NZ256254A

NZ256254A - Polyamines as anti-cytomegaloviral agents

Info

Publication number: NZ256254A
Application number: NZ256254A
Authority: NZ
Inventors: Albert Stanley Tyms; Ronald Duayne Synder; Terry Lynn Bowlin
Original assignee: Merrell Dow Pharma
Priority date: 1992-10-05
Filing date: 1993-09-10
Publication date: 1997-04-24
Also published as: KR950703335A; AU4854793A; HUT72663A; HU9500982D0; CA2146319A1; WO1994007480A1; EP0662829A1; GB9220921D0; JPH08512279A

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number £56254 New Zealand No. 256254 International No. PCT/US93/08517 Priority Dat©(s): Compter Sp*ciflci»lion Fii^tl: 4;,f?3.. C**#c: PufcflcaAkm Dsrt#: P.O. Journal No: . NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION ™e °f '""Stives as anti-cytomegaloviral agents Polyamme derivatives, a Name, address and nationality of applicant(s) as in international application form: MERRELL DOW PHARMACEUTICALS INC., of 2110 East Galbraith Road, Cincinnati, Ohio 45215jp United States of America, (obca WO 94/07480 PCT/US93/08517 * 2562 54 •1- 5 POLYAMINE DERIVATIVES AS ANTI-CYTOMEGALOVIRAL AGENTS 10 BACKGROUND OF THE INVENTION Human cytomegalovirus (CMV) infections occur in a range 15 of severity, from a passive silent infection without consequences to diseases that are manifested by fever, hepatitis, pneumonitis, and after congenital or neonatal infection may result in severe brain damage, stillbirth, or perinatal death. 20 Cytomegalovirus infections frequently occur through out life, with the incidence of seropositivity most frequent in the elderly. Intrauterine infection with CMV has a high frequency of infection, with CMV the leading cause of 25 congenital viral infection, occurring in about 0.4 to 2.2% of all births. CMV infections can have serious effects in diseases in immunocompromised patients with a high frequency of morbidity and mortality, and is the major cause of sight-threatening infections in AIDS patients. 30 CMV is ubiquitously transmitted in all populations. However, risk to CMV infections varies considerably in different geographic areas. Infected individuals may transmit the virus in urine, saliva, cervical secretions, 35 semen, feces, milk, or infected blood and organs. WO 94/07480 PCT/US93/08517 -2- Molecular analysis of the DNA of CMV isolates reveals minor strain-specific differences that are useful markers in epidemiological investigation. CMV is highly host-specific and cannot be propagated in laboratory animals or 5 in most non-human cell cultures. Therefore, experiments designed to be predictive of CMV activity generally are restricted to human cell lines that have resident the CMV virus or have become infected with CMV. 10 It is well known that aliphatic polyamines, such as spermine and spermidine, play a role in cell growth and proliferation. These naturally occurring polyamines are found in animal cells and are produced in a biosynthetic pathway involving putrescine as a precursor. Putrescine is 15 formed by a decarboxylation of ornithine by ornithine decarboxylase (ODC) and this is a highly regulated stage in the biosynthesis of spermidine and spermine. The first indication that polyamines may have a role in 20 certain viral function was discovered when putrescine and spermidine were found to be present in the bacteriophage T2. Later the polyamines, putrescine and spermidine, were found in other viruses, including human cytomegalovirus, poxvirus, vaccinia virus, and human type 5 adenovirus to 25 name a few. However, the fact that polyamines are present in several animal viruses does not directly indicate a role for polyamines in viral replication. Unusually, CMV infection of cells in vitro is antagonised by inhibition of ODC by specific inhibitors of the enzyme (Tyms et al., J. 30 Antimicrobial Chemotherapy 22, 403-427 1988). The human cytomegaloviruses are a subgroup of agents within the herpes group of viruses, all of which have the propensity for remaining latent in man. No specilic 35 • therapy is generally available for CMV infections. Although ganciclovir and foscovir are both licensed for"use against some infections in the immunocompromised host, both WO 94/07480 PCT/US93/08517 25 6 25 4 compounds can induce major side effects. It is a feature of the present invention that the CMV agents afford less cell and/or tissue toxicity compared to other agents in use. 5 CMV, compared to other herpes viruses, herpes simplex type 1 and type 11 and varicella-zoster virus, is relatively resistant to the action of acyclovir. Research in this area is primarily focused toward identifying agents 10 which would be therapeutically effective in humans. A further feature of the present invention is that the compounds described herein provide a CMV agent that selectively acts against CMV compared to other Herpes viruses. 15 It has now been found that certain polyamine derivatives are effective therapeutic agents when used against cell cultures infected with CMV. An object of the present invention is the use of the describe polyamine 20 derivatives as therapeutic agents against CMV. SUMMARY OF THE INVENTION AND DETAILED DESCRIPTION 25 Synthesis of compounds of the present invention as anticancer agents has been described in part in U.S. Patent No. 5109024 and EP 311 068 which are herein incorporated by reference. Synthesis of compounds of the present invention as potentiating cell-mediated immunity has been described in 30 part in EP 399 519 which is herein incorporated by reference. This invention relates to methods of use of certain polyamine derivatives in the treatment of patients suffering from CMV disease states and to pharmaceutical compositions 35 containing these polyamine derivatives. _ 4 - 256 254 More specifically, this invention relates to use of a compound of the formula (I): HN-Z-NH- (CH2) m-NH-Z-NH I I (I) R R for the manufacture of a medicament for inhibiting a cytomegaloviral infection or for the treatment of patients suffering from a cytomegalovirus (CMV) disease state; wherein said compound of the medicament is at an effective concentration of less than .1 micromolar; and wherein Z is a C2-C0 saturated straight or branched chain alkyl moiety; m is 7 or 8; each R group independently is hydrogen, a C!-C6 saturated or unsaturated hydrocarbyl, or - (CH2) x-(Ar)-X wherein Ar is phenyl or napthyl, X is H, C^Q alkoxy, halogen or Cx-C^ alkyl, wherein x is an integer 0, 1, or 2; with the proviso that both R groups cannot be hydrogen or if m is 7 then R cannot be benzyl; or said compounds of formula I can be a pharmaceutical^ acceptable acid addition salt thereof. As a furtner aspect of the present invention, it has been found that compounds of formulae (I) provide an anti-CMV effect in patients in need thereof. Compounds of formulae I generally produce effective treatment without similar delayed toxicity effects produced by other agents. In a still further aspect the present" invention provides a pharmaceutical composition for use in the treatment of a cytomegaloviral infection or cytomegalovirus (CMV) disease state in a patient in need thereof comprising an effective N.Z. P.VYM I 9 FEB 1997 R-ictr.:: concentration of less than .1 micromolar of a compound of formula HN-Z-NH- (CH2) w-NH-Z-NH R R wherein Z is a C2-C6 saturated straight or branched chain alkyl moiety and each Z is the same; m is 7 or 8; each R group independently is hydrogen, a C!-C6 saturated or unsaturated hydrocarbyl, or -(CH2)x-(Ar)-X wherein Ar is phenyl or napthyl, X is H, Cj-Cg alkoxy, halogen or Ci-C4 alkyl, wherein x is an integer 0, 1, or 2; with the proviso that both R groups cannot be hydrogen or if m is 7 then R cannot be benzyl; or said compounds can be a pharmaceutically acceptable acid addition salt thereof. The present invention relates to the use of compounds of the formula (I) for CMV infections, or more specifically to the compounds of formula (la) and (lb). The compounds of formula (la) are of the formula: wherein Zx is a branched chain C2-C6 alkyl moiety; m is 7 or 8; and each R group independently is hydrogen, a Ci-C6 saturated or unsaturated hydrocarbyl, or -(CH2) x-(Ar)-X wherein Ar is phenyl or napthyl, X is H, Cj-Cg alkoxy, halogen or Cj-C^ alkyl, wherein x is an integer 0, 1, or 2; with the proviso that both R groups cannot be hydrogen or if m is 7 then R cannot be benzyl; or said compounds of formula la can be a pharmaceutically acceptable acid addition salt thereof. The compounds of formula (lb) are of the formula: HN-Zx-NH- (CHzh-NH-Zi-NH (la) R R (followed by page 5a) HN-Z2-NH- (CH-.) ln-NH-Z?-NH R R 256254 wherein Z2 is a straight chain (C2-C6) alkyl moiety; m is 7 or 8; and each R group independently is hydrogen, a C!-C6 saturated or unsaturated hydrocarbyl, or -(CH2) x-(Ar)-X wherein Ar is phenyl or napthyl, X is H, Ci-Cc. alkoxy, halogen or Ci-C4 alkyl, wherein x is an integer 0, 1, or 2; with the proviso that both R groups cannot be hydrogen or if m is 7 then R cannot be benzyl; or said compounds of formula lb can be a pharmaceutically acceptable acid addition salt thereof. The center alkylene moiety of compounds of the formula (I) is a saturated, straight-chain hydrocarbyl radical comprising 7 or 8 carbon atoms, i.e., "(CH_)" or "(CH,)/. A., used herein, the term "Z" is understood to mean a saturated hydrocarbylene radical of straight (Z.) or branched-chain WO 94/07480 PCT/US93/08517 * -6- atoms including, but not limited to, -CH2CH2-, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, -CH2(CH2)4CH2-, -CH(CH3)CH2-, -CH(CH3)CH2CH2~, -CH2CH(CH3)CH2- and the like. In those instances where R is an unsaturated hydrocarbyl 5 moiety, such compounds include straight, branched, or cyclized hydrocarbyl moieties such as -CH2CH=CH2, -CH2CH2CH=CH2, -CH2CH£3CH, and -CH2CH=C=CH2. Compounds of the formula (I) can be used according to 10 the present invention as pharmaceutically acceptable acid addition salts thereof. The term "pharmaceutically acceptable acid addition salt" encompasses both organic and inorganic acid addition salts including, for example, those prepared from acids such as hydrochloric, hydrofluoric, 15 sulfuric, sulfonic, tartaric, fumaric, hydrobromic, glycolic, citric, maleic, phosphoric, succinic, acetic, nitric, benzoic, ascorbic, p-toluenesulfonic, benzenesulfonic, naphthalenesulfonic, propionic, and the like. The hydrochloric acid addition salts are preferred. 20 The selection and preparation of pharmaceutically acceptable non-toxic acid addition salts are within the ability of one of ordinary skill in the art utilizing procedures and techniques well known and appreciated in the art. 25 In general, the compounds of formula (I) may be prepared by chemical reactions analogously known in the art. The choice of any specific route of preparation is dependent upon a variety of factors. For example, general 30 availability and cost of the reactants, applicability of certain generalized reactions to specific compounds, and so forth, are all factors which are fully understood by those of ordinary skill in the art and all contribute to the choice of synthesis in the preparation of any specific 35 compound embraced by formula (I). WO 94/07480 PCT/US93/08517 -7- A preferred route for the synthesis of compounds of the formula (I) wherein Z is -CH2CH2CH2~, but also applicable by analogy for other compounds of formula (I) wherein Z is an alkyl-substituted propyl group (such as -CH(CH3)CH2CH2-), is presented in Reaction Scheme A. Reaction Scheme A 10 H2N(CH2)mNH2 + h2c=chcn EtOH NC (CH2) 2NH (CH2) mNH (CH2) 2CN 2 15 2 + h2 Pt02 H2N (CH2) 3NH (CH2 ) mNH (CH2) 3NH2*4HC1 HCl/AcOH ° ° Na0H/H20 HN(CH2)3N-(CH2)mN-(CH2)3NH ► I 2 + o-c-o-c-o thf 20 | | y y Boc Boc Boc Boc 4 4_+ Kt-BuO ^ RN(CH2)3N(CH2)mN(CH2)3NR 25 dmf n ' Boc Boc Boc Boc 5 Et2° ^ - RNH(CH2)3NH(CH2)mNH(CH2)3NHR 4HC1 EtOH 30 6 35 WO 94/07480 PCT/US93/08517 -8- wherein m is 7 or 8, R is as defined in formula (I) except when R is X-(Ar)-(CH)x, x cannot be zero. In reaction scheme A Boc is the t-butoxycarbonyl protecting group, and 5 Y is tert-butyl. The initial step of this process entails an N-alkylation of the appropriate diamine with 2 equivalents of acrylonitrile by heating reactants, either in a suitable 10 solvent or neat, according to standard conditions well known in the art. The resulting cyano derivatives (2) are chemically reduced by reaction with hydrogen in the presence of a catalyst (Pt02) in a suitable solvent, such as acetic acid containing 8 equivalents of hydrochloric or 15 hydrobromic acid, to produce the resulting hydrohalic salts according to standard procedures well known in the art. Of course, other reducing systems, e.g., reduction with lithium aluminum hydride, may also be utilized to produce compounds of formula (3). Following the preparation of 20 these compounds the hydrohalic salts are neutralized with base and the nitrogen atoms are protected, preferably with di-t-butyldicarbonate according to standard operating conditions well known in the art. The tetra N-protected amines (4) are alkylated by reacting (4) with the 25 appropriate alkyl halides (chloro or bromo) in the presence of potassium butoxide according to standard alkylation procedures well known in the art. When it is desired to provide compounds of the formula (I) wherein both R groups are the same, about 3 equivalents of the alkyl halide is 30 reacted. When it is desired to provide compounds of the formula (I) wherein the R groups are not the same, monosubstitution of compounds of formula (4) is effected by reacting about 1 to about 1.5 equivalents of the alkyl halide with subsequent isolation of the monosubstituted 35 compound according to standard procedures well known in the art and optionally further reacting the monosubstituted4 WO 94/07480 PCT/ US93/08517 -9- compound with the desired different alkyl halide. Following alkylation the N-protective groups of compound (5) are removed by standard procedures, e.g., treatment with acid, preferably HC1, in the presence of a suitable 5 solvent or solvent system, e.g., diethyloxide in ethanol, to obtain the desired products (6). Alternatively, compounds of formula (3) and their otherwise prepared homologs may be subjected to a reductive 10 alkylation using an appropriate aldehyde. The reduction is effected by hydrogenation in the presence of Pt02 or sodium cyanoborohydride according to well known procedures. This procedure does not generally require protection of the nitrogen atoms of the intermediates. 15 A preferred route for the preparation of compounds of formula (I) wherein Z is -CH2(CH2)2CH2~, but which is also applicable by analogy to those compounds wherein Z is any straight chain, is presented in Reaction Scheme B. 20 25 30 35 WO 94/07480 -10- PCT/US93/08517 f 20 25 30 Reaction Scheme B H2N(CH2)nOH Rj-CHO RHN(CH2)nOH Pt02/H2 7 8 N-Protect RN (CH2 ) nOH MsC1 RN-(CH2)nOMs 10 Boc pyridine Boc NH(CH2)mNH M I | Boc Boc ! 5 R-N-(CH2)n-N-(CH2)m-N-(CH2)n"N-R Boc Boc Boc Boc 35 ii wherein m is 7 or 8, n is an integer 2 to 6 describing a straight chain alkylene moiety, Boc is the t-butoxycarbonyl protecting group, R is as defined in formula (I), Ms is mesyl and Ri is hydrogen, methyl or ethyl. This synthesis is initiated by reductive alkylation techniques well known in the art using an amino alcohol (7) and an appropriate aldehyde to form R- substituted amino alcohols (8). The nitrogen atom is protected, preferably with di-t-butyldicarbonate, according to standard operating conditions well known in the art, to yield the N-protected amino alcohols (9) which are converted to their mesylates (10) by known reaction conditions, e.g., reaction with mesylchloride in the presence of pyridine, preferably in a solvent such as CH2C12. WO 94/07480 PCT/US93/08517 -lilt) The resylate is subjected to alkylation with an N-protecceO diamine (i.e., BocNH(CH2)mNHBoc) in the presence of potassium t-butoxide in a solvent such as DMF. The so-produeed tetra N-protected tetramines (11) are deprotected as in Scheme A. In essence the foregoing reductive alkylation, N-protection, mesylation, alkylation and ^protection procedures all employ techniques and reaction conditions which are well known in the art. In those instances wherein it is desired to prepare compounds of formula (I) wherein Z is -CH2-CH2-, it is preferred to employ Reaction Scheme C to obtain the necessary intermediates (14) which could be subjected to 15 the alkylation procedures discussed above in Scheme A wherein m is 7 or 8. Reaction Scheme C 20 H2NCH2CH2NH2+ Br (CH2)mBr ► H2N(CH2)NH(CH2),nNH(CH2) 2NH2 12 13 14 The foregoing N-alkylation entails the reaction of an appropriate dihaloalkane (13) with excess quantities (lOx) of ethylene diamine (12) by heating the reactants at reflux temperatures in a suitable solvent, e.g., ethanol. Preparation of the final products bearing the desired R 30 substituents on the terminal nitrogen atoms of the intermediates (14) may be effected by N-protection, alkylation with the appropriate alkyl halide, and deprotection in an analogous manner to that described for Reaction Scheme A. Preferably, the alkylation can be 35 carried out by the reductive alkylation procedures without N-protection as alternatively described for Reaction Scheme A. WO 94/07480 PCT/US93/08517 -12- In general, compounds of the formula (la) can be prepared in an analogous manner to that described in Reaction Scheme D. ^ Reaction Scheme D H2N-Z1-OH Rj-CHO ^ R-N-Zi-OH Pt02/H2 10 15 15 20 25 30 H 16 1 e; or ifi N-Protect^ R-N-Zi-OH — — ► | Boc 17 17 + MsCl pyridine" R-N-Zi-OMs Boc 18 18 + HN-(CH2)m-NH ► R-N-Zi~NH- (CH2)m-NH-Zi-N-R II II II Boc Boc Boc Boc Boc Boc 19 19 + HC1 Et2° » NH-Z!-NH-(CH2)m-NH-Zi-NH • 4HC1 EtOH I I R R 20 35 WO 94/07480 PCT/US93/08517 -13- wherein m is 7 or 8, R and Zi are as generically defined for formula (la), and Ri is hydrogen. The appropriate primary amino alcohol (15) containing a 5 branched chain hydrocarbyl moiety (i.e., Zi) is prepared by standard procedures well known in the art. If desired, the primary amine can at this point be converted to a secondary amine (16), by a reductive alkylation with the appropriate aldehyde. The amino alcohol is reacted as described in 10 Reaction Scheme A by standard conditions well known in the art to effect protection of the amines with an appropriate N-protecting group such as Boc (17). The mesylates of the N-protected amino alcohols (18) are prepared and are alkylated with the appropriate N-protected diamine (i.e., 15 BocNH(CH2)mNHBoc) using standard procedures well known in the art as discussed for Reaction Scheme B. The so-produced tetra N-protected tetramines (19) are deprotected as in Scheme A to yield compounds of the formula (la). In essence, the foregoing reductive alkylation, N-protection, 20 mesylation, alkylation and deprotective procedures all employ techniques and reaction conditions which are well known in the art. Where it is desired to provide a compound of the 25 formula (la) wherein each R group is not the same, the substituted mesylates (18) are prepared separately and monoalkylation of the appropriate N-protected diamine (i.e., BocNH(CH2)mNHBoc) is effected by reacting the diamine with about 1.0 to 1.5 equivalents of one of the mesylates 30 (18) with subsequent isolation of the monosubstituted compound and optionally further reacting the monosubstituted compound with the desired differently substituted mesylate (18). 35 WO 94/07480 -14- PCT/US93/08517 i In those instances in which it is desired to prepare compounds of the formula (la) wherein Zi is an alkyl-substituted propylene group such as *-CH(Q)CH2CH2- wherein Q 5 is a saturated alkyl radical comprising 1 to 3 carbon atoms of straight or branched chain configuration, Reaction Scheme E can be used to obtain intermediates of the formula (25) which can be subjected to alkylation of the N-terminal groups in a manner analogous to that described in Reaction 10 Scheme A prior to de-protection, 15 20 25 30 35 WO 94/07480 PCT/US93/08517 § 10 15 20 25 30 -15- Reaction Scheme E h2n-(ch2)m-nh2 + 0 II 0-c 1 h H2/Pt02 EtOH hn(ch2)m-nh I ch2 I 0 I ch2 I 0 21 + h2c=ch-c MeOH 21 O O II II c-{ch2)2-n-(ch2)m-n-(ch2)2-c 22 23 nh2oh • hc1 NaOH q ch2 ch2 I I 0 0 23 n-oh n-oh 11 II c-(ch2 )2-n-(ch2)m-n-(ch2)2-c I I I I q ch2 ch2 q 0 0 24 24 lah/aici3 h2n-ch-(ch2)2-n-(ch2)m-n-(ch2)2-ch-nh2 ► | I I 1 thf « q ch2 I 0 ch2 I 0 25 wherein m is 7 or 8, 0 is phenyl, and Q is as defined above. The initial step of the process entails a reductive 35 alkylation wherein the appropriate diamine is reacted with hydrogen gas and 2 equivalents of benzaldehyde in the * presence of a catalyst such as Pt02 to yield the N-protected WO 94/07480 PCT/US93/08517 r -16- diamine (21) under standard conditions well known in the art. The N-protected diamine (21) is then alkylated with 2 equivalents of the appropriate vinyl ketone (22) in a suitable solvent such as methanol using standard 5 techniques. The resulting N-substituted diamine (23) is further reacted under standard conditions with hydroxylamine hydrochloride in the presence of base such as NaOH in a suitable solvent such as ethanol/water. The resulting oximes (24) are reduced to the corresponding N-10 protected di-primary amines (25) by reaction with lithium aluminum hydride (LAH) in the presence of AICI3 in a suitable solvent such as THF according to standard procedures. The N-protected di-primary amines (25) can be further alkylated with an appropriate aldehyde prior to 15 deprotection in a manner analogous to that described for Reaction Scheme A. Compounds of formula (la) wherein Zi is *-CH(CH3)CH2CH2- or *-CH(C2H5)CH2CH2- are generally preferred 20 in their end-use application. Compounds of formula (la) wherein each R group is the same moiety are also preferred. Compounds of formula (la) wherein each R group is methyl or ethyl are particularly preferred. 25 Of course, it is appreciated that in those instances wherein a compound of formula (la) possesses one or more chiral centers, the individual stereoisomers as well as mixtures of stereoisomers are included within the scope of the present invention. For example, the following 30 compounds are specifically included within the scope of formula (I) and (la): (R,R)-N,N'-bis[3-(methylamino)butyl]-l,7-diaminoheptane (S,S)-N,N'-bis[3-(methylamino)butyl]-1,7-diaminoheptane (R,S)-N,N'-bis[3-(methylamino)butyl]-l,7-diaminoheptane. 35 A preferred method for preparing compounds of formula [I] wherein -(CH)x-(Ar)-X represents phenethyl or WO 94/07480 PCT/US93/08517 -17- naphthylethyl, particularly wherein Z is 3 and m is 8, is the reaction of an aroylchloride according to the method depicted in Reaction Scheme F wherein depicted in Scheme F Bn is benzyl, $ is phenyl, and LAH is lithium aluminum hydride. REACTION SCHEME F 10 15 H2N(CH2)3N-Bn (CH2)8 H2N(CH2)3N-Bn (f>CH2C-CI

[27]

[28] 20 25 30 O ct>CH2 C-CNH(CH2)3N-Bn (CH^s <J)CH2C-GCNH(CH2)BN-Bn O

[29] H2 Pd/C LAH Reduction <t>CH2CH2NH(CH2)3N -Bn (CH2)8 <|>CH2CH2NH(CH2)3N-Bn [30] <J>CH2CH2NH(CH2)3NH (CH2)8 35 <J)CH2CH2NH(CH2)3NH * 131] PCT/US93/08517 18- As stated above, the foregoing reaction is a preferred method for the preparation of one particular compound which entails N-alkylation of a partially protected intermediate 5 [27] with an arylacetyl chloride [28] in the presence of triethylamine, using an inert solvent, to form an amide [29] which is chemically reduced, preferably with LAH, and the resulting product [30] is catalytically de-benzylated (H2Pd/C) to form the desired end product [31]. These steps 10 entail reaction techniques and procedures well known in the art. Of course the same reaction scheme can be applied for the preparation of other compounds of formula [I]; adoption of the technique being with the usual caveats well understood by those of ordinary skill in the art. 15 In those instances wherein -(CH)x-(Ar)-X represents an aromatic moiety (X-phenyl or X-naphthyl) which is attached directly to the terminal nitrogen atoms (i.e., x is zero) then such compounds may be prepared according to the 20 general reactions of Reaction Scheme G. 25 30 WO 94/07480 35 4 WO 94/07480 i PCT/US93/08517 -19- 10 C|>NH(CH2)2CN

[32] REACTION SCHEME G LAH or H2 4>NH(CH2)3NH 133] N-Protect 15 20 4>n(ch2)3nh Boc Boc [34] l(CH2)nl NaH DMF <|)N(CH2)3N-Boc Boc (CH2)m <t>N(CH2)3N-Boc Boc

[35] 25 30 35 The foregoing reaction scheme depicts the preparation of compounds wherein Ar is phenyl, the first step of which is a LAH reduction effected according to procedures published is the art (Bui. Soc. Chim. Fr., Part 2, 165-7 (1979)). Of course this reaction scheme can be expanded to include napthyl and X-substituted intermediates which will not be adversely affected by the reaction conditions. Preferably the N-protection uses the t-butoxycarbonyl protecting groups which are put on and taken off according to standard techniques already mentioned hereinabove. The N-protected compounds are alkylated by reaction with an appropriate dihaloalkane using standard and well known * procedures. WO 94/07480 PCT/US93/08517 -20- In those instances wherein it is desired to prepare compounds of formula [I] which contain an unsaturated hydrocarbyl moiety, i.e., acetylenic, allenic, or allylic 5 moiety-containing compounds, it is preferred to use the techniques of Reaction Scheme H wherein as shown R2 is an appropriate unsaturated hydrocarbyl moiety, Bn is benzyl, MsCl is methanesulfonyl chloride, and Boc is the t-butoxycarbonyl protecting group. 15 20 25 30 35 WO 94/07480 PCT/US93/08517 BnNH(CH2)mNHBn [363 -21- REACT10N SCHEME H CI-Z-OH ^ nBnOH Nai HO-Z-N(CH2)mN -Z-OH Bn Bn

[37] 10

[37] Debenzylate HO-Z-NH(CH2)mNH-Z-OH 15

[38] N-Protect HO-Z-fs

[38] (CH2)mNH-Z-OH Boc Boc 20

[39] MsCI CH2CI2 Pyridine

[39] MsO-Z-N(CH2)mN -Z-OMs Boc Boc 25 30

[40]

[41] R2NHBoc NaH, Nal DMF HCI/EtOH

[40] R2N-Z-N (CH2)mN -Z-N R2 Boc Boc Boc Boc

[41] R2NH-Z-NH(CH2)mNH-Z-NHR2

[42] 35 WO 94/07480 PCT/US93/08517 i -22- In the foregoing reaction a dibenzylated diamine [36] is N-alkylated by a simple displacement reaction to form compounds [37] which are sequentially benzylated [38] and N-protected. These steps are effected according to well 5 known and standard procedures. The resulting bishydroxyamino-alkanes [39] are mesylated and the mesylates [40] are alkylated with two equivalents of an N-protected amine bearing an appropriate unsaturated hydrocarbyl moiety, e.g., N-(t-butoxycarbonyl)-2,3-10 butadienylamine. A so-obtained tetra protected tetramine [41] is then readily de-protected to produce the desired compounds [42]. In those instances wherein it is desired to convert an 15 alkylthio substituent to one of its higher oxidation states the alkyl thioether is treated with a peracid according to known conditions. Suitable oxidizing agents are H2O2 and NaI04, but meta-chloroperoxybenzoic acid is preferred. In effecting the oxidation to a sulfinyl derivative 1 molar 20 equivalent (per alkylthioether moiety) is used and 2 molar equivalents of the peracid will yield the sulfonyl derivatives. The oxidations are effected at temperatures of about OoC to room temperature in solvents which themselves are not susceptible to oxidation. Preferred 25 solvents are CH2C12, CHCI3, acetic acid, and ethyl acetate. The foregoing reaction scheme I depicts a method of preparation of the compounds of formula I. In scheme I the following abbreviations are used: Ts is a tolunesulfonyl 30 substituent, DEAD is diethyl azodicarboxylate, THF is tetrahydrofuran, TFA is trifluoroacetic acid, and Boc is the t-butoxycarbonyl protecting group. It is intended that the reaction scheme is illustrative, and not limiting, of the chemistry depicted, wherein it is understood the 35 reaction scheme can be expanded to include variations of m, Z, and R as defined in formula I. For instance, the * reaction scheme can be expanded to include the variations WO 94/07480 PCT/US93/08517 -23- of Ts protecting group by various other arylsulfonyl groups beside toluenesulfonyl, such as mesitylenesulfonyl, benzylsulfonyl and the like. 5 Initial step (a), of scheme I, is the formation of compound [44] by reaction of N-t-butyloxycarbonyl-p-tolunesulfonamide with a chloroalkyl alcohol in the presence of triphenylphosphine (TPP) and DEAD to form the compound of [44]. Reactions involving step (a) is 10 especially preferred to step (g) when Z=4. Step (b) involves the reaction of two equivalents of Compound [44] with a suitably protected diaminoalkane, e.g., as shown a di-tolsyl protected alkylydiamine 15 (TsNH(CH2)mNHTs) can be used to form intermediate [45]. An alternative route (step (g)) for forming intermediate [45] can be accomplished by reaction [43] with the approximately 1 equivalent of the alkyldiol shown in 20 the presence of TPP and DEAD in a suitable solvent, such as THF to form intermediate [49]. Intermediate [49] can then be reacted with a protected diaminoalkane, e.g., as shown, the ditosyl protected alkyldiamine (TsNH(CH2)mNHTs), to form intermediate [45]. This route of synthesis, step (g), is 25 suitable in those instance when Z is not 4. Further reaction of compound [45] can occur by deprotection of the Boc protecting groups with a suitable acid, as for example with trifluoroacetic acid in an 30 aprotic solvent to form intermediate [46]. Further deprotection with a stronger acid, e.g. HBr, can be used to form structures of compound [48] where in the substituted R group is hydrogen. Rather than a two step treatment of acid by steps (c) and (d), the more general route of 35 deprotection would be to directly treat [45] with a strong acid, e.g. HBr, to directly form [48] wherein R is" * hydrogen. WO 94/07480 PCT/US93/08517 -24- Optional substitution of compound [48] wherein R is desired to be other than hydrogen, can be accomplished in either of two ways. First, compounds of formula [45] can 5 be reacted with a R-alkyl alcohol in the presence of TPP and DEAD in an appropriate solvent (step e). Alternatively, intermediate [46] can be reacted with a R-alkyl halide in the presence of hydride ion and sodium iodide to give the compound of formula [47]. Compounds of 10 formula [47] can then be deprotected upon treatment with acid, to form the compounds of formula [48], wherein R is other than hydrogen. 15 20 25 30 35 REACTION SCHEME I TsN(CH2)z ■ HO(CH2)zCI TsNH(CH2)mNHTs lB0(: TsNHBoc »- TsN(CH2)2CI (a) 1800 (b) TPP, DEAD, NaH, DMF, Nal TsNfCH^z* LBoc 43 THF 44 -NTs tca TsN(CH2)2 j H (CH2)m —>- TsNfCH^ H NTs -NTs I -NTs HO(CH2)ZOH TsNfCH^OH LBoc TsNH^H^mNHTs TPP. DEAD, THF (h) TsN—(ch-;7— I R Ts N — (CH^ (d) HBr 47 HONfCH^z ■*" N H I (CH^m i RHNCCH^z —' N H 48 i to ui I WO 94/07480 PCT/US93/08517 { -26- 10 15 25 30 35 REACTION SCHEME J (a) HCHO H2N NH-(CH)m-NH NH2 —► SOS HN^ N-(CH2)m-N ^^H (b) Ac20 . .N N-(CH2)m-N N. . (C) LAH (d) CH3CH2-N N^CH^m-N N-CH2CH3 HCI CH3CH2-NH NH-(CH2)m-NH NH-CH2CH3 54 The foregoing reaction scheme J depicts the preparation of N,N1-Bis[3-(ethylamino)propyl]1,7-heptanediamine WO 94/07480 PCT/US93/08517 -27- tetrahydrochloride. Of course, certain parts of the reaction scheme can be expanded to include the variations of m and R of formula I, however, Z is generally limited to being propyl, wherein Z is 3. The R group introduced in 5 scheme J is shown as ethyl, but the reaction scheme is not limited only to ethyl derivatives but may include those R groups of formula I to form corresponding R substituted derivatives. 10 The initial step (a) of scheme J is a protection of the nitrogens of formula [50] by reaction with aqueous HCHO using standard and well known procedures to give the corresponding diaza ring systems in compound [51]. 15 In step (b) the N-protected amines [51] are alkylated by reacting with the appropriate acid anhydride according to standard alkylation procedures known to those skilled in the art. When it is desired to provide compounds of the formula (I) wherein both R groups are the same, about 3-6 20 equivalents of the acid anhydride is reacted. When it is desired to provide compounds of the formula (I) wherein the R groups are not the same, monosubstitution of compounds of formula [52] is effected by reacting about 1 to about 1.5 equivalents of the acid anhydride with subsequent isolation 25 of the monosubstituted compound according to standard procedures well known in the art and optionally further reacting the monosubstituted compound with the desired different acid anhydride or alkyl or aryl halide. 30 The resulting acetylated derivatives of [52] are then chemically reduced (step c) by reaction with lithium aluminum hydride in anhydrous conditions according to standard procedures well known in the art. Of course, other reducing systems, e.g., reduction with H2 and Pt02, 35 may- also be utilized under appropriate conditions to produce compounds of formula [53]. " * WO 94/07480 PCT/US93/08517 -28- Following reduction, the N-protective groups of compound [53] are removed by standard procedures, e.g., treatment with acid, preferably HC1, in the presence of a suitable solvent or solvent system, to obtain the desired 5 products of formula [54]. As is well known in the art of pharmaceutical inventions wherein generic classes of compounds are involved, certain specific compounds are more effective in 10 their end use applications than other members of the generic class. The following compounds are preferred in the method of use described by the present invention: 1,18-Bis[(phenyl)methyl]-l,5,14,18-tetraazaoctadecane* 4HC1, 15 1,20-Bis[(phenyl)methyl]-l,16,15,20-tetraazaeicosane*4HCl, N,N'-Bis(3-aminobutyl)-1,8-octanediamine, N,N'-Bis(3-ethylamino)butyl]-l,7-diaminoheptane tetrahydrochloride, 1,4,13,16-tetra(t-butoxycarbonyl)-20 l,4,13,16tetraazahexadecane, 1,18-Bis[(2-phenyl)ethyl]-l,5,14,18-tetraazaoctadecane* 4HC1 , 1,18-Bis(phenyl)-1,5,14,18-tetraazaoctadecane, 1,18-Bis(2,3-butadienyl)-1,5,14,18-tetraazaoctadecane 25 tetrahydrochloride. Especially preferred are the following compounds: 3,7,15,19-tetraazaheneicosane tetrahydrochloride, 3,17-dimethyl-2,6,14,18-tetraazanonadecane 30 tetrahydrochloride, and 4,16-dimethyl-2,6,14,18-tetraazanonadecane tetrahydrochloride. 35 WO 94/07480 PCT/US93/08517 -29- Based upon standard laboratory experimental techniques and procedures well known and appreciated by those skilled in the art, as well as upon comparisons with compounds of 5 known usefulness, the compounds of formula (I) can be used in the treatment of patients suffering disease states for CMV infections. Of course, one skilled in the art will recognize that 10 not every compound of formula (I) will be effective against each of the CMV disease states, and that selection of the most appropriate compound is within the ability of one of ordinary skill in the art and will depend on a variety of factors including assessment of results obtained in 15 standard animal tumor models. As used herein, the term "patient" refers to a warmblooded animal such as a mammal which is afflicted with a neoplastic disease state. It is understood that dogs, 20 cats, rats, mice, horses, bovine cattle, sheep, and humans are examples of animals within the scope of the meaning of the term. The term "cytomegaloviral infection" as used herein 25 refers to an abnormal state or condition characterized by an active or latent CMV infection or state whenever the patient has virus residing active or dormant in the patients tissues, fluids, or body cavities. 30 Treatment of a patient afflicted with a CMV disease state comprises administering to such patient an amount of a compound of the formula (I) which is therapeutically effective in controlling the symptomatic and/or associated viral states of CMV beyond that expected 35 in the absence of such treatment. WO 94/07480 PCT/US93/08517 -30- As used herein, "controlling CMV infection" refers to slowing, interrupting, arresting or stopping a CMV infection and does not necessarily indicate a total elimination of the virus. It is believed that the 5 prolonged survivability of a patient with the instant compounds indicates control of a CMV infection. In effecting treatment of a patient afflicted with a CMV infection or prophylatic treatment to prevent infection 10 a compound of formula (I) can be administered parenterally in any manner which makes (I) bioavailable in effective amounts including for example, by intraperitoneal (i.p.), subcutaneous (s.c.), or intravenous (i.v.) injection. Administration by intravenous injection is preferred. 15 A therapeutically effective dose or amount can readily be determined by the attending diagnostician and is a function of a number of factors including, but not limited to, the species of mammal, its size, age and general 20 health, the specific CMV infection involved, the stage of the CMV infection, the compound selected and mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, and use of concomitant medication. The correct amount for any 25 specific situation can be readily determined by those skilled in the art using conventional range finding techniques and analogous results observed under other circumstances. A therapeutically effective amount of (I) will vary from about 1 milligram per kiloqram of body 30 weight per day (mg/kg/day) to about 500 my/kg/day and preferably will be about 5 mg/kg/day to about 50 mg/kg/day. It is believed that compounds of the formula (I) administered at the above doses to a patient suffering from a CMV infection are therapeutically effective in 35 controlling the growth of one or more disease states a patient may have so as to prolong the survivability of the WO 94/07480 PCT/US93/08517 -31- patierit beyond that expected in the absence of such treatment. Another embodiment of the present invention relates to 5 pharmaceutical compositions for parenteral administration for compounds of the formula (I). These pharmaceutical compositions comprise a therapeutically effective amount of one or more compounds of the formula (I) in an admixture with one or more pharmaceutically acceptable excipients, "0 Such compositions are prepared in conventional manner well known in the art of pharmaceutical science. The amounts of the active ingredient(s) in a unit dosage form and the dosage regimen are adjusted to provide a sustained pharmacologic effect at the dose regimen selected. 15 Pharmaceutically acceptable excipients are substances that are chemically inert to the active compound(s) and have no detrimental side effects or toxicity to mammals under the conditions of use. Suitable excipients include 20 solvents such as water, alcohol, and propylene glycol, surface active agents, suspending agents, lubricants, flavors, colorants, and the like. Such carriers and excipients are known to those in the art and are disclosed, for example, in texts such as Remington's Pharmaceutical 25 Manufacturing, 13th Edition, Mack Publishing Co., Easton, PA (1965), Injectable dosage forms of a solution or suspension of (I) can be prepared, for example, in a physiologically 30 acceptable diluent with a pharmaceutical carrier which can be a sterile liquid such as water and oils with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. Illustrative of oils which can be employed in these preparations are those of petroleum, 35 animal, vegetable or synthetic origin, for example, peanut oil, soybean oil and mineral oil. In general, water, * saline, aqueous dextrose and related sugar solution WO 94/07480 PCT/US9j. 08517 -32- ethanols and glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. 5 As is well known in the art of pharmaceutical inven tions wherein generic classes of compounds are involved, certain subgeneric and certain specific compounds are more efficient in their end-use applications than other members of the generic class. In this invention, those compounds 10 wherein Z is -CH2CH2CH2- or -CH(CH3)CH2CH2- are most preferred. In all instances it has been shown that the symmetrical compounds are preferred. Compounds for which each R is independently methyl or ethyl are preferred for this method of use and compounds for which both R groups 15 are methyl or both R groups are ethyl are preferred. Compounds for which both R groups are the same moiety are generally preferred. Further compounds of formula I may have m = to 7 or 8 20 The following compounds are preferred in the method of use described by the present invention: N,N' -bis[3-(methylamino)butyl]-l,7-diaminoheptane; N,N1-bis[3-(ethylamino)propyl]—1,7-diaminoheptane (MDL28,314); 25 N,N1-bis[3-(methylamino)propyl]-1,7-diaminoheptane; N,N'-bis[3-(methylamino)-2-(methyl)propyl]-l,7-diaminoheptane. Amongst the listed compounds, and the respective 30 substituent which are structurally part their of may be used to form a more preferred grouping of compounds. 35 For instance, of the compounds listed, N,N'-bis[3-(ethylamino)propyl]-l,7-diaminooctane and N,N'-bis[3-'.ethylaminojpropyl]-l,7-diaminoheptane are most preferred. WO 94/07480 PCT/US93/08517 In order to illustrate the preparation of compounds of formulas (la) and (lb), the following examples are provided. The examples are illustrative only and are not 5 intended to limit the invention in any way. All temperatures are in degrees Celsius and the following abbreviations are used: (g) is grams, (mol) is moles, (ml) is milliliters, (1) is liters, (lb/in2) is pounds per square inch, (TLC) is thin layer chromatography, (THF) is 10 tetrahydrofuran, (DMF) is dimethylformamide, (mp) is melting point, (mm/Hg) is pressure expressed as millimeters of mercury, (bp) is boiling point. CHEMICAL EXAMPLES 15 EXAMPLE 1 N,N- Bis((3-methylamino)propyl)-1,8-octanediamine tetrahydrochloride Step As N,N'-Bis(2-(cyano)ethyl)-1,8-octanediamine 20 Dissolve 14.4 g (0.1 mol) of 1,8-diaminooctane and 14.5 ml (0.22 mol) of acrylonitrile in 100 ml of ethanol and reflux overnight. Remove the solvent at reduced pressure. Analysis showed the title compound to be >98% pure. 25 Step B: N,N'-Bis(3-famino)propyl)1,8-octanediamine tetrahydrochloride Combine 14.4 g (0.057 mol) of the product of Step A, 200 ml of acetic acid, 30 ml of conc. HC1, and 1.2 g Pt02 and treat the mixture with H2 at 45 lbs/in2 in a shaker flask until H2 30 is no longer being reacted. Filter the mixture and remove the solvent at reduced pressure. 22.5 g of the title compound is obtained after purification. (R£ is 0.17 for TLC on silica gel developed with 40% conc. ammonia/methanol). 35 WO 94/07480 PCT/US93/08517 -34- Step Cs 1,5,14,18-Tetra(t-butoxycarbonyl)-1,5,14,18-tetraazaoctadecane Combine 22.5 g (0.052 mol) of the product of Step B with 5 8.83 g (0.22 mol) of NaOH, 100 ml H20 and 500 ml THF and stir until a homogenous solution is obtained. To this solution add 48.13 g (0.22 mol) of di-t-butyldicarbonate and stir the resulting mixture overnight. Pour the mixture into 1 1. of ethyl acetate, separate the organic layer, and 10 dry over anhydrous MgS04. Remove the solvent at reduced pressure. Purify the residue by flash chromatography (silica gel), eluting with 25% ethyl acetate/hexane to yield 13.5 g of the title compound (Rf is 0.28 for TLC on silica gel developed with 25% ethyl acetate/hexane). 15 Step D: 1,18-Bis(methyl)-1,5,14,18-tetra(t-butoxy-carbonyl)-!,5,14,18-tetraazoctadecane Combine 4.3 g (0.0068 mol) of the product of Step C, 0.94 ml (0.015 mol) of iodomethane, 1.69 g (0.015 mol) of 20 potassium t-butoxide, and 15 ml DMF and stir overnight. Remove the solvent at reduced pressure and dissolve the residue in 500 ml ethyl acetate and 200 ml of H2O. Wash the organic layer with 100 ml H2O (2x) and dry over anhydrous MgSC>4. Remove the solvent at reduced pressure and purify 25 the residue by flash chromatography (silica gel), eluting with 20% ethyl acetate/hexane to yield 4.4 g of the title compound (Rf is 0.20 for TLC on silica gel developed with 20% ethyl acetate/hexane.) 35 4 WO 94/07480 -35- PCT/US93/08517 Step E: N,N1-Bis(3-(methylamino)propyl)-1,8-octanediamine tetrahydrochloride Dissolve 4.4 g (0.0065 mol) of the product of Step D in 3 5 ml ethanol and treat the solution with 50 ml of 2N HCl in diethyl ether stirring overnight. Filter the resulting mixture and crystallize the residue from methanol/isopropanol/water (20/60/20,v/v/v) at reduced temperature. Filter and dry the product at 79°C over P2O5 10 at 0.1 mmHg to yield 2.08 g of the title compound (mp >300°C). Elemental analysis: calculated, C-44.44, H-9.79, N-12.86, Cl-32.80; Found C-44.44, H-9.82, N-12.95,. EXAMPLE 2 15 N,N'-Bis(3-(ethvlamino)propyl)-1,8-octanediamine tetrahydrochloride Step A: 1,18-Bis(ethyl)-1,5,14,18-tetra(t-butoxv-carbonyl)-1,5,14-18-tetraazaoctadecane 20 Combine 9.5 g (0.0144 mol) of the product of Step C in Example 1, 2.91 g (0.026 mol) of potassium t-butoxide, and 45 ml of DMF and cool to 0°C. Add 2.1 ml (0.026 mol) of iodoethane and stir at 0°C for 4 hours. Allow the mixture to warm slowly to room temperature and stir overnight. 25 Remove the solvent at reduced pressure and partition the residue between 1400 ml ethyl acetate and 200 ml H2O. Wash the organic layer with 100 ml H20 (2x) and dry over anhydrous MgS04. Remove the solvent under reduced pressure and purify the residue by flash chromatography (silica gel) 30 eluting with 20% ethyl acetate/hexane to yield 3.3 g of the title compound (Rf is 0.26 for TLC on silica gel developed with 20% ethyl acetate/hexane.) 35 WO 94/07480 -36- PCT/US93/08517 ( Step B: N,N'-Bis(3-(ethvlamino)propyl)-1,8-octanediamine tetrahydrochloride hemihvdrate Dissolve 3.3 g (0.0046 mol) of the product of Step A in 7 5 ml ethanol and treat with 70 ml of 2 N HCl in diethyl ether stirring overnight. Filter the mixture and dry the residue at 70°C at reduced pressure to yield 1.95 g of the title compound, mp >300°C. Elemental analysis: Calculated, C-46.09, H-10.10, N-11.95, Cl-30.24; Found C-46.23, H-9.94, 10 N-12.11, Cl-29.99. EXAMPLE 3 N-(3-Aminopropyl)-N'-13-fethvlamino)propyl)-1,8-octanediamine tetrahydrochloride 15 Step A: 1-Ethvl-l,5,14,18-tetra-(t-butoxycarbonvl)-1,5,14,18-tetraazaoctadecane Follow the procedure described in Step A of Example 2 to yield 2.5 g of the title compound after flash 20 chromatography (Rf is 0.17 for TLC on silica gel developed with 20% ethyl acetate/hexane). Step B: N-(3-Aminopropvl)-N'-(3-(ethvlamino)propyl)-1,8-octanediamine tetrahydrochloride 25 Dissolve 2.5 g (0.0036 mol) of the product of Step A in 5 ml of ethanol and treat with 60 ml of 2 N HCl in diethyl ether stirring overnight. Filter the mixture and dry the residue to yield 1.35 g of the title compound, mp >300°C. Elemental analysis: Calculated, C-43.54, H-9.82, N-12.69, 30 Cl-32.13; Found, C-43.43, H-9.60, 9.55; N-12.60, 12.62; Cl-32.30. WO 94/07480 -37- PCT/US93/08517 EXAMPLE 4 N,N'-Bis[3-(methylamino)butyl1-1,7-diaminoheptane tetrahydrochloride 5 Step A: N,N'-Bisf(phenyl)methvl1-l,7-heptanediamine Combine 1,7-diaminoheptane (65.0 g, 0.5 mol), benzaldehyde (106 gm, 1 mol) and platinum oxide (PtC>2)[2.0 g] in ethanol (800 ml) and treat the mixture with hydrogen gas (45 lb/in2) 10 until the uptake of gas ceases. Remove the catalyst by filtration and remove the solvent in vacuo. Purify the residue by bulb to bulb distillation to yield 99.4 g of the title compound (bp 191-195°C @ 1.0 mm/Hg). 15 Step B: N,N'-Bisf(3-oxo)butyl]-N,N'-bisf(phenyl)methyl] -1,7-diaminoheptane Dissolve N,N'-bis[(phenyl)methyl]-l,7-heptanediamine (9.3 g, 0.03 mol) in methanol (120 ml) and while stirring the mixture introduce methyl vinyl ketone (5.6 ml, 0.066 mol) 20 in a stream of nitrogen gas. Stir the mixture for 18 hours to yield the title compound. Step C: N,N'-Bis[(3-hvdroxyimino)butyl]-N,N'-bis[(phenyl) methyl1-1,7-diaminoheptane 25 Cool the reaction mixture obtained in step B to 0°C and to this mixture add a solution of hydroxylamine hydrochloride (4.38 g, 0.063 mol) and sodium bicarbonate (5.54 g, 0.066 mol) in water (40 ml). Stir the mixture at 0°C for 30 minutes and then stir at ambient temperature for 2 hours. 30 Remove the solvent in vacuo and partition the residue between water (200 ml) and dichloromethane (200 ml). Wash the aqueous layer 3 times with 200 ml of dichloromethane each time. Combine the organic layers and dry over anhydrous MgS04. Remove the solvent in vacuo to yield 14.4 35 g of the title compound. Rf is 0.53 for TLC on silica gel developed with ethyl acetate. " * WO 94/07480 PCT/US93/08517 -38- Step D: N,N'—Bisf3-(amino)butvl1-N,N'-bisf(phenyl) methyl 1-1,7-diaminoheptane Add a solution of N,N'-bis[(3-hydroxyimino)butyl]-N,N'-bis[(phenyl)methyl]-l,7-diaminoheptane (14.4 g, 0.03 mol) 5 in THF (70 ml) to a mixture of lithium aluminum hydride (5.8 g, 0.15 mol) in THF (250 ml) and reflux the mixture overnight. Cool the mixture and quench slowly with water (5.8 ml), followed by 15% NaOH (5.8 ml), followed by water (17.4 ml). Filter the mixture and wash the filtrate 3 10 times with 100 ml of THF each time. Combine the organic layers and remove the solvent in vacuo to obtain 13.4 g of the title compound as a clear viscous oil. Rf is 0.33 for TLC on silica gel developed with 4% conc. ammonia in methanol. 15 Step E: 2,16-Bis(methvl)-l,5,13,17-tetra(t-butoxvcarbonyl)-1,5,13,17-tetraazaheptadecane Combine N,N1 -bis[3-(amino)butyl]-N,N1-bis[(phenyl)methyl]-1,7-diaminoheptane (13.4 g, 0.029 mol), Pearlman's Catalyst 20 (2.0 g) and ethanol (90 ml) and treat the mixture with hydrogen gas at 45 lb/in2 until gas uptake ceases. Remove the catalyst by filtration and remove the solvent in vacuo to obtain 7.7 g of N,N'-bis[3-(amino)butyl]-l,7-diaminoheptane (Rf is 0.37 for TLC on silica gel developed 25 with 40% conc. ammonia in methanol). Dissolve the residue in dichloromethane (90 ml) and treat the mixture with di-t-butyldicarbonate (26.2 g, 0.12 mol) for 3 hours. Remove the solvent in vacuo and purify the residue by flash chromatography on silica gel eluting with 25% ethyl acetate 30 in hexane to yield 17.1 g of the title compound as a clear oil. Rf is 0.35 for TLC on silica gel developed with 25% ethyl acetate in hexane. 35 WO 94/07480 PCT/US93/08517 -39- Step P: 1,2,16,17-Tetramethvl-l,5,13,17-tetra(t-butoxvcarbonvl)-1,5.13,17-tetraazaheptadecane 5 Combine 2,16-bis(methyl)-l,5,13,17~tetra(t-butoxycarbonyl)-1,5,13,17-tetraazaheptadecane (8.5 g, 0.0126 mol) and sodium hydride (60% in oil)[1.21 g, 0.03 mol] in DMF (75 ml) and stir until hydrogen evolution ceases. To this mixture add methyl iodide (1.88 g, 0.03 mol) and stir for 2 10 hours. Remove the solvent in vacuo and partition the residue between ethyl acetate (400 ml) and water (200 ml). Dry the organic layer over anhydrous MgS04 and remove the solvent in vacuo. Purify the residue by flash chromatography on silica gel eluting with 22% ethyl acetate 15 in hexane to yield 3.8 g of the title compound as a clear oil. Rf is 0.22 for TLC on silica gel developed with 20% ethyl acetate in hexane. Step G: N,N'-Bisf 3-(methylamino)butyl]-l,7-diaminoheptane 20 tetrahydrochloride Add IN HCl in methanol (50 ml) to 1,2,16,17-tetramethyl-1,5,13,17-tetra(t-butoxycarbonyl)-1,5,13,17-tetraazaheptadecane (3.8 g, 0.0054 mol) and stir overnight. Remove the solvent in vacuo and recrystallize the residue 25 two times from inethanol/acetonitrile (40/60, v/v) to yield 0.74 g of the title compound as a white solid (mp 238-9 °C). Rf is 0.31-for TLC on silica gel developed with 40% conc. ammonia in methanol. 35 A WO 94/07480 PCT/US93/08517 ( -40-EXAMPLE 5 N,N'-Bis[3-(ethvlamino)butvl1-1,7-diaminoheptane tetrahydrochloride 5 Step A: 1,17-Diethvl-2,16-dimethyl-l,5,13,17-tetra(t-butoxvcarbonvl1-1.5.13,17-tetraazaheptadecane Combine 2,16-bis(methyl)-1,5,13,17-tetra(t-butoxycarbonyl)-1,5,13,17-tetraazaheptadecane (8.5 g, 0.0126 mol), made as 10 described in Example 5, and sodium hydride (60% in oil)[1.21 g, 0.03 mol] in DMF (75 ml) and stir until hydrogen evolution ceases. To this mixture add ethyl iodide (4.68 g, 0.03 mol) and stir for 2 hours. Remove the solvent in vacuo and partition the residue between ethyl 15 acetate (400 ml) and water (200 ml). Dry the organic layer over anhydrous MgS04 and remove the solvent in vacuo. Purify the residue by flash chromatography on silica gel eluting with 22% ethyl acetate in hexane to yield 3.9 g of the title compound as a clear oil. Rf is 0.31 for TLC on 20 silica gel developed with 20% ethyl acetate in hexane. Step B: N,N'-Bis[3-(ethvlamino)butvl]-1,7-diaminoheptane tetrahydrochloride Add IN HCl in methanol (50 ml) to l,17-diethyl-2,16-25 dimethyl-1,5,13,17-tetra(t-butoxycarbonyl)-1,5,13,17- tetraazaheptadecane (3.9 g, 0.0054 mol) and stir overnight. Remove the solvent in vacuo and recrystallize the residue two times from methanol/acetonitrile (40/60, v/v) to yield 0.90g of the title compound as a white solid (mp 249-50 30 °C) . Rf is 0.56 for TLC on silica gel developed with 40% conc. ammonia in methanol. WO 94/07480 PCT/US93/08517 -41- EXAMPLE 6 N ,N1-Bis[3-(ethvlamino)propyl1-1, 7-heptanediamine 5 Steps A and B: 1.5.13.17-Tetraazaheptadecane tetrahydrochloride Prepare the title compound by the method of Israel et al., J. Med. Chem. 7, 710 (1964). 10 Step C: 1.5,13.17-Tetra(t-butoxycarbonyl)-l.5.13,17-tetraazaheptadecane Combine_l,5,13,17-tetraazaheptadecane tetrahydrochloride (3.9 gm, 0.01 mol) and sodium hydroxide (1.76 gm, 0.44 mol) 15 in water (44 ml) and stir until homogeneous. To this mixture add di-t-butyldicarbonate (9.6 gm, 0.044 mol) in THF (88 ml) and stir for 3 hours. Dilute the mixture with ethyl acetate (EtOAc) [300 ml] and separate the organic layer. Dry the organic layer over anhydrous MgS04 and 20 evaporate in vacuo to obtain a viscous oil. Purify the residue by flash chromatography (silica gel) eluting with 25% EtOAc/hexane to yield 3.0 gm of the title compound. Rf is 0.20 on silica gel plates eluted with 25% EtOAc/hexane. 25 Step D: 3,7,15,19-Tetra(t-butoxycarbonyl)-3,7,15,19-tetraazaheneicosane Combine 1,5,13,17-tetra(t-butoxycarbonyl)-1,5,13,17-tetraazaheptadecane (3.0 gm, 0.0046 mol) and sodium hydride (50% in oil) [0.45 gm, 0.011 mol] in DMF (9 ml) and stir 30 the mixture until hydrogen evolution ceases. Add ethyl iodide (0.9 ml, 0.011 mol) and stir the mixture for 18 hours. Evaporate the DMF in vacuo and partition the residue between ethyl acetate (600 ml) and water (200 ml). Separate the organic layer, dry the organic layer over 35 WO 94/07480 PCT/US93/08517 ( -42- anhydrous MgS04 and evaporate in vacuo. Purify the residue by flash chromatography (silica gel) eluting with 20% EtOAc/hexane to yield 1.68 gm of the title compound. Rf is 0.5 on silica gel plates eluted with 25% EtOAc/hexane. 5 Step Es N,N'-Bis[3-(ethvlamino^propyl 1-1,7-heptanediamine Treat 3,7,15,19-tetra(t-butoxycarbonyl)-3,7,15,19-tetraazaheneicosane (1.68 gm, 0.0024 mol) with HCl in methanol (50 ml, 1.0 N) and stir overnight. Filter the 10 mixture and recrystallize the title compound from methanol/water (20:80, v/v) to yield 0.5 gm of the title compound. Rf is 0.39 on silica gel plates eluted with 40% ammonia (concentrated) in methanol; mp 322-23°C with degradation. 15 EXAMPLE 7 1,18-Bis[(phenyl)methyl11,5,14,18-tetraazaoctadecane* 4HC1 Step A: N,N'-Bis-f 2,2'-bis(cyano)ethyl1-1,8-diamino-octane 20 Dissolve 28.8 gm (0.2 mol) of 1,8 diaminooctane in 250 ml of EtOH. Add 27 ml (0.41 mol) of acrylonitrile and gently reflux the mixture overnight. Remove the solvent at reduced pressure. Analysis shows desired material to be >95% pure. 25 Step B: 1,5,14,18-Tetraazaoctadecane tetrahydrochloride Combine 50.0 gm of the product of Example 1, 2.0 gm Pt02, 133 ml of conc. HCl at 45 lbs./sq. in. in a shaker flask until hydrogen is no longer taken up. Filter the resulting 30 mixture, evaporate the solvent and triturate the product with 1 liter of EtOH. Filter and dry the product to obtain 51.6 gm of the title compound, Rf is 0.17 (silica gel plates eluted with 40% conc. NH3/CH3OH). 35 WO 94/07480 -43- PCT/US93/08517 Step C: 1,5»14,18-Tetra(t-butoxvcarbonyl)-1,5,14,18-tetraazaoctadecane Treat 28.0 gm (0.069 mol) of the product of Step B with 5 10.99 gm (0.274 mol) of NaOH in 120 ml H20. When a homogenous solution is obtained add 65.7 gm (0.307 mol) of di-t-butyldicarbonate in 750 ml of THF and stir the resulting mixture for 16 hours. Separate the layers, remove and wash (2x) the aqueous layer with 500 ml CH2CI2. 10 Combine and dry (MgS04) the organics, filter and evaporate (invacuo) the solvents and flash chromatograph the residue (silica gel), eluting with 25% EtOAc/hexane to yield 30.2 g of the desired product. Rf is 0.33 on silica gel plates eluted with 25% EtOAc/hexane). 15 Step D: l,18,-Bis[(phenyl)methyl1-1,5,14,18-tetra(t-butoxvcarbonvl)-1,5,14,18-tetraazaoctadecane Dissolve 20.0 gm (0.03 mol) of the product from Step C in 30 ml DMF and treat with 7.5 gm (0.067 mol) KtBuO and 7.96 20 ml (0.067 mol) BnBr, with stirring for 18 hours. Evaporate the volatiles (0.5 mm and 45°C) and take up the resulting residue in 1400 ml of EtOAc and water-wash (2x, 500 ml). The organic layer is then dried (MgSO^) and the solvent is evaporated (in vacuo). Flash chromatography on silica gel 25 eluted with 20% EtOAc/hexane yields 12.4 gm (50%) of desired product as a clear viscous oil. Rf is .42 (silica gel plates eluted with 25% EtOAc/hexane). Step E: 1,18-Bis-[(phenyl)methyl1-1,5,14,18-tetraaza-30 octadecane»4HCl Dissolve 12.4 g (0.0147 mol) of the product of Step D in 14.7 ml of anhydrous EtOH and treat with 160 ml of 2N HCl in Et20 with stirring overnight. Filter, wash the filter cake with Et20, and dry to obtain 7.2 gm of the desired 35 compound, mp >300°C. Rf is 0.24 (from silica gel eluted with 10% conc. NH3/CH3OH). - * WO 94/07480 PCT/US93/08517 ( -44-EXAMPLE 8 1,20-Bis[(phenyl)methvl1-1.16,15,20-tetraazaeicosane* 4HC1 Step A: N,N'-Bis(t-butoxvcarbonyl)-1,8-octanediamine 5 Dissolve 10.8 gm (0.075) of diaminooctane in 200 ml CH2CI2 and 100 ml CH3OH, add 32.7 gm (0.156 mol) of di-t-butyldicarbonate and stir the mixture overnight. Evaporate, in vacuo, and crystallize the residue from hexane to obtain 20.2 gm of the desired compound, mp 9(j-97°C. 10 Step B: 4-[[(Phenyl)methvl]amino1-butan-l-ol Combine 4-amino-butan-l-ol (8.9 gm - 0.L mol), benzaldehyde (10.6 gm - 0.1 mol), EtOH (100 ml) and Pt02 (0.3 gm), and hydrogenate the mixture at 45 lbs./sq.in. until H2 is no 15 longer taken up. Filter, evaporate the solvent (in vacuo) to yield 17.7 gm of the desired compound. Rf is 0.70 (eluted from silica gel with 10% conc. NH3/CH30H). Step C: 4- [ N-(t-butoxycarbonyl) -N- [ (phenyl ) methyl 1 amino 1 20 butan-l-ol Combine the butanol of Step B (17.7 g - 0.1 mol) and di-tbutyldicarbonate in 100 ml of CH2CI2 and stir the mixture overnight. Evaporate off the solvents, in vacuo, and flash chromatography of the residue, eluting from silica gel with 25 25% EtOAc/hexane to obtain the desired compound. Rf is .27 (silica gel plates eluted with 20% EtOAc/hexane). Step Ds 4-[N-(t-butoxycarbonyl)-N-[(phenyl )methvl1-amino 1 -lnethansulfonyl butane 30 Cool (ice-bath) a mixture containing the product of Step C (21.8 gm - 0.078 mol), 250 ml CH2Cl2 and 9.7 ml pyridine (0.12 mol), add in a dropwise fashion (20 minutes) mesylchloride (6.65 ml - 0.086 mol) in 6.6 ml CH2CI2 and allow the mixture to warm to room temperature, stirring the 35 mixture for 2 hours. Pour the resulting mixture into 200 ml CH2CI2, wash with 500 ml 0.5 N HCl, saturated NaHC03, dry over MgS04, evaporate (in vacuo) and flash chromatograph WO 94/07480 PCT/US93/08517 -45- eluting from the silica gel with 25% EtOAc/hexane to obtain 10.7 g of desired product, Rf is ).36 (silica gel plates eluted with 25% EtOAc/hexane). 5 Step Es 1,20-Bisf(phenyl)methyl1-1,16.15,20-tetra-(t-butoxvcarbonyl)-1,6.15 ,20-tetraazaeicosane Admix the products of Step A (5.16 gm - 0.015 mol) and of Step D of this example (10.7 g - 0.032 mol), Kt-BuO (3.92 gm), Nal 0.2 gm), and 60 ml DMF and stir the mixture for 10 72 hours at room temperature. Evaporate the solvent (in vacuo), take up the residue in 600 ml EtOAc and wash (2x) with 200 ml water. Dry the organic layer (MgSOO, evaporate the solvents, and flash chromatograph the viscous residue on silica gel eluting with 20% EtOAc/hexane to obtain the 15 desired product, Rf is 0.22 (silica gel plates eluted with 20% EtOAc/hexane). Step F: 1,20-Bis[(phenyl)methyl]-1,6,15,2Q-tetraeicosane» 4 HCl 20 Dissolve the product of Step E (4.7 gm) (0.0054 mol) in 5 ml EtOH and treat with 54 ml of 2N HCl in Et02, stir the mixture overnight, filter and recrystallize to so-obtained solids from isopropanol/water. Cool, filter and dry the desired product, mp >300°C, Rf is 0.47 (eluted from silica 25 with 10% conc. NH3/CH3OH). EXAMPLE 9 N,N'-Bis(3-aminobutyl)-l,8-Octanediamine 30 Step As N,N' -Bis( (phenyl)methyl)-1,8-octanediamine Combine 14.4 g (0.1 mol) of 1,8-octanediamine, 20.3 ml (0.2 mol) of benzaldehyde, 0.3 g Pt02 and 150 ml ethanol and treat the mixture with H2 at 45 lb/in2 in a shaker flask until no more gas is taken up. Remove the catalyst by 35 filtration and remove the solvent at reduced pressure to yield the title compound. - * WO 94/07480 PCT/US93/08517 -46- Step B: N,N'Bis((3-oxo)butvl)-N,N'-bis((phenyl)methvl)-1,8octanediamine Dissolve the product obtained in Step A in 1400 ml of methanol and introduce 21.6 of methyl vinyl ketone on a 5 stream of N2 gas. Stir for 16 hours to yield the title compound. Step C: N,N'-Bis((3-hvdroxvimino)butvl)-N,N'-Bis-((phenyl)methvl)-1,8-octanediamine 10 Combine 18.07 g hydroxylamine hydrochloride, 10.4 g of NaOH and 40 ml of H20 and add to the solution obtained in Step B. Reflux the mixture for 3 hours, then cool and evaporate the solvent. Pour the reaction mixture into 300 ml of ethyl acetate and wash with 300 ml H20. Wash the aqueous layer 15 with 300 ml of ethyl acetate (2x). Combine the organic layers and dry over anhydrous MgS04. Remove the solvent at reduced pressure. Purify the product by flash chromatography (silica gel), eluting with ethyl acetate to yield 34.8 g of the title compound (Rf is 0.42 for TLC on 20 silica gel developed with ethyl acetate). Step D: N,N'-Bis((3-amino)butyl)-N,N'-Bis((phenyl)-methyl)-1,8-octanediamine Add 34.8 g of the product of Step C in 100 ml THF to 12.10 25 g (0.310 mol) of lithium aluminum hydride in 540 ml THF and reflux the mixture while stirring overnight. Cool the mixture and slowly add 15 ml H20 followed by 45 ml IN NaOH and stir the mixture for 6 hours. Filter the mixture to remove a white granular precipitate and remove the solvent 30 at reduced pressure. Subject the residue to short path distillation to yield 17.0 g of the title compound (bp 230-235°C at 0.1 mmHg). Step E: N,N'-Bis((3-amino)butyl)-l,8-octanediamine 35 Combine 5.0 g (0.01 mol) of the product of'Step D, 0.5 g of 20% Pd(OH)2 on carbon (Pearlman's Catalyst), and 50-ml os ethanol and treat the mixture with H2 at 45 lb/in2 in a WO 94/07480 PCT/US93/08517 -47- shaker flask until no more gas is taken up. Remove the catalyst by filtration and remove the solvent at reduced pressure. Subject the residue to short path distillation to yield 1.59 g of the title compound (bp 145-148°C at 0.012 5 mmHg). EXAMPLE 10 N,N'-Bis f 3-(methylamino)butvl]-1,7-diaminoheptane tetrahydrochloride 10 Step A: N,N'-Bis[(phenyl)methyl1-1,7-heptanediamine Combine 1,7-diaminoheptane (65.0 g, 0.5 mol), benzaldehyde (106 gm, 1 mol) and platinum oxide (Pt02)[2.0 g] in ethanol (800 ml) and treat the mixture with hydrogen gas (45 lb/in2) 15 until the uptake of gas ceases. Remove the catalyst by filtration and remove the solvent in vacuo. Purify the residue by bulb to bulb distillation to yield 99.4 g of the title compound (bp 191-195°C § 1.0 mm/Hg),. 20 Step B: N,N'-Bis[(3-oxo)butyl1-N,N'-bis[(phenyl)methyl]-1,7- diaminoheptane Dissolve N,N'-bis[(phenyl)methyl]-l,7-heptanediamine (9.3 g, 0.03 mol) in methanol (120 ml) and while stirring the mixture introduce methyl vinyl ketone (5.6 ml, 0.066 mol) 25 in a stream of nitrogen gas. Stir the mixture for 18 hours to yield the title compound. Step C: N,N'~Bis[(3-hydroxyimino)butyl1-N,N'-bis[(phenyl) methyl1-1,7-diaminoheptane 30 Cool the reaction mixture obtained in step B to 0°C and to this mixture add a solution of hydroxylamine hydrochloride (4.38 g, 0.063 mol) and sodium bicarbonate (5.54 gf 0.066 mol) in water (40 ml). Stir the mixture at 0°C for 30 minutes and then stir at ambient temperature for 2 hours. 35 Remove the solvent in vacuo and partition the residue between water (200 ml) and dichloromethane (200 ml). Wash the aqueous layer 3 times with 200 ml of dichloromethane WO 94/07480 PCT/US93/08517 c -48- each time. Combine the organic layers and dry over anhydrous MgS04. Remove the solvent in vacuo to yield 14.4 g of the title compound. Rf is 0.53 for TLC on silica gel developed with ethyl acetate. 5 Step D: N,N'-Bis[3-(amino)butvl]-N,N'-bis[(phenyl) methyl]-1,7-diaminoheptane Add a solution of N,N'-bis[(3-hydroxyimino)butyl]-N,N'bis[(phenyl)methyl]-l,7-diaminoheptane (14.4 g, 0.03 10 mol) in THF (70 ml) to a mixture of lithium aluminum hydride (5.8 g, 0.15 mol) in THF (250 ml) and reflux the mixture overnight. Cool the mixture and quench slowly with water (5.8 inl), followed by 15% NaOH (5.8 ml), followed by water (17.4 mi). Filter the mixture and wash the filtrate 15 3 times with 100 ml of THF each time. Combine the organic layers and remove the solvent in vacu . to obtain 13.4 g of the title compound as a clear viscous oil. Rf is 0.33 for TLC on silica gel developed with 4% conc. ammonia in methanol. 20 Step Es 2,16-Bis(methyl)-1,5,13,17-tetra(t-butoxvcarbonvl)-1,5,13,17-tetraazaheptadecane Combine NfN'-bis[3-(amino)butyl]-N,N'-bis[(phenyl)methyl]-1,7diaminoheptane (13.4 g, 0.029 mol), Fearlman's Catalyst 25 (2.0 g) and ethanol (90 ml) and treat the mixture with hydrogen gas at 45 lb/in2 until gas uptake ceases. Remove the catalyst by filtration and remove the solvent in vacuo to obtain 7.7 g of NrN'-bis[3-(amino)butyl]-l,7-diaminoheptane (Rf is 0,37 for TLC on silica gel developed 30 with 40% conc. ammonia in methanol). Dissolve the residue in dichloromethane (90 ml) and treat the mixture with di-t-butyldicarbonate (26.2 g, 0.12 mol) for 3 hours. Remove the solvent in vacuo and purify the residue by flash chromatography on silica gel eluting with 25% ethyl acetate 35 in hexane to yield 17.1 g of the title compound as a clear oil. Rf is 0.35 for TLC on silica gel developed with 25% ethyl acetate in hexane. WO 94/07480 -49- PCT/US93/08517 Step P: 1,2,16,17-Tetramethvl-l.5,13,17-tetra(t-butoxv-carbonvl)-1,5.13.17-tetraazaheptadecane Combine 2,16-bis(methyl)-1,5,13,17-tetra(t-butoxycarbonyl) 5 1,5,13,17-tetraazaheptadecane (8.5 g, 0.0126 mol) and sodium hydride (60% in oil)[1.21 g, 0.03 mol] in DMF (75 ml) and stir until hydrogen evolution ceases. To this mixture add methyl iodide (1.88 g, 0.03 mol) and stir for 2 hours. Remove the solvent in vacuo and partition the 10 residue between ethyl acetate (400 ml) and water (200 ml). Dry the organic layer over anhydrous MgS04 and remove the solvent in vacuo. Purify the residue by flash chromatography or silica gel eluting with 22% ethyl acetate in hexane to yield 3.8 g of the title compound as a clear 15 oil. Rf is 0.22 for TLC on silica gel developed with 20% ethyl acetate in hexane. Step G: N,N'-Bis[3-(methylamino)butyl]-1,7-diaminohepta.i? tetrahydrochloride 20 Add IN HCl in methanol (SO ml) to 1,2,16,17-tetramethyl-1,5,13,17-tetra(t-butoxycarbonyl)-1,5,13,17-tetraazahepta-deoane (3.8 g, 0.0054 mol) and stir overnight. Remove the solvent in vacuo and recrystallize the residue two times from methanol/acetonitrile (40/60, v/v) to yield 0.74 9 of 25 the title compound as a white solid (mp 238-9 °C). Rf is 0.31 for TLC on silica gel developed with 40% conc. ammonia in methanol. EXAMPLE 11 30 N, N'-Bis[3-(ethvlamino)butyl)-1,7-diaminoheptane tetrahydrochloride Step As 1,l7-Diethyl-2,16-dimethyl-l,5,13,17-tetra(t-butoxvcarbonyl)-1,5,13,17-tetraazaheptadecane 35 Combine 2,16-bis(methyl)-1,5,13,17-tetra(t-butoxycarbonyl)-1,5,13,17-tetraazaheptadecane (8.5 g, 0.0126 mol), made ate described in Example 5, and sodium hydride (60% in WO 94/07480 PCT/US93/08517 -50- oil)[1.21 g, 0.03 mol] in DMF (75 ml) and stir until hydrogen evolution ceases. To this mixture add ethyl iodide (4.68 g, 0.03 mol) and stir for 2 hours. Remove the solvent in vacuo and partition the residue between ethyl 5 acetate (400 ml) and water (200 ml). Dry the organic layer over anhydrous MgSC>4 and remove the solvent in vacuo. Purify the residue by flash chromatography on silica gel eluting with 22% ethyl acetate in hexane to yield 3.9 g of the title compound as a clear oil. Rf is 0.31 for TLC on 10 silica gel developed with 20% ethyl acetate in hexane. Step B: N,N'-Bis[3-(ethvlamino)butvl1-1,7-diaminoheptane tetrahydrochloride Add IN HCl in methanol (50 ml) to l,17-diethyl-2,16-15 dimethyl-1,5,13,17-tetra(t-butoxycarbonyl)-1,5,13,17- tetraazaheptadecane (3.9 gf 0,0054 mol) and stir overnight. Remove the solvent invacuo and recrystallize the residue two times from methanol/acetonitrile (40/60, v/v) to yield 0.90g of the title compound as a white solid (mp 249-50 20 °C). Rf is 0.56 for TLC on silica gel developed with 40% conc. ammonia in methanol. 25 30 35 rf WO 94/07480 -51- PCT /US93/08517 EXAMPLE 12 1,4,13,16-Tetra (t-butoxvcarbonvl) -1,4-13', 16-tet raazahexa-decane 5 Combine 4.75 gm 1,8-dibromooctane (0.017 mol), 20 ml EtOH and 9.32 ml of ethylene diamine and reflux the mixture overnight. Cool and treat the mixture with 1.4 gm NaOH. Evaporate off the solvent and triturate the residue with CH2CI2 (200 ml 2x), filter. Treat the filtrate with 66.6 gm 10 of di-t-butyldicarbonate and stir the mixture overnight. Remove the solvent and subject the residue to flash chromatography, eluted with 25% EtOAc/hexane to yield the desired product. Rf is 0.64 eluted from silica gel with 50% EtOAc/hexane. 15 The foregoing may be bis-N-alkylated and the product deprotected by methods analogous to Steps D and E of Example 7 to produce desired compounds of the Formula R1HN(CH2)2N(CH2)8^(CH2)2NHR' , e.g., 1,16-20 Bis[(phenyl)methyl]-1,4,13,16-tetraazahexadecane* 4 HCl, EXAMPLE 13 1,18-Bis[(2-phenvl)ethyl1-1,5,14,18-tetraazaoctadecane* 4HC1 25 Step A: 1,18-Bis[[(phenvl)methyllcarbonyll-5,14-bis-[(phenyl)methyl1-1,5,14,18-tetraazaoctadecane Chill a solution of 5,14-bis[(phenyl)methyl]-l,5,14,18-tetraazaoctadecane (2.2 g, 5 mmole) and triethylamine (2 g, 20 mmole) in chloroform (100 ml) in an ice bath. Add a 30 solution of phenylacetyl chloride (2.3 g, 15 mmole) in chloroform (10 ml) dropwise. Remove the ice bath and stir the mixture at ambient temperature for 18 hours. Extract the reaction mixture with aqueous sodium bicarbonate, dry the organic layer and evaporate. Chromatograph the residue 35 on a flash silica gel column (ethyl acetate) to give 3 g of the desired product as a thick oil. - * WO 94/07480 PCT/US93/08517 f -52- Step Bs Add a solution of the product of Step A in THF (150 ml) dropwise to a suspension of LAH (0.5 g) in THF (500 ml). Stir the mixture for 48 hours at ambient temperature. Decompose the excess reducing agent by dropwise addition of 5 1 ml of water, 1 ml of 15% NaOH then 3 ml of water. Filter the mixture and evaporate the filtrate. Take the residue up in ethanol (100 ml) and add anhydrous HCl gas to convert the product, 1,18-bist(phenyl)ethyl]-5,14-bis-[(phenyl)methyl]1,5,14,18-tetraazaoctadecane, to its 10 tetrahydrochloride salt. Hydrogenate this product in ethanol (150 ml) in the presence of Pearlman's catalyst (0.3 g) at 43 psi on a Parr hydrogenation apparatus for 24 hours. Filter off the catalyst and evaporate the filtrate. Crystallize the residue from 2-propanol to give the product 15 l,18-bis-[(phenyl)ethyl]-l,5,14,18-tetraazaoctadecane tetrahydrochloride salt hemihydrate, mp 228-231°C. EXAMPLE 14 1,18-Bis(phenyl)-1,5,14,18-tetraazaoctadecane 20 Step A: N-(Phenyl-N,N'-bis(t-butoxvcarbonvl)propanediamine Cool 200 ml of anhydrous Et20 in an ice bath and add lithium aluminum hydride (8.74 gm -0.23 mol). Add, in a dropwise fashion over 30 minutes, 3-anilinopropionitrile (14.6 gm) 25 in 50 ml of Et20, remove the ice bath, and reflux the resulting mixture overnight. Sequentially add 8.7 ml of water, 1.5 g of NaOH (in 10 ml of water) and 25 ml of water. Filter the resulting ppt, rinse with 200 ml of Et20 and remove the solvent, in vacuo, and treat the resulting 30 N-(phenyl) propanediamine with 43.6 g of di-t- butyldicarbonate in 600 ml of CH2Cl2. After stirring overnight, evaporate off the solvent and subject the residue to flash chromatography from silica gel eluting with 17% EtOAc/hexane to produce the desired compound. Rf 35 is 0.50 (eluted from silica gel with 25% EtOAc/hexane). WO 94/07480 PCT/US93/08517 -53- Step B: 1,18-Bis(phenyl)-1,5,14,18-tetra(t-butoxvcarbonvl)-1,5,14,18-tetraazaoctadecane Stir a mixture containing the product of Step A (13.0 gm), diiodooctane 3.70 gm) and 4.14 g of potassium t-butoxide in 5 200 ml of DMF for about 16 hours. Evaporate the solvent at 0.5 mm and 45°C, take up the residue in 800 ml of EtOAc. Wash (2x) with 300 ml of water, dry (MgS04) and remove the solvent invacuo. Subject the so-obtained viscous oil to flash chromatography, eluting with 15% EtOAc from silica 10 gel to yield 5.7 g of the desired product. Rf of 0.36 (eluted from silica gel with EtOAc/hexane). Remove the N-boc protecting groups according to the procedure of Step E of Example 7 to produce the title compound of this example as its hydrochloride salt, mp 264-267°C. 15 EXAMPLE 15 1,18-Bis (2,3-butadienvl) -1,5,14,18-tetraazaoctadecane tetrahydrochloride 20 Step A: N- (t-Butoxvcarbonyl)proparqylamine In a dropwise fashion, add propargylamine (25 gm) in 25 ml of CH2CI2 to a stirring mixture of di-t-butyldicarbonate (99.18 gm) in 900 ml of CH2Cl2» After 2 hours, remove the solvent, in vacuo, to obtain 70 gm of the desired N-25 protected propargylamine. Step B: N- (t-Butoxvcarbonyl)-2,3-butadienylamine Reflux a mixture containing N-(t-butoxycarbonyl)-propargylamine (70 gm), 93.5 ml of 32% formaldehyde, 76.4 30 ml of diisopropylamine, 19.66 gm of cuprous bromide and 860 ml of p-dioxane for 12 hours. Cool and dilute the resulting mixture with 3000 ml of Et20, wash with 500 ml of water, 1000 ml acetic acid, 500 ml of water (2x), 200 ml sat'd. sodium chloride, dry (MgS04) and evaporate in vacuo. 35 Flash chromatograph the residue eluting from silica gel with 10% Et20/hexane to yield 40.8 g of the desired * WO 94/07480 PCT/US93/08517 -54- compound. Rf is 0.31 (eluted from silica gel with 10% EtOAc/hexane). Step C: N,N-Bis[(phenyl)methyl3-1,8-diaminooctane 5 Combine 14.4 gm of diaminooctane, 20.3 ml of benzaldehyde and 0.66 gm of Pt20 in 100 ml of ethanol. Treat the resulting mixture with hydrogen at 45 lbs./sq.in. until no further hydrogen is taken up. Filter, evaporate the solvent (in vacuo), and distill the rendered material to 10 obtain 25.5 gm of the desired product, bp 185-190°C at 0.1 mm. Step D: 1,18-Bis(hydroxy)-5,14-bis[(phenyl)methvl1-5,14-diazaoctadecane 15 Reflux a mixture containing 25.5 g of the product of Step C, 13.2 ml of 3-chloro-l-hydroxy-propane, 50.4 gm of Na2C03 and 1.19 gm of sodium iodide in 40 ml of n-butanol for 18 hours. Cool the mixture and pour into 700 ml of ethylacetate, wash with water, dry over MgS04 and remove 20 the solvent (in vacuo) to obtain a residue which upon distillation yields 30.0 gm of the desired product, bp 250-252°C at 0.1 mm. Step E: 1,18-Bis(hydroxy)-5,14-diazaoctadecane Hydrogenate a mixture containing 3.0 gm of the product of Step D, 30 ml of AcOH and 0.6 gm of palladium oxide at 45 lbs./sq.in. until no further hydrogen is taken up. Filter and remove the solvent (in vacuo) to yield 1.77 gm of the desired product, Rf is 0.37 (eluted from silica gel with 10% conc. NH3/CH3OH)-. 25 30 35 * O 94/07480 PCT/US93/08517 -55- Step Fs 1,18-Bis(hydroxy)-5,14-bis-(t-butoxvcarbonvl)-5,14-diazaoctadecane Stir a mixture containing 1.77 gm of the product of Step E, 5 2.97 gm (0.0136 mol) of di-t-butyldicarbonate, 3 ml of triethylamine and 50 ml of CH2CI2 overnight. Dilute the mixture with 200 ml of CH2C12, wash with 200 ml of 0.5N HCl, and then 100 ml of sat'd NaCl, dry (over MgS04) and remove the solvent (in vacuo). Flash chromatograph the residue, 10 eluting from silica gel with 75% EtOAc to obtain the desired product, Rf 0.29, (eluted from silica gel with 75% EtOAc/hexane). Step G: 1,18-Bis(methanesulfonvl)-5,14-bis(t-15 butoxycarbonyl)- 5,14-diazaoctadecane Cool to 0°C a mixture containing 3.0 gm of the product of Step F, 3.3 ml of triethylamine and 70 ml of CH2Cl2. In a dropwise fashion add 1.22 ml of mesylchloride in 10 ml of CH2Cl2 and stir the resulting mixture at 0°C for 1% hours. 20 Pour the mixture into 100 ml of CH2CI2, wash with 200 ml of IN AcOH, 100 ml of water, 100 ml of sat'd sodium bicarbonate, dry over MgS04 and remove the solvent invacuo. Flash chromatograph the residue, eluting from silica gel with 60% EtOAc/hexane to obtain 3.5 gm of the desired 25 product. Rf is 0.39. Step H; 1,18-Bis(2,3-butadienyl)-l,5,14,18-tetra-(t-butoxvcarbonyl)-1,5,14,18-tetraazaoctadecane Combine a mixture containing 3.5 gm of the product of Step 30 G, 1.74 gm of sodium iodide, 0.51 gir of hexane washed sodium hydride (60% in oil) in 12 ml of DMF with 2.16 gm of N-(tbutoxycarbonyl)-2,3-butandienylamine (i.e., the product of Step B) and allow the resulting mixture to stand for 2 hours. Remove the solvent (in vacuo), add 350 ml of ethyl 35 acetate to the residue, wash with 50 ml of "water (4x), 1Q.0 ml sat'd sodium chloride and dry over MgS04. Remove the solvents (in vacuo) and flash chromatograph the residue WO 94/07480 PCT/US93/0851 ? -56- from silica gel eluting with 30% EtOAc/hexane to yield 0.5 gm of the desired product, as a viscous oil. Rf is 0.39 (eluting from silica gel with 25% EtOAc/hexane). 5 Step Is It18-Bis(2,3-butadienyl)-1,5,14,18-tetraazaoctadecane* 4HC1 Dissolve 0.5 gm of the product of Step H in 2 ml of EtOH and while stirring treat the mixture with 10 ml of 2N HCl in Et20. Stir the resulting mixture overnight, filter and 10 dry the solids (in vacuo) to obtain 0.22 gm of the desired product, mp 283-284°C dec. EXAMPLE 16 N, N' -Bis [ 3- (ethvlamino) propyl ] -1,7-heptanediamine 15 tetrahydrochloride (MDL 28314QA) Step A: 1,7-Bis(hexahvdropyrimidin-1-yl)heptane (MDL 102533) A solution of N,N'-bis(3-aminopropyl)-l,7-heptanediamine 20 4HCl (MDL 26752QA, 10.0 g, 25.6 mmol)* in 1 N NaOH (103 mL, 4 eq) was cooled to 5°C (ice-water bath) and aqueous HCHO (37 wt%, 3.8 mL, 50.8 mrnol) was added in one portion. The solution was stirred for 1 h at 0-5°C and then 1 h at room temperature. The reaction solution was extracted wit* CH2CI2 25 (3 x 100 mL). The extracts were combined, dried (K2CO3) and concentrated to give crude MDL 102533 (6.9 g, 100%) as a light yellow solid. This material was used without further purification. Purification of a sample by flash chromatography using eluant A gave analytically pure MDL 30 102533 as a white solid: mp 52-55°C; TLC (eluant B) Rf 0.46?1H NMR (CDCI3) 6 1.3 (m, 6), 1.46 (br pentet, 4), 1.61 (pentet, 4, J=5.5 Hz), 1.7 (br s, 2, NH), 2.21 (dd, 4, J=7.6, 8.6 Hz), 2.56 (br t, 4, J=4.8 Hz), 2.81 (t, 4, J=5.5 Hz), 3.37 (s, 4); "C NMR (CDCI3) 5 26.79, 27.05, 27.58, 35 29.39, 45.07, 53.12, 55.66, 69.84; mass spectrum (CI/CH4), wo 94/07480 PCT/US93/08517 -57- m/z(rel intensity) 270(21), 269(100), 97(18), 83(19), 79(14). Anal. Calcd for C15H32N4 (268.45): C, 67.11; H, 12.02; N, 5 20.87. Found: C, 66.65; H, 12.85; N, 20.69. Step B: 1,7-Bis(3-acetvlhexahydropvrimidin-l-yl)heptane (MDL 44868) 10 A stirred solution of crude bis(hexahydropyrimidine) MDL 102533 (5.0 g, 18.7 mmol) in EtOAc (40 mL), Et3N (7.6 g, 75 mmol) and Ac20 (7.6 g, 75 mmol) was heated at reflux for 8 h under N2. The reaction solution was cooled and concentrated (50°/10 mm). The concentrate was partitioned in CH2CI2 (75 15 mL) and 1 N NaOH (40 mL), the layers were separated and the aqueous layer was extracted with CH2CI2 (2 x 75 mL), The CH2CI2 layers were combined, dried (K2CO3) and concentrated to give crude MDL 44868 (7.2 g, 110%) as an orange oil. This material was used without further purification. 20 Purification of a sample by flash chromatography using eluant C gave analytically pure MDL 44868 as a colorless oil: TLC (eluant C) Rf, 0.35; *H NMR5 (CDCI3) S 1.3 (m, 6), 1.5 (m, 4), 1.63/1.70 (overlapping, complex pentets, 4, J=5.6/5.6 Hz), 2.09/2.11 (s, 6), 2.4 (m, 4), 2.72/2.73 (two 25 overlapping t, 4, J=6.3/6.3 Hz), 3.48/3.60 (t, 4, J=5.6/5.6 Hz), 4.04/4.23 (s, 4); 13C NMR (CDCI3) 6 20.99, 21.12, 23.00, 23.06, 23.40, 26.99, 27.21, 29.16, 41.45, 45.96, 51.71, 51.82, 52.21, 53.37, 53.46, 62.46, 62.54, 67.78; mass spectrum (CI/CH4), m/z(rel intensity) 381(21), 354(28), 30 353(100), 351(10). Anal. Calcd for CigH36N402 (352.52): C, 64.74; H, 10.29; N, 15.89. Found: C, 63.13; H, 10.59; N, 15.52. 35 Step C: 1,7-Bis(3-ethylhexahydropyrlmldln-l-yl)heptane (MDL 45692) WO 94/07480 PCT/US93/08517 -58- A solution of crude bis(acetylhexahydropyrimidine) MDL 44868 (2.0 g, 5.7 mmol) in anhydrous THF (40 mL) was added to a stirred suspension of LAH (0.85 g, 22.7 mmol) in anhydrous THF (60 mL) under N2. The reaction mixture was 5 heated at reflux for 16 h and then allowed to cool to room temperature. The reaction mixture was stirred vigorously and quenched by the cautious addition of saturated aqueous Na2S04 (5 mL) at room temperature. It was necessary to stir the mixture for 16 h to ensure total quench. The mixture 10 was filtered (Celite) and the filter cake was washed with THF (3 x 10 mL). The filtrate and washings were combined and concentrated, the residue was dissolved in CH3CN (50 mL) and was concentrated again to give crude MDL 45692(1.6 9,87%) as a yellow oil. This material was used without further 15 purification. Purification of a sample by flash chromatography using eluant D gave analytically pure MDL 45692 as a light yellow oil: TLC (eluant D) Rf 0.56; 1 H NMR (CDCI3) 6 1.07 (t, 6, J=7.2 Hz), 1.3 (m 6), 1.46 (br pentet, 4, J=6.3 Hz), 1.67 (pentet, 4, J=5.6 Hz), 2.30 20 (overlapping dd, 4, J=7.6, 7.6 Hz), 2.39 (q, 4, J=7.2 Hz), 2.4-2.5 (m, 8), 3.08 (br s, 4); "c NMR (CDCI3) 6 12.23, 23.67, 27.10, 27.42, 29.39, 49.00, 52.03, 52.44, 55.38, 76.13; mass spectrum (CI/CH4), m/z(rel intensity) 353(16), 326(22), 325(100), 324(57), 323(94). 25 Anal. Calcd for C19H40N4 (324.56): C, 70.31; H, 12.42; N, 17.26. Found: C, 69.50; H, 12.75; N, 16.70. 35 4 WO 94/07480 PCT/US93/08517 -59- Step D: N,N' -Bis[ 3-(ethvlamino)propyl 1-1,7-heptanediamine tetrahydrochloride (MDL 28314QA) 5 To a stirred solution of crude bis(ethylhexahydropyrimidine) MDL 45692 (0.50 g, 1.5 mmol) in MeOH (20 mL), conc. HCl (5 mL) was added in one portion. The reaction solution was heated at reflux for 3 h, using a nitrogen sweep to remove HCHO/MeOH distillate (MDL 28314QA 10 precipitated from the reaction solution shortly after heating began). The loss in reaction volume was periodically adjusted to the original level by addition of MeOH. The reaction mixture was allowed to cool to room temperature and then was filtered. The solid was washed 15 with MeOH (2x5 mL) and air-dried to give MDL 28314QA (0.40 g, 58%) as an off-white solid. Recrystallization from H2O (0.8 mL) and i-PrOH (2.9 mL) gave pure MDL 28314QA (0.32 g, 80% recovery) as a white solid: mp 313°C (dec); TLC (eluant E) Rf 0.39; 1H NMR (D20) 6 1.30 (t, 6, J=7.3 20 Hz), 1.4 (m, 6), 1.70 (br pentet, 4, J=7.3 Hz), 2.1 (m, 4), 3.1 (m, 4), 3.13 (q, 4, J=7.3 Hz), 3.15 (m, 8); 13c NMR (D20) 5 13.30, 25.50, 28.21, 28.25, 30.48, 45.87, 46.71, 47.17, 50.61; mass spectrum (CI/CH4), m/z(rel intensity) 329(17), 302(21), 301(100), 300(22), 299(30), 197(8), 25 159(6), 75(10). Anal. Calcd for C17H40N4 4HCI (446.38): C, 45.74; H, 9.94; N, 12.55; CI, 31.77. Pound: C, 45.49; H, 10.48; N, 12.33; CI, 31.20. 30 35 WO 94/07480 -60- PCT/US93/08517 r EXAMPLE 17 1,6 >14,19-Tetraazanonadecane Tetrahydrobromide 5 Step As N-t-Butvloxvcarbonyl-N-4-chlorobutvl-p-toluenesulfonamide A solution of DEAD (17.4 g, 0.1 mole) in THF (20 ml) was added dropwise to a solution of 25 (27.1 g, 01. mole), triphenylphosphine (26.2 g, 0.1 mole) and 4-chloro-l-10 butanol (10.8 g, 0.1 mole) distilled to remove HCl) in THF (600 ml). After 4 hours at ambient temperature the mixture was evaporated and the residue was chromatographed (toluene) to give the product (29.4 g, 81%) as an oil. IR (film) 2980/ 1728, 1456, 1394, 1356, 1292, 1256, 1186, 15 1156, 1088, 10000, 814, 772, 722, 674, 598, 576 and 546 cm-1. NMR (CDC13) 1.35 (s, 9H), 1.8-2.0 (m, 4H), 2.45 (s, 3K), 3.6 (t, J=7.5 Hz, 2H), 3.86 (t, J=7.5 Hz, 2H), 7.33 (J=7.5 Hz) and 7.78 (d, J=7.5 Hz, 2H). MS (CI/CH4) 362 (M + H) . Anal Calcd for C16H24CINO4S 1/8 toluene: C, 54.28; 20 H, 6.75; N, 3.75. Found: C, 54.39; H. 694; N, 3.70. Step B: 1,7-bis-p-Toluenesulfonamidoheptane A mixture of dichloromethane (600 ml), aqueous sodium bicarbonate (600 ml) and 1,7-diaminoheptane (25 g, 0.19 25 mole) was stirred while p-toluenesulfonyl chloride (109 g, 0.57 mole) was added in portions over 1 hour. The layers were separated, the organic layer was extracted with IN HCl and evaporated. The residue was triurated with toluene to give the product (58g, 70%) as a white solid mp. IR (KBr) 30 3256, 2942, 2864, 1430, 1328, 1306, 1160, 1094, 1078, 808, 706, 668, 578, 552 and 522 cm-1. NMR (DMSO-d6) 1.1-1.2 (m, 6H), 1.25-1.3 (m, 4H), 2.4 (s, 6H), 2.6-2.7 (m, 4H), 7.37 (d, J=7.5 Hz, 4H), 7.45 (t, J=7.5 Hz, 2H) and 7.75 (d, J=7.5 Hz, 4H). MS (CI/CH4) 439 (M + H). Anal. Calcd for 35 C21H30N2O4S2: C, 57.50; H, 6.89; N, 6.39. Found: C, 57.87 H, 7.13; N, 6.20. WO 94/07480 PCT/US93/08517 -61- Step C: 1,6 ,14,19-Tetraaza-l,19-bis-t-butvloxycarbonvl-1,6>14,19-tetra-p-tol-uenesulfonylnonadecane Sodium hydride (0.8 g, 16.8 mmol, 50% mineral oil dispersion) was added in portions to a solution of 27 (3 g, 5 8.4 mmol) in DMF (300 mL) and the mixture was stirred for 1 h at ambient temperature. Sodium iodide (0.1) and a solution of 26 (5.2 g, 16.8 mmol) in DMF (50 mL) were added and the mixture was heated at 55 C for 18 h. The mixture was cooled, methanol (10 mL) was added to decompose any 10 unreacted sodium hydride, and the mixture was evaporated to dryness. Dichloromethane (300 mL) and water (300 mL) were added, the organic layer was separated, dried and evaporated. Chromatography (toluene/ethyl acetate 6/1) gave the product (4.4 g, 51%) as a thick oil. IR (film) 15 2980, 2934, 1726, 1458, 1394, 1348, 1306, 1286, 1258, 1186, 1156, 1090, 1006, 814, 756, 722, 674, 654, 598, 576 and 548 cm-1. NMR (CDCL3) 1.25 (s, 6H), 1.32 (s, 18H), 1.42-1.153 (m, 4H), 1.55-1.65 (m, 4H), 1.7-1.8 (m, 4H), 2.55 and 2.56 (s,together 12H), 3.02-3.18 (m, 8H), 4.82 (t, J=7.5 Hz, 20 4H), 7.25-7.35 (m, 8H), 7.68 (d, J=7.5 Hz, 4H) and 7.77 (d, J=7.5 Hz, 4H). MS (FAB) 1089 (M+). Anal: Calcd for C53H76N4O12S4, C, 58.42? H, 7.03? N, 5.14. Found: C,60.50: H,7.25; N, 5.08. 25 Step Ds 1,6,14,19-Tetraaza-l,6,14,19-tetra-p-toluenesulfonvlnonadecane A solution of 28 (20 g, 18 mmol) in a mixture of dichloremethane (500 mL) and TFA (20 mL) was stirred overnight at ambient temperature. The mixture was 30 evaporated, toluene (400 mL) was added and the mixture was evaporated to dryness. The residue was chromatographed (toluene/ethyl acetate 3/1) to give the product (14.3 g, 89%) as an oil. IR (film) 3286, 2936, 2864, 1598, 1454, 1426, 1330, 1306, 1290, 1268, 1158, 1092, 1040, 816, 736, 35 704, 656, 570 and 550 cm-1. NMR (CDCL3) 1.25 (s, 6H), I.45-1.6 (m, 12H), 2.42 (s, 12H), 2.86-2.95 (m, 4H),~ 3.0-3.1 (m, 8H), 4.88 (t, J=7.5 Hz, 2H), 7.15-7.3 (m, 8H), 7.43 WO 94/07480 PCT/US93/08517 -62- (D, J=7.5 Hz, 4H) and 7.72 (d, J=7.5 Hz, 4H). MS (CI/CH4) 889 (M + H). Anal: Calcd for C43H60N4O8S4 3/4 toluene: C, 60.38; H, 7.15; N, 5.70. Found: C, 60.48; H, 7.17; N, 5.57. 5 Step E: 1,6,14,19-Tetraazanonadecane Tetrahvdrobromide A solution of 29 (12.2 g, 13.7 mmol) in 48% aq HBr (500 mL) was heated in a 100 C oil bath for 2 4 h, cooled and evaporated to dryness. The residue was recrystallized 10 (ethanol/H20) to give the product (5.2 g, 63%) as a white solid, mp 303 C. IR (KBr) 2952, 2874 and 2810 cm-1. NMR (D20) 1.4 (s, 6H), 1.6-1.85 (m, 12H) and 3.0-3.1 (m, 12H) . MS (CI/CH4) 273 (M + H). Anal: Calcd for C15H36N4 4HBr:C, 30.22; H, 6.76; N, 9.40. Found: C, 30.60; H, 6.90; N, 15 9.09. Test compound numbers relate to the following 20 compounds: MDL19,190 = NH=C(NH2)-N=C(CH3)—CH=N-NH-C(NH2)=NH 2,21 -(1-methyl-l,2-ethanediylidene)-bis[hydrazinecarboximidamide], (methyl-glyoxal 25 bis(guanylhydrazone)) MDL27,393 = CH3CH2-NH-(CH2)3-NH-(CH2)8-NH-(CH2)3-NH-CH2CH3 N,N'-Bis(3-(ethylamino)propyl)-1,8-octanediamine 30 MDL27,616 = OCH2-NH- (CH2) 3-NH- (CH2) 3-NH-CH2<t> N-(phenylmethyl)-N'[3-[(phenyImethylamino)propyl]-1,3- propanediamine 35 WO 94/074S0 PCT/US93/08517 -63- MDL27,695 = OCH2-NH-{CH2)3-NH-(CH2)7-NH-(CH2)3-NH-CH20 1,18-Bis[(phenyl)methyl 31,5,14,18-tetraazaoctadecane 5 MDL28,314 =CH3CH2-NH-(CH2)3-NH-(CH2)7-NH-(CH2)3-NH-CH2CH3 N,N'-Bis(3-(ethylamino)propyl)-1,7-heptanediamine 10 MDL28,454 =CH3CH2CH2-NH-(CH2)3-NH-(CH2)7-NH-(CH2)3- NH-CH2CH2CH N,N'-bis[3-(propylamino)propyl]-l,7-diaminoheptane gaciclovir = 9-[(2-ydroxy-l-hydroxymethylethoxy)-15 methyl]guanine BIOLOGICAL EXAMPLES 20 EXAMPLE 18s ANTIVIRAL ACTIVITY The antiviral effects of, polyamine analogues was determined by a standard plaque reduction assay as described by Tyms et al. (J. Antimicrobial Chemotherapy 8, 65-72, 1981). Monolayers of human embryo fibroblasts (MRC-25 5 strain) were formed in the presence or absence of varying concentrations of compound and infected with CMV strain AD169 or Towne (lOO pfu/105 cells) in the presence or absence of compound and incubated in the presence or absence of compound for ten days at 37°C. For incubation 30 post infection, cell monolayers were overlaid in maintenance medium (Eagles MEM with 2% bovine fetal calf serum) which contained 0.5% agarose, and fixed and stained with methylene blue at the appropriate time (7-10 days post infection). From the percent reduction in plaque formation compared to untreated controls, a dose response plot was made and the 50% inhibitory concentration (IC50) _ „ determined. A range of IC50 values for various polyamine analogues are shown in Table A. The preferred compound, WO 94/07480 PCI7US93/08517 -64- MDL27, 393, consistently gave IC50 values about 10 pmolcs/litre when cells were also pre-exposed to compound prior to infection. Using these protocols, the IC50 values for MDL 27,393 were about 100,000 lower than those 5 recorded for the current therapies for CMV infections, ganciclovir and foscovir (Table B). With similar protocols using MRC-5 cells (Table C) infected with HSV type 1 (17i strain) but using a three day 10 post-infection incubation period, IC50 values for MDL27,393 (TABLE CI) and MDL28,314 (TABLE C2) were batween 1-10 pmoles/litre with the pre-post treatment protocol. No antiviral activity was observed in MRC-5 cells even at 100 yM with treatment post-infection only. In vero cells 15 (simian origin) treatment with MDL 27,393 (£100 pM), pre-post or post-only, failed to inhibit the growth of HSV-1 (strain 17i). This was evidence that the activity of the analogues was related to cellular or virus specific events involved in CMV but not HSV replication. 20 Natural polyamines putrescine, spermidine or spermine when presented to infected cells, as described in the above protocol, failed to inhibit CMV growth at concentrations < lOpM moles/litre. 25 Similar experiments were carried out with the same pretreatment protocol as above but at high multiplicity infection (approximately 105/pfu/105 cells): monolayers were maintained in the absence of agarose in the maintenance 30 medium. Production of progeny virus and the synthesis of "late" viral proteins were measured as previously described Tyms et al (J. Gen. Virol, 68, 1563-1573, 1987). Virus production was inhibited by the preferred compound, MDL27,393, by 90% at 1 nmoles/litre and 95% at 35 lOnmoles/litre with a corresponding reduction in the synthesis of the three major "late" viral proteins the * major capsid protein (153KD) tegument protein (69KD) and a WO 94/07480 PCT/US93/08517 -65- DNA-binding protein (51KD). This was confirmed in experiments in which numbers of CMV infected cells expressing the "late" antigens under these conditions, identified by a specific monoclonal antibody to a late 5 protein, were shown to be reduced. These results were interpreted to mean that the production of infectious progeny virus in infected cells was curtailed which limited the spread of infection in cell monolayers when infected at low multiplicity of infection. This was considered due to 10 an interference with the synthesis of CMV "late" viral proteins by the preferred compound. EXAMPLE 19s CYTOTOXICITY MRC-5 cells were seeded at low plating density and 15 grown in the presence or absence of the preferred compound, MDL 27,393, or MDL 28,314 and cell numbers were determined at five days postseeding. The results showed that growth of these cells was not inhibited at concentrations of 0.5 ymoles/litre or less although the time to confluency of the 20 monolayers was longer compared to untreated controls at concentrations between 1 and 5 pmoles/litre (data not shown). On the basis of these results the therapeutic window, 25 for the preferred compound MDL 27,393, established by the amount of compound required to inhibit virus growth by 50% compared to the concentration required to inhibit cell growth by 50%, was greater than 10,000. 35 WO 94/07480 m -66- PCT/US93/08517 TABLE A ANTIVIRAL EFFECT OF NATURAL POLYAMINES AGAINST CMV INFECTION 5 REF: PA219 10 15 TABLE B ANTIVIRAL EFFECT OF POLYAMINE ANALOGUES AGAINST CMV INFECTION 20 IC50 Values Compound Pre/Post Post only MDL 27,393 <0.lnM <100nM MDL 28,314 10nM 100nM MDL 27,484 <1nM 100nM MDL 29,587 ClOOnM 1pM MDL 27,666 1nM <1pM MDL 27,667 10nM >1pM MDL 27,394 <10nM <1pM Ganciclovir 1pM <10pM Conc |iM Spermine Mean % VC Spermidine Mean % VC DHPG Mean % VC 100 45,52,0 47% 99,91 93% 0,0 0% 10 106,107,0 100% 99,92 96% 10,17 12% 1 -AsVC- 100% -AsVC- 100% 68, 67 57% 0.1 - As VC - 100% -AsVC- 100% 106,95 91% 0.01 -AsVC- 100% - As VC- 100% - As VC- 100% VC 103,99, 105 100% 103,99, 105 100% 106,129, 99 100% Plaque reduction assay - Towne strain grown in MRC-5 cells Plaque reduction assay - Towne strain grown in MRC-5 cells. 35 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> WO 94/07480 PCT/US93/08517 -67-TABLE C EFFECT OF POLYAMINE ANALOGUES AGAINST HSV TYPE II INFECTION: REF: PA 241 MDL 27. 393: TABLE C1 10 15 Conc pM Pre/Post Mean % VC Post Mean % VC 100 0,0,0 0% 68,71,71 77% 10 16,17,20 19% 91,90,92 100% 1 30,27,30 32% As VC 100% 0.1 49, 50,61 59% As VC 100% 1x10-2 75,71,60 76% As VC 100% 1x10*3 81,63,70 78% As VC 100% 1x10*4 65,80,80 81% As VC 100% VC 95, 87,91 100% 90,89,92 100% 20 MDL 28.314: TABLE C2 25 30 Conc MM Pre/Post Mean % VC Post Mean % VC 100 0,0,0 0% 90,76, 82 81% 10 24,20,15* 22% As VC 100% 1 58,65,51 64% As VC 100% 0.1 63, 65,59 69% As VC 100% 1x10-2 78,71,60 77% As VC 100% 1x10-3 - As VC - 100% As VC 100% 1x10*4 - As VC - 100% As VC 100% VC 87,97,89 100% 103,99,105 100% 35 25 6 2 5 4 68 WHAT IS CLAIMED: 1 . A use of a compound of the formula: HN-Z-NH- (CH2) m-NH-Z-NH R R for the manufacture of a medicament for inhibiting a cytomegaloviral infection; wherein said compound of the medicament is at an effective wherein Z is a C2-C6 saturated straight or branched chain alkyl moiety and each Z is the same; m is 7 or 8; and each R group independently is hydrogen, a Ci-C6 saturated or unsaturated hydrocarbyl, or -(CH2) x-(Ar)-X wherein Ar is phenyl or napthyl, X is H, Ci-Cg alkoxy, halogen or Cj-C^ alkyl, wherein x is an integer 0, 1, or 2; with the proviso that both R groups cannot be hydrogen or if m is 7 then R cannot be benzyl; or said compounds of formula I can be a pharmaceutically acceptable acid addition salt.
2. A use of Claim 1 wherein the compound is Ii,N' -Bis (3-(ethylamino)propyl)-1,8-octanediamine.
3. A use of Claim 1 wherein the compound is 1,18-Bis[(phenyl)methyl]1,5,14,18-tetraazaoctadecane.
4. A use of Claim 1 wherein the compound is N,N'-Bis(3-(ethylamino)propyl)-1,7-heptanediamine.
5. A use of Claim 1 wherein the compound is N,N'-bis[3-(propylamino)propyl]-1,7-diaminoheptane.
6. Use of a compound of the formula: concentration of less than .1 micromolar; and HN-Z-NH- (CH2) m-NH-Z-NH R R 1 9 FEB 1937 256 25 4 - 69 - for the manufacture of a medicament for the treatment of patients suffering from a cytomegalovirus (CMV) disease state; wherein said compound of the medicament is at an effective concentration of less than .1 micromolar; and wherein Z is a C2-Cb saturated straight or branched chain alkyl moiety and each Z is the same; m is 7 or 8; each R group independently is hydrogen, a Ci-C6 saturated or unsaturated hydrocarbyl, or -(CH2) x-(Ar)-X wherein Ar is phenyl or napthyl, X is H, C^C* alkoxy, halogen or Ci-C4 alkyl, wherein x is an integer 0, 1, or 2; with the proviso that both R groups cannot be hydrogen or if m is 7 then R cannot be benzyl; or said compounds of formula I can be a pharmaceutically acceptable acid addition salt thereof.
7. A use of Claim 6 wherein the compound is N,N'-Bis(3-(ethylamino)propyl) -1,8-octanediamine.
8. A use of Claim 6 wherein the compound is 1,18-Bis [(phenyl)methyl]1,5,14,18-tetraazaoctadecane.
9. A use of Claim 6 wherein the compound is N,N'-Bis(3-(ethylamino)propyl)-1,7-heptanediamine.
10. A use of Claim 6 wherein the compound is N,N'-bis[3-(propylamino)propyl]-1,7-diaminoheptane.
11. A pharmaceutical composition for use in the treatment of a cytomegaloviral infection or cytomegalovirus (CMV) disease state in a patient in need thereof comprising an effective concentration of less than .1 micromolar of a compound of formula HN-Z-NH- (CH2) m-NH-Z-NH I I R R wherein Z is a C2-Cb saturated straight or branched chain alkyl moiety and each Z is the same; m is 7 or 8; each R group independently is hydrogen, a Ct-Q saturated or unsaturated - 70 - 25 6 2 5 4 hydrocarbyl, or -(CH2)(Ar)-X wherein Ar is phenyl or napthyl, X is H, Ci-C6 alkoxy, halogen or Ci-C4 alkyl, wherein x is an integer 0, 1, or 2; with the proviso that both R groups cannot be hydrogen or if m is 7 then R cannot be benzyl; or said compounds can be a pharmaceutically acceptable acid addition salt thereof; together with one or more pharmaceutically acceptable excipients or carriers.
12. A pharmaceutical composition according to claim 11 wherein the compound is N,N'-Bis(3-(ethylamino)propyl)-1,8-octanediamine.
13. A pharmaceutical composition according to claim 11 wherein the compound is 1,18-Bis[(phenyl)methyl]1,5,14,18-tetraazaoctadecane.
14. A pharmaceutical composition according to claim 11 wherein the compound is N,N'-Bis(3-(ethylamino)propyl)-1,7-heptanediamine.
15. A pharmaceutical composition according to claim 11 wherein the compound is N,N'-bis[3-(propylamino)propyl]-1,7-diaminoheptane.
16. Use as defined in Claim 1 of a compound of the general formula as defined therein substantially as herein described with reference to any example thereof.
17. Use as defined in Claim 6 of a compound of the general formula as defined therein substantially as herein described with reference to any example thereof.
18. A pharmaceutical composition as defined in claim 11 substantially as herein described with reference to any example thereof. END OF CLAIMS iNCr,- By the authorised agents A. J fiark & Son </div>