CA2147608C - Topoisomerase ii inhibitors and therapeutic uses therefor - Google Patents

Abstract

Azatoxin and derivatives thereof of formulae B-D are illustrative of a new class of antitumor drugs that are topoisomerase II (top 2) inhibitors. The pharmacophore inhibits the catalytic activity of the purified enzyme but does not unwind relaxed or supercoiled DNA. It is nonintercalative and has at least two domains: a quasi-planar polycyclic ring system, which may bind between DNA base pairs, and a pendant substituent thought to interact with the enzyme, with the DNA grooves or with both. In SV40 and C-myc DNA, azatoxin induces numerous double-strand breaks according to a cleavage pattern which differs from those of known top (2) inhibitors. Azatoxin also is a potent inhibitor of tubulin polymerization.

Description

2~.47~~8 'O 94/10175 PCT/US93/09629 TOPOISOMERASE II INHIBITORS
AND THERAPEUTIC USES THEREFOR
Backctround of the Invention Enzymes categorized under the rubric of "DNA
topoisomerase" control the topology of DNA over the course of conformational and topological changes which occur during many cellular processes. For example, DNA
topoisomerases are involved in DNA replication, RNA
transcription and :recombination.
Two kinds of DNA topoisomerases are recognized generally. Type T and type II enzymes catalyze topological changes in DNA by transiently breaking one stand or two strands of the DNA helix, respectively. The relaxation of superhelical DNA is a characteristic reaction catalyzed by a topoisomerase I ("top 1"), while a topoisomerase IT ("top 2") catalyzes the passing of two DNA segments i;n a manner leading to such topoisomerization reactions of DNA as supercoiling/relaxation, knotting/unknotting and catenation/decantenation.
DNA topoisomerase II has been implicated as the chemotherapeutic target for a diverse group of antitumor agents, including epipodophyllotoxins, anthracyclines, acridines, anthracenediones and ellipticines. See Macdonald et a1 . , in DNA TOPOISOMERASES IN CANCER 199-214 (Oxford University Press 1991) (hereafter "Macdonald (1991) ") . Under the influence of such drugs, top 2 is believed to cleave DNA and form a concomitant covalent association with the broken strands) of duplex DNA. The formation of such "c:leavable complexes" of drug, DNA and top 2 enzyme has been attributed to the stabilization by the drug of a covalE~nt, DNA-bound catalytic intermediate in the cleavage-resealing sequence of the enzyme. Id.
New inhibitors of top 2 have been identified after they were noted fox- their antitumor properties. Some, such as amonafide, genistein and the terpenoides, act in ~~47oos -2_ the manner of the above.-mentioned drugs, by trapping cleavabla Complexes. Antitumor compounds like marbarone and fostriecin, by contrast, inhibit top 2 activity without stabilizing cleavable complexes. -while attempting to elucidate mechanistic issues in this field, including the nature of binding sites) fox top 2 inhibitors ir. the ternary complex, Macdonald ( 1991) formulated a composite l0.odsl by superi,z~posing structural subunits of top 2 inhibitors from the give classes of to compounds mentioned previously, namely, epipodophyllatoxins, anthracyclines, acridines, anthracenediones arid elliptici,nes. The composite, three-domain pharmaaophore encompassed, inter alia, a family of hybrid structures which incorporated, respectively, substructural elements from each class of top 2 inhibitors. The °unitied pharmacophore" model was not refined sufficiently, however, to allow fwr a priori predictions of any certainty regarding the activity, if any, of actual molecules deemed within the ambit of the 2o composite.
Hulkenbarg et al., 8P 357,122 discloses beta-carbolines that 2~re rtported as having good cytostatic pzoperties. Hulkenberg do net describe the effects the compounds described therein have on tubulin inhibition or topoisomerase II catalytic activity. Lateurtre et al., Cancer Research, Vol 52(16), 4478-83 (1992) discloses azatoxin as exhibiting topoisomerase II inhibition activity, and is described in detail below.
Snmmarv Of the T,nV~nt3on It is an object of the present invention, therefore, to provide top 2 inhibitors which display properties that not only distinguish theM from known inhibitor compounds but also recommend them for therapeutic uses in anticancer and antiviral contexts, In accomplishing tris object and others there has been provided, in accordance ~rith one aspect of fiche present invention, co~apounds that are represented by the following formulae (A) - (D):
Ar~~~o~~ s~~
L~2~

'~~ WO 94/10175 _3_ ~ ~ ~~ ~ ~ ~ PCT/US93/09629 wherein (i) Y represents the formula i R~ R3 R1 and R3 are the same or different and denote, respectively, hydrogen or methoxy, and R2 denotes hydroxyl;
and (ii) R4 denotes hydrogen, hydroxyl, alkyl ether or hydroxyalkyl ether;
In a preferred embodiment, R1 and R3 both denote methoxy and R4 denotes hydrogen;
Rs (B) w ERs H3t a wherein (i) X denotes NH, S or O;
(ii) R5 denotes COOCH3, COCH3, COCHZOH;
(iii) R6 denotes F, Cl, Br, CN, OH, H or NH2; and (iv) W and W' are the same or different and denote, respectively, H or F;

2~.476U~
WO 94/10175 _4 _ PCT/US93/096?~
(C) Rs w wherein (i) X is the same as above;

(ii) W and W' are the same as above;

(iii) R6 is the same as above; and (iv) R~ denotes H, OH, the formula R
-NH

wherein R denotes H, OH F, Br, C1, NO2, NH2, CN, OCH3, or COZCHZCH3;
the formula -T- ( CHZ ) a Z
wherein T denotes NH or O;
Z denotes NHZ, OH, N (CH;) 2, or N ( CH2CH2C1 ) 2;
n is 2, 3 or 4; and R~ may be derivatized with a 4,6 o-protected sugar;

(D) ~, wherein (i) X is the same as above;
(ii) W and W' are the same as above;
5 (iii) R4 is the same as above;
(iv) R~ is the same as above; and (v) R4 denotes H or OH.
The aforesaid compounds are usefull to inhibit topoisomerase II catalytic activity.
Advantageously, the invention relates to a compound of formula (B):
Rfi (B) in which W', W, X, RS and Rb are as defined hereinabove. Preferably, R6 may be F or Br.
Advantageously, the invention relates to a compound of formula (C):
RF
Vv (C) in which W', W, X, R~ and R~ are as defined hereinabove. Preferably, R~ may be H;

-Sa-and/or R7 may be selected from the group consisting of -NH~
and -NH(CH2)ZN(CH3)2.
Advantageously, the invention relates to a compound of formula (D):

w R
Kg 11 ~ 3 C °---~~ o H0~ ~OCN

in which W', W, X, R6, R7 and R8 are as defined hereinabove. Preferably, R~
may be H; and/or R7 may be - ~ ~
-NN
The term "4,6 o-protected sugar" includes etoposide analogs such as o glucosyl sugars protected with a conventional protecting group such as a methyl acetal or a thiophene acetal group.
It has surprisingly been found that compounds of formula (C), preferably when R6 is H, R~ is -N i-1 and R is F, have increased topoisomerase II catalytic activity when compared to compounds of formula (A). The activity is increased by a factor of at least 3, and preferably by at least 5, when compared to compounds of formula (A). The compounds of formula (A) are not claimed in the present invention. Those skilled in the art readily recognize that the dosage amount required for compounds having such an increase in activity are respectively decreased to maintain the same or a similar effect.

m Sb-In accordance with another aspect of the present invention, a pharmaceutical composition is provided that comprises a tumor-affecting amount of a compound represented by formula (B), (C) or (D), and a physiologically compatible carrier for that compound. In one preferred embodiment, the pharmaceutical composition is an injectable, or infusible preparation.
In accordance with yet another aspect of the present invention, a use and a method is provided for treating cancer in a mammal, which method comprises the step of bringing a pharmaceutical composition as described above into contact with cancerous tissue in a mammal suffering from y. r . . W v ~ cr n - ~.. W G. W .. .a... . ~.
2~47~pg a tumor, such that neoplastic development in the cancerous tissue is retarded or arrested. Thus, preferred modes of administration in the context of such a method are those that maximize contact between -_ cancerous tissues and the active agent of the pharmaceutical composition.
other objects, features and advantages of thA
present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the inventions, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention Will become apparent to those skilled in the art rrom this detailed description.
Brief Description ~ha Dra,~inas Figure s is a graphical representation of results generated by testing the inhibitory activity of azatoxin against panel of tumor cell lines which is a standard of the National Cancer Institute Developmental Therapeutics Program (GI5a: "50% azowth inhibition"; TGI: ~~tumor growth inhibition~~; LCSQ: ~~50% lethal concentration's ] .
Figure 2 depicts, at left, the structural formula of a less preferred compound, azatoxin and, at right, a stereochemical superimposition of the top 2 poison demethyldesoxy--podophyllotoxin (DIP; dashed lines) and azatoxin (solid lines).
t d a cri t o t a fsr ed Efibodiments A new class of top 2 poisens has been discovered which, dsapite certain structural similarities to demethylepipodophyllotoxins and other known top 2 inhibitors, are distinguishable in terms of DNA cleavage pattern and structure-activity relationships which could not have been predicted from any structural superiraposition of known anthracycl;ne, acridine', epipodophyllotoxin and antriracenedione structures.
Archetypical of the new class of inhibitors i.s the compound azatoxin and derivatives thereof, SR,llas-3-one-~,?~~F~~;DtD S~tEcT

tc~ v . v uw : tr.y-:w t:wrtt.v t . ~+- 1 1 -a'~ ~ __. ~ m ~ -uy, wtW ;3'.i--+~.a ti:3 '':35:J4-~b5 : tt 1() 1H,6H,-5,4,11,11a-tetrahydra-5-(3,5-dimethoxy-4-AMENDED ShE~T

'YO 94/10175 PCT/US93/09629 hydroxyphenyl)-oxazolo[3',4':1,6]pyrido[3,4-b]indole, which is represented by formula (A) above when R1 and R3 are methoxy, R2 is hydroxyl and R4 is hydrogen.
Compared to other top 2 inhibitors, azatoxin induces the largest number of top 2 cleavage sites both in SV40 and c-myc DNA, and is very active in inducing protein linked DNA breaks in cells. Accordingly, azatoxin and other pharmacophores of the present invention should be useful as reagent: in the context of mapping top 2 sites in chromatin.
Azatoxin also displays a pattern of differential growth inhibition against human tumor cell lines which is indicative of an antitumor drug action reminiscent of that of top 2 poisons like the nonintercalative epipodophyllotoxins, VP-16 (etoposide) and VM-26 (teniposide). See: Yang et al., Cancer Res. 45: 5872-76 (1985), and Liu (1989), supra, at 361-63. More specifically, azatoxin evidenced significant inhibitory activity when screened against a panel of sixty human tumor cells lines representing leukemia and melanoma, as well as cancers of the lung, colon, kidney, ovary and central nervous system. Pursuant to the convention of Paull et al., J. Nat'3 Cancer Inst. 81: 1088-92 (1989), Figure 1 depicts these results, i.n terms of parameters conventional to cancer research, by graphs which are centered at the arithmetic mean of the logarithm of each parameter. See also Monks et al., J. Nat'1 Cancer Inst.
83: 757-66 (1991), and Boyd, Principles & Practice of Oncology 3: 1-12 (:1989) .
Since a right-extending bar in such a "means graph"
indicates a sensitivity by the cell line to the test substance, Figure 1 evidences the cytotoxicity of aratoxin to cells associated with disseminated cancers (leukemias) as well as several consolidated cancers (non-small cell lung and colon) . It also has been discovered, using a conventional in vitro tubulin polymerization assay, that azatoxin is a potent tubulin inhibitor in the manner of several compounds, such as vineristine, -8 - PCT/US93/096?~'~
vinblastine and taxol, that are very active in cancer chemotherapy. Thus, azatoxin effectively prevents tubulin polymerization in vitro at concentrations in the range of 1 to 10 ~M.
Compounds of the present invention can be synthesized via a modified Pi.ctet-Spengler reaction. More specifically, the inventive compounds are obtained by reacting a corresponding pendent-group dimethylacetal with a precursor carbamate in the presence of a catalytic amount of para-toluenesulfonic acid.
Because the azatoxin skeleton is synthetically accessible, a large number of derivatives are readily prepared and screened for top 2 inhibitory activity in accordance with the present invention. Such screening can be effected by means of an in vitro assay which employs purified top 2 and 3~P-end-labelled DNA. In such an assay, top 2 inhibition by a test substance results in DNA fragmentation which can be detected by agarose gel electrophoresis, as described by Fesen and Pommier, J.
Biol. Chem. 19: 11354-59 (1989); by the filter-binding assay described by Pommier et al., Biochem. 24: 6410-16 (1985); or by the sodium dodecylsulfate precipitation assay employed by Nelson et al., Proc. Nat'1 Acad. Sci.
USA 81: 1361-65 (1984).
Although a myriad of azatoxin derivatives may be synthesized, the structure-activity relationships illuminated in this description indicate that those derivatives falling within the present invention will conform to certain guidelines of molecular design. Thus, with reference to formula (A) and Figure 1, there should be a conservation of the relative spatial orientation between the polycyclic ring system and the pendant ring (Y) in azatoxin. The R/S stereochemistry of the polycyclic ring system also should be carried over from azatoxin. Methoxy groups at the 3' and 5' positions (R1 and R3, respectively) enhance top 2 inhibition, while a 4' hydroxyl group (R2) is essential for inhibitory activity. In contrast to R2, greater flexibility is _ 2~ ~ 7~0~
~~ WO 94/10175 PCT/US93/09629 realized at the 11R position (R4) , where substitutions of the preferred hydrogen can be a hydroxyl group, an alkyl ether group or a hydroxyalkyl ether group such as ethoxyethyl and hydroxypropyl.
The present invention also contemplates the use of compounds according to formulae (A), (B), (C) and (D) in a pharmaceutical composition which further comprises a physiological compatible carrier for the compound. The phrase "physiological compatible carrier" here denotes a solid, liquid or gaseous material that can be used as a vehicle for administering a formula (A), (B), (C), or (D) compound as a medicament because the material is inert or otherwise medically acceptable, as well as compatible with the active agent,. in a particular context of administration. In addition to a suitable excipient, a physiologically compatible carrier can contain conventional additives such as diluents, adjuvants, antioxidants and other preservatives, solubilizing agents, and the like.
The preferred routes for administering a pharmaceutical composition of the present invention are those that maximize introduction of the active agent into the immediate region of the tumor under treatment. It is advantageous, therefore, that the pharmaceutical composition should be an injectable or infusible preparation, the formulation of which would typically require initial solubilization in a nonaqueous solvent, such as dimethyl sulfaxide (DMSO) , that is employed in the field to accommodate hydrophobic anticancer agents.
Similarly, intraarterial administration is preferred for therapy when the tumor is supplied by a known artery, while intrathecal administration can be used for tumors located in the brain.
Intradermal and intracavitary administration is feasible with tumors that are restricted to areas close to a particular skin region and particular portion of the body cavity, respectively. By the same token, an active agent of the present invention can be administered via 5 ~ ~ ~ ~ ~ ~ PCT/US93/0962' application to a mucosal lining (sublingually, for example), when the tumor is accessible through the lining, or via inhalation or insufflation with a gaseous carrier, when the tumor is accessible to the respiratory tract. It is possible, furthermore, to administer an active agent of the present invention in a topical preparation applied to a superficial tumor or, more generally, to a lesion associated with a viral infection against which compounds of the present invention prove effective.
Alternatively, an inventive pharmaceutical preparation can be in a form suitable for oral administration (cachet, tablet, hard- and soft-gelatin capsule, etc.). This route takes advantage of the targeting afforded by the particular sensitivity of proliferating, neoplastic cells to the cytoxic effects of the inventive compounds.
A pharmaceutical composition of the present invention also can take the form of a solid dosage preparation (implant) for introduction surgically into the tumor or its immediate vicinity. A so-called "implantation tablet" would be made up primarily of the active substance and, hence, could be absorbed completely. On the other hand, an implant featuring a non-absorbable skeleton (plastic matrix) or coating would effect controlled release of the inventive compounds upon implantation and then would be removed from the tissues after ceasing to exert an antitumor influence.
A pharmaceutical composition within the present invention preferably contains an inventive compound in an amount that itself is tumor-inhibiting or, in the context an infusion regimen, that permits accumulation in the tumor of a cytotoxic level of the active agent. Since an inventive compound typically inhibits both topoisomerase II activity and tubulin polymerization, it is possible to realize a therapeutic spectrum combining aspects both of a top 2 poison like doxorubicin and a tubulin inhibitor like vincristine. See GOODMAN AND GILLMAN'S THE

"V0 94/10175 -11- PCT/US93/09629 PHARMACOLOGICAL BASIS OF THERAPEUTICS (7th ed. 1985), at pages 1279-85. Symptoms of clinical toxicity associated with these two types of anticancer agents also may be observed and, if so, will require remedial countermeasures which are conventional to the field of cancer therapy.
From these considerations it will be apparent that the optimum dosage of the inventive compound will vary with the particular case. The relevant pharmacokinetics can be determined routinely in the clinical context, which may be therapeutic or prophylactic. "Therapeutic treatment" in this context means treatment intended to kill a maximum number of neoplastic cells, while a "prophylactic treatment",is one aimed at retarding or preventing re-establishment of a proliferating neoplastic population and, hence, a relapse in the disease, once remission has been achieved. It is anticipated that a typical dosage regimen will be similar to that of etoposide (VP-16), i.e., on the order of 100 mg/m2 per day.
Without further elaboration, it is believed that those skilled in the art, informed by the preceding description, can utilize the present invention fully.
Accordingly, the following examples are presented for purposes of illustration only. The materials and methods employed in the examples are described below:
Chemicals, and enzymes: Azatoxin and its derivatives, as well as the demethyldesoxypodo phyllotoxin (DMDP) and demethylepipodophyllotoxin (DMEP), were synthesized by conventional methods. The compounds m-AMSA and mitoxantrone were obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute, Bethesda, MD. The compounds VP-16 and VM-26 were obtained from Bristol-Myers Company (Wallingford, CT) . Drug stock solutions were made in dimethylsulfoxide at 10 mM. Further dilutions were made in distilled water. Various other reagents, including simian virus 40 (SV40) and c-myc human DNA inserts in plasmid pBR322, 2~4760~
WO 94/10175 PCT/US93/0962' restriction endonucleases, DNA topoisomerase I, T4 polynucleotide kinase, calf intestine phosphatase, agarose and polyacrylamide/bis, were purchased from Bethesda Research Laboratories (Gaithersburg, MD), from the American Type Culture Collection (Rockville, MD) or from New England Biolabs (Beverly, MA) . [Gamma 32P]ATP
was purchased from New England Nuclear Research Products (Boston, MA).
DNA topoisomerase II was purified from mouse leukemia L1210 cell nuclei as described, for example, by Pommier et a1. (1985), supra, and was stored at -70°C in 40%
(v/v) glycerol, 0.35 M NaCl, 5 mM MgCl2, 1 mM KHZP04, 0.2 mM dithiothreitol and 0.1 mM phenylmethanesulfonyl fluoride (pH 6.4). The purified enzyme yielded a single 170 kDa band after silver staining of SDS-polyacrylamide gels, in accordance with the description of Pommier et al., Cancer Res. 46: 3075-81 (1986).
Preparation of end-labeled DNA fragments: DNA
fragments were 5' end-labeled as described, for example, by Fesen and Pommier (1989), supra. Briefly, native DNA
was first linearized with a restriction enzyme, then the 5'-DNA was first linearized with a restriction enzyme, then the 5'-DNA termini were dephosphorylated with calf alkaline phosphatase and labeled with [gamma-3zP]ATP using T4 polynucleotide kinase. For double-strand breaks assays using HL-60 nuclear extract, SV40 DNA was digested with BclI endonuclease and labeled at both DNA termini.
For sequencing experiments, SV40 and c-myc DNAs were first 5'-end labeled at the XhoII and XbaI restriction sites, respectively. Then, in order to generate uniquely 5'-end-labeled fragments, labeled DNA was subjected to a second enzyme digestion, PfIMI for SV40, and EcoRI plus HindIII for c-myc DNA. The resulting DNA fragments were separated by agarose gel electrophoresis and isolated by electroelution. Purification by phenol-chloroform extraction and ethanol precipitation were included between each step and at the end of the labeling ~-~ ~ 760 4~V0 94/10175 -13- PCT/US93/09629 procedures, pursuant to Pommier et al., J. Molec. Biol.
222: 909-24 (1991).
Topoisomerase II-induced DNA cleavage reactions: DNA
fragments were equilibrated with or without drug in 10 mM
Tris-HC1, pH 7.5, 50 mM KC1, 5 mM MgCl2 0.1 mM EDTA, 1 mM
ATP and 15 ~cg/ml bovine serum albumin for 5 minutes before addition of purified topoisomerase II (40 to 70 ng) or HL-60 nuclear extract in 20 ~1 final reaction volume. Reactions were stopped by adding sodium dodecyl sulfate (SDS) to a final concentration of 1% and proteinase K to 400 ~.g/ml, followed by incubation for 1 hour at 40°C.
For agarose gel analysis, 3 u1 (10 X) loading buffer (0.3% bromophenol blue, 16% Ficoll, 10 mM Na2HP04) was added to each sample which was then heated at 65°C for 1 2 minutes before loading into an agarose gel made in (1X) TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH8), in accordance with Fesen & Pommier (1989), supra. Agarose gel electrophoresis was conducted overnight at 2 V/cm.
The quantification of drug-induced DNA double-strand breaks in the presence of HL-60 nuclear extract was effected as described .in the next paragraph.
Radioactive gels were counted in betascope 603 blot analyzer. For each lane radioactivity then was measured in the DNA cleavage products (C) (size between 600 5243bp), and in the total DNA present in the lane with a size superior to 600 by (T). Drug-induced cleavage was expressed as:
C/T - C./T.
Percent DNA cleaved = 100 x 1 - C./T.
where C. and T. are the counts for cleaved and total DNA, respectively, in presence of nuclear extract without drug.
For DNA sequence analysis, samples were precipitated with ethanol and resuspended in 2.5 ~,l loading buffer (80% formamide, 10 mM NaOH, 1 mM EDTA, 0.1% xylene cyanol and 0.1% bromophenol blue). Samples were heated to 90°C and immediately loaded into DNA sequencing gels (6% polyacrylamide; 19:1, acrylamide:bis) containing 7 M
urea in (1X) TBE buffer. Electrophoresis was at 2500 V (60 W) for 4 hours. Gels were dried on 3M paper sheets and autoradiographed with Kodak* XAR-2 film. See Pommier et al.
(1991), supra.
EXAMPLE 1. SYNTHESIS OF AZATOXIN AND OTHER COMPOUNDS
1H NMR spectra were taken on a General Electric QE300 Spectrometer at 300 MHz. Mass spectra were recorded on a Finnegan MAT4 615 GC/MS/DS instrument using chemical or electron impact ionization techniques. Elemental analyses were determined by Atlantic Microlab Inc. (Norcross, GA). Melting points were determined on a Thomas-Hoover UNI-MELT* apparatus and are uncorrected.
Thin-layer chromatography was performed using E. Merck glass plates pre-coated with silica gel 60 F-254 and visualized with a phosphomolybdic acidlethanol solution. Woelm silica 32-63 was employed for column chromatography which was carried out using.a modified shortlflash column technique.
Tetrahydrofuran was distilled from sodium benzophenone immediately prior to use. Dichloromethane was distilled from CaH2 immediately before use. All chemicals were purchased from Aldrich Chemical Company except for D and L-tryptophan methyl ester-HCL, which was purchased from Sigma Chemical Company.
All reactions were carried under an argon atmosphere.
48- (1H-Indol-3-ylmethyl)-2-oxazolidone (Compound 1A):
Sodium (1.56 g, 68.1 mmol) was dissolved in absolute ethanol (150 ml) and 1-trptophanol (12.94 g, 68.02 mmol) in ethanol (100 ml) and diethyl carbonate (8.83 g, 74.8 mmol) were added. The solution was heated at reflux for 5 hours and was concentrated after cooling. Saturated NH3C1 (100 ml) and CHZC12 (200 ml) were added, and after mixing well the layers were separated. The organic layer *Trademark was washed with CHZC12 (2 X 100m1) and the organic fractions were combined, dried over Na2S04, and concentrated. Recrystallization from MeOH/H20 yielded 11.66 g (79%) of a white solid: mp 155oC; 1NMR (CDCL3) 8.17(sb, 1H) 7.57(d, J=7.94 Hz, 1H), 7.40(d, J=8.06 Hz, 1H) , 7 .19 (m, 2H) , 7 . 08 (d, J=2 . 2 Hz, 1H) , 5. 22 (sb, 1H) , 4.50(m, 1H), 4.21(m, 2H), 3.04(m, 2H). [g]~°D +8.4 (c 9.4, MeOH) . Anal. (C~ZH12N202) C, H, N.
4R-(1H-Indol-3-ylmethyl)-2-osazolidone:
This compound was prepared in a manner analogous to the preparation of Compound la. [a]~'D -8.3 (c 1.25, MeOH) , Anal. (C12H,2N202) C, H, N.
General Method for the Preparation of Dimethvl Acetals To a solution of the aromatic aldehydes (1 g) in trimethyl orthoformate (7 ml) a catalytic amount of p-TSOH (40 mg) was added and the reaction was followed to completion by TLC. The solvent was removed under reduced pressure and the remaining oil was dissolved in CHC12 and filtered through a plug of silica. The solvent was again removed under reduced pressure and the remaining oil was stored in a desiccator until use.
Preparation of 5,lla-trans-aza toxins:
Method A
To a solution of Compound la (2 mmol) and the corresponding aldehyde (2 mmol) in a CHZCIz/MeOH (9:1) solution (6 ml) was added concentrated HZS04 (4 mmol).
The reaction was followed to completion by TLC (20%
acetone in CHCL3). The solution was added to sat NaHC03, the layers were separated, and the aqueous layer was washed with CHZC12 (3X). The combined organic fractions were dried over Na2S04, filtered and concentrated. The product was purified by flash chromatography (acetone-CHZC12) .

WO 94/10175 PCT/US93/096"

Method B
To a solution of the corresponding dimethyl acetal ( 2 . 2 mmol ) and Compound 1a ( 2 mol ) in CHC13 ( 8 ml ) , p-toluenesulfonic acid (.2 mmol) was added . The reaction was followed to completion by TLC. If the reaction proceeded too slowly the reaction was heated at reflux.
Saturated NaHC03 was added. The layers were separated, and the aqueous layer was washed with CH2C13 (2 x 20 ML).
The organic layers were combined, dried over Na2S04, filtered and concentrated. The product was purified by f lash chromatography ( acetone-CHZC12) .
Method C
To a solution of the corresponding dimethyl acetal (3 mmol) and Compound la ~(2 mmol) in anhydrous THF
(8 ml), anhydrous TFA (10 mmol) was added and the solution was heated at reflux. The reaction was followed to completion by TLC. After cooling, the solution was added to saturated NaHC03, the layers were separated, and the aqueous layer was washed with CHZC12 (3 X 20 ml) . The combined organic fractions were dried over Na2S04, filtered and concentrated. The product was purified by flash chromatography (acetone-CHZC12).
5R,11x8-3-One-5,4,11,i1a-tetrahydro-5-(3,5-dimethouy-4-hydro$yphenyl)-1H,6H-oxazolo[3',4':1,6]pyrido[3,4-b]indole (Compound 1) This compound was prepared as described in method C.
Purification by flash chromatography (12% acetone in CHC13, Rf=.28) produced a white solid in 91% yield: mp (decomposed slowly around 175°C) ; 1NMR (CD3CN) 7.94 (sb, 1H), 7.51(d, J-7.91 Hz, 1H), 7.30(d, J=7.57 Hz, 1H), 7.09(m, 2H), 6.59(s, 2H), 6.27(s, 1H), 5.88(d, J=1.7 Hz, 1H), 4.54(dd app t, J=8.3 Hz, 1H), 4.33(m, 1H), 4.21(dd, J=8.5 Hz, 4.7 Hz, 1H), 3.75(s, 6H), 3.16,(dd, J=15 Hz, 4.6 Hz, 1H), 2.76(ddd, J=15 Hz, 10.38 Hz, 1.73 Hz, J=1.73 Hz, 1H) , [a]ZZD=-139.6. Anal. (CZ~,HZO,Nz,05) C, H, N.

~'VO 94/10175 -17- PCT/US93/09629 58,iiaR-3-One-5,4,11,i1a-tetrahydro-5-(3,5-dimethogy-4-hydrogyphenyl)-1H,6H-oxazolo[3,4,:1,6]pyrido[3,4-b]indole This campound was prepared in a manner analogous to the preparation of Compound 1. [a]nD=+139.4. Anal.
(Cz~~Hzo~Nz.Os) O~ H~ N.
5R,11a8-3-One-5,4,ii,iia-tetrahydro-5-(3-methosy-4-hydrouyphenyl)-iH,6H-oxazolo[3',4':1.6]pyrido[3,4-b]indole (Compound 2) This compound was prepared as described in method A.
Purification by flash chromatography (15% acetone in CHC13, Rr=.30) gave a white solid in 40% yield: 'NMR
(CD3CN) 8.94(s, 1H), 7.5~1(d, J=7.59 Hz, 1H), 7.29(d, J=7.95 Hz, 1H), 7.09(m, 2H), 6.91(d, J=1.7 Hz, 1H,), 6.79(d, J=8.08 Hz, 1H), 6.74(dd, J=8.08 Hz, 1.7 Hz 1H), 6.55(sb, 1H), 5.90(d, J=1.5 Hz, 1H), 4.52(dd app t, J=7.94 Hz, 1H), 5.27(m.lH), 4.20(dd, J=8.21 Hz, 4.83 Hz, 1H), 3.78(s, 3H), 3.16(dd, J=14.93 Hz, 8.48 Hz, 1H), 3.77(ddd, J=14.91 Hz, 10.07 Hz, 1.59 Hz, 1H). Anal.
(CzoH~aN20a) C. H~ N.
SR,ilaB-3-one-5,4,ii,ila-tetrahydro-5-(4-hydrogyphenyl)-iH,6H-ogazolo[3~,4':1,6]
pyrido[3,4-b]indole (Compound 3) This compound was prepared as described in method C.
Purification by flash chromatography (20% acetone in CHC13, Rf=.28) followed by recrystallization from CH3CN
gave a white solid in 89 % yield: ~NMR (db-DMSO) 9.47 (s, 1H), 7.43(d, J=7.56 Hz, 1H), 7.24(d, J=7.91 Hz, 1H), 6.99(m, 4H), 6.70(d, J=8.6 Hz, 2H), 5.81(s, 1H), 4.49(dd app t, J=8.03 Hz, 1H), 4.14(m, 2H), 3.10(dd, J=12.14 Hz, 4.69 Hz, 1H), 2.68(dd, J=14.3 Hz, 10.6 Hz, 1H). Anal.
(Cl9.Hi6~Nz.03/CH3CN) C, H, N.
SR,ila8-3-one-5,4,i1,ila,tetrahydro-5-(3,4,5-trimethoxyphenyl)-iH,6H-ouazolo[3',4':1,6]pyrido[3,4-b]indole (Compound 4) WO 94/10175 ~ ~ ~ ~ ~ ~ -18- PCT/US93/096~~
This compound was prepared as described in method C.
Purification by flash chromatography (7% acetone in CHC13, Rf=.30) yielded a white solid in 91% yield: mp 203°C, ~NMR (CD3CN) 8.95(s, 1H), 7.51(d, J=7.64 Hz, 1H), 7 . 31 (d, J=7. 93 Hz, 1H) , 7 . 09 (m, 2H) , 6. 61 (s, 2H) , 5. 89 (s, 1H) , 4 . 57 (dd app t, J=8. 24 Hz, 1H) , 4 . 35 (m, 1H) , 4.23 (dd, J=8.42 Hz, 4.68 Hz, 1H), 3.74(s, 6H), 3.70(s, 3H), 3.17(dd, J=15.02 Hz, 4.48, Hz, 1H), 2.77(dd, J=14.85 Hz, 10.41 Hz, 1H).
Methyl-3-(1-benzenesulfonyl-indol-2-yl)-2-amino-propanoate (Compound Sa) This compound was prepared, according to the method of Schollkopf et al., Angew, Chem. Int. Ed. Engl. 18: 863 (1979), using (1-benzenesulfonyl)-2-chloromethyl-indole (Compound X) and 2,5-diethoxy-3,6-tetrahydropyrazine (Compound Y) as starting materials. 'H NMR(CDC13) 8,19(d,lH), 7,79(d, 2H), 7,55-7,18(m, 6H), 6.51(s,lH), 4,19(q, 2H), 4,00(dd, 1H), 3.55(dd, 1H), 1.18(t, 3H).
3-(1-benzenesulfonyl-indol-2-yl)-2-aminopropanol (Compound 8b) To a well-stirred solution of 0.44g (4.1 eq.) NaBH4 in 20 mL 75 % ethanol, 0. 85g (8a) in 10 mL 75% ethanol was added and the solution heated at reflux 15 hours. TLC
showed no starting material. The solution was allowed to cool and then it was diluted with 20 mL water and the ethanol was removed by rotary evaporation. The residue was extracted with ethyl acetate (4 X 20mL), dried over sodium sulfate, and concentrated to yield 0.56g (56%) of Compound 8b as a clear oil which was used without further purification. 'H NMR (CDC13) 8.18(d, 1H), 7.71(d, 2H), 7.60-7.19(m, 6H), 6.50(s, 1H), 3.70(m, 1H), 3.50(dd, 1H), 3.45(dd, 1H), 3.25(dd, 1H), 2.90(dd, 1H).
iH-Indol-2-ylmethyl-2-oxazolidone (Compound 8c) To a solution of 0.07g (1.5 eq) Na dissolved in 5 mL
absolute ethanol, 0.67g of Compound 8b in 10 mL absolute ethanol and 0.36g (1.5 eq) diethyl carbonate were added ~~~'O 94/10175 and the solution was heated at reflux overnight (16 hours). TLC showed a higher Rf spot(80:20) ethyl acetate: hexane, Rt=0.35). After cooling the ethanol was evaporated by rotary evaporation and the residue was diluted with 15 mL saturated ammonium chloride and extracted with CHZC12 (3 X 20 mL). Purification by flash chromatography using an 80:20 ethyl acetate: hexane mixture as eluent yielded 0.22g (71%) of Compound 8c as a tan solid. 'H NMR(CDC13) 8.85(s, 1H), 7.55(d, 1H), 7.30(d, 1H), 7.15(m, 2H), 6.45(s, 1H), 6.20(g, 1H), 4.30(m, 1H), 4.00(m, 2H), 2.82(d, 2H).
S,iia-traps-3-one-5,,4,11,i1a-tetrahydro-5-(3,5-dimethouy-d-hydrogyphenyl-1H, lOH-ouazolo[3',4':1,6]pyrido[4,3-b]indole (Compound 8) To a solution of 0.7g (1.5 eq) syringealdehyde dimethyl acetal and a few grains p-TSOH in 2 mL CHZC12 was added 0.04 g of Compound 8c. The solution was stirred for 2 hours. TLC showed no starting material. The solution was concentrated and purified by flash chromatography using a 15% acetone in chloroform solution as eluent to yield 0.05g( 69%) of Compound 8 as a beige solid. 'H NMR(CDCN) 9.28(bs, 1H), 7.40(d, 1H), 7.15(t, 1H), 7.00(d, 1H), 6.92(t, 1H), 6.58(s, 2H), 6.22(bs, 1H), 5.98(s, 1H), 4.47(t, 1H), 4.31(m, 1H), 4.16(q, 1h), 3.69(s, 6H), 3.11(dd, 1H), 2.91(ddd, 1H).
Raaemic methyl-3-(acenapth-1-yl)-2-aminopropanoate (Compound 7a) This compound was synthesized according to the method of Schollkopf et al., Liebigs Ann. Chem. 1987: 393-97, using acenapthylene-1-carboxaldehyde (Compound Z) and 2,5-diethoxy-3,6-tetrahydropyrazine (Compound Y) as starting materials. The final product was synthesized in an identical manner as described for Compound 8. Due to the substitution in the 4-position, however, the intermediates existed as an inseparable mixture of -2 0- PCT/US93/096"

diastereomers and 'H NMR analysis proved impossible, except for identification of important groups.
S,ila-trans-3-one-5, 4, 11, ila-tetrahydro-5-(3,5 dimethosy-4-hydroxyphenyl)-iH, 10H-ouazol [3', 4' :1,6]
pyrido[1, 2-b] acenapthylene (Compound 7) 0.01 mL BP3oEtz was added to a solution of 0.03g of Compound 7d and 0.01 mL triethyl silane in 1mL CH2C12 at -78 °C. The solution was warmed slowly to room temperature over a period of 2 hours. TLC showed no starting material. 5 mL Hz0 was added, the layers were separated, and the aqueous layers were extracted with 2 X 5 mL
portions of CHZC12, dried and concentrated. The residue was purified by flash chromatography using a 15% acetone in chloroform solution as eluent to yield 19.4 mg (70%) of Compound 7. 'H NMR(CDC13) 7.81(d, 1H), 7.71(d, 1H), 7 . 65-7 . 51 (m, 2H) , 7. 38 (t, 1H" ) , 7 .11 (d, 1H) , 6. 72 (s, 2H) , 6.14(d, 1H), 5.52(s, 1H), 4.58(t, 1H), 4.25(m, 2H), 3.77(s, 6H), 3.24(dd, 1H), 2.90(ddd, 1H).
iR,3R-1-(3-5-dimethoxy-4-hydroayphenyl)-3 methosycarbonyl-1,2,3,4-tetrahydro-~-carboline(Compound 9A) To a solution of 1-tryptophan methyl ester hydrochloride (9.52 g, 37.4 mmol) in CHC13 (150 ml) was added 14% ammonium hydroxide (30 ml). The biphasic mixture was allowed to stir for 1 hour. The layers were separated and the aqueous layer was extracted with CHC13 (2 X 100 ml). The organic layers were combined, dried over Na2S04, and concentrated to yield a yellow oil. The oil was dissolved in benzene (200 ml). Syringaldehyde (6.818, 37.4 mmol) and NaZS04 (10 g) were added, and the solution was allowed to stir for 60 hours. A white precipitate formed. The mixture was again concentrated, and anhydrous CHZCIz (150 ml) and anhydrous TFA (5.76 ml, 74.8 mmol) were added at 0°C. The solution was allowed to stir at 0°C for 12 hours. The mixture was again 2.~~'~~0~
"~O 94/10175 -21- PCT/US93/09629 concentrated, and the remaining solid was added to a biphasic mixture of saturated NaHC03 and ether. The mixture was allowed to stir for 1.5 hours and the white solid that formed was collected in a sintered glass funnel. The solid was washed with water, dried in a vacuum oven, and recrystallized from CH3CN/water to produce 13.34 g (93%) of a white solid: Anal.
(C21iH22iN2i~3) Ci Hr N.
18,38-1-(3,5-dimethoay-4-hydrosyphenyl)-3-methouycarbonyl-1,2,3,4-tetrahydro-~-carboline This compound was prepared in a manner analogous to the preparation of Compound 9A. Anal . (c21, Hue, N2, OS) C, H, N.
iR,3R-1-(3,5-dimethoxy-4-hydro8yphenyl)-3-hydrosymethyl-1,2,3,4-tetrahydro-~-carboline (Compound 98) To a solution of Compound 9a (2.01 g, 5.31 mmol) in 1:1 dioxane/water (20 ml) NaBH~ (1.00 g, 26.6 mmol) was added, and the solution was allowed to stir at room temperature for 3 hours. The solvent was removed under reduced pressure and the remaining solid was redissolved.
The product was precipitated by the addition of NaCl, and collected by filtration and dried in a vacuum dessicator to yield 1.43 g (76~) of a white solid which was used without further purification.
18,38-i-(3,5-dimethouy-4-hydroxyphenyl)-3-hydrosymethyl-1,2,3,4-tetrahydro-f~-carboline This compound was prepared and analyzed in a manner analogous to the preparation of Compound 9B.

WO 94/10175 PCT/US93/096' SR,iiaR-3-One-5,4,ii,iia-tetrahydro-5-(3,5-dimethosy-4-hydrosyphenyl)-iH,6H-osazolo[3'4':1,6']pyrido[3,4-b]indole (Compound 9) To a suspension of the amino-alcohol (1.31 g 3.70 mmol) in THF (10 ml), carbonyl diimidazole (1.79 g, 11.1 mmol) was added and the suspension was allowed to stir for 5 hours. The suspension was concentrated and 10%
NaOH was added. After stirring for an additional 3 hours, the solution was carefully acidified to pH 6 by the addition of concentrated HC1 and the resulting mixture was extracted with EtOAc (3 X 50 ml), dried over Na2S04 and concentrated. ~NMR (CDC13) 7.58 (s, 1H) , 7. 51 (d, J=8. 37 Hz, 1H) , 7.19 (m, 3H) , 6. 57 (s, 2H) , 5. 52 (s, 1H) , 5.24 (s, 1H) , 4. 63 (t, J=6. 65 Hz, 1H) , 4.22 (m, 2H) , 3.83 (s, OH), 3.22(m, 1H), 2.92(ddd, J=16.4 Hz, J=10.1 Hz, J=1.8 Hz, 1H) . Anal (C21, HZO, N2, OS) C, H, N.
58,i1a8-3-One-5,4,ii,ila-tetrahydro-5-(3,5-dimethouy-4-hydroxyphenyl)-iH, GH-oxazolo[3'4':1,6]pyrido[3,4-b]indole (Compound i0) This compound was prepared in a manner analogous to the preparation of Compound 9 . Anal . (CZ" H2o, NZ, OS) C, H, N.
SR,iiR,iiaB-3-One-ii-methosy-5,4,ii,iia-tetrahydro-5 (3,5-dimethoxy-4-hydrogyphenyl)-iH,6H
oaazolo[3'4':1,6]pyrido[3,4-b]indole (Compound 1C) To a solution of syringaldehyde dimethyl acetal (.50 g) and catalytic p-toluenesulfonic acid in anhydrous CHzCl2/MeOH 9 : 1 ( 8 ml ) , Compound 1B was added at 0 ° C in small portions. After stirring for 4 hours the precipitate that formed was collected by filtering the reaction mixture through a sintered glass funnel, and was dried in a vacuum desiccator to yield the pure product in 47% yield. ~NMR (db-DMSO) 8.42(sb, 1H), 7.65(d, 1H), 7.31(d, 1H), 7.06(m, 2H), 6.48(s, 2H), 5.85(s, 1H), ~'O 94/10175 ~ ~ ~ ~ ~ -23- PCT/US93/09629 4.63(d, J=1.8 Hz, 1H), 4.43(m, 3H), 3.64(s, 6H), 3.28(s, 3H).
SR,iiR,ilaB-3-One-ii-hydrosy-5,4,ii,iia-tetrahydro-5 (3,5-dimethouy-4-hydrosyphenyl)-iH,6H
Oaazolo[3'4:1,6]pyrido[3,4-b]indole (Compound 6) To a suspension of Compound Ic (1 mmol) in water (8 ml ) KIOH ( 2 mmol ) was added, and the solution was brought to reflux and immediately cooled. The solution was then acidified to ph 7 with 10% HC1, and the precipitate that formed was collected by filtration in a sintered glass funnel and dried in a vacuum dessicator.
5R,liR,ilaB-3-One-ii-methoxy-5,4,ii,iia-tetrahydro-5-(3,5-dimethosy-4-hydrouyphenyl)-iH,6H-oxazolo[3'4~:1,6]pyrido[3,4-b]indole (Compound 1C) To a solution of syringaldehyde dimethyl acetal (.50 g) and catalytic p-toluenesulfonic acid in anhydrous CHZC12/MeOH 9 :1 ( 8 ml ) , Compound 1B was added at 0 ° C in small portions. After stirring for 4 hours the precipitate that formed was collected by filtering the reaction mixture through a sintered glass funnel, and was dried in a vacuum desiccator to yield the pure product in 47% yield. ~NMR (db-DMSO) 8.42(sb, 1H), 7.65(d, 1H), 7.31(d, 1H), 7.06(m, 2H), 6.48(s, 2H), 5.85(s, 1H), 4.63(d, J=1.8 Hz, 1H), 4.43(m, 3H), 3.64(s, 6H), 3.28(s, 3H).
SR,iiR,iiaB-3-One-1i-methouy-5,4,ii,ila-tetrahydro-5-(3,5-dimethouy-4-O-carbomethoxyphenyl)-iH,6H-osazolo[3',4':1,6]pyrido[3,4-b]indole (Compound 1D) Compound 1C (1.96g, 4.77 mmol) was suspended in 10 ml of CH2C12 and 6.7 ml (47.7mmo1) TEA. In the suspension, 3.69 ml (47.7 mmol) of methyl chloroformate was added dropwise at 0 °C, the solution was diluted with CHZC12 and washed with water. The organic fraction was dried over Na2S04, filtered and concentrated.
Purification by flash chromatography eluting with 8%

PCT/US93/0962' acetone in CHC13 yielded 2.09 g (94%) of a white solid.
An analytical sample was obtained by recrystallization from ethyl acetate.
5R,11R,liaS-3-One-11-hydrouy-5,4,ii,ila-tetrahydro-5 (3,5-dimethouy-4-O-carbomethouyphenyl)-iH,6H
ouazolo[3',4':1,6]pyrido[3,4-b]indole (Compound 1E) To a solution of 1.88 g (7.00 mmol) of compound 1D
in a 9:1 dioxane/water solution (30 ml), 130 mg (0.70 mmol) of paratoluenesulfonic acid (p-TSOH) was added.
The solution was followed to completeion by TLC and diluted with CHC13. The resulting mixture was washed with saturated NaHC03, dried over NaZS04, filtered and concentrated. Purification by flash chromatography eluting with 20% acetone in CHC13 yielded 1.21 g (66%) of a white solid.
5R,11R,i1a8-3-One-11-(4-fluoroanilino)-5,4,ii,ila-tetrahydro-5-(3,5-dimetho$y-4-O-carbomethosy)-iH,6H-osazolo[3',4':1,6]pyrido[3,4-b]indole (Compound 1F) To a solution of 0.33 g (0.73 mmol) of Compound 1E
in anhydrous dioxane (4 ml) , 350 ~1 (2.5 mmol) of TEA and 160 ~1 (2.1 mmol) of acetyl chloride were added. After stirring for 15 minutes, the solvent and unreacted acetyl chloride were removed under reduced pressure. The reaction vessel was recharged with dioxane, (diethylene dioxide DDO), 1.4 mmol of 4-fluoro aniline and 1.4 mmol TEA. The reaction mixture then was heated at 50°C and stirred for 6 hours. To this solution, CHC13 was added and the resulting solution was washed with water, dried over Na2S04, and concentrated. Purification by flash chromatography eluting with 10% acetone in CHC13 yielded 62 mg (16%) of a white solid.
5R,ilR,liaB-3-One-li-(4-fluoroanilino)-5,4,ii,ila-tetrahydro-5-(3,5-dimethosy-4-hydrouyphenyl)-1H,6H-oxazolo[3',4':1,6]pyrido[3,4-b]indole (Compound 11) 214'~~~&
''O 94/10175 PC'T/US93/09629 To a solution of sodium methoxide (90 ~mol) in 1 ml methanol, 16.5 mg (30 ~,mol) of Compound 1F was added and the reaction was followed to completion by TLC.
Saturated NH4C1 (200 ~1) was added followed by addition of CHZC12. The layers were separated and the organic layer was dried over Na2S04, filtered and concentrated.
Purification by flash chromatography eluting with 20%
acetone in CHC13 yielded 10.8 mg (73%) of a white solid.
Representative syntheses for the aforementioned compounds are depicted below:
Compound 1A:
C02Me H
~NH3C1 \ \ NaBH4 -N~ EtOH/H20 H H
(known (Et0)2C=0 EtONa, EtOH

~~ 0 \ H
N
H
( 1A~

WO 94/10175 2, ~ ~~ ~ ~ ~ PCT/US93/0962'' Compounds 1-5:
~o ArCH[OMe~2 CH2C12, p-TSOH
H Ar H

C 1A]
Wherein Ar can be Y, as defined above.
Compound 6:
O Ac0 O

H
DDD ~ ~ ~ H ARCH[OAAe)2 / H HOAC/THF / N CH2C12, AIEOH
H -79'C H p-TSOH
C 1A) ( 18]
OH
04a KOH
H
H ~ H2p, a - 0 /
/ Na OMe AIeO OMe OH
OH
(6) C ~C) °'~O 94/10175 PCT/US93/09629 Compounds X, Y and Z have the following structures:
OEt CHO
CI
\ \N I
I ~ N~--~
SOzPh OEt CX~ ~Y~ CZ~

WO 94/10175 PCT/US93/096:' Compound 7:
OMEAI
OMEM
COZEt NaBH ~ ~ OH CO~H30)ZC=0 C H ONe 75% EtOH ' ~ ~ J NH2 2 5 NH2 4 a [ 7n] C 7b) OAIEIA OIAe O
H P'TSOH I ~ ~0 Syringaldehyde 0 dlmethyl acetal O
[7c) 1A6 / OMe OH
[ 7d) OMe ~0 Et3St H
BF3'OEtz /
Me0 ~ home /
Me0 OMe OH
OH
7d) 'V0 94/ I 0175 ~ _ _2 9 _ PCT/US93/09629 Compound 8:
SOzOH S020H
\ Na BH
~ N EtONe/EtOH
/ ~ C02Ne EtOH/H 0 ~ / ~ [Et0]zC=0 ( BA] [ 88) Mo0 OMe H
N
HN ~e0 home 0 OH Me p p-TSOH, CHZC~
(BC]
(8]
Compound 9 cozue ~NH3C 1 ~Me \ ~ 1) NH3, CHC1~
/ N 2]SyrlnOe aldehyde, NatSO~
H 3) TFA
[9A]
NeBH~ Carbonyl dllmldazole dfoXene/ THF
H?0 (98) [8) WO 94/10175 'Z ~ ~ _3 ~_ PCT/US93/096' Compound 11:

0 ~O
H DDO ~ \ ~ H A~CH(OMe]2 / N HOAc/THF / CHpCl2, AIeOH
H -78'C H p-TSOH
[1A) (1B]
ONe OMe .O

CICOOMe H ~ p-TSOH
H ~ O~C 0 CHC13 0 \
/ , Me OMe Me0 OM8 OCOZMe OH
( 1 D]
(1C]

'O 94/10175 ~ ~ ~ ~ -31-F
OH NH
0 TEA, CH3COC1 ~N ~0 DDO, C HqFNH H
6 2 p \ ~ \
/ /
Me0 OMe Me0 OMe OCOZMe OC02Me (1E] [1F]
F
NH
D

i Me0 OMe OH
[ 11]
EXAMPLE 2. INDOCTION OF DNA DOOBLE-STRAND BREARS BY
AZATO%IN IN THE PRESENCE OF HL-60 NOCLEAR E%TRACT
Drug-induced DNA double-strand breaks were measured first in SV40 DNA in the presence of HL-60 nuclear extracts. SV40 DNA was chosen because it is a natural substrate of top 2 and is cleaved at many sites by other WO 94/10175 ~ ~, ~ ~ ~ ~ -32- PCT/US93/096' top 2 inhibitors. See, for example, Fesen & Pommier (1991), supra. The smallest azatoxin concentration that produced detectable cleavage was 5 to 10 ~M. Above 10 ~M, cleavage occurred at many sites and was proportional to the logarithm of azatoxin concentration. The potency of azatoxin was comparable to that of VP-16 and, as in the case of VP-16, azatoxin-induced DNA cleavage was not suppressed at high drug concentrations (up to 1 mM), consistent with azatoxin's not intercalating into DNA
(see below).
EXAMPLE 3. SEQUENCING OF TOPOISOMERASE II CLEAVAGE SITES

Induction of top 2 cleavage by azatoxin was tested directly with the use of purified murine leukemia top 2.
Since the SV40 nuclear matrix-associated region has been shown to be cleaved preferentially by top 2, see Pommier et a1. (1991), supra, this region was chosen for analysis. Sites of cleavage were also determined by DNA
sequencing in the 5' flank of c-myc first intron.
Azatoxin induced many cleavage sites both in the SV40 and the c-myc DNA fragments. In general, azatoxin induced more cleavage sites than mitoxantrone, m-AMSA, VM-26 or VP-16. Thus, azatoxin was shown to be a potent top 2 inhibitor, with a cleavage pattern differing from those induced by other top 2 inhibitors.
The cleavage pattern of azatoxin also was compared to that of epipodophyllotoxin derivatives the structures of which seem quite similar (for drug structures and abbreviations, see table below). The compound 4'-demethyl-4-desoxypodophyllotoxin (DMDP), with a structure most similar to azatoxin, induced less cleavage that azatoxin and at distinct cleavages sites. The ~-4-hydroxy derivative of DMDP, 4'-demethylepipodophyllotoxin (DMEP) , was at least as potent as VP-16, and its cleavage patterns, while similar to that of VP-16 with some local differences, was different from that of azatoxin.

i !i EXAMPLE 4. EFFECTS ON TOPOISOMERASE ACTIVITY
Two different assays were conducted to illuminate the nature of azatoxin's effects vis-a-vis DNA relaxation. To study the inhibition of top 2 catalytic activity, topoisomerase reactions were performed with 0.4 ~,g native SV40 DNA in 30 w1 reaction buffer for 30 minutes at 37°C and stopped by adding SDS to a final concentration of 1%
and proteinase K to 400 ~g/ml, followed by incubation for 1 hour at 42°C, essentially as described by Fesen & Pommier (1989), supra. Agarose gel electrophoresis vas performed in 1 % gels made in Tris-Acetate-EDTA (TAE) buffer (40 mN Tris-Acetate, pH 7.6, 10 mM NaZEDTA). Gels were run at 2 V/cm overnight, washed in water and then stained with 1 ~,M ethidium bromide for 45 minutes. After an additional 45 minutes destaining in 1 mM MgZS04 the DNA was visualized under UV light and photographed with a Polaroid* type 57 film.
To assess DNA unwinding, see Pommier et al., Nucleic Acids Res. 15: 6713-31 (1987), the DNA was relaxed first by treatment for 15 minutes with top 1 (20 units), after which the test agent was added. These steps were carried out at 37°C.
DNA-agent-top 1 mixtures were incubated for an additional 30 minutes and then stopped as described above. Samples were then subjected to agarose gel electrophoresis as described above.
From the assay data it was determined that azatoxin inhibits top 2-mediated relaxation of native SV40 DNA. At the same time, azatoxin was observed to produce a substantial amount of linear DNA without significant increased of nicked DNA.
The DNA unwinding assay, with excess topoisomerase I and relaxed SV40 DNA, was employed to assess azatoxin intercalation in accordance with Pommier et al., Nucleic Acids Res. 15: 6713-31 (1987). In fact, azatoxin did not induce detectable DNA
unwinding even at drug concentrations as high as 1 mM. This was also the case for the 2 azatoxin isomers, 8 and 10, and for the *Trademark WO 94/10175 ~ ~ ~ ~ ~ ~ -34- PCT/US93/096,"
demethylepipodophyllotoxins, DMDP and DMEP. Similar results were obtained with supercoiled DNA. The lack of unwinding by azatoxin strongly indicated that the drug does not intercalate into DNA.
EXAMPLE 5. EFFECTS OF AZATO$IN BTRDCTORAL MODIFICATIONS
ON TOPOISOMERASE II INHIBITION
Three isomers and six azatoxin derivatives, the synthesis of which is described in Example 1, were tested for drug-induced cleavage efficiency in the presence either of HL-60 extract or of purified top 2. The compounds and test results are set out in the table below.

'O 94/10175 ~ ~ ~~ _35_ PCT/US93/09629 R~
1'1a \ 1~a \ \
1= I T 0 2=_ I 0 s ~ / s Y Y

~~n a= / \ I s ~0 r~.~~~~0 Y
Y= I \
R~ R3 '1 1 n , 1=_ ~ \ I ~ .0 s Y

~~ .'~ Gag WO 94/10175 PCT/US93/096.

r s~~n~mia~y Topoi.oounx a i hibi i on n t COMPOUND STRUCTURER1 ~ ~ R4 5 lla AZATOXINS

Autooin I OCH3 OH OCH3 H R S +++

6 1 OCH3 OH OCH3 OH R S +

1p 1 OCH3 OH OCH3 H S S O

EPIpODOPHYLLOTOXINS

DMDP 2 OCH3 OH OCH3 H R S +

DMEP 2 OCH3 OH OCH3 OH R S +++

VP-16 2 OCH3 OH OCH3 a R S +++

VM-26 2 OCH3 OH OCH3 6 R S ++++

a t: H b H H

OH OH
Azato$in Isomers The three azatoxin isomers (compounds 8_-10 in the table) were found not to be active as top 2 inhibitors in DNA cleavage assays. The finding that the two diastereoisomers (9 and 10) were inactive demonstrated that a strict stereochemical relationship between the polycyclic ring system and the pendant aromatic ring must exist for activity. The inactivity of isoazatoxin (8_) was surprising and indicated the great sensitivity of the binding site for these agents to minor structural modification. Thus, azatoxin and isoazatoxin (8_) differ only in the orientation of the tetrahydrooxazolopyrido ring fusion into the indole ring; this change in .1~V0 94/10175 -3,~- PCT/US93/09629 orientation imparts (1) only a subtle differential "curve" to the tetracyclic nucleus of the molecule, without altering the spatial relationship between the indole and phenyl ring systems, and (2) a change in orientation of the nitrogen indole.
Azatouin Derivatives The results from testing the six azatoxin derivatives for top 2 inhibition also are set out in table. Two of derivatives were modified on the polycyclic ring system and the others were modified on the pendant ring.
Hydroxylation at position 11 (R4) of the azatoxin polycyclic ring system yielded a compound (6) that was structurally similar to DMEP and that displayed measurable but not strong t,op 2 activity. This 4-hydroxy substitution differentiates azatoxin from the 4'-demethylpodophyllotoxin framework, since hydroxyl substitution at this site significantly decreases top 2 activity of azatoxin (_1 versus 6) and increases activity of the podophyllotoxins (DMDP, DMEP). Differing from azatoxin by its polycyclic ring system, Compound 7 is inactive, again indicating that the structure of the polycyclic ring system is critical for azatoxin activity.
Modification to the pendant (Y) ring gave the following results. Monodemethoxylation (2_) reduced top 2 activity by a factor 10, while didemethoxylation (3) abolished the top 2 activity. Position 4' (R2) was also crucial as methylation of the hydroxyl residue (4_) abolished top 2 inhibition. These results are not inconsistent with those obtained for demethyl-epipodophyllotoxins. See, for example, Sinha et al., Eur. J. Cancer 26: 590-93 (1990). Notably, compound 5_, in which the azatoxin pendant ring had been placed by that of AMSA was inactive. These results differentiate the azatoxin family of top 2 inhibitors from the m-AMSA
family of inhibitors.

Claims (14)

The embodiments of the invention, in which an exclusive property or privilege is claimed, are defined as follows:

1. A compound that is represented by one of the following formulae (B)-(D):
(D) wherein (i) X denotes NH, S or O;
(ii) R5 denotes COOCH3, COCH3 or, COCH2OH;
(iii) R6 denotes F, Cl, Br, CN, OH, NH2 or H;
(iv) R7 denotes OH, the formula wherein R denotes H, OH, F, Br, Cl, NO2, NH2, CN, OCH3 or CO2CH2CH3;
or R7 denotes the formula -T-(CH2)n Z
wherein T denotes NH or O;
Z denotes NH2, OH, N(CH3)2, or N(CH2CH2Cl)2;
n is 2, 3 or 4; and R7 may be derivatized with a 4,6 o-protected sugar;
(v) R8 denotes H or OH; and (vi) W and W' are the same or different and denote, H or F, provided that if R6 is H, then R7 is selected from wherein R denotes H, OH, F, Br, Cl, NO2, NH2, CN, OCH3 or CO2CH2CH3 and -T-(CH2)n Z

wherein T denotes NH or O;
Z denotes NH2, OH, N(CH3)2, or N(CH2CH2Cl)2;
n is 2, 3 or 4.

2. A compound according to claim 1, wherein said compound is represented by formula (B) in which W', W, X, R5 and R6 are as defined in claim 1.

3. A compound according to claim 2, wherein R6 denotes F.

4. A compound according to claim 2, wherein R6 denotes Br.

5. A compound according to claim 1, wherein said compound is represented by formula (C) in which W', W, R6 and R7 are as defined in claim 1.

6. A compound according to claim 5, wherein R6 is H.

7. A compound according to claim 5, wherein R7 is and wherein R denotes F.

8. A compound according to claim 5, wherein R6 is H, and R7 is NH(CH2)2N(CH3)2.

9. A compound according to claim 1, wherein said compound is represented by formula (D) in which W', W, X, R6, R7 and R8 are as defined in claim 1.

10. A compound according to claim 9, wherein R6 is H.

11. A compound according to claim 10, wherein R7 is wherein R denotes F.

12. A pharmaceutical composition comprising a tumor-affecting amount of a compound according to claim 1 and a physiologically compatible carrier therefor.

13. A pharmaceutical composition according to claim 12, wherein said composition is an injectable or infusible preparation.

14. Use of a compound as claimed in claim 1, for treating cancer in a mammal.