

1. A compound according to the formula R;, •A B R, R Rio where A is oxygen or sulfur coupled to the ring by a single or double bond; R2 is selected from the group consisting of hydrogen, aryl, and lower aliphatic substituents; R 4 -R 7 are independently selected from the group consisting of alkoxy, amino including aliphatic amines such as aminoalkyl, acyl, aliphatic, aliphatic 10 alcohols, aliphatic nitriles, cyano, nitro, carboxyl, halogen, hydrogen, hydroxyl, imino, a, p unsaturated ketones and epoxy, including aliphatic epoxys such as epoxyalkyl; R 8 -R 1 1 are independently selected from the group consisting of aliphatic, aliphatic alcohols, alkoxy, acyl, cyano, nitro, epoxy, haloalkyl groups, halogen, hydrogen, hydroxyl sulfoxide, sulfone and aminosulfonyl; R 1 2 is selected 15 from the group consisting of aliphatic, aliphatic alcohols, carboxylic acids and hydrogen, and wherein: S* at least one of R 4 R 7 is aminoalkyl or epoxy, and/or at least one of R 8 R 1 1 is sulfoxide, sulfone or aminosulfonyl.
2. The compound according to claim 1 wherein A is oxygen in a double bond.
3. The compound according to claim 1 or claim 2 wherein R 2 is selected from the group consisting of H, -CH 2 COOCH 3 -CH 3 and -CH 2 Ph.
4. The compound according to claim 3 wherein R 2 is hydrogen. \\mclb files\homcS\suzannct\Keep\Spci45714-99. I Divisional SPECI.doc 16/01/01 -72- The compound according to any one of claims 1 to 4 wherein R 4 -R 7 are independently selected from the group consisting of alkoxy, amino including aliphatic amines such as aminoalkyl, acyl, alkyl, alkenyl, alkinyl, cyano, nitro, carboxyl, halogen, hydrogen, hydroxyl and imino, lower aliphatic alcohols, lower aliphatic nitriles, a, P unsaturated ketones and epoxy, including aliphatic epoxys such as epoxyalkyl.
6. The compound according to any one of claims 1 to 5 wherein R 4 -R 7 are independently selected from the group consisting of-H, -OH, -C(=NH)NH 2 -CO 2 H, -Br, -OCH 3 cyanoethyl, 3-hydroxy-l-propinyl, 3-oxo-1-butenyl, and 2-(1- hydroxycyclohexyl)-ethinyl.
7. The compound according to any one of claims 1 to 6 wherein R 8 -RI1 are S 15 independently selected from the group consisting of alcohols, alkoxy, acyl, alkyl, alkenyl, alkinyl, cyano, nitro, epoxy, haloalkyl, halogen, hydrogen, hydroxyl, lower alkyl, sulfoxide, sulfone and aminosulfonyl.
8. The compound according to any one of claims 1 to 7 wherein R8-R 1 are 20 independently selected from the group consisting of-H, halogens, -OH, -CH 2 0H, CH 2 CHOCH 2 -CH 2 CH 2 CHOCH 2 -CF 3 and -OCH 3
9. The compound according to any one of claims 1 to 8 wherein R 12 is selected from the group consisting of alcohols, cyano, nitro, carboxylic acids, hydrogen and 25 lower alkyl groups. The compound according to any one of claims 1 to 9 wherein R 1 2 is selected from the group consisting of-H, -CH 2 CH 2 0H, -CH 3 and -CH 2 CH 3
11. A composition comprising an effective amount of a compound according to the formula: \\nilbfilc\homc$\sannet\Kecp\Spcc45714-99 Divisional SPECIdoc 16/01/01 -73- A B R;, Rio where A is oxygen or sulfur coupled to the ring by a single or double bond; R 2 is selected from the group consisting of hydrogen, aryl, and lower aliphatic substituents; R 4 -R 7 are independently selected from the group consisting of alkoxy, amino including aliphatic amines such as aminoalkyl, acyl, aliphatic, aliphatic alcohols, aliphatic nitriles, cyano, nitro, carboxyl, halogen, hydrogen, hydroxyl, imino, a, p unsaturated ketones and epoxy including aliphatic epoxys such as epoxyalkyl; Rs-RII are independently selected from the group consisting of aliphatic, aliphatic alcohols, alkoxy, acyl, cyano, nitro, epoxy, haloalkyl groups, halogen, hydrogen, hydroxyl sulfoxide, sulfone and aminosulfonyl; R 1 2 is selected from the group consisting of aliphatic, aliphatic alcohols, carboxylic acids and hydrogen, and wherein: at least one of R 4 R 7 is aminoalkyl or epoxy, and/or at least one of R 8 R 11 is sulfoxide, sulfone or aminosulfonyl.
12. The composition according to claim 11 wherein A is double bonded to oxygen.
13. The composition according to claim 11 or claim 12 wherein R 2 is selected from the group consisting of H, -CH 2 COOCH 3 -CH 3 and -CH 2 Ph.
14. The composition according to claim 13 wherein R 2 is hydrogen. The composition according to any one of claims 11 to 14 wherein R 4 -R 7 are independently selected from the group consisting of alkoxy, amino including \\melbfiles\hom$\suzannet\Kep\Speci45714-99.1 Divisional SPECI.doc 16/01/01 -74- aliphatic amines such as aminoalkyl, acyl, alkyl, alkenyl, alkinyl, cyano, nitro, carboxyl, halogen, hydrogen, hydroxyl, imino, lower aliphatic alcohols, lower aliphatic nitriles, ao, P unsaturated ketones epoxy including aliphatic epoxys such as epoxyalkyl.
16. The composition according to any one of claims 11 to 15 wherein R 4 -R 7 are independently selected from the group consisting of -OH, -C(=NH)NH 2 -CO 2 H, -Br, -OCH 3 cyanoethyl, 3-hydroxy-l-propinyl, 3-oxo-l-butenyl, and 2-(1- hydroxycyclohexyl)-ethinyl.
17. The composition according to any one of claims 1 to 16 wherein R 8 -R 1 1 are independently selected from the group consisting of alcohols, alkoxy, acyl, alkyl, alkenyl, alkinyl, cyano, nitro, epoxy, haloalkyl, halogen, hydrogen, hydroxyl, lower alkyl sulfoxide, sulfone and aminosulfonyl.
18. The composition according to any one of claims 1 to 17 wherein R 8 -R 1 1 are %t independently selected from the group consisting of halogens, -OH, -CH20H, CH 2 CHOCH 2 -CH 2 CH 2 CHOCH 2 -CF 3 and -OCH 3
19. The composition according to any one of claims 11 to 18 wherein R 12 is selected from the group consisting of alcohols, carboxylic acids, hydrogen and lower Salkyl groups. *20. The composition according to any one of claims 11 to 19 wherein R 12 is 25 selected from the group consisting of-H, -CH 2 CH 2 0H, -CH 3 and -CH 2 CH 3
21. The composition according to any one of claims 11 to 20 and further comprising inert carriers, excipients, diagnostics, direct compression binders, buffers, stabilizers, fillers, disintegrants, flavors, colors, lubricants, other active ingredients, other materials conventionally used in the formulation of pharmaceutical compositions, and mixtures thereof. \\mclbfiles\homcS\suzannet\Keep\Spci\45714-99.1 Divisional SPECI.doc 16/01/01
22. A method for inhibiting the proliferation of living cells in a subject, comprising: providing a compound, or a composition comprising the compound, according to the formula I ,A R R i o wherein A is oxygen or sulfur coupled to the ring by a single or double bond; R 2 is **selected from the group consisting of hydrogen, aryl, lower aliphatic substituents, particularly alkyl and lower alkyl ester; R 4 -R 7 are independently selected from the group consisting of alkoxy, amino including aliphatic amines such as aminoalkyl, acyl, aliphatic, aliphatic alcohols, aliphatic nitriles, cyano, nitro, carboxyl, halogen, hydrogen, hydroxyl, imino, a, 1 unsaturated ketones and epoxy, including aliphatic epoxys such as epoxyalkyl; R 8 -R 1 are independently selected from the group 15 consisting of aliphatic, aliphatic alcohols, alkoxy, acyl, cyano, nitro, epoxy, haloalkyl groups, halogen, hydrogen, hydroxyl sulfoxide, sulfone and aminosulfonyl; R 12 is selected from the group consisting of aliphatic, aliphatic alcohols, carboxylic acids and hydrogen, and wherein: at least one of R 4 R 7 is aminoalkyl or epoxy, and/or at least one of R 8 R 1 is sulfoxide, sulfone or aminosulfonyl administering an effective amount of the compound, or composition comprising the compound, to the subject to inhibit the proliferation of living cells in the subject. \\nxIb_ fies\homS\suannet\Kcep\Spec 4S?14-99.1 DivisiomI SPECIdoc 16/01/01 -76-
23. The method according to claim 22 wherein A is oxygen.
24. The method according to claim 22 or claim 23 wherein R 2 is selected from the group consisting of H, -CH 2 COOCH 3 -CH 3 and -CH 2 Ph. The method according to claim 24 wherein R 2 is hydrogen.
26. The method according to any one of claims 22 to 25 wherein R4-R 7 are independently selected from the group consisting of alkoxy, amino including aliphatic amines such as aminoalkyl, carboxyl, halogen, hydrogen, hydroxyl, imino, lower aliphatic alcohols, lower aliphatic nitriles, ca, 3 unsaturated ketones and epoxy, including aliphatic epoxys such as epoxyalkyl.
27. The method according to any one of claims 22 to 26 wherein R 4 -R 7 are 15 independently selected from the group consisting of -OH, -C(=NH)NH 2 -CO 2 H, -Br, -OCH 3 cyanoethyl, 3-hydroxy-l-propinyl, 3-oxo-l-butenyl, and 2-(1- hydroxycyclohexyl)-ethinyl.
28. The method according to any one of claims 22 to 27 wherein R 8 -R 1 1 are 20 independently selected from the group consisting of alcohols, alkoxies, epoxies, haloalkyl groups, halogen, hydrogen, hydroxyl, lower alkyl groups sulfoxide, sulfone and aminosulfonyl.
29. The method according to any one of claims 22 to 28 wherein R 8 -R 1 1 are 25 independently selected from the group consisting of-H, halogens, -OH, -CH 2 0H, CH 2 CHOCH 2 -CH 2 CH 2 CHOCH 2 -CF 3 and -OCH 3 The method according to any one of claims 22 to 29 wherein R 1 2 is selected from the group consisting of alcohols, carboxylic acids, hydrogen and lower alkyl groups. \\mcIbfil\hoS\uznct\Kecp\SpccUS7I14-99 I Divisional SPECIdoc 16/01/01 -77-
31. The method according to any one of claims 22 to 30 wherein R 1 2 is selected from the group consisting of-H, -CH 2 CH20H, -CH 3 and -CH 2 CH 3
32. The method according to any one of claims 22 to 31 wherein the step of administering comprises administering compound or composition topically, orally, intramuscularly, intranasally, subcutaneously, intraperitoneally, intralesionally or intravenously.
33. The method according to any one of claims 22 to 32 wherein the effective amount comprises from about 0.1 gram to about 3.0 grams of the compound.
34. The method according to any one of claims 22 to 33 wherein the effective amount comprises from about 30 mg/kg of subject/dose to about 400 mg/kg of subject/dose.
35. The method according to any one of claims 22 to 34 wherein the cells are hyperproliferative.
36. The method according to any one of claims 22 to 35 wherein the cells are a neoplasm.
37. The method according to any one of claims 22 to 35 wherein the cells are a malignant tumor. 25 38. A compound according to claim 1 selected from the group consisting of 2- (2,3-Epoxypropyl)-9-trifluoromethyl-7,12-dihydroindolo[3,2-d][1]benzazepin- 2-(Epoxyethyl)-9-trifluoromethyl-7,12-dihydroindolo[3,2- d] [1 ]benzazepin-6(5H)-one, 9-Bromo-2-epoxyethyl-7,12-dihydroindolo[3,2- d][l1]benzazepin-6(5H)-one, 9-Bromo-2-(2,3-epoxypropyl)-7,12-dihydroindolo[3,2- d][1]benzazepin-6(5H)-one and 2-(2-oxopropyl)-9-trifluoromethyl-7,12- dihydroindolo[3,2-d][ 1 \\neIbilc\honie$szannetKeepSpcc?4 14-99.1 Divisional SPECIdoc 16/01/01 -78-
39. A composition comprising an effective amount of a compound according to claim 38. A method as claimed in claim 22 wherein the compound is selected from the group consisting of 2-(2,3-Epoxypropyl)-9-trifluoromethyl-7, 12-dihydroindolo[3 ,2- d] [1 ]benzazepin-6(5H)-one, 2-(Epoxyethyl)-9-trifluoromethyl-7, 12- dihydroindolo[3 [1]benzazepin-6(5H)-one, 9-Bromo-2-epoxyethyl-7, 12- dihydroindolo[3 [1]benzazepin-6(5TH-one, 9-Bromo-2-(2 ,3 -epoxypropyl)-7, 12- dihydroindolo[3,2-d] [1 ]benzazepin-6(5TH-one and 2-(2-oxopropyl)-9- trifluoromethyl-7, 12-dihydroindolo[3 [1 ]benzazepin-6(5H)-one, or a composition comprising this compound.
41. A compound substantially as herein described with reference to Examples eq..XI to GO::42. A composition including a compound substantially as herein described with reference to Examples X1 to
43. A method for inhibiting the proliferation of living cells in a subject ~.20 substantially as herein described with reference to Examples XI to Goo* \\fficbfics\home$\suzannc\Kecp\Spc~i\4571 4-99.1 Divisional SPECIdoc 6/0110
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