AU640135B2 - Hetero polycyclic biocidal compounds and their use in medicine - Google Patents

Description

OPI DATE 21/10/91 AnJP DATE 21/11/91.

APPLN. ID 53552

PCT

PCT NUMBER PCT/GB90/00424 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 C07D 491/044, 495/04, 493/04 C07D 487/04, A61K 31/40 A61K 31/38, 31/34 (CO7D 491/044, 307/00, 209/00) Al (C07D 495/04, 333/00, 209/00) (C07D 493/04, 307/00, 307/00) (C07D 487/04, 209/00, 209/00) (C07D 495/04. 333/00. 209/00) (1I) International Publication Number: WO 91/14688 640135 (43) International Publication Date: 3 October 1991 (03.10.91) (21) International Application Number: PCT/GB90/00424 (22) International Filing Date: 20 March 1990 (20.03.90) (71)Applicant: THE WELLCOME FOUNDATION LIMIT- ED [GB/GB]; 160 Euston Road, London NWI 2BP

(GB).

(72)Inventor: BAIR, Kenneth, Walter 342 Wesley Drive, Chapel Hill, NC 27514 (US).

(74) Agent: ROLLINS, The Wellcome Foundation Limited, Langley Court, Beckenham, Kent BR3 3BS (GB).

(81) Designated States: AU, FI, HU, JP, KR, MC, NO, SU.

Published With international search report.

(54) Title: HETERO POLYCYCLIC BIOCIDAL COMPOUNDS AND THEIR USE IN MEDECINE

R

6 1 R 10 c- R 7 I l 2 I 3 R_4 (6) 11 14 (57) Abstract Heterotetracyclic aromatic compounds having biocidal activity containing two hetero atoms selected from oxygen, sulphur and nitrogen and that are substituted by an aminoalkanol group of the formula ArCH 2 NHR or a monomethyl or monoethyl ether thereof, the compound of formula including these ethers containing no more than 29 carbon atoms in total, or an ester or a salt thereof; wherein Ar is a fused tetracyclic hetero aromatic ring system of formula in which Z is oxygen, sul.

phur or a group NRI wherein R I is hydrogen, methyl or ethyl and is a bicyclic aromatic ring system comprising a phenyl ring and a 5-membered ring system which contains one heteroatom ZI selected from oxygen, sulphur or a group NR 2 wherein R2 is hydrogen, methyl or ethyl; the tetracyclic ring system being optionally substituted by one or two substituents; and R contains not more than eight carbon atoms and is a group or wherein m is 0 or 1; R 6 is hydrogen or alkyl optionally substituted by hydroxy; R" and R 8 are the same or different and each is hydrogen or alkyl; R 9 is hydrogen, methyl or hydroxymethyl; is a five- or six-membeed saturated carbocyclic ring; Rlo, Rll and RI 2 are the same or different and each is hydrogen or methyl; Ris is hydrogen, methyl, hydroxy, or hydroxymethyl; R 14 is hydrogen, methyl, hydroxy or hydroxymethyl. Processes for preparing the compounds, intermediates in their preparation, pharmaceutical compositions containing them and their use in medicine are also disclosed.

WO 91/14688 PC/GB9/00424 1 Hetero polycyclic biocidal compounds and their use in medicine The present invention relates to heteropolycyclic aromatic alkanol derivatives which have been found to have biocidal activity.. More specifically the invention concerns aminoalkanol derivatives containing a heteropolycyclic aromatic ring system, methods for the synthesis thereof, novel intermediates thereof, pharmaceutical formulations thereof and the use thereof as biocidal agents, particularly antitumor agents.

We have discovered a novel class of heteropolycyclic aromatic alkanol derivatives which have biocidal activity. Accordingly, in a first aspect, the present invention provides a compound of the formula 4I) ArCH2NHR (I) or a monomethyl or monoethyl ether thereof, the compound of formula including these ethers containing no more than 29 carbon atoms in total, or an ester or a salt thereof; wherein Ar is a fused tetracyclic hetero aromatic ring system of the formula: in which Z is oxygen, sulphur or a group NR wherein R is hydrogen, methyl or ethyl and rv y is a bicyclic aromatic ring system PCT/GB 9 0 00424 13 05 92 13 May 19 PB1125 comprising a phenyl ring and a 5-membered ring system which contains one heteroatom Z 1 selected from oxygen, sulphur or a group NR 2 wherein

R

2 is hydrogen, methyl or ethyl; the tetracyclic ring system being optionally substituted by one or two substituents; said substituents containing not more than four carbon atoms in total when taken together and are the same or different each being selected from halogen; cyano; C 1 4 alkyl or C 1 4 alkoxy, each optionally substituted by hydroxy or C12 alkoxy; halogen substituted C12 alkyl or C12 3 3 alkoxy; a group S(O) R wherein n is an integer 0,1 or 2 and R is C-2 alkyl optionally substituted by hydroxy or C alkoxy; or Ar is optionally substituted by a group NR R 5 containing not more than carbon atoms wherein R 4 and R 5 are the same or different and each is a 45

C

3 alkyl group or NR4 R forms a five-or six-membered heterocyclic ring optionally containing one or two additional heteroatoms; R contains not more than eight carbon atoms and is a group 6 R C R I R 12 C R C (CH2) m 11 2 m or 14 R- C OH

OH

wherein m is 0 or 1;

R

6 is hydrogen or C13 alkyl optionally substituted by hydroxy; 7 8

R

7 is hydrogen, C1-3 alkyl or CH2OH and R 8 is hydrogen or C 3 alkyl;

R

9 is hydrogen, methyl or hydroxymethyl; is a five-or six-membered saturated carbocyclic ring; R and R1 are the same or different and each is hydrogen or methyl; PWC/JM/llth May 1992 r-i Uh cc. o t O.TaffSe SUBSTITUTE SHEET PCT Atrication WO 91/14688 PCT/GB90/00424 3

R

13 is hydrogen, methyl, hydroxy, or hydroxymethyl; R 14 is hydrogen, methyl, hydroxy or hydroxymethyl.

Suitably the tetracyclic aromatic ring system Ar is unsubstituted or has only one substituent. Preferably the aromatic ring is unsubstituted.

Suitably not more Suitably Suitably ArCH2NHR or a monomethyl or monoethyl ether thereof contains than 28 carbon atoms in total.

m is 0.

R is O 16 C R 1 6 H R 17 H C R or

OH

wherein R 1 5 is CH2OH, CH(CH 3 )OH or CH2CH20H, 16 R is hydrogen, C1.

3 alkyl or CH2OH, 17 R is hydrogen or methyl.

Suitably R 15 is CH20H or CH(CH3)OH.

methyl, ethyl or CH 2

OH.

Preferably R is Suitably R 17 is hydrogen, CH OH H R wherein R 17 is hydrogen or methyl and R 18 is hydrogen, methyl or ethyl, preferably methyl.

WO 91/14688 WO 9114688PCT/G B90/00424 -4- Tetracyclic heteroaromatic ring systems of compounds of the present invention have the following nomenclature: Z Z1 NMe, 10-methyl-l1ll- [l)benzothieno[3,2-k]) indol-6-yl 2 Z Z1 NMe, Q lO-Methyl-l1ll-[l)benzothieno[3,2-bI z3 indol-3-yl 51 Z 0, Z -NMe, 2- (10-methyl- l01-benzofuro- 2-b) indol-6-yl Z 0, z1- 0, 2- (benzofuro(5 ,6-lz)benzofuran-4-yl, Z 0, Z1- 0 2- (benzofuro(5 ,6-bk)benzofuran-8-yl-, z 0, 2- (benzofuro(5 ,6-bk)benzofuran-2-yl-, 2 7.1 7 6 5 z 0, z 1 0, 2- (benzofuro(5 ,4-h)benzofuran-2-y1-, Z 0, Z- 0 2- (benzofuro(5 ,4-D)benzofuran-9-yl-, z 0, zI- 0, 2- (benzofuro(5 ,4-ka)benzofuran-4-yl, Z 0, Z1- 0, 2- (benitofuro(5,4-bh)benzofuran-8-yl., WC 91/14688 WO 9114688PCT/GB9O/J0424 Z S, Z- NMe, 1-Methyl-iji- t]benzothieno[2,3-g] indol-3-yl- 9. Z 0, Z1 N'e, I -Methyl-lH-benzofuro[2,3-g] indol- 9-l 0 0 8 z- 41 7 6 5 Z NEt, Z -NMe, 6-Etyl-16-dihydro-1-methylpyrrolo (3 ,2-R]carbazol-3-yl.

Z S, ZI NMe, 3-Methyl-3lj- [1)benzothienoj2,3-e) indol-1-yl- Z NMe, 9. S 7 4 0 1 lOMethyl-10i-thieno[3,2-a]carbazol- 8 Z 2yl 10-lMethyl-i1iI-thieno[3,2-p]carbazol- 4 .yl- Specific compounds within the scope of formula include: 2 (((10-Methyl-10j- i~benzothieno[( 3, 2-h] indol- 3-yl)methyl) -amino) -2 methyl.1, 3-propanediol, 2-(((10-Hethyl-10li[1]benzothieno[3,2-k]indol-6-y)methyl)amino)-2methyl-i, 3-propanediol, 2-Methyl-2-(((10-methyl-l1jj-benzofuro(3,2-k)indol-6-yl)methyl)amino)- 1,3 -propanediol, WO 91/14688 PCT/GB90/00424 -6- 2-(((Benzofuro(5,6-b)benzofuran-4-yl)methylamino)-2-methyl-1,3-propanediol, 2-(((Benzofuro(5,6-k)benzofuran-8-yl)methyl)amino)-2-methyl-1,3propanediol, 2-(((Benzofuro(5,6-k)benzofuran-2-yl)methyl)amino)-2-methyl-1,3-propanediol, 2-(((Benzofuro(5,4-b)benzofuran-4-yl)methyl)amino)-2-methyl-1,3-propanediol, 2-(((Benzofuro(5,4-k)benzofuran-9-yl)methyl)amino)-2-methyl-1,3-propanediol, 2-(((Benzofuro(5,4-b)benzofuran-8-yl)methyl)amino)-2-methyl-1,3-propanediol, 2-(((Benzofuro(5,4-b)benzofuran-2-yl)methy)amino-2-methyl-1,3-propanediol, and monomethyl or monethyl ethers, esters, and addition salts thereof.

Salts included within the scope of the present invention are those of compounds of formula and ethers and esters thereof.

Esters and non-pharmaceutically useful salts of the compounds of the formula are useful intermediates in the preparation and purification of compounds of the formula and pharmaceutically useful salts thereof, and are therefore within the scope of the present invention. Thus, salts of the compounds of the formula (I) useful in the present invention include but are not limited to those derived from inorganic acids, such as hydrochloric, hydrobromic, sulfuric and phosphoric acids, and organic acids such as isethionic (2-hydroxyethylsulfonic), maleic, malonic, succinic, salicylic, tartaric, lactic, citric, formic, lactobionic, pantothenic, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, naphthalene-2-sulfonic, and ascorbic acids, and amino acids such as glycine.

Pharmacologically and pharmaceutically acceptable salts are preferred, particu'arly those that are soluble in solvents suitable for parenteral administration, for ixample, hydrochloridec, mqthanesul- WO 91/14688 PCT/GB90/00424 7 fonates and isethionates.

Esters of compounds of formula are derived from acids known to those skilled in the art to be suitable for ester formation, and are conveniently those derived from 01-6 alkanoic acids or alkanoic acid derivatives, for example acetic acid, propionic acid, n-butyric acid and iso-butyric acid. The esters may be formed from all or only some of the hydroxy groups contained in the compounds of formula The compounds of formula and their ethers, esters, and salts thereof may be prepared by any method known in the art for the preparation of compounds of analogous structure. Thus, the compounds of formula may, for example, be prepared by any of the methods defined below.

1. The reduction of a compound Ar-CH-NR (II) wherein Ar and R are as hereinbefore defined or an appropriately protected derivative thereof followed by deprotection where appropriate.

The conditions and reagents for such a reaction are well known to those skilled in the art, and any such conditions/reagents may be employed that will not reduce the aromatic ring system. The conversion of (II) or suitably protected derivatives thereof may be carried out by a reducing agent followed by deprotection if necessary.

The reduction is conveniently carried out by a metal hydride such as lithium aluminum hydride, sodium borohydride, sodium cyanoborohydride, or by catalytic hydrogenation, conveniently by hydrogen in the presence of a metal catalyst such as palladium or platinum, or equivalent reagerts as outlined by J, March, Advanced Organie Chemistry, 2nd ed., pages 819-820, McGraw Hill, New York, 1977. The reduction is suitably carried out with Ar-CH-NR in solution in an inert solvent or mixture of solvents compatible with the reducing WO 91/14688 PCT/GB90/00424 8 agent, at a non-extreme temperature, for example, between 0° and 80 0

C,

conveniently at room temperature.

In the case of lithium aluminum hydride and like reagents, suitable solvents include ethers (for example tetrahydrofuran, diethyl ether and 1,2-dimethoxyethane) optionally in the presence of a hydrocarbon cosolvent (for example toluene, benzene or hexane).

In the case of sodium borohydride and like reagents, suitable solvents include alcohols (for example ethanol, methanol or isopropanol) optionally in the presence of a hydrocarbon cosolvent (for example toluene, benzene or hexane) or an ether cosolvent (for example diethylether or tetrahydrofuran).

In the case of sodium cyanoborohydride and like reagents, suitable solvents include those described for sodium borohydride and the reduction is conveniently carried out in the presence of an acid, conveniently glacial acstic acid or ethanolic hydrochloric acid as outlined in, for example, R. Hutzhins et al., Organic Preparations and Procedures International 11, 201 (1979).

In the case of catalytic hydrogenation, suitable solvents include alcohols (for example methanol and ethanol) optionally in the presence of a hydrocarbon cosolvent (for example toluene or benzene), or ether cosolvent (for example diethyl ether or tetrahydrofuran) optionally in the presence of an acid (for example glacial acetic acid or ethanolic hydrochloric acid), or glacial acetic acid.

Protected derivatives of compounds ArCH-NR are conveniently used when lithium aluminum hydride is employed as the reducing agent.

Convenient protecting groups are compatible with the reducing agent utilized and are readily removed under nondestructive conditions, for example benzyl, tetrahydropyranyl and isopropylidene ethers.

WO 91/14688 PCT/GB90/00424 9 It is often convenient not to isolate the compound ArCH-NR but to react a compound ArCHO with a compound NH2R; wherein Ar and R are as defined in and to reduce the compound ArCH-NR so formed in situ.

The reaction of the compounds ArCHO and NH 2 R is again suitably carried out using conditions and reagents which are well known to those skilled in the art, for example in the presence of an acid, such as a sulfonic acid, i.e. E-toluenesulfonic acid, in an appropriate inert solvent, such as an aromatic hydrocarbon, suitably toluene, with azeotropic removal of water followed by treatment with the reducing agent in an appropriate solvent, suitably ethanol or methanol.

Alternatively, ArCH-NR formed under equilibrium conditions in appropriate solvents can be reduced in situ with an appropriate reducing agent, suitably sodium cyanoborohydride.

The compound ArCHO may be in the form of a protected aldehyde, for example an acetal, which liberates the aldehyde functionality under the reaction conditions. In turn, ArCHO can be synthesized by reacting the appropriate aromatic polyheterocycle optionally substituted with a carbalkoxy group with a formylating agent such as that generated by the reaction between SnCI 4 and C12CHOCH 3 or equivalent reagents, for example, according to the method of A. Reiche et al., Che. Ber, 93, 88 (1960), or with other standard formylating reagents/procedures known to the art, for example, the Gatterman-Koch reaction (CO/HC1/AlCl3/CuCl), the Gatterman reaction (HCN/HC1/ZnCl 2 and the Vilsmeier reaction (POCl 3 /PhN(Me)CHO or POC1 3 /Me 2 NCHO) March, vide supra pages 494-497).

The compounds ArCHO may also be prepared from an appropriate aromatic heteropolycycle substituted by a suitable functional group and converting this functional group to an aldehyde group by methods well known to those skilled in the art. Suitable functional groups include 19 19 CHBr 2

CH

3

COR

19 wherein R 19 is a primary or secondary C 1 -6 alkyl group, COOH or a derivative thereof such as an ester, amide or acid chloride or CN.

WO 91/14688 PCT/GB90/00424 10 Where the aromatic heteropolycycle bears substituents, ArCHO may be prepared by a variety of methods known in the art of organic chemistry depending on the nature of the substituent on the ring. For example, if the substituent(s) is a halogen, the starting materials may be prepared by direct treatment of the aromatic heteropolycycle ring with a halogenating agent C1 2 Br 2 or S0 2 C1 2 or indirectly by such routes as the Sandmeyer reaction Hodgson, Chem. Rev. 0, 251 (1947). If the substituent(s) is alkyl, the aromatic heteropolycycle may be reacted with the appropriate reagents under Friedel-Crafts reaction conditions Olah, Friedel Crafts and Related Reactions, Vols. 1-3, Interscience, New York, NY, 1963-1965).

The compounds NH2R also may be prepared by methods known in the art, for example when R is as hereinbefore defined by the reaction of 19 19 NO2R wherein R 19 is a group

R

6 1 R7 CH R 6 9 R R

I

OH

and R to R and m are as hereinbefore defined with an appropriate aldehyde, conveniently acetaldehyde or formaldehyde (as in B.M. Vanderbilt and H.B. Hass, Ind. En. Chem 32, 34 (1940)) followed by reduction (as outlined in J. March, vide supra, pages 1125-1126), conveniently by hydrogen and a metal catalyst (for example, a platinum containing catalyst) in an appropriate solvent, conveniently glacial acetic acid.

2. The reduction of a compound Ar.CO.NHR; wherein Ar and R are as hereinbefore defined and the hydroxy groups are optionally protected, followed by deprotection of the hydroxy groups where PrT-- g fI nn 4 4 PB1125 1 1 C2 Jun 02 6 92 appropriate, The reduction may be carried out by standard reducing agents known for carrying out this type of reduction that will not reduce the aromatic ring system (as outlined in J.

March, vide suvra, page 1122), for example, a hydride reagent such as lithium aluminium hydride in an inert solvnt, such as an ether, i.e. tetrahydrofuran, at a non-extreme temaperature, for example, at between 00 and 100°C and conveniently at the reflux temperature of the ether.

The compound Ar.CO.NHR may be formed by the reaction of the appropriate acid (ArCOOH) or a suitable reactive acid derivative thereof as outlined in J. March, yide Su.E pages 382-390) for example, an acid halide in an inert solvent, with an amine hN2R in which the hydroxy groups are optionally protected, for example, when the compound NH 2 R is a diol, by an isopropylidene group. The compound Ar. CO. NHR so formed is suitably reduced in situ and deprotected if necessary to give a compound of formula The compounds of the formula ArCOOH can be prepared by methods well known to those skilled in the art.

3. The reaction of a compound ArCH 2 L (wherein Ar is as hereinbefore defined and L is a leaving group), with a compound NH2R as hereinbefore defined. Suitable leaving groups are those defined by J. March, vide supra pages 325-331, and include halogens such as chlorine or bromine and sulfonic acid derivatives such as -toluenesulfonate. The reaction is suitably carried out in an appropriate solvent, such as a dipolar aprotic solvent or alcohol at a non-extreme temperature, for example 50-1000. The compounds of the formula ArCH 2 L can be prepared by methods well known to those skilled in the art.

There is therefore provided, as a further aspect of the invention, a method for the preparation of a compound of formula comprising any method known for the preparation of analogous compounds, in particular those methods defined in to hereinabove.

AJR/JJ/6th March, 1990.

1 ,3 i t .S.C.C j fT PCT/GB 9 0 0 0 4 24 PB1125 12 02 June 1992 U 2 06 92 In a further aspect, the present invention pro/ides novel chemical intermediates of the formulae Ar.CH-NR, Ar.CO.NHR or ArCH 2 L as herein before defined.

Compounds of the formula in which one or more hydroxy groups are protected, for example by benzyl or trityl groups or by an isoproplylidene group aro also useful intermediates in the preparation of compounds of the present invention.

The compounds o" this invention have biocidal activity, e.g. are toxic ro certain living cells which are detrimental to mammals, for example pathogenic organisms and tumours.

This toxicity to pathogenic organisms has been demonstrated, for example, by activity against one or more of the following: viruses Herpes simplex 1/vero), fungi Candida albicans), protozqa Eimeria tenella and Trichomonas vaginalis), bacteria (e.g.

Mvcoplasma sme gmatis and Streptoocccus pogenes), and helminths (e.g.

Nippostrongvlus brasiliensls and Brugai pahangi). The antitumour activity of compounds of formula I has been demonstrated in a number of recognized screens and primarily by activity against ascitic P388/0 leukaemia.

Preferred compounds of the formula are those which have antitumour activity. The activity against ascitic tumours, including P388/0, is evidenced by reduction of tumour ceUl number in mammals (for example, mice bearing ascitic tumours) and their consequent increase in survival duration as compared to an untreated tumour bearing control group. Antitumour activity is further evidenced by measurable reduction in the size of solid tumours following treatment of mammals with the compounds of this invention compared to the tumours of untreated control tumour bearing animals. Compounds of formula (I) are active against murine tumours such as lymphocytic leukaemia P388/0, lymphocytic leukaemia L1210, melanoti, melanoma B16, P815 mastocytoma, HDAY/D2 fibrosarcoma, colon 38 adenocarcinoma, M5076 rhabdomyosarcoma and Lewis lung carcinoma.

AJR/JJ/6th March, 1990.

.1 WO 01/14688 PCT/GB90/00424 13 Activity in one or more of these tumour tests has been reported to be indicative of antitumour activity in man Goldin et al. in Methods in Cancer Research ed. V.T. DeVita Jr. and H. Busch, 16, 165, Academic Press, N.Y. 1979).

There are sublines of P388/0 which have been made resistant to the following clinically useful agents: cytosine arabinoside, doxorubicin, cyclophosphamide, 1-phenylalanine mustard, methotrexate, actinomycin D, cis-platin and bis-chloroethylnitrosourea. Compounds of this invention show potent activity against these drug-resistant tumours using the procedure for P388/0 above.

Compounds of formula have also been found to be active against human tumour cells in primary cultures of lung, ovary, breast, renal, melanoma, unknown primary, gastric, pancreatic, mesothelioma, myeloma, and/or colon cancer. (As used herein "cancer" is to be taken as synonymous with "malignant tumour" or more generally "tumour" unless otherwise noted.) This is a procedure in which the prevention of tumour cell colony formation, i.e. tumour cell replication, by a drug has been shown to correlate with clinical antitumour activity in man Von Hoff et al., Cancer Chemotherapy and Pharmacology 6, 265 (1980); S. Salmon and D.D. Von Hoff, Seminars in Oncology, 8, 377 (1981)).

Compounds of formula I which have been found to have antitumour activity intercalate in vitro with DNA (this property is determined by viscometric methods using the procedure of W. D. Wilson et a Nucleic Acids Research 4, 2697 (1954)) and have a log P as calculated by the method of C. Hansch and A. Leo in Substituent Constants for Correlation Analysis in Chemistry anrd Biology, John Wiley and Sons, New York, 1979, lying in the range between -2.0 and As has been described above, the compounds of the present invention are useful for the treatment of tumours. The invention thus further provides a method for the treatment of tumours in animals, including WO 91/14688 PCT/GB90/00424 14 mammals, especially humans, which comprises the administration of a clinically useful amount of compound of formula in a pharmaceutically useful form, once or several times a day or other appropriate schedule, orally, rectally, parenterally, or applied topically.

In addition, there is provided as a further, or alternative, aspect of the invention, a compound of formula for use in therapy, for example as an antitumour agent.

The amount of compound of formula required to be effective as a biocidal agent will, of course, vary and is ultimately at the discretion of the medical or veterinary practitioner. The factors to be considered include the condition being treated, the route of administration, and nature of the formulation, the mammal's body weight, surface area, age and general condition, and the particular compound to be administered. A suitable effective antitumour dose is in the range of about 0.1 to about 120 mg/kg body weight, preferably in the range of about 1.5 to 50 mg/kg, for example 10 to 30 mg/kg.

The total daily dose may be given as a single dose, multiple doses, two to six times per day or by intravenous infusion for selected duration. For example, for a 75 kg mammal, the dose range would be about 8 to 9000 mg per day, and a typical dose would be about 2000 mg per day. If discrete multiple doses are indicated, treatment might typically be 500 mg of a compound of formula I given 4 times per day in a pharmaceutically useful formulation.

Whilst it is possible for the active compound (defined herein as compound of formula or ether, ester, or salt thereof) to be administered alone, it is preferable to present the active compound in a pharmaceutical formulation. Formulations of the present invention, for medical use, comprise an acti.ve compound together with one or more pharmaceutically acceptable carriers thereof and optionally other therapeutical ingredients. The carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof, WO 01/14688 PP/GB0/00424 15 The present invention, therefore, further provides a pharmaceutical formulation comprising a compound of formula (in the form of the free base, ether, or ester derivative or a pharmaceutically acceptable acid addition salt thereof) together with a pharmaceutically acceptable carrier therefor.

There is also provided a method for the preparation of a pharmaceutical formulation comprising bringing into association a compound of formula an ether, ester, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier therefor.

Whilst the antitumour activity of the compounds of formula is believed to reside in the free base, it is often convenient to administer an acid addition salt of a compound of formula The formulations include those suitable for oral, rectal or parenteral (including subcutaneous, intramuscular and intravenous) administration. Preferred formulations are those suitable for oral or parenteral administration.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active compound into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into assocation with a liquid carrier or a finely divided solid carrier or both and then, if necessary, shaping the product into desired formulations.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablats or lozenges, each containing a predetermined amount of the active compound; as a powder or granules; or a suspension in an aqueous WO 91/14688 PCT/GB90/00424 16 liquid or non-aqueous liquid such as a syrup, an elixir, an emulsion or a draught.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a freeflowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent.

Moulded tablets may be made by moulding in a suitable machine, a mixture of the powdered active compound with any suitable carrier.

A syrup may be made by adding the active compound to a concentrated, aqueous solution of a sugar, for example sucrose, to which may also be added any accessory ingredients, Such accessory ingredient(s) may include flavourings, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredient, such as a polyhydric alcohol for example glycerol or sorbitol.

Formulations for rectal administration may be presented as a suppository with a conventional carrier such as cocoa butter.

Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably isotonic with the blood of the recipient, Such formulations suitably comprise a solution of a pharmaceutically and pharmacologically acceptable acid addition salt of a compound of the formula that is isotonic with the blood of the recipient. Thus, such formulations may conveniently contain distilled water, dextrose in distilled water or saline and a pharmaceutically and pharmacologically acceptable acid addition salt of a compound of the formula that has an appropriate solubility in these solvents, for example the hydrochloride, isethionate and methanesulfonate salts, preferably the latter.

WO'91/14688 PCf/GB90/00424 17 Useful formulations also comprise concentrated solutions or solids containing the compound of formula which upon dilution with an appropriate solvent give a solution suitable for parenteral administration as above.

In addition to the aforementioned ingredients, the formulations of this invention may further include one or more accessory ingredient(s) selected from diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like.

The following examples are provided by the way of illustration of the present invention and should in no way be construed as a limitation thereof.

General Comments All solvents were reagent grade and used without further purification with the following exceptions. Tetrahydrofuran (THF) was dried by distillation from Na/K alloy under nitrogen (N 2 and used immediately.

Toluene (PhCH 3 was distilled from CaH 2 under N 2 and stored over 3A molecular sieves. Chemicals used were reagent grade and used without purification unless noted. The full name and address of the suppliers of the reagents and chemicals is given when first cited. After this, an abbreviated name is used.

Preparative HPLC was carried out on a Waters Prep LC/System 500A machine using two 500 g silica gel (SiO2) cartridges unless otherwise noted, Plugs of Si02 used for purificationi were "flash chromatography" silica gel (Merck Co., Inc., Merck Chemical Division, Rahway, NJ, 07065 silica gel 60, 230-400 mesh). An appropriate volume sintered glass funnel was filled approximately 3/4 full with the SiO 2 and packed evenly by tapping the outside of the funnel. A piece of filter paper was then placed on top of the SiO 2 and a solution of the material to be purified applied evenly to the top. Gentle suction WO 91/114688 PCr/GB90/00424 18 through a filter flask moved the eluting solvent through the plug rapidly. The appropriate size fractions were combined as needed and further manipulated.

General procedures are described in detail. Analogous procedures show melting point recrystallization solvents, and elemental analyses (all elements analyzing within a difference of 0.4% of the expected value). Any changes to the procedure such as solvent, reaction temperature, reaction time, or workup are noted.

NMR (1H, 13C), IR, MS data of all new products were consistent with the expected and proposed structures. The positions assigned to structural isomers wore unequivocally determined by a number of NMR techniques. All final products were dried in a vacuum oven at 20 mm Hg pressure at the temperature indicated overnight (12-16 All temperatures are in degrees Celsius.

Example 1 2-Methyl-2-(f(10-methvl-10H-benzofuro(3.2-b)indol-6-vlhmethvl)amino)- 1.3-propanediol 1A 2-(2-Nitrophenvl)benzofuran To a RB flask equipped with a magnetic stirring bar, rubber septum and N 2 inlet line was added a solution of benzofuran (Aldrich Chemical Co., P.O. Box 2060, Milwaukee, WI, 53201, 34.26 g, 0.29 mol) in Et20 (800 ml). A solutic I of n-BuLi (Aldrich, 20.5 g, 0.32 mol) in hexane (200 ml) was added to the flask via cannula. The mixture was rtirred for 30 min. at RT. The mixture was then added via cannula to a RB flask equipped with a magnetic stirring bar, rubber septum and N 2 inlet line that contained a solution of 2-fluoronitrobenzene (Aldrich, 37,26 g, 0.264 mol) in (800 ml) cooled to -40' (CH 3 CN/dry ice bath). The resulting mixture was stirred overnight at RT. H20 (40 ml) was then added WO 91/14688 PCT/GB90/00424 19 to the flask to destroy excess n-BuLi. The mixture was then further diluted with H20 (1 the two layers separated and the layer extracted with Et20 (2x500 ml). The organic layers were combined, dried (Na 2

SO

4 and concentrated to give a dark red oil. This material was passed through a plug of SiO 2 using hexane/EtOAc as the eluting solvent, The appropriate fractions were combined and the solvent removed to give 34.0 g (49.7% yield) of crude solid that was used in the next step without additional purification. A portion was washed with hexane to give 2-(2-nitrophenyl)benzofuran, mp 79-80.5" 1B 10H-Benzofuro(3.2-b)indole To a RB flask equipped with magnetic stirring bar, reflux condenser and N 2 inlet line was added 2-(2-nitrophenyl)benzofuran (1A,70.0 g,0,29 mol) and triethylphosphite (Aldrich, 98.0 g, 0.59 mol). The resulting mixture was heated at 100-110 for 7 h, The mixture was then dissolved in EtOAc (500 ml) and passed through a plug of SiO2 using EtOAc as the eluting solvent, The appropriate fractions were combined and concentrated to give a crude material which was dissolved in a mixture of hexane/EtOAc and passed through a plug of SiO 2 using hexane/EtOAc (9:1) as the eluting solvent. The appropriate fractions were combined and concentrated to give 53.5 g (89% yield) of crude solid that was used in the next stop without additional purification. A portion was recrystallized from EtOAc/hexane to give 10H-benzofuro(3,2-b)indole, mp 197.5-198 6 IC 10-Methvl-10H-benzofuro(3.2-b)indole To a RB flask equipped with magnetic stirring bar, reflux condenser, rubber septum and N 2 inlet line was added a 60% oil dispersion of NaH (Aldrich,5,78 g,0.151 mol). The oil was removed from the dispersion by washing with hexane (3x100 ml).

Dry THF (200 ml) was then added to the flask via cannula to cover WO 91/14688PC/G9!44 PCr/GB90!9M24 20 the Nail. A solution of lORj-benzofuro(3,2-b)indole (lB,28,4 g,0.137 mol) in THF (200 ml) was added to the flask via cannula.

The mixture was stirred for 15 min. at RT. Dimethyl sulfate (Aldrich,19.0 g, 0,151 mol) was then added to the flask by syringe and the resulting mixture was stirred 90 min. at RT.

H2 0(10 ml) was added to the flask to destroy excess NaH, The mixture was diluted with 0.1 N HUl (I L) and extracted with EtOAc (1 The organic layer was washed with H 2 0 (2x1 dried (Na 2 so 4 and concentrated to give 35.6 g of crude material. This material was passed through a plug of SiO 2 using PhCH 3 as the eluting solvent. The appropriate fractions were combined and the solvent removed to give after drying 29.0 g (96.0% yield) of 10-methyl-l0li-benzofuro-(3,2-b)indole, mp 114.115' ID1) 0-Methyl-10H-benzofuro(3.2-b~indole-6-carbaldehyde To a RB flask equipped with a magnetic stirring bar, rubber xeptum, and N 2 inlet line was added a solution if IOkj-benzofuro(3,2-b)indole (1C,10.0 g,44 mmol) in dry THF (1 L).

The mixture was cooled to then a solution of §j-BuLi (Aldrich,l.3 H in cyclohexsne, 8.8 mmol,6.8 ml) was added by syringe. The resulting mixture was allowed to warm to -30' then recooded to -78' before adding DMF (Aldrich,0.67 g,9.2 mxnol) by syringe. The resulting mixture was stirred overnight at RT, H12 0 ml) was added to the flask to destroy excess a.BuLi, The mixture was concentrated and the residue was partitioned between EtOAc (1 L) and 0.2 N HCl (I The organic layer was washed with satd. NaCt solution, dried, (Na 2 so 4 and concentrated to give a yellow solid which was triturated with Et 2 O0 (300 ml) yield after drying 9.4 g of l0-methyl-l011-benzofuro- (3,2-b)indole-6-carbaldehyde, mp 114.5-115.5' 1E 2 -hl 2 LUAP0meth1Hbnouo32b'lo..y~ehl ani4n~ 1 -rpndo.Hdohod WO 91/14688 PCT/GB90/00424 21 To a 3-necked RB flask equipped with a magnetic stirring bar, condenser, thermometer, Dean-Stark trap and N 2 inlet line was added 10-mpthyl-10H-benzofuro(3,2-b)indole-6-carbaldehyde (ID, 9.3 g,37.3 mmol), 2-amino-2-methyl-l,3-propanediol (Aldrich,7,84 g,74.6 mmol), p-toluenesulfonic acid monohydrate (Aldrich,O.l g) and PhCH 3 (300 ml). The mixture was stirred at reflux with azeotropic removal of H 2 0 for 2.5 h. Most of the PhCH 3 was then removed by distillation. The mixture was then cooled in an ice bath and diluted with abs. EtOH (300 ml) and further cooled.

Solid NaBH 4 (Aldrich,2.82 g,74,6 mmol) was added in one portion to the reaction mixture. The ice bath was then removed, the reaction mixture allowed to warm to RT and stirred overnight.

The crude reaction mixture was concentrated to dryness, vigorously stirred with H20 (500 ml) and filtered, After thorough washing with H 2 0 and drying, the resulting solid crude free base was converted to its HC1 salt by treatment with a 5M solution of g. HC1 in absolute EtOH. The solution was filtered through a medium porosity sintered glass funnel and precipitated by addition of Et20 (final volume 2 Recrystallization twice (CH3OH/Et 2 0) followed by filtration and drying gave 5.7 g (40.8% yield) of 2-methyl-2-(((10-methyl-10-benzofuro(3,2-b)indol-6.yl) methyl)amino)-1,3-propanediol hydrochloride, mp 258-258.50, (C,H,N,Cl).

Additional commehts: Azetropic removal of H20 is continued until complete. This process normally takes h. If the crude reaction mixture does not form an easily filterable solid, it can be extracted with EtOAc (2x500 ml), The organic layers are then combined, washed with H 2 0 (2x500 ml), satd, NaCl solution (2x500 ml), dried (K 2 C0 3 filtered and the solvent removed to give the solid crude free base which is converted to its HCl salt by the method described above. Normally CH3OH, EtOH and occasionally i-PrOH ii used to produce the solution. Alternatively, in cases where the HC1 salt is not soluble enough in H 2 0, the CH 3 S0 3 I salt is made by treating the free base with 1.1 equivalents of CH 3 SoH WO 91/14688 WO 9114688PCT/G B90/ 00424 22 Alfa Products, P.O. Box 8247, Ward Hill, MA 01835-0747) dissolved in abs. EtOH (10% v/v) in an appropriate alcohol solvent (CH 3 OH,EtQH,i-PrOH), After dissolution, the mixture is filtered through a sintered glass frit and diluted with Et 2 O0.

The resulting solid is recrystallized once or twice to give the pure final product.

1,3-nroganediol 2A 10-Methvl-10H-thieno(3.2-a)carbazole Using the procedure outlined in Example 1C, l01j-thieno(3,2-a) carbazole (Cambridge Chemicals, Inc., 202 East Smith Street, Milwaukee, WI 53207) and dimethyl sulfate (Aldrich) gave a 96.0% yield of l0-methiyl-lOjj-thieno(3,2-a)carbazole. This material was used without further purification, 2B 10-Mothyl-IOH-thienoe3,2-a)carbnzole.4-carbaldehyde 10-Miethyl-l0ij-thieno(3,2-a)carbazole (2A,30,0 S,126 nmol) Was formylated using the procedure of A. Rieche et al., gbeM D~.r.

2_3, 88 (1960). The crude reaction product was passed through a plgofSo2 uig CH 2 C1 2 as the eluting solvent. The appropriate fractions were combined and the solvent removed to give after drying 6.04 (18% yield) of lO-methyl-l1li-thieno 2-a)carbazolc.4-carbaldehyde.

Ushing the procedure ouatlined in Example 1E, lO-mothyl-l0l-thiono- (3,2-a~c~rbato16-4-carbaldehyde (2B) and 2.amino-2-methyl-1,3- 23 propanediol (Aldrich) gave a 44.0% yield of 2-methyl-2-(((10methyl-l1jj-thieno(3,2-a)carbazol-4-yl)methyl)amino) 3-propanedial methanesulfonate, mp 187-l88.5*, (EtOH/Et 2 O0), Example 3 2-Methvl-2-(((10-methyl-IOH-thieno(3.2-a)carbazol-2-lnmethylmir.o L 1. 3-nroanediol 3A 10-Methyl-10H-thieno(3.2-a)carbazole-2-carbaldehyvde Using the procedure outlined in Example 1D, l0-methyl-10ii-thieno- (3,2-a)carbazolel (2A.30.75 g,1311.8 mmol) was formylated to give 36.92 g of crude solid. Trituration in hot PhCH 3 followed by recrystallization from EtOAc/hexane gave~ after drying 10.90 g (30.7% yield) of 10-methyl-l01-thieno(3,2-a)arbazole-2-carbaldehyde, mp 207-208,5* 3B 2-Methyl-2-(((10-methyl-JOH-thieno(3.2-acarbzol.2-yl)methyl)amino) 3-pronanediol methanesulfonate Using the procedure outline in Example 1E, l0-mothyl-l0]i-thieno (3,2-a)carbazole-2-carbaldehyde (3A) and 2-amino-2-methyl-1,3propanediol (Aldrich) gave a 68.2% yield of 2-mothyl.2-(((10methyl-IOH-thieno(3,2-a)carbazol-2-yl)meth-,l)anino)-1,3-propanediol methanesulfonate, mp 219-220*, 24A h- (B -o2r -(Ri 4-enfurnon 8~fe thy)ain)- -eevA3 WO 91/14688 PCT/GB90/004'1 24 To a 2 L 3-necked RB-flask equipped with mechanical stirrer, reflux condenser, addition funnel and N 2 inlet line was added 2-hydroxydibenzofuran (Aldrich,250 g,0.814 mol), K 2

CO

3 (Mallinckrodt, Inc., 2nd Hallinckrodt Street, St. Louis, MO 63147,247.61 g,1.79 mol) and dry acetone (500 ml), The mixture was stirred vigorously for 45 min. at RT. BrCH 2 COOEt (Aldrich 136 g,0.814 mol) was then added dropwise to the flask over 0.5 h. The mixture was then refluxed for 2.5 h. The mixture was cooled to RT, filtered and the solvent removed from the resulting filtrate to give a dark brown semisolid. This material was stirred vigorously with hot hexane (3 The light yellow solution was decanted from a dark brown oil. The solution was decolorized R R with Norit filtered through a Celite plug, concentrated to a volume of 2 L and allowed to stand at RT overnight. The off-white crystals that formed were removed by filtration. A second crop of product was obtained from the filtrate after refrigeration overnight. The two crops were combined and dried affording 103 g (46.8% yield) of crude product. This material was used without further purification. Additional crystallation from hexane gave pure ethyl 2-(2-dibenzofuranyloxy)acetate, mp 51.5-53*,(C,H).

4B Ethyl 2-((l-Formyl-2-dibenzofuranvl)oxy)acetate 4C Ethyl 2-((3-Formvl-2-dibenzofuranyl)oxv)ycetate Ethyl 2-(2-dibenzofuranyloxy)acetate (4A,182.0 g, 0.673 mol) was formylated using the procedure of A, Rieche et al., CheM. Ber.

93, 88 (1960). The reaction mixture was passed through a plug of Si0 2 using CH2Cl 2 as the eluting solvent affording after combination of the appropriate fractions and removal of solvent 151.3 g of crude material shown to contain two isomeric aldehyde esters by NMR, The mixture was again chromatographed on SiO 2 using CH 2 C12 as the eluting solvent. The fractions containing isomerically pure aldehyde esters were combined. Fractions WO'91/14688 PC/GB90/00424 25 containing mixtures of the two aldehyde esters were also combined and rechromatographed until the mixture was completely separated.

A total of 71.3 g (35.5% yield) of the faster eluting (Rf 0.24, Si02/CH 2 C1 2 aldehyde ester isomer shown by NMR to be the 1-CHO derivative was obtained and used without further purification.

Recrystallization (EtOAc) of a portion of this material gave pure ethyl 2-((l-formyl-2-dibenzofuranyl)oxy)acetate, mp 100.5-101.5°, A total of 81.2 g (40.4% yield) of the slower eluting aldehyde ester isomer (Rf 0.14,SiO 2

/CH

2 C12) shown by NMR to be the 3-CHO derivative was obtained and used without further purification. Recrystallization (EtOAc) of a portion of this material gave pure ethyl 2-((3-formyl-2-(dibenzofuranyl)-oxy)acetate, mp 177-178*, 4D Ethyl Benzofuro(5.4-h)benzofuran-2-carboxylate To a 1 L 3-necked RB flask equipped with mechanical stirrer, reflux condenser, thermometer and N 2 inlet line was added ethyl '2-((l-formyl-2-dibenzofuranyl)oxy)acetate (4B,5628 g,0.189 mol), K2CO3 (Mallinckrodt, 31.29 g,0.226 mol) and dry DMF (600 ml), The mixture was heated to 150" over 20 min. and then stirred for 1 h. 'The reaction was cooled to RT and filtered through a fritted glass funnel. The solvent was removed to give 62.1 g of crude brown oil. This material was partitioned between EtOAc L) and H 2 0 (1 The EtOAc layers were combined and washed with H 2 0 (750 ml), satd, NaCI (750 ml) and then dried (Na 2 S04).

The mixture was filtered and the solvent removed to give after drying 54.2 g of off-white solid. The solid was dissolved in warm PhCH 3 (400 ml) and passed through a plug of SiO2 using PhCH 3 as the eluting solvent. The appropriate fractions were combined and the solvent removed to give 35.9 g of ethyl benzofuro(5,4-b)benzofuran-2-carboxylate, mp 176.5-178.5", 4E Ethyl 8-Formvl-benzofuro(5.4-b)benzofuran-2-carboxvlate WO 91/14688 PCT/GB90/004'24 26 4F Ethyl 9-Forivl-benzofuro(5.4-b'benzofuran-2-carboxylate 0.1 CH 2Cl 2 4G Ethyl 5-Forml-benzofuro(5.4-b)benzofuran-2-carboxylate 4H Ethyl 4-Formyl-benzofuro(5 .4-b~benzofuran-2-carboxylate Ethyl benzofuro(5,4-b)benzofuran-2-carboxylate (4D,63.2 g, 0.225 mol) was formylated using the procedure of A. Rieche et al., hem. Ber. 93, 88(1960). The crude reaction product was chromatographed on SiO 2 using CH 2 C1 2 as the eluting solvent several times until the mixture was resolved into four aldehyde esters: Ethyl 8-formylbenzofuro(5,4-b)benxofuran-2-carboxylate, 4.64 g yield), mp 214-2150, isolated directly from chromatography, Rf 0.62 (SiO 2

/CH

2 /C1 2 Ethyl 9-formylbenzofuro(5,4-h)benzofuran.'2-carboxylate 0.1 CH2 Cl 2 19.8 g (27.7% yield), mp 176.178.50, isolated directly from chromatography, Rf 0,60 (SiO 2

/CH

2 Cl 2 Ethyl 5-formylbenzofuro(5,4.k)benzofuran-2-carboxylate, 0.05 g (0.07% yield), mp 201-202.5, (PhCH 3 Rf 0.55 (Sio 2

CH

2 C1 2 Ethyl 4-formylbenzofuro(5,4-b)benzofuran-2-carboxylate, 16.6 g (23.9% yield), mp 2250 (dec), (CHC113), Rf 0.35 (Sio 2

/CH

2

%A

2 41 Ethyl 8-(Hvdroxvmethvl)benzofuro(5.4-b)benzofuran-2-carboxvlate To 1 L RB flask equipped with magnetic stirring bar and N inlet line was added ethyl 8-formylbenzofuro(5,4-b)benzofuran-2-carboxylate (4E,3,70 g,12.0 mmol) and dry THF (600 ml). The mixture was warmed until the starting material was completely dissolved.

27 The solution was cooled to 0° and NaBH 4 (Aldrich, 0.45 g,12.0 mmol) was added in one portion. After stirring for 30 mins. at the reaction mixture was treated with H 2 0 (300 ml) and acidified to pH 1 with 1 N HC1. The mixture was extracted with EtOAc (3x500 ml), satd. NaCl solution (3x500 ml) and dried (NaS0 4 After filtration the solvent was removed and the resulting white solid dried in a vacuum oven. A total of 3.80 g (-100% yield) of ethyl 8-(hydroxymethyl)benzofuro(5,4-b)benzofuran-2-carboxylate, mp 172-173*, was obtained and used directly in the next step.

4J 8-(Hydroxymethyl)benzofuro(5 4-b)benzofuran-2-carboxylic acid To a 500 ml RB flask equipped with magnetic stirring bar and N 2 inlet line was added ethyl 8-(hydroxymethyl)benzofuro(5,4-h)benzofuran-2-carboxylate (41,3.70 g,11.92 mmol), THF (150 ml) and a solution of 1 N NaOH solution (12 ml,12 mmol) in EtOH (25 ml).

The solution turns milky white instantly. After 20 min. H 2 0 ml) was added to the mixture. The resulting white solid was filtered and washed with H 2 0 (3x200 ml) and dried overnight in a vacuum oven at 90* after drying 3.17 g (93.2% yield) of 8-(hydroxymethyl)benzofuro(5,4-b)benzofuran-2-carboxylic acid, 0.2 H 2 0, mp 260-2620 (dec), 4K Benzofuro(5.4-b)benzofuran-8-methanol To a 500 ml 3-necked RB flask equipped with magnetic stirring bar, reflux condenser, thermometer and N 2 inlet line was added 8-(hydroxymethyl)benzofuran-2-carboxylic acid 0.2 H 2 0 (4J,3.13 g, 11.1 mmol), Cu20 (Aldrich, 4.76 g,33.27 mmol) and quinoline (Aldrich, 225 ml). The reaction mixture was warmed to 200-205° and stirred for 30 min. After cooling, the quinoline was removed by distillation leaving a dark green oil. This material was dissolved in CH 2 C12 (150 ml) and passed through a plug of Si02 using CH2C12 as the eluting solvent. The appropriate fractions I2 WO 91/14688 WO 914688PT/GB90/00424 28 were combined and the solvent removed by rotary evaporation to give after drying, 2,26 g (85.6% yield) of benzofuro(5,4-b-)benzofuran-8-methanol, mp 137.138*, 4L Benzofuro(5 .4-b)benzofuran-8-carbaldehyde To a 500 ml RB flask equipped with magnetic stirring bar, reflux condenser and N 2inlet line was added benzofuro(5,4-b)benzofuran.

8-methanol (4K,2.26 g,0.29 mmol), BaMnO 4 (Aldrich, 4.76 g,18.6 mmoi) and CH 2 Cl 2 (300 ml). The mixture was refluxed for 2 h, fill-ered through a plug of Celite Rand the solvent removed to give after drying 2.08 g of crude product. This material was dissolved in CH-2 Cl 2(50 ml) and passed through a plug of SiO 2 using PhCH 3 as the eluting solvent. The appropriate fractions were combined and the solvent removed to give 2.06 g of (94.1%) of benzofuro(5,4-k)benzofuran-8-carbaldehyde, mp 156.5-158', 4M 2(((Benzofuro(5.4-b~benzofuran-8-l)methy)amino)-2-methl- 1. 3-proganediol Hydrochloride Using the procedure outlined in Example 1E, benzofuro(5,4-bk)benzofuran-8-carbaldehyde and 2-amino-2-methyl-l,3-propanediol (Aldrich) gave a 57.3% yield of 2-(((benzofuro(5,4-b)benzofuran.

8-yl)methyl)amino)-2-methyl -1,3-propanediol ibydrochloride, mp 262-263.50, (CH 3 Ol/Et 2 O0), (C,H,N,Cl).

Example 2-(((Benzofuro(5.4-b'benzofuran-9-vl~hiethyl~aminoP-2-methyl- 1.3-tRropanediol Ethyl 9- (Hydroxyme hy2.).bnz furo(54-b~henzofMI: n-2-carboxylate WO 91/14688PC/B9042 PCr/GB90/00424 29 Using the procedure outlined in Example 41, ethyl 9-formylbenzofuro(5,4-b)benzofurar.-2-carboxylate 0.1 CH 2

CI

2 gave a 88.9% yield of ethyl 9-(hydroxymethyl)benzofuran(5,4-b)benzofuran-2-carboxylate, nip 138-l40*, SB 9-(Hydrox'nnethyl~benzofuro(5.4-b~benzofuran-2-carboxvlic acid Using the procedure outlined in Example 4Q, ethyl 9-(hydroxymethyl) -benzofuro(5,4-ka)benzofuran-2-carboxylate (5A) gave a 98.6% yield of 9-(hydroxyxnethyl)benzofuro(S,4-b)benzofuran-2carboxylic acid, nip 295*, (0,11).

Benzofuran(S .4-b~benzofuran-9-methanol Using the procedure outlined in Example 4K, 9-(hydroxymethyl)benzofuro(5,4-1k)benzofuran-2-carboxylic acid (5B) gave a 75,9% yield of benzofuro(5,4-b)benzofuran-9-methanol, nip 132-133', (0,11).

Benzofuro(5.4-b)benzofuran-9-carbflldehyde Using the procedure outlined in Example 4L, benzofuro(5,4-b)benzofuran-9-methanol (5C) g-ave a 92.3% yield of benzofuro- (5,4-b)benzofuran-9-carbaldihyde, nip 183-180, (0,11).

2-(((BenZofuro(5.4-b~benzofuran-9-v1'methyl~amino)-2-methyl- 1 .3-propanediol Hydrochloride Using the procedure outlined in Example 1E, benzofuro(5,4-b)benzofuran-9-carbaldehyde (5D) and 2-amino-2-niethyl-1,3-propanediol (Aldrich) gave a 34.2% yield of 2-(((benzofuro(5,4-k)benzofuran-9-yl)methyl)amino)-2-methyl-l1,3-pr-opanediol hydrochloride, nip 243-244.56, (EtOH/E 2 (C,H,NCl).

WO 91/14688 WO 9114688PCT/GB90/00424 30 2- (Benzofuro(5 .4-b)benzofuran-4-yl)methyl~amino) -2-methyl- 1. 3-provanediol 6A 4-Formyl-benzofuro(5 .4-b)benzofuran-2-carboxylic acid Using the procedure outlined in Example QJ, ethyl 4-formylbenzofuro(5,4-ka)benzofuran-2-carboxylate gave a 81.8% yield of 4-formylbenzofuro(5,4-b)benzofuran-2-carboxylic acid which was directly in the next step without further purification.

6B Benzofuro(5 .4-b)benzofuran 6C Benzofuro(5 .4-b'benzofuran-4-inethanol 4-Formyl-benzofuro(5,4-k)benzofunan-2-carboxylic acid was decarboxylated (6A,13.60 q,48.5 mmol) using the procedure outlined for the hydroxymethyl tearboxylic acid derivative in Example 4K. Two major products were formed in this reaction and crudely separated by column chromatography (SiO 2 using CH 2 Cl 2 as the eluting solvent. The fractions containing the faster eluting material were collected the solvent removed and the residue chromatographed (SiO 2 using PhCH 3 as the eluting solvent, The appropriate fractions were combined and the solvent removed to give 2.61 g (25.8% yield) of benzofuro(5,4-b_)benzofuran, mp 66-67*, C,l1, The fractions containing the slower eluting material were also combined and the solvent removed to give 3.79 g (32.7% yield) of benzofuro(5,4-la)benzofuran-4-mthanol), mp 129-131*, C,H. The aldehyde carboxylic acid starting materials undergoes a Cannizaro type reaction unlike the hydroxymethyl carboxylic acid (4Q).

6D fenzofuro(5 .4-b'benzofuran-4-carbaldehvde Using the procedure outlined in Example 4L, benzofuro(5,4-k)benzofuran-4-methanol (6C) gave a 78.6% yield of benzofuro- WO 01/14688 PC1/GB90/00424 31 (5,4S.)benzofuran-4-carbaldehyde, mp 170.5-171.5*, 6E 2-(((Benzofuro(5.4-b'benzofuran-4-vl)methl)amino)-2-methvl- 1.3-propanediol-Hydrochloride Using the procedure outlined in Example 1E, benzofuro(5,4-b)benzofuran-4-carbaldehyde (6D) and 2-amino-2-methyl-l,3-propanediol (Aldrich) gave a 52.0% yield of 2-(((benzofuro(5,4-k)benzofuran-4-yl)methyl)amino)-2-methyl-1,3-propanediol hydrochloride, mp 263-2656 (dee), (CH 3 OH/Et 2 (C,H,N,Cl).

Exarnnule 7 4-b)benzofuran-2-vl)methl)amino)-2-methyl- 1.3-propanediol 7A Benzofuro(5.4-b'benzofuran-2-carbaldehyde To a 300 ml RB flask equippad with magnetic stirring bar, rubber septum and N 2 inlet line was added benzofuro(5,4-k)benzofuran (6B,2.46 g,11.81 mmol), dry THF (125 ml) and N,N,N',N'-tetramethylethylenediamine (Aldrich, 3.16 g,27.17 mmol,4,10 ml) (distilled from CaH 2 The mixture was cooled to p-BuLi (Aldrich, 1,3 M in cyclohexane, 27.17 mnol, 20.9 ml) was added dropwise to the mixture by syringe. The mixture was stirred at -78* for I h, warmed to -20' for 45 min, and then recooled to -780, Dry DMF (Hallinckrodt, 3.20 g,41.3 mmol,3.l ml) was then added dropwise by syringe to the flask, The mixture was stirred at -780 for 10 min. and then allowed to warm to RT (over 1 h), The reaction was quenched with H 20 (25 ml) and acidified with 1 N HC11 The THF was removed by rotary evaporation and the residue further diluted with H 2 0 (300 ml). This material was extracted with EtOAc (3x50 ml), The organic layers were combined and washed with H 2 0 (2x500 ml), satd. NaCi solution (1 L) and dried (Na 2 So 4 The solvent was removed to give 2.49 g of yellow WO 91/14688 PCT/GB90/00424 32 solid. This material was chromatographed (Si02) using PhCH 3 as the eluting solvent. The appropriate fractions were combined and the solvent removed to give 1.52 g (54.5% yield) of benzofuro- (5,4-b)benzofuran-2-carbaldehyde, mp 193-194.5", 7B 2-(((Benzofuro(5.,4-b)benzofuran-2-vl)methl)amino)-2-methyl- 1.3-propanediol hydrochloride Using the procedure outlined in Example 1E, benzofuro(5,4-b) benzofuran-2-carbaldehyde (7A) and 2-amino-2-methyl-,3-propanediol (Aldrich) gave a 41,5% yield of 2-(((benzofuro(5,4-b)benzofuran-2-yl)methyl)amino)-2-methyl-1,3-propanediol hydrochloride, mp 230-230.5' (dec), (EtOH/Et 2 (C,H,N,C1), Example 8 2-(((Benzofuro(5.6-b)benzofuran-8-vl)methvl)amino)-2-methvl- 1.3-propanediol 8A Ethyl Benzofuro(5 .6-b)benzofuran-2-carboxvlate To a 1 L 3-necked RB flask equipped with mechanical stirrer, reflux condenser, thermometer and N2 inlet line was added ethyl 2-((3-formyl-2-dibenzofuranyl)oxy)acetate (40,67,32 g,0.225 mol),

K

2 C0 3 (Mallinckrodt, 37.43 g,0.271 mol) and dry DMF (800 ml).

The mixture was heated to 155' for 1.5 h. The reaction was cooled to RT and filtered through a fritted glass funnel. The solvent was removed to give 82.1 g of crude brown oil. This material was partitioned between EtOAc (1,5 L) and H 2 0 (2x750 ml), satd. NaC1 (2x1 L) and then dried (Na 2

SO

4 The mixture was filtered and the solvent removed to give after drying 53.2 g of a yellow solid, The solid was dissolved in warm PhCH 3 (550 ml) and passed through a plug of Si0 2 using PhCH 3 as the eluting solvent.

The appropriate fractions were combined and the solvent removed to give 40,4 g (53.2% yield) of material that was used without 33 further purification. Recrystallization of a small amount of material from PhCH 3 gave ethyl benzofuro(5,6-b)benzofuran-2carboxylate, mp 123.5-l24.5*, 8B Ethyl 8-Formyl-benzofuro(5 ,6-b~benzofuran-2-carboxylate 0,05 CH Cl 8C Ethyl 4-Forrnvl-benzofuro(5 .6-b'benzofura'n-2-carboxylate Ethyl benzofuro(5,6-k)benzofuran-2-carboxylate (8A,36.0 g, 0.132 mol) was formylated using the procedure of A. Rieche et L.

Chem, Ber. 93, 88(1960). The crude reaction mixture was passed through a plug of SiO 2 using CH 2 Cl 2 as the eluting fraction several times until the mixture was resolved into two aldehyde esters: Ethyl 8-for-myibenzofuro(5,6-ka)benzofuran-2-carboxylate 0.05 CH 2 Cl 2 6.18 g yield), mp 227*, isolated directly from chromatography, R f 0.23 (Sio 2 /CH 2

CY

2 Ethyl 4-formylbenzofuro(5 ,6-k2)benzofuran-2-carboxyla-te, 26.2 g (64.4% yield), (PhC 3 F) R f 0.63 (SiO 2 /CH 2 C'l 2 8D Ethyl 8- (Hvdroxvinethyl~benzof-uro(5 .6-b~benzcfuran-2-carboxylatA Using the procedure outlipned in Example 41, ethyl 8-formyl-benzofuro(S,6-)2)benzofuran-2-carboxylate(8B) gave a 93.3Vs yield of ethyl 8- (hydroxymethyi)benzofuro(5,6-h)benzofuran-2-carbcxylate, mp 186- 8E 8- (Hydroxym thyl)-henzof~r-o(5-.-6-b~bnnzofurarn-2-car-boxvlic Aqcid Using the procedure outlined in Example 4J, ethyl 8-(hydro.

AjR/jj/6th March, 1990.

WO 91/14688 WO 9114688PCT'/GB90/00424 -34 methyl)benzofuro(5,6-1a)benzofuran-2-carboxylate (8D) gave a 92.7% yield of 8- (hydroxymethyl)benzofurD(5,6-b)benzofuran-2-carboxylic acid, mp >2700, (0,11).

8F Benzofuro(5 .6-b)benzofuran-8-methanoI Using the procedure outlined in Example 4K, 8-(hydroxynethyl)benzofuro(5,6-ka)benzofuran-2-carboxylic acid (8E) gave a 74.8% yield of benzofuro(5,6-1a)benzofuran-8-methanol, mp 170.1720, (C,11).

BG Benzofur2(5 .6-b'benxofuran-8-carbaldehvde Using the procedure outlined in Example 4L, benzofuro(5,6-h)benzofuran-8-uiethanol gave P 89.7% yield of benzofuro(5,6-]a)benzofuran-8-carbaldehyde, mp 188-l89*, (C,11), 8H1 2(((Benzofuro(5,6-b~beirzofuran-B-v1Amethv32~amino)-2-meth.l- 1. 3-propanediol Hydoloride~ Using the procedure outlined in Example IE, benzofuro(5,6-k)benzofuran-8-carbaldehyde (8G) and 2-amino-2-methyl-l,3-propane.

diol (Aldrich) gave a 66,3% yield of 2-(((benzofuro(5,6-)benzo-.

furan-8..yl)iethyl)amino)-2-methyl-1,3-propanediol hydrochloride, rnp 235-236.5' (doe), (CH 3 OH/Et 2 O0), (C,H.N,Cl).

(((Menzofuro .6-h benzofumrap-4:al'mothjl) amino)--2--methyl 9A 4 ormyll. zoturo L O. bnofuon Using the procedure outlined in Example 43, ethyl 4-formyl-bentofuravi-2-carboxylic. (8C) gave a 98.'9% yield of 4-formyl-bentofuro- WO 91/14688 WO 9114688PCI'/GB9O/00424 (5,6-h)benzofuran-2-carboxylic acid, that was used directly in the next step without further purification.

9B Denzofuro(5.6-b~benzofuran Benzofuro(5.6-b~benzofuran-4-niothanol 4-Formyl-benzofuro(5,6-b)benzofuran-2-carboxylic acid (9A,25.2 g, 81.7 mmol) using the procedure outlined for the hydroxymethyl carboxylic acid derivative in Example 4K. The two major products formed in this reaction were crudely separated by column chromatogtaphy (SiO 2 using CH 2 C1 2 as u.he eluting solvent. The fractions containing the faster eluting material were collected, the solvent removed and the resid e chromatographed (SiO 2 using PhCH 3 as the eluting solvent, The appropriate fractions were combined and the solvent removed to give 6,20 g (33.4% yield) of benzofuro(5,6.k2)furan, mp 90-1001, The fractions containing the slower eluting material were also combined and the solvent removed to give 3.91 8 (18.4% yield) of benzofuro(S,6.h).

benzofuran-4-methanol, mp 152-153'. As mentioned for Examples 6B and 60, the aldehyde carboxylic acid starting material undergoes a Cannizaro type reaction .nlika the hydroxymethyl carboxylic acid 8E.

9D Aenzofuro(.,6-b~benzofura.4carbadehd.

Using the procedure outlined in Example 4K, benzofuro(5,6-b)benzofuran-4-methanol (9C) gave a 96.5% yield of benzofuro- (5,6.k)bentofuran.4-carbaldehyde, mp 167.5-1691, 9E 2.(-((Benzof-uro(5-.6.b)benz-of-uran*.4.v1)methvl~amlnoP2-m-ethvl Using the procedure outlined in Example IE, bentofuro(5,6-h).

benzofurn-4carbaldthyde (90) and 2-aino'.2-nmethyl1,3-propane- 36 -PB1125 diol (Aldrich) gave a 46.9% yield of 2-(((benzofuro(5,6-h)benzofuran-4-yl)methyl)amino) -2-methyl-l,3-propanediol hydrochloride, nip 224,,2250 (dec), (EtOH/Et 2 O0), 2-(((Benzofuro(5.6-b)benzof-uran-2-ylethl~aiino-2-mthl- 1. 3-pro~anediol Benzofuro(S 6-b~henzofuran-2-cgrbaldehyde Using the procedure outlined in Example 7A, benzofuro(5,6-]Z)benzofuran (9B) gave a 82.8% yield of benzofuro(5,6-1a)benzofuran- 2-carbaldehyde, isp 194-195.5*, (0,11).

lOB 2-(((Benzofuro(5.6-b~benzoouran-2-vlIhiethvl~hmino)-2-methyl- L-.rropanediol Hydrochloride Using the procedure outlined in Example 1E, benzofuro(5,6-h-) benzofuran-2-carbaldehyde (10A) and 2-amino -2-methyl- 1,3-propanediol (Aldrich) gave a 50.3% yield of 2-(((banzofuro- 6-k)benzofuran-2-yl)nethyl)anino) -2-methyl- 3 -propanediol hydrochloride, nip 231-232.5* (dec), (CH 3 01/Et 2 (C,H,N4,Cl).

JEXample 11 2--Mothyl-2-U((l-nethyl-lH- rlbenzothienor2 3 -gl indol-3-yl)moetl) aminoA- 3 -propanediol 11A thvf -e -1Ihe t Lenl23 y.Iidg. g co y ae Using the procedure outlined in Example 10, ethyl t'heino-12,3-zlindolt-2-carboxylate Pars Laboratories, Inc., 763 Concord Ave,, Cambridge, lHllbenzo- Pharmaceutical MA 02138) and \AJRVjj/6th March, 1990.

PB1125 dimethyl sulfate (Aldrich) gave a quantitative yield ol ethyl l-methyl-lHj- l]benzothieno[2,3-g]-indole-2-carboxylate, mp 85-86*, (PhCH 3 11B Ethyl 3-Formvl--1-methvl-lllrllbenzothienor2.3-pl'indole-2carboxylate Ethyl 1-methyl-li- (llbenzotheino(2,3-glindole-2-carboxylate (11A) was formylated by the procedure of A. Rieche g~ al. Ob&. Ber.

93, 88(1960) to give a 58.7% yield of ethyl 3-formyl-l-methyl-lj- [l]benzothieno(2,3-g]indole-2-carboxylate, mp 145-147", (CH 2 Cl 2 hexane), 11C 3-Formvl-l-methvl-lH-rllbenzothienor2.3-glindo.e-2-carboxylic Using the procedure outlined in Example 4J, ethyl 3-for:myl-lmethyl-lli- (l]benzothieno(2,3-g] indole-2-carboxylate (11B) gave a 91.0% yield of 3-forrnyl-l-nethyl-li-(11-benzothieno(2,3-g1indole- 2-carboxylic acid, which was used directly without further purification.

l1D 1-Methv1-1H-rll-benzothienor2.3-y-lindole-3-carbaldehvde Using the procedure outlined in Example 4K, 3-formyl-l-methyllj- 1]-benzothieno(2,3-.g]indole-2-carboxylic acid (110) gave a 35.3% yield of 1-methyl-1H-benzothieno(2,3-g~indole-3-carbalde.

hyde, mp 192-194", (CH Cl 2 /ptroleum ether), (C,3H,N).

wethyl) amino) 1. 3 -r~rovanediol Hydrochloride Using the procedure outlined in Example 1E, l-methyl-1fi-tl)benzoA~hieno- 3-g] -indole-3-carbaldehyde (11D) and 2-amino-2methyl-1,3-propanediol (Aldrich) gave a 56.7% yield of 2-methyl- 'A ]R/J3/6th March, 1990, PB 1125 38 2-(((l-methyl-ljj-[l~benzothieno[2,3-.g~indol-3-yl)methyl)amino)- 1,3-propanediol hydrochloride, mp, 228-23Oe (dec), (CH 3 OH/Et 2 0), (C,H,N,Cl).

Example 12 2-(((6-Ethyl-1.6-dihvdro-l-methylvvrrolof3.2-clcarbazol-3-Yj), methyl~amino'i-2-methyl-1 .3-Droaanediol 12A Ethyl 6-Ethyl-1.6-dihydro-l-rnethylpyrrolor3-.2-clcarbazole-2carboxylate Using the procedure outlined in' Example 1C, ethyl 6-ethyl-l,6dihydro-pyrrolo(3,2-.q]carbazole-2-carboxylate Pars Pharmaceutical Laboratories, Inc.) gave a 90.5% yield of ethyl 6-ethyl- 1, 6-dihydro-l-methylpyrrolo(3,2-.qjcarbazole-2-carboxylate, mp 115.5-116*, (CH 2 Cl 2 /hexane), 12B 6 -Ethyl-I 6_-dihydro-1-methlprrlo r 3.2-c-1carbazole 3 Using the procedures outlined in Examples 411, *4Q and 4K, ethyl 6 -ethyl 6-dihydro -1-methylpyrrolo [3,2 q)carbazole- 2-carboxylate (12A) gave a 9.3% yield of 6-ethyl-1,6-dihydro-l-methylpyrrolo- E3,2-g]carbazole-3-carbaldehyde, mp 144-146*, (CH1201l2), (C,HN), 12C 2- (U6-thv-1.6-dihydr-l-iiethyvlhyrlor3.2.clcarbazol-3-yl) methyl) -amino)-24ethyl-l. 3-protpnedi .olHydrochloride_ 0.6 H120 Using the procedure in Example 1E, 6-ethyl-l,6-dihydro.4-methylpyrrolo[3,2-.qjcarbazole-3-carbaldhyde (12B) and 2-axnino.2-mtthyl- 1,3-propanediol (Aldrich) gave a 48.3% yield of 2-[[6-ethyl-l,6dihydro-l-methylpyrroloi3,2.q]carbazol-3-yl)methyl~amino] -2xnethyl-1,3-propanediol hydrochloride 0.6 H12 0, nip 265-267*, (EtOH/Et 2 O0), (C,H,N,Cl).

AJR/JJ/6th March, 1990.

PB1125 Example -13 2 -e thl-2- hyl-1H-benzofurqf 2.3-C Iindol-3-y1)methyl) -amino) 1. 3-propanediol 13A Ethyl l-Miethvl-lH-benzofuro23-flindole-2-carboxylate Using the procedure outlined in Example 1C, ethyl llj-benzofuro- [2 ,3-&]indole-2-carboxylate Pars Pharmaceutical Laboratovies, Inc.) gave a 86.3% yield of ethyl l-methyl-1lH-benzofuro- [2,3-,glindole-2-carboxylate, mp 114-ll6*, (EtOAc), 13B Ethyl 3-Formyl-l-rethl- H-enzofuror2.3-lindole-2-carbgat Ethyl 1-methyl-lll-benzofuro[2,3-glindole-2-carboxylate (13A.) was formylated according to the procedure of A. Rieche g&t Al., Chemn.

Ber 9, 88(1960) to give and 82.5% yield of ethyl 3-formyl-lmethyl-lli-benzofuro[2,3.gj-indole-2-carboxylate, mp 190-192', (CH 2 Cl 2 /hexane), 13C 3-omllmty-Hbnouo2.-1idl-tcroyi ai Using the procedure outlined in Example'4.., ethyl 3-forriyl-lmethyl-ljj-benzofuro(2,3-gC]indole carboxylate (13B) gave a 90,3% yield of 3-formyl-l-methyl-lIH-benzofuro[2,3-Z]indole-2-,carboxylic acid, which was used direci~y without further purification.

13D 1-Methyl-lli-benzofuror2.3-g'1indole.-3-carbadehyde Using the procedure outlined in Example 4K, 3-formyl-l-methyl-i1benzofuro[2,3-.g]indole-2-carboxylic acid (13C) gave a 40.1% yield of l-meth~yl-lH-benzofuro(2,3-.g~indole-3-carbaldehyde, mp 163- 165-, (CH 21 C1 2 /Petroleum ether), (C,H,N1).

AJR/JJ/6th March, 1990.

WO 91/14688 WO 9114688PC'/G B90/00424 13E 2-Methyl-2-(f,(l-methvl-lH-benzofuror2.3-,,1indole-3-yl~methyl) amino)-..3-vroipanediol-Hydrochiloride Using the procedure outlined in Example 1E, 1-methyl-ljj-benzofuro[2,3-g])-indole.3-carbaldehyde (13D) and 2-amino-2-methyl-l,3propanediol (Aldrich) gave a 63.9% yield of 2-methyl-2.-(((l.

rnethyl-ljj-benzofuro- [2,3-g&)indol.3-yl)rnethyl)amino)-l,3-propanediol hydrochloride, mp 234-2360, (EtOH/Et 2 O0), (C,H,N,Cl).

Examp~le 14 2-Methyl-2-(((3-methyl-3H-[llbenzothieno(2.3.elindol-l-vl)methyl) aminO~ 3-nropanediol) 14A 3-Methyl-3H--fllbenzothienaor2.3-elindole-l-carbaldehyde 0 .25 H 2 7Q Using the procedure outlinoed in Example 1C, ethyl 3li-[l)benzothieno[2 indole..2-carboxylate Pars Pharmaceutical Laboratories, Inc.) and dimethyl sulfate (Aldrich) gave a nearly quantitative yield of crude ethyl 3-methyl-3fl-(l)benzothieno- [2,3-elindole-2-carboxylate. This material was formylated using the procedure of A. Rieche et al., §hm Ber. j2., 88(1960) ti: give crude mixture of aldehydes which was hydrolyzed and decarboxy~l.ated without purification using the procedure outlined in ExamplL 15B to give a 19.0% yield of 3-methyl-31i.(l)benzothieno[2 ,3-&q]indole-l-carbaldehiyde 0.25 H 2 0, mp 223.5-2240, (CH- C1 2 /hexane), 14B 2-Methyl-2-(((3-methyl-3H-rllbenzothienor2,3-elindoI l.vl~methyj'L amino) 1, 3 -pro anediol-Hy-dr-ochloride Using the procedure described in Example 1E, 3-methyl-311-1l1.

benzothieno[2,3-Ijindole-l-carbaldehyde (14A) and 2-amino-2methyl-1,3-propanediol (Aldrich) gave a 41.4% yield of 2-inethy~l- 2-(((3-methyl-311-Ellbenzothieno[2,3-&lindol-1-yl)methyl)amino).- WO 91/14688 WO 9114688PCT/GB9O/00424 41 1,3-propanediol hydrochloride, mp 228-2300, (CH 3 QH/Et 2 0),

(C,H,N,CI).

Exam~le 2-(((lO-Methvl-10H-fllbenzothienol3.2-blindol-6-yl)methyl)mino)- 2-methyl-i. 3-propanediol 10-Methyl-IOH-[llbenzothieno[3.2-blindole Using the procedure outlined in Example IC, I1lj-[Ilbenzothienof3,2-b]indole (prepared by the method of K.E. Chippendale and B.

Iddon, ja Pelkin Trans. 1 2023 (1972) gave and dimethyl sulfate (Aldrich) a 91.9% yield of lO-mxethyl-10HkI]benzothieno[3,2-]] indole, isp 175-176', benzofuro(5,6-]k)berzofuran-2-carbaldehyde.

150 lO-Methl-OI-f r1benzothienor3.21*r,1indole-3-carbaldehvde 10-Methyl-l0u- (l~benzothieno(3,2-2] indole (15A) was formylated according to the procedure of A, Rieche et Al. Chm D&I. j9., 88 (1960) to give a low yield of a mixture of two aldehydes.

Chromatography (SiO 2 /PhCH 3 gave a 6.7% yield of 10-methyl-1IU.

[l~benzothieno- indole-6-carbaldehyde 0.1 2 0 mp 128-130', (CH 2 Cl 2 f/hexane), R f 0,39 (SiO 2 /PhC4 3 and a 22.3% yield of 10-methyl-10kH-[I~benzothieno[3,2-b]indole- 3-carbaldehyde (150), isp 188-189.5*, (PhCH 3 /hexane), (C,H,N4,S), R f 0,11 (SiO 2 /PhCH 3 2-(((10O-Methyv1-1OI4-rllbenzothienor3.2-blinlol-6-vyl)methv-l~hmino)- 2-methyl- 3-nrtanediol Hydrochloride Using the procedure outlined in Example 1E, l0-methyl-lOjj-[l~benzothieno(3,2-hlindole-6-carbaldehyde (15B) and 2-amino-2- WO 91/14688 WO 9114688PCT'/GB9O/00424 42 methyl-l,3-propanediol (Aldrich) gave a 26.9% yield of methyi-i0k1-tl]benzoth-ieno-(3,2-]a]indo.-6-yl)methyl)anino)-2methyl-i, 3-propanediol hydrochloride, mp 249.5-250*, (CH 3

OH/

Et 20), (C,H,N,S,Cl).

Example 16 2- ((lO-Methyl-IOH- tllbenzothienot3 .2-blindol-3-yl'~methyl'amino)- 2-methyl-i .3-nronanediol hydrochloride Using the procedure outlined in Example 1E, thieno(3,2-klindole-3-carbaldehyde (15C) and 2-amino..2-methyl-l,3propanediol (Aldrich) gave a 39.4% yield of 2-(((10-methyl-10H.

(l]benzothieno[3,2-ka]indol-3-yl)methyl)amino)-2-methyl-i,3-propanedio1 hydrochloride, mp 285.5-259.5*, (CH 3

OH/ET

2 O0), (C,H,N,S,Cl).

Antitumor Screening~ Results Methods for evaluating the antitumnor activity of these compounds are essentially those used in the Tumour Panel by the Development Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, A. Goldin, et AlMtosi Cne eerh Vol. XVI, p.165, Academic Press (1979). Some modifications, in dose level and schedule have been made to increase the tes~ting efficiency, Lymphocytic Leukemia P388/0 -Ij CD2-F I mice, of the same sex weighing within 20±3g, are used for this test. Control and test animals are injected intraperitoneally with a suspension of 10 6 viable P388/0 tumour cells on day 0. in each test several dose levels which bracket the LD 20 for the compound are, evaluatedl each dose level group contains 6 animals. The test compounds are prepared either in physiologic saline containing 0.05% Tween 80 or distilled water containing 5% dextrose and are administered intraperitoneally on days, 1, 5, and 9 relative to tumor WO 91/14688 PC~r/GB90/00424 43 implant. Doses are on a mg/kg basis according to individual animals' body weights. the day of death for each animal is recorded, the median identified for each group and the ratios of median survival time for treated (T)/control groups are calculated. The criterion for activity is T/C x 100 >120%. Results of P388/0 testing are summarized in Table I below.

TABLE I Activity of Compounds Against P388 Lymphocytic Leukemia Compounds of Formula LD20 (mg/kg) Optimal Dose (mg/kg)

T/C*

Survivors 1E 2C 3B 4M 6E 7B 8H 9E 11E 12C 13E 14B 16 150 375 375 >450 175 60 175 300 75 >675 250 100 115 150 225 250 250 350 275 550 150 45 175 300 75 600 300 100 100 125 300 250 210 195 195 117 117 213 138 200 192 100 135 113 120 215 140 170 0/6 0/6 0/6 0/6 0/6 1/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 5/6 0/6 *Excluding 30 day survivors WO 91/14688 PCT/GB90/00424 44 Formulation Examples A. TABLET Compound of Formula I (as hydrochloride) 500.0 mg Pr-gelatinised Corn Starch 60.0 mg Sodium Starch Glycolate 36,0 mg Magnesium Stearate 4.0 mg The Compound of formula is finely ground and intimately mixed with the powdered excipients, pregelatinised corn starch and sodium starch glycolate. The powders are wetted with purified water to form granules. The granules are dried and mixed with the magnesium stearate. The formulation is then compressed into tablets weighing approximately 600 mg each, B. TABLET Compornd of formula 500.0 mg Corn Starch 70.0 mg Lactose 83,8 mg Magnesium Stearate 4.2 mg Polyvinylpyrrolidone 14,0 mg Stearic Acid 28.0 mg The Compound of formula is finely ground and intimately mixed with the powdered excipients, corn starch and lactose. The powders are wetted with a solution of polyvinylpyrrolidone dissolved in purified water and denatured alcohol to form granules. The granules are dried and mixed with the powdered stearic acid and magnesium stearate, The WO 01/14688 POr/GB90/00424 45 formulation is then compressed into tablets weighing approximately 700 mg each.

C. CAPSULES Compound of formula (I) Corn Starch Magnesium Stearate 500.0 mg 50.0 mg 3.0 mg The finely divided compound of formula is mixed with powdered corn starch and wetted with denatured alcohol to densify the powder. The dried powder is mixed with stearic acid and filled into hard-shell gelatin capsules.

Compound of formula (I) Ethanol Glycerin Sucrose Flavouring Agent Colouring Agent Preserving Agent 0.1% Purified Water 250.0 mg 250.0 mg 500.0 mg 3,500.0 mg q.s.

q.s. to 5.0 ml The Compound of formula is dissolved in the ethanol, glycerin, and a portion of the purified water, The sucrose and preserving agent are dissolved in another portion of hot purified water, and then the colouring agent is added and dissolved. The two solution are mixed and cooled before the flavouring agent is added. Purified water is added to final volume. The resulting syrup is throughly mixed.

WO 91/14688 PC/GB90/00424 46 E. IV INJECTION Compound of formula (I) Glycerin Preservative Hydrochloric Acid or Sodium Hydroxide Water for Injection 5.0 mg q.s. for isotonicity 0.1% as needed for pH adjustment q.s. to 1 ml The compound of formula and preservative is added to the glycerin and a portion of the water for injection. The pH is adjusted with hydrochloric acid or sodium hydroxide. Water for injection is added to final volume and solution is complete after thorough mixing. The solution is sterilised by filtration through a 0.22 micrometer membrane filter and aseptically filled into sterile 10 ml ampoules or vials.

Claims (10)

1. A compound of the formula (I) ArCH 2 NHR (I) or a monomethyl or monoethyl ether thereof, the compound of formula including these ethers containing no more than 29 carbon atoms in total, or an ester or a salt thereof; wherein Ar is a fused tetracyclic hetero aromatic ring system of the formula: O z in which Z is oxygen, sulphur or a group NR 1 wherein R 1 is hydrogen, methyl or ethyl and "Cy is a bicyclic aromatic ring system which consists of a phenyl ring and a membered ring system which contains one heteroatom Z 1 selected from oxygen, sulphur or a group NR 2 wherein R 2 is hydrogen, methyl or ethyl; the tetracyclic ring system being optionally substituted by one or two substituents; said substituents containing not more than four carbon atoms in total when taken together and are the same or different each being selected from halogen; cyano; Cz. 4 alkyl or Cz. 4 alkoxy, each optionally substituted by hydroxy or CI. 2 alkoxy; halogen substituted CI. 2 alkyl or C1. 2 alkoxy; a group S(0)nR 3 whe:rein n is an integer 0,1 or 2 and R3 is C3. 2 alkyl optionally substituted by hydroxy or CI., alkoxy; or Ar is 1 PCT/GB 9 0 0 0 4 13 15 92 13 May i PB1125CL optionally substituted by a group NR R 5 containing not more than carbon atoms wherein R 4 and R 5 are the same or different and each is a C1- 3 alkyl group or NR4R 5 forms a five-or six-membered heterocyclic ring optionally containing one or two additional heteroatoms; R contains not more than eight carbon atoms and is a group R 6 1 7 12 I 1 R 1 S,13 (CH 2 )m or 13 9o1 C -8 R C I I H OH OH wherein m is 0 or 1; R is hydrogen or C1- 3 alkyl optionally substituted by hydroxy; R is hydrogen, C1-3 alkyl or CH 2 OH and R is hydrogen or C1-3 alkyl; R 9 is hydrogen, methyl or hydroxymethyl; is a five-or six-membered saturated carbocyclic ring; R 11 and R are the same or different and each is hydrogen or methyl; R13 in hydrogen, methyl, hydroxy, or hydroxymethyll R14 in hydrogen, methylhydoxyo hydroxy or hy doxymethyl R i 2) A compound according to claim 1 in which the totracyclic aromatic ring system At is unsubstituted. 3) A compound of the formula according to either claim I or 2 in which R is a groupt PWC/JH/lith May 1992 United -orM P otf;e SUBSTITUTE SHuET PCT In., .aal A;,icationl WO!)1/14688 PCT/GB90/00424 49 I C 1 o -C- 6 H OH- wherein R5is CH 2 0H, CH(CH 3 )OH or CI1 2 CH 2 01, 1is hydrogen, C 1 3 alkyl or CH 2 OH, and R 17 is hydrogen or methyl. 4) A compound of the formula according to any one of claims 1 to 3 in which R is a group: CH OHR 1 H C 17 wherein R7is hydrogen or methyl and R 8is hydrogen, methyl or ethyl. A compound of the formula according to any one of claims 1 to 4 in which Ar is a group: 0 C, CzA zI z or r WO 91/14688 icr/GB90/00424 6) A compound of the formula according to any one of claims 1 to selected from

2-(((1O-Methyl-l1OH-[l)benzothieno[3,2-bJindo1-3-yl)methyl)- amino)-2-methyl-1 ,3-propariediol, 2-(((1O-Methyl-10jj-[1Jbenzothieno[3,2-.bjindol-6-yl)methyl)amino)- 2- tethyl-1,3-propanediol, 2-Methyl-2-(((1-methyl-Oji-benzofuro(3,2-bk)indol-6-yl )methyl) amino)1 ,3-propanediol, 2-(((Benzofuro(5,6-.k)benzofuran-4-yl)methylamino)-2-methyl-1 .3- propanediol, 2-(((Benzofuro(5,6-.b)benzofuran-8-yl )methyl)amino)-2-methyl-1 .3- propanediol, 2-(((Benzofuro(5,6-.k)benzofuran-2-yl)methyl)amino)-2-methyl-1,3- propanediol, 2-(((Benzofuro(5,4-)beni±ofuran-4-yl)methyl)aiino)-2-niethyl-1 .3- propanediol, 2-(((Benzofuro(5,4-)benzofuran-9-y)uethyl)amino)-2-methyl-1e3- propanediol, 2-(((Benzofuro(5,4-b,)benzofuran-8-yl )methyl)amino)-2-methyl-1 .3- propanediol, 2-(((Benzofuro(5,4-,k)benzofuran-2-yl)methyl)amino-2-methyl-1 .3- propanediol, and monomethyl or manethyl ethers, esters, and addition salts thereof. 7) A process for the preparation of a compound of the formula (I) according to any one of claims 1 to 6 which comprises: the reduction of a compound Ar-CH=NR (11) or an appropriately protected derivative thereof followed by deprotection where appropriate. 51 (ii) the reduction of a compound ArCO.NHR or a derivative thereof in which the hydroxy groups are protected, followed by deprotection where appropriate, or (iii) the reaction of a compound ArCH 2 L with a compound NH 2 R wherein L is a leaving group; wherein Ar and R are as hereinbefore defined.

8. A novel chemical intermediate of the formula Ar CH=NR, ArCONHR or ArCH 2 L (wherein L is a leaving group and Ar and R are as hereinbefore defined), involved in the preparation of a compound of the formula

9. A pharmaceutical formulation which comprises a compound of the formula according to claim 1 together with a pharmaceutically acceptable carrier. A method for treatment of tumours, viral infection or bacterial infection in a subject comprising administering to the subject an effective amount of a compound of formula a pharmaceutically acceptable salt or ester thereof.

11. A method according to claim 10 for the treatment of leukaemia.

12. A method according to claim 10 for the treatment of solid tumours.

13. A compound of formula pharmaceutically acceptable ester or salt thereof substantially as herein described with reference to any one of examples 1 to 16. S:15431CGI26.5.93 52

14. A process of preparing a compound of formula pharmaceutically acceptable ester or salt thereof substantially as herein described with reference to any one of examples 1 to 16.

15. A formulation comprising a compound of formula (I) or a pharmaceutically acceptable salt or ester thereof substantially as herein described with reference to any one of the formulation examples A- E. Dated this 26th day of May 1993 THE WELLCOME FOUNDATION LIMITED By their Patent Attorney Griffith Hack Co. *o S 16431CG/20.B.93 INTERNATIONAL SEARCH REPORT International Application No PCT/GB 90 /00424 1. CLASSIFICATION OF SUBJECT MATTER (it several classiication symbols! apply, indicate all) I Accorditig to International Patent Classification (IPC) or to both National Classification and IPC 5C07 D 491/044, C 07 D 495/04, C 07 D 493/04, C 07 D 487/04 IC:A 61 K 31/40, A 61 K 31/38, A 61 K 31/34, II 11, FIELDS SEARCHED Minimum Documnentation Searched Classilication System I Clasalication Symbol* IPC C 07 D 491/00, C 07 D495/00, C 07 D493/00, Documentation Searched other thah Minimum Docu~mentation to the Extent that such Documents are Included In the Fields SearchedI Ill. DOCUMENTS CONSIDERED TO 51 RELEVANT Categoy Citatirmn of Document, 11 with lndir~atlcin, where appropriate, of the relevant passagest 1 Relevant to Claim No, %3 A EP, A, 0182608 (WELLCOME) 1,10 28 May 1986 see claims 1,2; pages 17-20 Special categories of cited documenta- to "T later document published affer the International filing dale document defining the general alati of the art which Is not or priority date and not in Conflict withI the application but conaicieled to be of pvrticular relelaonce cited to understand the principle or theory underlying the invention sefller document but published on or alter the IntetrnatIonal OX document ot Particular relevance: Ithe claimed Invention( filing date cannot be Considered novel or cannot be Considered to document which may throw doubts on priority claim(s) or Involve an Inventive step which is Cited to establish the publicationl date 0f another *Y document of Particular tolovancot; the claimed Invention Citation or other special I eason (as specified) cannot be Considered to Involve an Inventive Step when the document referring to an oral disclosure, use, exhibition or document is combined with one or moret other such docu- other means menta, such combination being Obvious to a P04r11, skilled document Published p tior to the Interinatlonal filing date but In the art later than the priority C!4% Claimed 6A" document member of the same patent family IV, CERTIFICATION Date of the Actual Completion 01 the International Search Date of Maeling of this International Search Reorm 21st November 1990 13 DEC 1990 Internastional Searching Authority Signature of AuthotIted Ons EUROPEAN PATENT OFFICE vrN.KJPR 1' Form PCTIISAI210 toeecond sheeit) (Jlanutary 11111S) INTERNATIONAL SEARCH REPORT International Application No PC:T/ GB 90 /00424 -2- 1. CLASSIFICATION OF SUBJECT MATTER (it several CISSSIflC3ion symbol& apply. indicate alit According to lnternativnai Patent Classifir.tion (IPC) or to both National Classification and IPC IP 5 (C 07 D 491/044, 307:00, 209;00), (C 07 D 495/04,

333-:QO 209!00) 07 fl 493/04, 307--000 117-710f) It. FIELDS SEARCHED Minimum Documentation Searched7 Ciassification System_ CassifIcalionSymbols___________ 1PC Documentation Searched other than Minimum Documentation to the Extent that such Documents are Inciuded In the Fields Searched 111, DOCUMENTS CONSIDERED TO BE Category Citation ot Document, It with Indication, where eppropriati, ot the relevant passages 13 Relevant to Claim No, 13 Speciai categories of cited documents, if later document published car the International tiling data document defining the Ceneral slate at the art which Is not or Priority date and not In conflict with the application but Considered to be of particular relevance C ited to understand the Principie or theory underlying the Invention earlier document but published on or after the International "X document of particular relevance-, the claimed Invention filing oate Cannot be considered r,.vsi or cannot be Considered to "L drocumeant which may throw double on priority cisim(s) or Involve an Inventive step which it cited to establish the Publication data of another document at particular relevance.' the claimed Invention Citation or other special resoon (s secified) cannot be considered to Involve an Inventive step when the document referring to an oral disclosure, use, eshibition or document is combined with one or more other such doCV* other means Monts, such combination being obvious to a person skilled document Published prior to the International filing date but In the Art. later then the priority date claimed document member of the same patent family IV. CERTIFICATION Data of the Actual Completion of the International Search Date of Maelling of this International Search Report International Searching Authority Signature ot Authorized Officer EUROPEAN PATENT OFFICE Form PCTJISA/21O rsecond sheet) ijanuary 119841 INTERNATIONAL SEARCH REPORT International Application No PCT/ GB 90 /00424 I. CLASSIFICATION OF SUBJECT MATTER (it several claaaific3tion symbols apply, indicate ll) According to International Patent Classification (iPC) or to both National Ciassification and IPC IP 5 (C 07 D 487/04, 209:00, 209:00),(C 07 D 495/04, 333:00, 209:00) 11. FIELDS SEARCHED Minimum Documentation Searched Ciassification System IClaaification Symbol& Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched a 111, DOCUMENTS CCJ4SIDERED TOmSE RELEVANT$ Category Citatio n of Document, I' with Indication. where appropriate, of the relevant passage* 12 Relevant to Claim No. 13 Special categories of cited documents: Is later document published car the International filing date "A"docmen deinig te gnerl tat 81theartwhih I no t priority d ate and not In conflict with the application but ""dcnde nin to e orl releae fteatwihI o ctd to understand the principle ar theory underlying the consdere tobe o pati~ust elevnceInvention earlier document but published on or ar the International document of particular reloeancot the claimed Invention filing data cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or Involve an inventive stop which Is cited to establish the publication date Of another "Y1" document of particular raeence-, the claimed Invention Citation or other special reason (as specified) cannot be considered to Invaivo an inventive stop when the document referring to an oral disclosure, use, exhibition or document Is combined with one or mote other such d*cu. other means ments. such combination being obvious to a person skilled document published prior to the Intrialionoli filing date but In the art. later than the priority dale claim"d "W document member of the saine patent family IV. UN~TIPICATION Date of the Actual Completion of the International Search Date of Malting of thia International Search Report International Searching Authority Signature of Authorized Offcer EUROPEAN PATENTr OFFICE form PCTIISA12t0 (second sheot) (Janueary 0W* International Application No. PCT/GB 90/0U424 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET V.X OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE This International search report has not been established in respect of certain claims under Article 17(2) for the following reasons: ir.i Claim numbers because they relate to subject matter not required to be searched by this Authority, namely: 2,L Claim numbers because they relate to parts of the International applicltion that do not comply with the prescribed require. ments to such an extent that no meaningful international search can be carried out, seciically. The intermediates to which claim 8 refers are not defined Claim because they are dependet chums and are not drafted In tccordnce with the second and third sentence of PCT Rule 6 4(a), OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING I This International Searching Authority found multiple Inventions In this International application as followel 1r As ill required additional search fees were timely paid by the applicant, this International sarch report covers all searchable claims of the nlternational application, 2.r As only some of the required addlllonal search feei were timely paid by the applicant, this International search rport covers only those claims of the International application for which feeI were paid, specifically claims: No required additional lserch fle* were timely paid by the appllcant Consequently, this International search report Is restricted to the Invention firnt mentioned In the claims It is covered by claim numberet 4 r As ill searchableclaims could be searched without effort justifying in additional fee, the Interntionl Searching Authority did not invite payment Of any additional e, Remark on Protest STYhe additional ellrch fees were accompanied by ipollcant's protest No protest accompanied the payment of additional search lles. Form PCTJISAt210 (supplemental ishet (January 1085) ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. GB 9000424 SA 35803 This annex lists the patent family members relating to the patent documents cited In the above-mtentioned international search report. The members are as contained in the European Patent Office EDP file on 04112/90 The European Patent Office is in no WaY liable for these particulars which are merely given for the purpose of information, Patent document Publication Patent family Publication cited In search report date member(s) 7date EP-A- 0182608 28-05-86 AU-B- 590527 09-11-89 AU-A- 4993085 22-05-86 CA-A- 1256114 20-06-89 JP-A- 2085252 26-03-90 J[P-A- 61158961 18-07-86 ror more detals about this annex ,see Official Journal of the European Patent Office, No. 121112