CA2165956A1 - Novel substituted purinyl derivatives with immunomodulating activity - Google Patents

Abstract

The present invention covers substituted purinyl compounds. In particular, the present invention concerns 6-substituted purinyl alkoxycarbonyl amino acids, more particularly arginine derivatives. The present invention further includes substituted purinyl compounds which stimulate the immune system and thereby help protect the body against pathogens and cancer.

Description

~ 1 65956 WO 95/35297 ~ ~ f~ d ~ ~ PCT/CA95/00356 NOVEII S~JB~ ll'lu ~ L~ P~JRINYh DERIVATIVES
WIT~I I r - Ul!~ U ATING A~: ~ 1 v 1 l Y

Field of the Invention The present invention covers substituted purinyl compounds. In particular, the present invention concerns 6-substituted purinyl alkoxycarbonyl amino acid compounds, more particularly arginine derivatives.

Bachy oul~d of the Invention 15 The primary function of the ;mmllne system relates to the protection of the body from disease. The immune system protects against not only those diseases which result from an attack by bacteria, viruses, and other pathogens, but also cancer, as well as disease states which result from immune imbalance, opportunistic infections, or auto;mmllne disorders.

Modulation of the ;mmllne system through p~A~mAceutically induced st;mlllAtion or suppression offers an important 25 approach to the control of disease. Compounds which non-specifically stimulate the ;mmllne system are of potentially significant medicinal importance and have been the object of a lengthy research effort. Often, the research results show that ;mmllnomodulating compounds are either weak ;mmllnostimulants~ and hence not very effective, or potent ;mmllnostimulants and, therefore, effective but toxic by virtue of this potent ;mmllnostimulating activity.

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2 1 6 5 9 5 6 PCT/CA95/00356 ~

Among the many classes of compounds which non-specifically stimulate the immune system are nucleosides which are well known in the art. For example, 7-thia-8-oxoguanosine has been described by D.F. Smee et al. in the Journal of Biological Response Modifiers, 9, 24-32, 1990 as an antiviral agent in mice. The activity of this compound is derived from its ability to activate NK and B cells in the immune system, and to induce interferon. However, subsequent antiviral studies in hnmAn~ as reported by P.G.
Higgins et al. in Antiviral Chemistry and Chemotherapy, 2, 61-63, 1991, have disclosed few encouraging results. One problem has been the lack of oral bioavailability.

Other nucleosides have been synthesized and studied in an effort to develop an improved medication. For example, D.F. Smee et al. report in Antimicrobial Agents and Chemotherapy, 35, 152-157, 1991, that 7-deazaguanosine has significant immunostimulatory and antiviral activity after oral A~m;n;.~tration. However, these results are prel; m; nAry . With many nucleoside compounds, toxicity is an important issue which must also be closely analyzed.

A particular class of nucleoside ;mmllnost;mlllAnts has arisen from inosine and other similar hypoxanthine-cont;7; n; ng compounds. A well know example is isoprinosine, an inosine-contA; n; ng complex. Isoprinosine has been thoroughly studied as an ;mml7nomodulator and referred to as a "gold stAn~Ard" by C.D. Simone et al. in Thymus, 19, 51-55, 1992. Some rationale for the activity of hypoxanthine- (inosine) contA; n; ng compounds arises from the observation that a lack of adenosine ~eAm;n~e~
the enzyme which converts adenosine to inosine, results in severe combined immunodeficiency disease (SCID).

' -; t~ 21 65956 W095/35297 ~ . PCT/CA95/00356 Although very nontoxic, isoprinosine is not an effective ;mmllno~odulator, and in order to improve its immunopharmacological properties, numerous analogues have been synthesized, as reported by J.W. Hadden et al. in International Journal of Immunopharmacology, 13, 49-~4, 1991 (suppl. 1). In particular, they describe a prodrug in the form of inosine 5'-monophosphate (inosine, unless complexed, has little in vivo activity) and methyl inosine monophosphate (MIMP). However, MIMP is not a very active immunomodulator.

In an effort to retain the nontoxic properties of isoprinosine, but enhance the immunostimulatory activity, an immunomodulator was synthesized which contained both hypoxanthine and the amino acid L-arginine covalently linked by a pentamethylene bridge. The compound, ST 789 (hypoxanthine pentyloxycarbonyl L-arginine, formerly PCF
39) has been thoroughly described in a recent issue of Thymus, 19, Sl-S112 (1992). L-Arginine was selected because it is known to play a role in immune activation and is present at the t~m;n-lc of many ;mml7nomndulatory peptides such as tuftsin, substance P, thymopentin, and splenopentin. ST 789 is further described in European Patent Application #91830284, publication ~464,009, published January 2, 1992. Analogues of ST 789 are also described in the European publication where oligopeptides compcsed of naturally occurring L-amino acids replace L-arginine. However, the purine base portion of the molecule r~m~; n~ hypoxanthine.

While no ;mmllnological comparison was made with isoprinosine, a similar pattern emerged. The compounds are nontoxic but, at best, moderate ;mmllnostimulants. For W095J3~297 ~f~)~ d ~ ~ 2 1 6 5 ~ 5 6 PCTICA95/00356 ~
example, there was no indication that ST 789, or analogues thereof, could st;m~ te an important immune cell subset such as cytotoxic T lymphocytes (CD8+ T cells). This subset plays a key role in the defense of the body from s viral infections and cancer.

P. Cornaglia-Ferraris describes still another analogue of ST 789 in International Journal of Immunopharmacology, 13, 1005-1012, 1991. In the published compound, L-arginine is replaced with the bombesin carboxy t~rmin~ dipeptide L-leucyl L-methionine. The purine base r~mA; n.~
hypoxanthine. In fact, in this class of compounds where a purine base is covalently linked by a methylene chain to an amino acid or an oligopeptide, very little data has been reported for compounds including a purine base other than hypoxanthine. Further, because of the requirement for physiologically active amino acids in m~mm~lian systems, all the work reported to date describes amino acids of the (natural) L-configuration. One brief description of the replacement of hypoxanthine with the naturally occurring purine bases A~n;ne and guanine is reported by R. Stradi et al. in Fl. Farmaco, 45, 39-47, 1990, but there is no indication of significant biological activity.
As noted above, adenosine ~e~m;nA.ce, and by implication inosine, is necessary to maintain normal immune status.
Therefore, in U.S. patent 5,272,151 issued December 21, 1993, M. Marzi et al. reported that in ST 789 the hypoxanthine is replaced with the xanthine oxidase inhibitor allopurinol. The result is ST 689, allopurinol pentanol. This substitution is expected to increase the concentration of inosine in vivo since inosine is catabolized to xanthine, and then uric acid in m~mm~ls in r .~ 2 1 6 5 9 5 6 WO9S/35297 ,~ PCT/CA95100~6 the presence of xanthine oxidase enzyme. However, allopurinol was noted to be immunosuppressive and ST 689 was not significantly more ir.lmunostimulatory than ST 789 in most of the immunology assays reported in the `151 patent.

Levamisole is another ;mml7noregulator agent used against malignant melanoma. It has now been found that levamisole induces serious thrombocytopenia after starting adjuvant levamisole therapy for malignant melanoma [Med. Pediatr.
Oncol. Apr 1995, 24 (4), 262-4].

The prior art indicates that there is a need for compounds which have the ability to stimulate a number of immune cell subsets and thereby possess significant ;mmllnomodulating activity, but, at the same time, lack toxicity.

Summary of the Invention In accordance with the present invention, there is provided a compound which possesses significant ;mmllnostimulatory capability both in vi tro and in vivo .

Specifically, there is provided a compound which possesses activity in increasing the amount of cytotoxic T cells in vi tro and in vivo .

In another aspect of the invention there is provided an immunomodulatory compound which does not have significant toxicity and, in particular, does not have the toxicity which is associated with significant or potent immunostimulation.
.

WO9S/35297 2 1 6 5 9 5 6 PCT/CA95/003S6 ~
In another aspect of the invention, there is provided an immunomodulatory compound possessing a purine derivative which is not a natural base.

In a further aspect of the invention, there is provided a compound which acts as a control against tumor growth.

The present invention includes compounds of formula (I):

~, (I) or phArmAceutically acceptable derivatives thereof, wherein Rl is selected from the group consisting of hydrogen; Cll6 alkyl; halogen; substituted or unsubstituted thiol;
unsubstituted or substituted aminoi and oR8 wherein R3 is selected from the group consisting of hydrogen, Cl_ 16 alkyl, Cl8 acyl, and C7_l8 aryl;
R2 and R3 are independently selected from the group consisting o~ hydrogen; Cl_4 alkyl; amino; substituted or unsubstituted thiol; and halogen; and R4 is selected from the group consisting of a linear or cyclic carbon chain of the formula (CHo_2) 0-20 -Xl2 optionally interrupted with one or more heteroatom, and optionally substituted with one or more =O, or =S, and wherein Xl2, is selected from the group consisting of hydroxy, an aminoalkyl group, an amino acid, or a peptide of 2-8 amino acids, with the proviso that, when Rl is NH2, and R4 is pentyloxy carbonyl-L-arginine, then R2 is not hydrogen, and when Rl is OH, and R4 is pentyloxycarbonyl-L-arginine, then R2 is not NH2-~ W095/35297 ~ ` 2 ~ ~ 5 q 5 6 PCT/CA95/0035~

The following definitions are used herein.The term "alkyl" as employed herein includes both straight and branched chain radicals, for example methyl, ethyl, s propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, the various branched chain isomers thereof. The chain may be saturated or unsaturated and may contain, for example, double and triple bonds. The alkyl may be interrupted or substituted with, for example, one or more halogen, oxygen, hydroxy, silyl, amino, or other acceptable substituents.

The term "aromatic or non-aromatic ring" as used herein includes 5 and 6 membered aromatic and non-aromatic rings uninterrupted or interrupted with one or more heteroatom, for example O, S, SO, SO2, and N, or the ring may be unsubstituted or substitute~ with, for example, halogen, alkyl, acyl, hydroxy, aryl, and amino, said heteroatom and substituent may also be substituted with, for example, alkyl, acyl, aryl, aralkyl.

The term "acyln as used herein refers to carbonyl groups of the formula -COR wherein R may be any suitable substituent such as, for example, alkyl, amino, halogen, thiol, oxygen, hydroxy, and hydrogen.

The term "aryl" as employed herein refers to monocyclic or bicyclic aromatic groups con~; n; ng from 6 to 10 carbons in the ring portion, such as phenyl, naphtyl, substituted phenyl, naphtyl, substituted phenyl or substituted naphthyl, wherein the substituent on either the phenyl or 2 ~ ~ 5 9 5 6 W095/35297 ~ PCT/CA95/00356 naphthyl may be for example Cl_4 alkyl, halogen, Cl_4 alkoxy, hydroxy or nitro.

The term "aralkyl" as used herein refers to alkyl groups as discussed above having an aryl substituent, such as benzyl, p-nitrobenzyl, phenethyl, diphenylmethyl, and triphenylmethyl.

The term "substituted amino" as used herein refers to an amino which may be substituted with one or more substituent, for example, Cl8 alkyl, Cl_8 acyl, C6l2 aryl, hydroxy, and hydrogen.

The term "amino acidN as employed herein includes and encompasses all of the naturally occurring amino acids, those amino acids in their D- and L-configurations, and the known non-native, synthetic, and modified amino acids, such as homocysteine, ornithine, norleucine and ~-alaline.
A list of non natural amino acids may be found in ~The Peptidesn, vol 5, 1983, Academic Press, Chapter 6 by D.C.
Roberts and F. Vellaccio.

The term Ul;neA~ or cyclic" when used herein includes, for example, a linear chain which may optionally be interrupted by an aromatic or non-aromatic ring. Cyclic chain includes, for example, an aromatic or non-aromatic ring which may be connected to, for exam.ple, a carbon chain which either precedes or follows the ring.

The term "p~ArmAceutically acceptable derivative" as employed herein, includes any p~rmAceutically acceptable salt, ester, or salt of such ester, of a compound of formula I or any other compound which, upon A~m;n;stration to the recipient, is capable of providing (directly or 2 ~ 6 5 9 5 6 indirectly) a compound of formula I or an active metabolite or residue thereof.

Brief Description of Drawi~gs .

Figure l illustrates the variations in tumor growth for mice treated with cyclophosphamide, or compound #l, or both.
Figure 2 illustrates the body weight variations for mice treated with the same regimen as in Figure l Figure 3 illustreates the variations in tumor volume for lS mice treated with Cytoxan, or compound #l , or both.

Figure 4 illustrates the body weight variations for mice treated with the same regimen as in Figure 3.

Figure 5 illustrates the variations in tumor volume for mice treated with 5FU, 5FU with levamisole, and 5FU with compound #l.

Figure 6 illustrates the growth curves of male Fisher rats treated with compound ~l at high doses.

Figure 7 illustrates the growth curves of female Fisher rats treated with compound #l at high doses.

Description of the Invention In one aspect of the present invention, there is provided a compound of formula (I) wherein R4 is (CHo_2)l_8~Xl2, wherein Xl2 is OH.

a ~ ~ 21 65956 W095/35297 PCT/CA9~/00356 In a further aspect of the invention, there is provided a compound of formula (I) wherein R4 is (CH2)- L -O-CO-Xl2, wherein L is a linear or cyclic carbon chain optionally interrupted with one or more ~, S, or NH.

Preferably, Xl2 can be (CH2)nNH2 wherein n is an integer between 1 and 6. More preferably, n is 2.

More preferably, Xl2 can be a naturally occuring amino acid in the D- or L- configuration. Preferably, these amino acids can be selected from the group consisting of:
arginine, glycine, A 1 An; ne, glutamic acid, valine, ornithine, or citrulline, or conservative substitutions thereof.

Still, more preferably, the amino acid is L-arginine.
Even more preferably, the amino acid is D-arginine.

In an alternative embodiment of the invention, Xl2 may be a peptide of 2 to 8 amino acids.

Preferably, such a peptide can be Val-Pro-Leu, or Ile-Pro-Ile, or conservative substitutions thereof.
In another embodiment of the invention, L can be selected from: ~(CH2)n-, ~(cH2)m-~-(cH2)m-~ and (CH2)m-C-C-(CH2)m-, wherein ~ is O, S, or NH, n is an integer between 1 and 6, and m is an integer between 1 and 3.
Preferably, L can be selected from: phenyl, cyclohexyl, dioxolanyl, oxathiolanyl, and cyclopentyl.

In an further alternative of the invention, when Rl is Cl~6 alkyl, Rl can be an aromatic or non aromatic ring opti.onally interrupted with one or more heteroatom, and optionally substituted with one or more heteroatom, s hydroxy, halogen, Cll6 alkyl, Cll6 acyl, C6l2 aryl, nitro, or substituted or unsubstituted amino.

More preferably, Rl can be OH, OCH3, SH or SCH3.

Alternatively, Rl can be selected from the group consisting of: hydrogen, halogen, Cl_6 alkyl, unsubstituted or substitued amino, 9H, and OCl6 alkyl, SH, or SCl6 alkyl.

Preferably, Rl can be chloro.

Alternatively, Rl can be represented by formula NR5R6 wherein R5 and R6 are independently selected from the group consisting of hydrogen, Cl_4 alkyl, Cl4 alkoxy, Cl4 acyl, substituted or unsubstituted amino, and C6l0 aryl.
Preferably, Rl can be selected from the group consisting o ~ :
-N(CH3)2, -NHNH2 ~
-NHCH3, -NH2, -N ( NH2 ) CH3, -NH-CH ( CH3 ) CH2-O- ( CO ) CH3, ~N , or ~20 NH , wherein R20 is H or methyl.

2 ~ ~ 5 ~ 5 6 Even more preferably, Rl can be: -N(CH3) 2 Even more preferably, Rl can be: -NHNH2.
Even more preferably, Rl can be: . -NHCH3, S Even more preferably, Rl can be: -NH2, and Even more preferably, Rl can be: -N(NH2)CH3.

Most preferably, Rl can be -N(CH3)2.

In a further alternative embodiment of the invention, R2 and R3 can be independently selected from the group consisting of: Cl, Br, I, and F.

Preferably, R2 and R3 can be independently Cl, or Br.
More preferably, R2 can be H, Cl, or NH2.

More preferably, R3 can be H, Br, or SH, or SCH3.

Most preferably, the compound of the invention is represented by formula (I) wherein Rl is N(CH3) 2 i R2 and R3 are both hydrogen; and R~ is pentyloxycarbonyl-D-arginine, or ph~rm~ceutically acceptable derivatives thereof.

Preferred compounds of the present invention are selected from:
Compound #III N-(6-Chloropurin-9-yl)-5-pentanol Compound #V N-(6-N,N-Dimethylaminopurin-9-yl)-pentanol Compound #l N,N-Dimethyl ~m; nopurinyl Pentoxycarbonyl D-Arginine Compound #2 N,N-Dimethylaminopurinyl Pentoxycarbonyl L-Arginine WO95/35297 ~ 2 1 6 5 9 5 6 PCTICA9S/00356 Compound #3 N-Monomethyl ~mi nopurinyl Pentoxycarbonyl D-Arginine Compound #3a N-(6-N-~ethyl-Aminopurin-9-yl)-pentanol Compound #4 N-Monomethylaminopurinyl Pentoxycarbonyl L-Arginine Compound #5 Aminopurinyl Pentoxycarbonyl D-Arginine Compound #5a N-(6-Aminopurin-9-Yl) 5-Pentanol 10Compound #6 Aminopurinyl Pentoxycarbonyl L-Arglnine Compound #7 Hydrazinopurinyl Pentoxycarbonyl D-Arginine Compound #7a N-(6-Hydrazinopurin-9-yl) 5-Pentanol 15Compound #8 Hydrazinopurinyl Pentoxycarbonyl L-Arginlne;
Compound #9 Chloropurinyl Pentoxycarbonyl D-Arginine;
Compound #10 Chloropurinyl Pentoxycarbonyl L-20Arginine;
Compound #11 Hydroxypurinyl Pentoxycarbonyl D-Arginine;
Compound #12 Mercaptopurinyl Pentoxycarbonyl D-Arginine;
25Compound #13 Mercaptopurinyl Pentoxycarbonyl L-Arginine;
Compound #14 N,N-Dimethyl~m; nopurinyl Pentoxycarbonyl Glycine;
Compound #15 N,N-(6-Dimethylaminopurin-9-yl)-7'-ethoxy-ethoxycarbonyl-D-arginine;
Compound #16 (2S,4S)-2-(N,N-dimethylaminopurin-9-yl)-4-(methyloxycarbonyl-D-arginine)-1,3-dioxolane;
Compound #17 N-(6-Dimethyl~m; no-8-bromopurinyl-Pentoxycarbonyl L-Arginine;

Compound $18 N-(6-dimethylamino-8-bromopurin-9-yl) 7-pentoxycarbonyl-D-arginine;
Compound $19 N-9-purinyl-5-pentanol;
Compound #20 N-9-purinyl-7-pentyloxycarbonyl-D-arginine;
Compound $21 N-9-purinyl-7-pentyloxycarbonyl-L-arginine;
Compound $22 N,N-Dimethylaminopurinyl Pentoxycarbonyl L-Valyl L-Prolyl L-Leucine;
Compound ~23 N,N-Dimethylaminopurinyl Pentoxycarbonyl L-Isoleucyl L-Prolyl L-Isoleucine;
Compound #24 N-(6-Cyclopropylaminopurin-9-yl)-5-pentanol;
Compound $25 N-(6-cyclopropylaminopurin-9-yl)-7-pentyloxycarbonyl-D-arginine;
Compound #26 N-(6-cyclopropylaminopurin-9-yl)-7-pentyloxycarbonyl-L-arginine;
Compound $27 N-(6-Azetidinepurin-9-yl)-5-pentanol;
Compound #28 N-(6-Azetidinepurin-9-yl)-7-pentyloxycarbonyl-D-arginine;
Compound ~29 N-(6-Azetidinepurin-9-yl)-7-pentyloxycarbonyl-L-arginine;
Compound ~30 trans-(N-6-chloropurin-9-yl)-4-methyl-cyclohexyl-methanol;
Compound $31 trans-(N-6-dimethylaminopurin-9-yl)-4-methyl-cyclohexyl-methanol;
Compound $32 trans-tN-6-dimethylaminopurin-9-yl)-4-methyl-cyclohexyl-methyloxycarbonyl-D-arginine;
Compound ~33 trans-(N-6-hydroxypurin-9-yl)-4-methyl-cyclohexyl-methanol;
Compound $34 trans-(N-6-methoxypurin-9-yl)-4-methyl-cyclohexyl-methanol;
Compound $35 cis-(N-6-dimethylaminopurin-9-yl)-4-methyl-cyclohexyl-methanol;

2 ~ 65956 WO 9S/35297 ,~ r~ PCT/CA95/00356 Compound #36 cis-(N-6-dimethylaminopurin-9-yl)-4-methyl-cyclohexyl-methyloxycarbonyl-D-arginine;
Compound #37 N-(6-dimethylaminopurin-9-yl) 7-pentoxycarbonyl-D-citrulline;
Compound #38 N-(6-methylaziridinepurin-9-yl)-5-pentanol;
Compound #39 racemic N-(6-methylaziridine purine-9-yl)-7-pentyloxycarbonyl-D-arginine;
Compound #40 N,N-(6-Dimethylaminopurinyl-9-yl)-7-thioethoxy-ethoxycarbonyl-D-arginine;
Compound #41 Meta-(N-6-dimethylaminopurinyl-9-yl) methyl-benzyloxycarbonyl-D-arginine;
Compound #42 5-(N-6-Dimethylaminopurinyl-9-yl)-3-pentynyl-l-oxycarbonylD-arginine;
Compound #43 Racemic N-[6-(1-methyl-2-acetoxy)-ethylaminopurin-9-yl]-5-pentanol;
Compound #44 Racemic N-[6-(1-methyl-2-acetoxy), ethyl ~mi nopurin-9-yl]-7-pentyloxy-carbonyl-D-arginine;
Compound *45 N-(2,6-Dichloropurin-9-yl)-5-pentanol;
Compound #46 N-(2,6-Dichloropurin-9-yl)-7-pentyloxycarbonyl-D-arginine;
Compound #47 N-(2,6-Dichloropurin-9-yl)-7-pentyloxycarbonyl-L-arginine;
Compound *48 N-(2-Amino, 6-N, N-Dimethylaminopurin-9-yl)-5-pentanol;
Compound #49 N-(6-dimethyl ~mi no-8-methylthiopurin 9-yl) 5-pentanol;
Compound #50 N-(6-dimethylamino-8-methylthiopurin-9-yl) 7-pentoxycarbonyl-D-arginine;
Compound #51 N-(6-methoxypurin-9-yl) 5-pentanol;
Compound *52 N-(6-methoxypurin-9-yl) 7-pentoxycarbonyl-D-arginine;

W095/3S297 2 1 6 5 9 5 6 PCT/CAg5/oo~
Compound #53 N-(2-chloro-6-methoxypurin-9-yl)-7-pentyloxycarbonyl-D-arginine;
Compound #54 N-(6-dimethylaminopurin-9-yl) 7-pentoxycarbonyl-D-ornithine;
S Compound #55 N-(6-dimethylaminopurin-9-yl) 7-pentoxycarbonyl-L-ornithine;
Compound #56 N-(6-dimethylaminopurin-9-yl) 7-pentoxycarbonyl-L-valine;
Compound #57 N-(6-dimethylamino-9-yl) 7-pentoxycarbonyl-D-valine;
Compound #58 N(N,N-dimethylaminopurin-9-yl)-7-pentyloxycarbonylethylamine hydrochloride;
Compound #59 N-(6-Mercaptopurin-9-yl)-pentanol;
Compound #60 N-(6,-N-Methylthiopurin-9-yl)-pentanol;
Compound #61 N-(6-chloropurin-9-yl) 4-butanol;
Compound #62 N-(6-dimethylaminopurin-9-yl) 4-butanol;
Compound #63 N-(6-dimethylaminopurin-9-yl)-6-butoxycarbonyl-D-arginine;
Compound #64 N-(6-dimethylaminopurin-9-yl)-6-butoxycarbonyl-L-arginine;
Compound #65 N-(6-chloropurin-9-yl)-6-hexanol;
Compound #66 N-(6-N,N-dimethylaminopurin-9-yl)-6-2~ hexanol;
Compound #67 N-(6-N,N-dimethylaminopurin-9-yl)-8-hexyloxycarbonyl-D-arginine;
Compound #68 N(6-N,N-dimethylaminopurine-9-yl)-8-hexyloxycarbonyl-L-arginine;
Compound #69 cis-(N-6-hydroxypurin-9-yl)-4-methyl-cyclohexyl-methanol;
Compound #70 cis-(N-6-hydroxypurin-9-yl)-4-methyl-cyclohexyl-methyloxycarbonyl-D-arginine;

. 2 1 65956 W095/35297 ~ PCTICA95/00356 Compound #71 trans-(N-6-hydroxypurin-9-yl)-4-methyl-cyclohexyl-methyloxycarbonyl-D-arginine;
Compound #72 N-(6-N,N dimethylaminopurin-9-yl)-5-pentylamine hydrochloride salt;
5Compound #73 N-(6-methylaziridinepurin-9-yl)-7-pentyloxycarbonyl-L-arginine;
Compound #74 (2S,4S)-2-(N,N-Dimethylaminopurin-9-yl)-4-hydroxymethyl-1,3-dioxolane;
Compound #75 (lS,3R) and (lR,3S)-l-(N-6-10Dimethylaminopurin-9-yl)methyl-3-cyclopentane methanol;
Compound #76 (lS,3R) and (lR,3S)-l-(N-6-Dimethylaminopurin-9-yl)methyl-3-(methyloxycarbonyl-D-arginine)cyclopentane;
15Compound #77 N,N-(6-Dimethylaminopurin-9-yl)-7-ethylaminoethanol;
Compound #78 N,N-(6-Dimethylaminopurin-9-yl)-7-ethylaminoethoxycarbonyl-D-arginine;
Compound #79 N,N-(6-Dimethylaminopurin-9-yl)-7-20ethyl Am; noethoxycarbon:yl-L-arginiIle;
Compound #80 5-(N-6-Dimethylaminopurin-9-yl)-3-pentyn-l-ol;
,Compound #81 5-(N-6-Dimethylaminopurin-9-yl)-3-pentynyl-l-oxycarbonyl-L-arginine;
25Compound #82 N,N-(6-Dimethylaminopurin-9-yl)-7-thioethoxy-ethanol;
Compound #83 N,N-(6-Dimethylaminopurin-9-yl)-7-thioethoxy-ethoxycarbonyl-L-arginine;
Compound #84 (2S,4S)and (2R,4R)-2-(N,N-30Dimethylaminopurin-9-yl)-4-(methoxycarbonyl-D-arginine)-1,3-oxathiolane;
Compound #85 N,N-(6-Dimethyl~m; nopurin-9-yl)-7-ethoxy-ethoxyethanol;

W095/35297 2 1 6 5 9 5 6 PCTICA9~J~D356 Compound #86 N,N-(6-Dimethylaminopurin-9-yl)-7-ethoxy-ethoxycarbonyl-D-arginine;
Compound #87 N,N-(6-Dimethylaminopurin-9-yl)-7-ethoxy-ethoxycarbonyl-L-arginine; and S Compound #88 N-(6-Dimethylamino-8-bromopurin-9-yl)-5-pentanol.

More pre~erably, the compound o~ the present invention is selected ~rom:
Compound #III N-(6-Chloropurin-9-yl)-5-pentanol Compound #V N-(6-N,N-Dimethylaminopurin-9-yl)-pentanol Compound #l N,N-Dimethylaminopurinyl Pentoxycarbonyl D-Arginine Compound #2 N,N-Dimethylaminopurinyl Pentoxycarbonyl L-Arginine Compound #3 N-Monomethyl ~m; nopurin Pentoxycarbonyl D-Arginine Compound #3a N-(6-N-Methyl-Aminopurin-9-yl)-pentanol Compound #5 Aminopurinyl Pentoxycarbonyl D-Arginine Compound #5a N-(6-Aminopurin-9-Yl) 5-Pentanol Compound #6 Aminopurinyl Pentoxycarbonyl L-Arginine Compound #7 Hydrazinopurinyl Pentoxycarbonyl D-Arginine Compound #7a N-(6-Hydrazinopurin-9-yl) 5-Pentanol Compound #8 Hydrazinopurinyl Pentoxycarbonyl L-Arginine;
Compound #ll Hydroxypurinyl Pentoxycarbonyl D-Arginine;
Compound #l9 N-9-purinyl-5-pentanol;

WO 95/35297 rr~ 2 1 6 5 9 5 6 PCT/CAg5J~356 Compound ~20 N-9-purinyl-7-pentyloxycarbonyl-D-arginine;
Compound ~51 N-(6-methoxypurin-9-yl) 5-pentanol;
Compound ~59 N-(6-Mercaptopurin-9-yl)-pentanol; and s Compound ~60 N-(6,-N-Methylthiopurin-9-yl)-pentanol.

Most preferably, the compound of the present invention is N,N-(6-dimethylaminopurin-9-yl)-7-pentoxycarbonyl-D-arginine .

The following abbreviations and definitions are usedherein:
PHA - phytohemagglutinin ConA - concanavalin A
CY - cyclophosphamide PWM - pokeweed mitogen LPS - lipopolysaccharide DEAD - diethylazodicarboxylate PBS - phosphate buffered saline TBDPSCl - tert-butyldiphenylsilyl chloride CTX - Cytoxan The term "conservative substitution" as e-mployed herein refers to modifications and substitutions of amino acids which are conservative ones, i.e. those having a m; n; m~l influence on the secondary structure and hydropathic nature of the amino acid or peptide. These include substitutions such as those described by Dayho~ in the Atlas of Protein Sequence and Structure 5, 1978, and by Argos in ~RO J., 8, 779-785, 1989. For example, amino acids belonging to the following groups represent conservative changes: ala, pro, gly, glu, asp, gln, asn, ser, thr; cys, ser, tyr, thr; val, ile, leu, met, ala, W095/35297 2 ~ 6 5 9 5 ~ PCT/CA95/00356 phe; lys, arg, his; and phe, tyr, trp, his. The preferred substitutions also include substitutions of D-isomers for the corresponding L-amino acids.

It has been surprisingly discovered that contrary to the well-established prior art, hypoxanthine or other naturally occurring purine bases such as adenine or guanine need not be used in the design of an ;mmllnostimulant of the type similar to ST 789. In fact replacement of hypoxanthine with a 6-substituted purine base that does not occur in biological systems can provide an equal or even greater degree of immunostimulation.
Further, it has been surprisingly discovered that the amino acid need not be of the (natural) L-configuration.

It will be recognized that the designation of a naturally occurring amino acid does not preclude the use of racemic mixtures or D-enantiomers and in one aspect of the invention, it is especially preferred to use amino acids in the D-configuration.

It has surprisingly been discovered that the compounds of the invention possess in vi tro and in vivo activity to increase the number of cytotoxic T lymphocytes in the 25 m~mm~ 1 being treated.

It has further been discovered that the compounds of the present invention are surprisingly active against tumor growth. The compounds of this invention represents a non-toxic substitute to levamisole in the treatment ofmalignant melanoma.

When tested in mice against a control group, the compounds of the present invention significantly inhibit tumor W095/3S297 .~ 5 9 5 6 PCT/CA95100356 growth when used in combination with cyclophosphamide or 5-fluorouracil, particularly against mAmm~y and colon carcinoma respectively.

s The compounds o~ the present invention may be prepared by the use of synthetic methods well known in the art. Thus, for example, it is possible to follow the synthetic procedure described by R. Stradi et al. in Il Farmaco, 45, 39-47, l990, with the provision that the chlorine atom from the chloropurine intermediate must be displaced by an appropriate substituent other than hydroxyl. However, it is preferred to carr,~ out a modi~ication o~ this synthetic procedure, as outlined in the following examples, wherein the purine ring is already constructed by use of 6-lS chloropurine as a starting material. This avoids the needto build the purine ring and thereby provides a more e~ficient and higher yield preparation o~ the desired immunost-m~ nt. This preferred synthetic pathway is outlined in Scheme l.
In Scheme l, R~' which is (CHo_2)l_8~0~CO~Xl2 as defined above, is reacted with a protecting group in the presence of a base such as NaH/THF to produce compound V. L
represents a leaving group well known to those skilled in the art. Any suitable leaving group can be used. Pg is a protecting group well known in the art. Any suitable protecting group can be used.

W095/35297 2 t- 6 5 9 5 6 PCT/CA95/00356 Rl . PG-L

N ~ N R~ V NaH/THF R~
IV 2 ¦ DEAD
~ PPh3/THF

Rl R2 ~ IN R3 III R~ PG

1 (C~Hs~NF/THF
AcOH

N ~ N

II R~ - OH
4 ClCOC1/THF
amino acid /H20 R~

Compound V is coupled with compound IV, for example, in the presence of DEAD and PPh3/THF to yield compound III.
Compound IV can be prepared using known techniques in the art. As well, Rl can be added before this step or at a later step using techniques well known in the art.

~ W095/35297 ~ S 2 1 6 5 95~ PCT/CA95/00356 Compound III is deprotected by using methodology well known to those skilled in the art, for example, with (C4Hg)4NF/THF and AcOH for a OTBDPS protecting group, to yield compound II. This compound is further optionally s reacted with an amino acid or a peptide group of 1-8 amino acids in length, for example, in the presence of ClCOCl/THF and H2O. The resultant compound is a compound of formula I.

Those skilled in the art will appreciate that compounds of formula 1 wherein R4 is not an amino acid or peptide chain can be synthesized by utilizing steps 1 to 3 without the addition step 4.

It will be appreciated by those skilled in the art that the compounds of the present invention include all pharmaceutically acceptable derivatives and analogues thereof, as well as all isomers and enantiomers.

Another aspect of the invention is the use of the compounds of formula I or pharmaceutical preparations for the manufacture of a medicament.

Another aspect of the invention is the method of treatment 2s of a m~mm~l, preferably a human, comprising the step of a~m;n;~tering a compound of formula I, a ph~rm~ceutical composition, or a ph~rm~ceutically acceptable derivative thereof for the treatment of immune deficiency or control of tumor growth.
It will be appreciated by those skilled in the art that the reference herein to treatment extends to prophylaxis as well as treatment of established infections or symptoms and therefore includes control of tumor outgrowth.

W095/35297 2 ~ 6 5 ~ ~ 6 PCTICA95/00356 ~

It will be further appreciated that the amount of a compound of the invention re~uired for use in treatment will vary not only with the particular compound selected but also with the route of a~m; n; stration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion o~
the attendant physician or veterinarian.

In general, however, a suitable dose will be in the range from about 0.1 to about 250 mg/kg o~ body weight per day.
Preferably, doses will range from about 1 to about 100 mg/kg/day. More preferably between about 2 to about 20 mg/kg. Most preferably about 2.5 mg/kg. Still, most preferably about 450 mg/m2.

The desired dose may conveniently be presented in a single dose or as divided doses ~m;n;stered at appropriate intervals, for example as two, three, four or more sub-doses per day.

The compound is conveniently ~m;n;.~tered in unit dosageform; for example cont~;n;ng 10 to 1500 mg, conveniently 20 to 1000 mg, most conveniently 50 to 700 mg of active ingredient per unit dosage form.

Ideally the active ingredient should be a~m;n;~tered to achieve peak plasma concentrations of the active compound.
This may be achieved, for example, by the intravenous injection of a solution of the active ingredient, optionally in saline, or a~m;n;stered as a bolus.
Desirable blood levels may be maintained by a continuous infusion or by intermittent infusions.

2 1 6 5 ~56 W095/352~7 ;~, ~l,J ¦~ PCT/CA95/003~6 While it is possible that, for use in therapy, a compound of the invention may be administered as the raw chemical, it is preferable to present-the active ingredient as a pharmaceutical formulation.

The invention thus further provides a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable derivative thereof together with one or more ~hArmAceutically acceptable carriers thereof and, optionally, other therapeutic ingredients.
The carrier(s) must be "acceptableN in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

Pharmaceutical formulations include those suitable for topical, oral, rectal, nasal, or parenteral (including intramuscular, sub-cutaneous and intravenous) a~m;n;.ctration or in a form suitable for a~m;n;stration by inhalation or insufflation. The formulations may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art o~ ~hArm~cy All methods include the step o~
bringing into association the active compound with li~uid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

For topical administration to the epidermis, the compounds according to the invention may be formulated as ointments, creams or lotions, or as a trans~rmAl patch. Such transdermal patches may contain penetration ~nhAncers such as linalool, carvacrol, thymol, citral, menthol and t-anethole. Ointments and creams may, for example, be formulated with an a~ueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may WO95/35297 21 6 5 9 5 6 PCT1CA95100356 ~
be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, sUsp~n~;ng agents, thickening agents, or colouring agents.

Pharmaceutical ~ormulations suitable ~or oral ~m; n; .ctration may conveniently be presented as discrete units such as capsules, cachets, or tablets. Each pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, _r other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.
Alternatively, for ~m;n;.ctration by inhalation or insufflation, the compounds according to the invention may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for exa-mple~ capsules or cartridges or, e.g., gelatin or blister packs from which the powder may be ~m; n; ctered with the aid of an inhalator or insufflator.
When desired, the above described formulations adapted to give sustained release of the active ingredient may be employed.

The compounds of the invention may also be used in com.bination with other therapeutic agents, for example, other immuomodulators or tumor control agents.

The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a ?~ 21659~6 WO95/35297 ~ ' PCT/CA9S/00356 physiologically acceptable derivative thereof together with another therapeutically active agent.

Such therapeutically active agents include cytotoxic s agents used to treat tumors. Such cytotoxic agents include cyclophosphamide, or 5-fluorouracil (5-FU).

Preferably, cyclophosphamide doses used in the treatment of tumors range from about 10 to 1000 mg/m2.
Morepreferably, from about 100 to about 500 mg/m2. Most preferably, about 350 mg/m2/day.

Also preferably, 5-fluorouracil doses used in the treatment of tumors ranges from about 0.1 to about 250 mg/kg. Preferably, between about 1 to about 50 mg/kg. More preferably, between about 5 to about 20 mg/kg. Most preferably, at about 12 mg/kg (500 mg/m2).

As will be recognized by people skilled in the art of cancer therapy, such doses will vary with the type of malignancy being treated, the stage of the disease, the responsiveness o~ the tumor, etc..

The combinations referred to above may conveniently be presented for use in the form of a phArm~ceutical composition and thus phArm~ceutical composition comprising a combination as defined above together with a pharmaceutically acceptable carrier thereof comprise a further aspect o~ the invention.
The individual components of such combinations may be a~m; n; .~tered either se~uentially or simultaneously in separate or com.~bined pharmaceutical formulations.

W095/35297 ~ a ~ 2 ~ 6 5 9 5 6 PCT/CA95/00356 ~
When the compound of formula (I) or a pharmaceutically acceptable derivative thereof is used in combination with a second therapeutic agent, the dose of each compound may be either the same or differ from that when the compound S is used alone. Appropriate doses will be readily appreciated by those skilled in the art.

In a further embodiment o~ the invention, there is provided a method of treatment of immune deficiencies or for the control of tumor growth comprising the step of A~m;n;Stering a pharmaceutically acceptable amount of a compound of the invention.

Preferably, such tumors include malignant melanoma, mAmmAry and colon carcinoma.

More preferably, there is provided a method for the treatment of mAmmAry carcinoma comprising the step of administering a phArmAceutically acceptable amount of a compound of the invention, in combination with cyclophosphamide.

Most preferably, there is provided a method for the treatment of colon carcinoma comprising the step of A~m;n;stering a phArmAceutically acceptable amount of a compound of the invention, in co-m-bination with 5-fluorouracil.

The invention will be further described by the following examples which are not intended to limit the invention in any way. All temperatures are in degrees Celsius.

W095/35297 ~ 3'~ ~ ' 2 1 6 5 95~ PCT/CA95/00356 EXA~qPIæS
The compounds of formula I were synthesized and tested for immunological activity using the procedures outlined below.
5 r _le la Synthesis of N,N-Dimethylaminopurinyl Pentoxycarbonyl D-Arginine -Compound #1 TBDPSCl ~N~N~ HO ~~OTBDPS N~/THF
I DEAD
PPh~ /ThF
Cl N~N~

II OTBDPS
(C,H, ) ,NF/ThF
AcO}I

N~N~

¦~ ~OH
III Clcocl/Toluene D~ inine/ H20 HN ( C}i3 ) 2 ' H20 N

N~N~I
~~O~Nh~COOH

~NHJ~NH

W095/35297 ~s~ S 21 6 5 9 5 6 PCT/CA95/00356 Step 1:
a) Synthesis of protected 1,5 - pentanediol 4.0 g., 36.3 mmole of pentanediol was dissolved in 75 ml dry tetrahydrofuran and stirred under argon flow.
Sodium hydride (1.4 g., 57.8 mmole) was added and the suspension was stirred for 30 minutes, and then tert-butyldiphenylsilyl chloride (8.0 ml, 30.8 mmole) dissolved in 25 ml dry tetrahydrofuran was added dropwise to the diol solution. The reaction was stirr~d at ambient temperature and under argon overnight. The suspension was then poured onto lOO
ml ether. The etheral suspension was washed with 10%
potassium carbonate (100 ml), brine (lOOml) and dried with magnesium sulfate. Removal of the solvent in va cuo gave 10.3 g., 30.1 mmole of product in 98~
yield which was used without further purification.

b) Coupling of Compound I with 6-chloropurine To a stirred solution of triphenylphosphine (4.7 g., 17.9 mmole), and 6-chloropurine (2.3g., 15.1 mmole) in lOO ml dry tetrahydrofuran, under argon flow, was added diethylazodicarboxylate (DEAD, 2.8 ml, 17.9 mmole). After lO minutes, compound I (4.7 g., 13.7 mmole) dissolved in 20 ml dry tetrahydrofuran was added dropwise to the reaction, which was then stirred at ambient temperature and under argon overnight. The solvent was L~IIOv~d in vacuo, and the crude product was purified by flash silica gel chromatography using 30% ethyl acetate-hexane as eluent (R = 0.30). The product, compound II, 3,7 g., 7.6 mmole, was obtained in 55~ yield as a colorless oil.

WO9S/35297 ~ `~; 2 ~ ~5q56 PCT/CA9S/00356 Ste~ 2: Removal of silyl protecting group 2.3g, 4.4 mmole of com~ound II was dissolved in 40 ml dry tetrahydrofuran and stirred under argon flow.
s Tetrabutylammonium fluoride (5.3 ml, 5.1 mmole) was added and the reaction was stirred at ambient temperature and under argon overnight. To the solution was added glacial acetic acid (90.31 ml. 5.3 mmole) and the solvent was l~l.lo~d in vacuo. The crude product was purified by flash silica gel chromatography using 10% methanol-ethyl acetate as eluent (Rf = 0.20). The product was taken up in m;n;mAl methylene chloride and filtered through celite to remove silica. The solvent was removed in vacuo, and the product dried, giving compound III, 1.0 g. 4.2 mmole, in 95~ yield.

Step 3: Coupling of compound III with D-arginine l.Og., 4.2 mmole of 6-chloropurinyl pentanol, compound III , was dissolved in 75 ml dry tetrahydrofuran and stirred under argon flow. Toluenic phosgene (4.4 ml., 8.3 mmole) was added and the reaction was monitored by TLC (developed in methanol) and continued until the int~rmP~;ate chloroformate was the pre~m;n~nt product (6-10 hours). The solvent was removed in vacuo, and the residue was taken up in 50 ml. dry tetrahydrofuran.
D-arginine (0.94 g., 5.4 mmole), dissolved in 5 ml.
water, was added to the chloroformate suspension.
Another 5 ml. aliguot of water was used to rinse the beaker which contained the arginine solution, and then added to the reaction. The reaction was stirred overnight at ambient temperature, and then extracted with toluene (60 ml.). The toluene was back extracted with water (60 ml.) and the combined agueous portions were brought to slightly alkaline pH by the addition of 5% sodium bicarbonate. Water was relllo~ed in vacuo and the residue was dissolved in methanol (10 ml.). After filtration, the methanolic solution was added dropwise ~ ~ u ~
W095/35297 2 i 6 5 9 5 6 PCT/CA95/00356 ~
to 500 ml vigorously stirred acetone. The precipitate was collected by filtration and washed several times with acetone. The filtrate contained unreacted III.
The precipitate was dried, and then dissolved in water (50 ml.). to the a~ueous solution was added dimethylamine (40% aqueous solution, 5.0 ml, 40 mmole) and the reaction was stirred for 3 hours at ambient temperature. The solvent was r~-llov~d in vacuo and the crude product was purified by flash silica gel chromatography, using methanol as eluent (Rf = 0.25).
The combined product fractions were reduced in volume (approximately 5 ml) and stored at 4DC for 2 hours. The solution was centrifuged for 10 minutes (375 x g) to remove silica, and the supernatant was added dropwise to 500 ml vigorously stirred ether. The precipitate was collected by filtration and dried to give N,N-dimethylaminopurinyl pentoxycarbonyl D-arginine, compound #1, 0.79 g., 1.8 mmole in 43% yield.
mp (softens 119C) = 123 - 125C
Rf silica (methanol) = 0.30 1HNMR (DMSO - d6, 300 MHz, ~ in ppm); 9.40 (lH, br, s, COOH); 8.20 (lH, s, purine); 8.15 (lH, s, purine); 8.0 - 7.3 (4H, b, guanidine); 6.33 (lH, d, NH); 4.13 (2H, t, N-C_2); 3.86 (2H,t, O-CH2); 3.63 (lH, m, CH)i 3.36 (6H, s, b, N-(CH3)2); 3.02 (2H, b, CaH); 1.8-1.2 (lOH, m, C~H, C~_,-(CH2)3 -). MS (high-resolution FAB, glycerol);
m/e, 450.25780; calculated for M+H+, (C1gH32O4Ng), 450.25773.

2~ 65956 W095/35297 ,`;~ PCTICA95/00356 _le lb Alternative Synthesis of N,N-Dimethylaminopurinyl Pentoxycarbonyl D-Arginine - Com~ound #1 N N
~ OTBDPS
II

HN(cH3j2 ~2 (C4Hg)4~.'THF
AcOH

N

N~N~J
V ~ OH

ClCOCl/Toluene D-Ar~inine/ H20 N

N~N~
O ~ NH ~ COOH

C __ ~ #l ~ ~

s Step 1: A modified synthesis of compound #1 was undertaken by reaction of protected 6-chloropurinyl pentanol, compound II (prepared as described in example la) with aqueous dimethylamine, followed by deprotection to yield compound V, and coupling with D-arginine (the coupling reaction is as described in example la) to WO 95/3~297 2 1 6 5 9 5 6 PCT/CA95/00356 ~
give the product. The spectral and chromatographic properties were identical to the product obtained from the synthesis described in example la.

S A typical example of the reaction of protected 6-chloropurinyl pentanol, compound II, with dimethylamine is as follows; to 0.13 g., 0.26 mmole of compound II
dissolved in 20 ml tetrahydrofuran was added dimethylamine (4096 aqueous solution, 0.5 ml, 10.0 mmole) The reaction was stirred for 18 hours at ambient temperature, and the solvent was removed in vacuo. the crude product was purified by flash silica gel chromatography, using 50% ethyl acetate-hexane as eluent (Rf = 0.27). The product compound V, 0.12 g.
0.30 mmole, was obtained in 9496 yield.

r _l e 2 Synthesis of N,N-Dimethyl~m;nopurinyl Pentoxycarbonyl L-Arginine - C _ _u--d #2 20 The L-enantiomer of compound #l,compound #2, was synthesized as described above in example lb to give 50 mg. of product as a white solid.
mp (softens 118C) = 123 - 125C.
Spectral properties were identical with compound #1 .
pl e 3 Synthesis of N-Monomethylaminopurinyl Pentoxycarbonyl D-Arginine - C _ u--d #3 Compound #3 was synthesized as described above in example lb, 30 except that dimethylamine was replaced with methylamine (40%
a~ueous solution) to give 6-methylaminopurinyl pentanol compound #3a. This was then coupled with D-arginine, as described in example la to give 32 mg. of product.
mp (softens at 127C) = 133C.
35 Rf silica (methanol) = 0.20 1HNMR (DMSO-d6, 300 MHz, ~ in ppm);

2 1 659~

W095/352~7 ~ PCTICA95/00356 9.30 (lH, b, COOH), 8.21 (lH, s, purine); 8.13 (lH, s, purine);
7.8-7.2 (4H, b, guanidine)i 6.29 (lH, d, NH); 4.13 (2H, t, N-CH2); 3.86 (2H, t, O-CH2); 3.61 (lH, m, CaH); 3.17 (lH, m, CH3-N-H); 3.02 (3H, b, HN-CH3); 2.97 (2H, b, C~H); 1.9-1.2 (lOH, m, 5 C~H~ CY_, - (CH2) 3-) -_le 4 Synthesis of N-Monomethylaminopurinyl Pentoxycarbonyl L-Arginine - ~_ ~.d #4 The L-enantiomer of compound #3 and compound #4 , was synthesized as described above in example 3 to give 44 mg. of product as a white solid.
mp (softens at 123C) - 132 - 134C.
Rf silica (methanol) = 0.20.
Spectral properties were identical with compound #3.

e 5 Synthesis of Aminopurinyl Pentoxycarbonyl D-Arginine - Compound #5 Compound #5 was synthesized as described above in example lb except that protected 6-chloropurinyl pentanol, compound II, was reacted with Ammnn;a gas instead of dimethylamine. The 6-aminopurinyl (A~n;ne) product was thus deprotected to give the alcohol compound #5a. This was then coupled with D-arginine to 2~ give 260 mg. of product as a white solid. A typical example of the reaction of compound II with Ammon;A is as follows; 0.42g, 0.88 mmole of compound II was dissolved in 75 ml absolute ethanol, and the solution was placed on an ice bath. Ammon;a gas was bubbled through the chilled solution for 10 minutes, and the saturated solution was transferred to a bomb (150 ml cylinder). Ammonia gas was bubbled through the solution for another minute, the bomb sealed, and the bomb was heated overnight in a 120C oil bath. Solvent was removed in vacuo, yielding 0.40 g., 0.88 mmole of product in 95% yield. This product was used without further purification.
Characteristics of compound #5;
mp(softens 143C) - 150C.

WO9S/35297 2 1 6 5 ~ 5 6 PCT/CA95/00356 ~
Rf silica (methanol) = 0.20 ~HNMR (DMSO-d6, 300 MHz, ~ in ppm); 9 40 (lH, b, COOH); 8.14 (lH, s, purine); 8.13 (lH, s, purine)i 8.0 - 7.0 (6H, m, guanidine,-NH2); 6.36 (lH, b, NH); 4.13 (2H, t, N-CH2); 3.87 (2H, t, O-CH2); 3.65 (lH, m, CaH); 3.03 (2H, b, CH); 1.9-1.2 (lOH, m, C~H, C~_, - (CH2) 3~

_le 6 Synthesis of Aminopurinyl Pentoxycarbonyl L-Arginine - Compound ~6 The L-enantiomer o~ compound ~5, compound # , was synthesized as described above in example 5 to give 93 mg of product as a white solid.
mp (softens at 143C = 153 - 155C.
Rf silica (methanol) = 0.22 Spectral properties were identical with compound #5.

_le 7 Synthesis of Hydrazinopurinyl Pentoxycarbonyl D-Arginine ~ ~.d #7 Compound #7 was synthesized as described above in example la, except that dimethylamine was replaced with hydrazine from the corresponding alcohol compound ~7a . Thus, in a typical example, 50 mg, 0.11 mmole of 6-chloropurinyl pentoxycarbonyl D-arginine, dissolved in 5 ml 95~ ethanol, was reacted withhydrazine hydrate ~12 ~l, 0.40 mmole) at ambient temperature overnight. The reaction was then slowly cooled to 0C for 3 hours, and the resulting crystals were collected by filtration, and washed with cold ethanol. The white solid product, 32 mg., 0.07 mmole, was obtained in 65~ yield.
mp(softens 130C) = 134C
Rf silica (m~th~nol) = 0.27 HNMR (DMSO-d6, 300 MHz, ~ in ppm); 9.20 (lH, b, COOH); 8.23 (lH, s, purine)i 8.14 (lH, s, purine); 7.4 (3H, b, guanidine);
6.6 (2H, b, N_2); 6.43 (lH, d, NH); 4.14 (2H, t, N-CH2); 3.87 - ~ - i 21 65 956 W095/352~7 ~ - PCT/CA95100356 (2H, t, O-CH2); 3.64 (lH, m, CaH); 3.04 (2H, b, C_); 1.8-1.3 (lOH, m, C~H, C~ (CH2)3-).

_le 8 Synthesis of Hydrazinopurinyl Pentoxycarbonyl L-Arginine - C _ ~-d #8 The L-enantiomer of compound #7, compound #8, was synthesized as described in example 7 to give 40 mg of product as a white solid.
10 mp (softens at 130C = 134C.
:f silica (methanol) = 0.27 Spectral properties were identical with compound #7.

_le 9 Synthesis of Chloropurinyl Pentoxycarbonyl D-Arginine - Compound #9 Compound ~9 was synthesized by the coupling reaction of 6-chloropurinyl pentanol, compound III , with D-arginine, as described in example la (with omission of the addition of dimethylamine after the coupling reaction). This gave 622 mg of product as a white solid.
mp(softens 137C) = 145 - 148C.
Rf silica (methanol) = 0.35 1HNMR (DMSO-d6, 300 MHz, ~ in ppm); 9.15 (lH, b, COOH); 8.78 (lH, s, purine); 8.74 (lH, s, purine); 7.8-7.2 (4H, b, guanidine); 6.33 (lH, d, NH); 4.29 (2H, t, N-CH2)i 3.88 (2H, t, O-CH2); 3.64 (lH, m, CH)i 3.04 (2H, b, C~H); 1.95-1.20 (lOH, m, C~H, C~_, - (CH2) 3-) -Exam~le 10 Synthesis of Chloropurinyl Pentoxycarbonyl L-Arginine - C _ ~d ~10 The L-enantiomer of compound #9, compound #10, was synthesized as described above in example 9 to give 65 mg of product as a white solid.
mp (softens at 137C) = 143-146C.
Rf silica (methanol) = 0.26 Spectral properties were identical with compound #9.

_le 11 Synthesis of HydrGxypurinyl Pentoxycarbonyl D-Arginine - C _ ~-d #11 Compound #11 was synthesized as described in example 9 above except that the 6-chloropurinyl pentanol intermediate, compound III, was first subjected to base catalyzed hydrolysis to yield 6-hydroxypurinyl (hypoxanthine) pentanol compound #lla prior to coupling with D-arginine Thus, in a typical example, 398 mg, 1.7 mmole of compound III was dissolved in 25 ml of water.
Sodium hydroxide (1.0 m, 3.4 ml) was added, and the reaction was refluxed for 90 minutes. Upon cooling, the reaction was acidified (5% hydrochloric acid), the solvent removed in vacuo, and the crude product purified by flash silica gel chromatography using 30% methanol-ethyl acetate as eluent (Rf =
0.32). The product, compound #lla , 290 mg, 1.3 mmole, was obtained in 79~ yield as a white solid. Subsequent coupling with D-arginine gave 148 mg of compound #11 as a white solid.
mp(softens 163C) = 182C.
Rf silica (methanol) = 0.22 HNMR (DMSO-d6, 300 MHz, ~ in ppm); 9.28 (lH, b, COOH); 8.10 (lH, s, purine); 8.04 (lH, s, purine); 7.8-7.2 (4H, b, guanidine); 6.38 (lH, d, NH); 4.32 (lH, b, OH); 4.13 (2H, t, N-CH2); 3.87 (2H, t,O-CH2); 3.61 (lH, m, C~H); 3.04 (2H, b, C~H);
1.8-1.1 (lOH, m, C~H, C~_, - (CH2) 3-) .

_le 12 Synthesis of Mercaptopurinyl Pentoxycarbonyl D-Arginine - Com~ound #12 Compound #12 was synthesized as described in example la, except that dimethylamine was replaced with thiourea. Thus, in a typical example, 80 mg, 0.18 mmole of 6-chloropurinyl pentoxycarbonyl D-arginine, dissolved in 5 ml absolute ethanol was reacted with thiourea, 16 mg, 0.21 mmole, under reflux for 5 hours. The reaction was then stored at 0C overnight and the resulting crystals were filtered and washed with cold absolute WO9~/352~7 `` ;~ ~ 2 1 6 5 9 5 6 PCTICA9S/00356 ethanol. The product was recrystallized from absolute ethanol to give a white solid, 48 mg, 0.11 mmole in 61% yield.
mp(softens 180C) = 200C.
Rf silica (methanol) = 0.50 lHNMR (DMSO-d6, 300 MHz, ~ in ppm); 9.15 (lH, b, COOH); 8.29 (lH, s, purine); 8.18 (lH, s, purine); 7.5-7.3 (4H, b, guanidine); 6.39 (lH, d, NH); 4.13 (2H, t, N-CH2); 3.87 (2H, t, O-CH~); 3.65 (lH, m, C~H); 3.04 (2H, b, C~H); 1.90-1.23 (lOH, m, C~H, CrH, - (CH2) 3-) .
_le 13 Synthesis of Mercaptopurinyl Pentoxycarbonyl L-Arginine - C __~.d #13 The L-enantiomer of compound #12, compound #13, was synthesized as described in example 12 to give 42 mg of product as a white solid.
mp (softens at 180C = 200C.
Rf silica (methanol) = 0.50 Spectral properties were identical with compound #12 .
ExamJ~le 14 Synthesis of N,N-Dimethylaminopurinyl Pentoxycarbonyl Glycine - Com~ound #14 Compound #14 was synthesized as described above in example lb except that the coupling reaction was undertaken on smaller scale, with glycine, 68 mg, 0.91 mmole, instead of arginine and the free base of glycine was generated in situ by the addition of 3 equivalents of sodium carbonate (relative to the alcohol).
The crude product was purified by flash silica gel chromatography, using 50~ methanol-ethyl acetate as eluent (Rf =
0.35). Silica was removed by dissolving the product in methylene chloride, followed by filtration. Removal of solvent in vacuo gave 30 mg of product as a white solid.
mp(softens 100C) = 126C.
Rf silica (1:1 methanol-ethyl acetate) = 0.35 HNMR (DMSO-d6, 300 MHz, ~ in ppm); 8.20 (s, lH, purine); 8.16 (s, lH, purine); 6.05 (lH, t, NH); 4.14 (2H, t, N-CH2); 3.86 W095/35297 ~ r~ ~ 2 1 6 5 9 ~ 6 PCTICA95~ ~C~ ~
(2H, t, O-CH2); 3.33 (6H, s, b, N-(CH3)2); 3.18 (2H, d, C H2);
1.80 (2H, m, CH2); 1.55 (2H, m, CH2); 1.26 (2H, m, CH2);

_le 15 N,N-(6-Dimethylaminopurin-9-yl)-7'-ethoxy-ethoxycarbonyl-D-aryinine - Com~ound #15 Ste~ 1: N,N-(6-Dimethylaminopurin-9-yl)-5-ethoxyethoxy-t-butyldiphenylsilane To a solution of alcohol (0.201 g, 1 eq) in anh. THF
(2.9 ml), at room tem~--ature, under argon, were added successively 6-chloropurine (90 mg, 0.58 mmol), Ph3P
(0.199 g, 1.3 eq) and DEAD (0.12 ml, 1.3 eq). The yellow solution was stirred at room temperature for 15 hours. The THF was evaporated and the residue was chromatographed (6:4, Hexanes/EtOAc) to give a mixture of (EtO2CNH) 2 and the coupled purine. To a solution of this mixture in THF (6 ml), at room temperature, was added 40% Me2NH/H2O (0.70 ml, 10 eq). The solution was stirred at room temperature for 45 minutes and was then poured in sat. aq. NaHCO3tCH2Cl2. The phases were separated and the aqueous phase was extracted with CH2Cl2(2x). The combined organic extracts were dried over MgS04, the solids filtered and the solvents evaporated. The residue was purified by flash chromatography (silica gel, ~:8 Hex/AcOEt) to give 0.16 g (55~) of the coupled dimethyl amino purine.

Step 2: N,N-(6-Dimethylaminopurin-9-yl)-5-ethoxyethanol -compound #85 To a solution of the silane (0.16 g, 0.32 mmol) in anh.
THF (3.2 ml), at room temperature, under argon, was added nBu4NF 1.0 M/THF (0.32 ml, 1.1 eq). The solution was stirred at room temperature for 3 hours and the solvent was evaporated in vacuo. The residue was ;mm~;ately purified by flash chromatography (silica J`~ 2 1 6 5 9 5 ~
WO9S/35297 ' ' - PCT/CA95/00356 gel, 4:1 AcOEt/MeOH) to give 72 mg (89%) of the alcohol compound #85 as a clear oil.

lHNMR (CDC13): ~ 8.29 (s, lH, purine), 7.80 (s, lH, S purine), 4.33 (t, 2H, CH2), 3.82 (t, 2H, CH2), 3.68 (t, 2H, CH2), 3.55 (t, 2H, CH2), 3.50 (m, 6H, N(CH3)2).

Ste~ 3: N,N-(6-Dimethylaminopurin-9-yl)-7-ethoxyethoxycarbonyl-D-arginine - C _,~,d #15 To a solution of the alcohol, compound #85 (72 mg, 0.29 mmol) in anh THF (4.8 ml), at room temperature, under argon, was added COC12/Toluene 1.93 M (0.30 ml, 2 eq) and the solution was stirred at room temperature for 5 hours. The THF was evaporated i~ vacuo and the residue was redissolved in THF (3.6 ml). To this solution was added a solution of D-arginine in water (65 mg, 1.3 eq/0.5 ml H2O). The flask cont~;n;ng the D-arginine solution was rinsed with 0.5 ml H2O and the reaction mixture was stirred at room temperature for 15 hours.
It was then extracted with toluene and the toluene phase was back-extracted with H2O. The combined aqueous layers were brought to pH 7.5-8.0 (NaHCO3 5%) and the water was evaporated. The residue was purified by flash chromatography (silica gel, 100% MeOH). The fractions cont~;n;ng the compound were evaporated and the residue was dissolved in a m; n; mllm ~uantity of MeOH. Et2O was then added and the solvents were decanted to give a white gum that was dried under high vacuum. The compound was obtained as a white solid (42 mg, 33%).

lH NMR (DMSO-d6): ~ 8.25 (s, lH, purine), 8.14 (s, lH, purine), 6.5 (bd, lH, NH), 4.37 (t, 2H, CH2 linker), 4.03 (m, 2H, CH2 linker), 3.81 (m, 2H, CH2 linker), 3.72 (m, lH, CaH), 3.60 (m, 2H, CH2 linker), 3.55-3.89 (m, W095/35297 }~ 2 1 ~ 5 9 5 6 PCT/CA9S/003S6 6H, N(CH3)2), 3.05 (m, 2H, C H2), 1.78-1.39 (m, 4H, C~H2, C H2 ) _le 16 (2S,4S)-2-(N,N-dimethyl ~m; nopurin-9-yl)-4-(methyloxycarbonyl-D-arginine)-1,3-dioxolane -com~ouna ~16 X ~ OH l ~ ~ OSitBuPh2 2 HO ~ OSitBuPh2 HO

BzO o ~ OSitBuPh2 Cl N
~N~ 4 N~ \>
~O
~--\ OSitBuPh2 N N
N~N> ~ NH2 6 N~ \>

~\oJ~HN COOH ~<~\OH
O O
Compound #16 Step 1: (4S)-2,2-dimethyl-1,3-dioxolane-4-t-butyldiphenylsilylmethanol s ~ 2 1 ~ 5 95~
W095/35297 -~ ~ PCT~CA95/00356 To a solution of (4s)-2,2-dimethyl-1,3-dioxolane-4-methanol (lg, 7.57 mmols) in anh. CH2Cl2 (76ml) , at room temperature, under argon, were added successively imidazole (1.03g., 2eq) and t-BuPh2SiCl (1.95 ml, 1.1 e~). A white precipate formed immediately. This suspension was stirred at room temperature for 1 hour and then poured in sat. aq.
NaHCO3. The phases were separated and the aqueous layer was extracted with CH2Cl2 (2x). The combined organic extracts were dried over MgSO4, the solids were filtered and the solvents evaported to give 2.80 g (100~) of the silane as a clear oil.

Step 2: (2S)-3-t-Butyldiphenylsilylpropanetriol To a solution of the silane (1.01 g, 2.73 mmols) in a 4:1 mixture of THF/H2O (15 ml), at room temperature, was added TFAA (0.5 ml, 2.4 eq) and the solution was heated at 50C for 5 hours. It was then poured in sat. aq. NaHCO3/CH2Cl2 and the phases were separated.
The aqueous layer was extracted with eH2Cl2 (2x) and the combined organic extracts were dried over MgSO4.
The solvents were evaporated and the residue was purified by flash chromatography (silica gel, 1:1 Hex/EtOAc) to give 0.62 g. (70~) of the diol as a clear oil.

Ste~ 3: (2S,4S)-2-benzoyloxymethyl-4-t-Butyldiphenylsilyloxymethyl-1,3-dioxolane To a solution of the diol (0.62 g, 1.89 mmol) and of the aldehyde (0.31 g, leq) in anh. toluene (19 ml), at room temperature, under argon, was added a cat.
amount of PPTS. The solution was refluxed for 18 hours, after which it was poured in sat. aq.
NaHCO3/CH2Cl2 . The phases were separated and the aqueous layer was extracted with CH2Cl2 (2x). The combined organic extracts were dried over MgSO4 and W095/3S297 ` `~ 2 ~ 6 5 9 5 6 PCTICA95/00356 the solvents were evaporated. The residue was purified by flash chromatography (silica gel, 9:1 Hex/EtOAc) to give 0.49 g (55~) of a 5:1 (cis/trans) mixture of the dioxolanes.
s Ste~ 4: (2S,4S)-2-Hydrox~methyl-4-t- =
butyldiphenylsilyloxymethyl-1,3-dioxolane To a solution of the benzoate (0.49 g, 1.03 mmol) in anh. MeOH (10.3 ml), at room temperature, under argon, was added MeONa/MeOH 4.37 M (24 ~l, 0.1 e~).
The solution was stirred for 18 hours after which it was poured in sat. aq. NH~Cl/CH2Cl2. The phases were separated and the aqueous phase was extracted with CH2Cl2 (2x). The combined organic extracts were dried over MgSO4, the solids were filtered and the solvents were evaporated. The residue was purified by flash chromatography (silica gel, 3:1 Hex/EtOAc) to give the cis-alcohol (0.26 g, 67~) as a clear oil.
Ste~ 5: (2S,4S)-2-(N,N-dimethylaminopurin-9-yl)-4-t-butyldiphenylsilyloxymethyl-1,3-dioxolane The compound was prepared using a similar method as in Example 15, step 1.

Step 6: (2S,4R)-2-(N,N-dimethylaminopurin-9-yl)-4-hydroxymethyl-1,3-dioxolane The compound was prepared using a similar method as in Example 15, step 2.

Purification: 10~ MeoH/EtoAc 1HNMR (CDCl3): ~ 8.32 (s, lH, purine), 7.75 (s, lH, purine), 5.33 (dd, lH, J=2.0, 6.6, H-2-dioxolane, 5.33 (bs, lH, OH), 4.45 (dd, lH, J=6.6, 14.3, CH2-~ WO 95/35297 ~ .t i ',~; ^ 2 1 6 ~ 9 5 ~ PCTICA95/00356 purine), 4.20 (dd, lH, J=2.0, 14.3, CH2-purine), 4.20 (m, lH, H-4-dioxolane), 4.05 (d, 2H, J-7.2, H-5), 3.78 (d, lH, J=13.0, CH2-OH) 3.53 (bs, 6H, (CH3)2N), 3.40 (d, lH, J-13.0, CH2-OH).

Ste~ 7: (2S,4S)-2-(N,N-dimethylaminopurin-9-yl)-4-(methyloxycarbonyl-D-arginine)-1,3-dioxolane -Compound #16 The compound #16 was prepared using a similar method as in Example 15, step 3.

Purification: MeoH 100%

lHNMR (DMSO-d6): ~ 8.43 (s, lH, purine), 8.11 (s, lH, purine), 6.6 (m, lH, NH), 5.28, (m, lH, H-2-dioxolane), 4.39 (m, 2H, CH2-purlne), 4.26 (m, lH, H-4-dioxolane), 3.97-3.81 (m, 3H, CaH, CH2-OCO-D-arginine), 3.71 (m, 2H, H-5-dioxolane), 3.39 (bs, 6H, (CH3)2N), 3.07 (m, 2H, CaH2), 1.70-1.45 (m, 4H, C~H2, C H2 ) _le 17 Synthesis of N-(6-Dimethylamino-8-bromopurinyl-9-yl)-Pentoxycarbonyl L-Arginine - C _ ~.~ #17 H2N~NH
~N~ HN~

~CN>-- OJ~NH~OH

H NMR (DMSO-d6, 400 MHz, ~ in ppm): 9.5 (lH, s, b, COOH), 8.19 (lH, s, purine), 8.1-7.2 (4H, b, guanidine), 6.30 (lH, d, NH), 30 4.11 (2H, t, N-CH2), 3.87 (2H, t, CH2-O), 3.64 (lH, m, CaH), 3.39 W095/35297 ~ 2 ~ 6 ~ 9 5 6 PCT~CAgs~ c~ ~
(6H, s, b, N-(CH3)2), 3.02 (2H, m, C~H), 1.8-1.2 (lOH, m, C~H, CrH , - ( CH2 ) 3 - ) -m.p. (softens 115-118) = 124-127C.
R~ silica (70% methanol-ethyl acetate) = 0.25 _le 18 Synthesis of N-(6-dimethylamino-8-bromopurin-9-yl)-7-pentoxycarbonyl-D-arginine - C~ _ ~.d #18 H2N~NH
~N HN~

~N OJ~NH~OH
~~

lH NMR (DMSO - d6, 400 MHz, ~ in ppm); 9.08 (lH, br s, COOH);
8.18 (lH, s, purine); 7.9-7.3 (4H, br s, guanidine); 6.34 (lH, d, NH); 4.10 (2H, t, N-CH2); 3.86 (2H, m, O-CH2); 3.55 (lH, m, CaH);
3.35 (br s, N-(CH3)2); 3.03 (2H, m, C~H2); 1.9-1.2 (lOH, m, (C_2)3, C~_2, C~H2) m.p. (softens 116C) = 122-125C.
Rf silica (70% methanol-ethyl acetate) = 0.25 `t ~ 3t~, 2165~56 W095/3529~7 ~ PCT/CA95/00356 ExEm~le 19 N-9-purinyl-5-pentanol - C __~.d #19 N EOanol N N 25C/overni~ht N IN
(CH2)5--OH (CH~)5--OH
lH NMR ~ (CDCl3 in ppm): 9.12 (s, lH, purine), 8.96 (s, lH, purine), 8.10 (s, lH, purine), 4.30 (t, 2H, CH2-O), 3.63 (t, 2H, CH2-N), 1.97 (m, 3H, CH2 and OH), 1.62 (m, 2H, CH2), 1.47 (m, 2H, CH2) 13C NMR (~ CDC13 in ppm): 153.09, 151.96, 149.15, 145.80, 134.60, 62.78, 44.38, 32.47, 30.27, 23.57.
Purification 5~ MeOH~AcOEt Rf (silica) 0.29 (5% MeOH/AcOEt).

_le 20 N-9-purinyl-7-pentyloxycarbonyl-D-arginine -~ __ #20 Cl N~C~N H2/Pd/C N~N
N Nl 1l 25C ~N IN O
(CH2)5--O--C--NH--ICH--COOH (CH2)5--O--C--NH--fH--COOH
( ICH2)3 ( ICH2)3 NH NH
~C~ HN~C`

lH NMR ~ (DMSO in ppm): 9.10 (s, lH, purine), 8.93 (s, lH, purine), 8.64 (s, lH, purine), 7.77 (bs, 4H, guanidine), 6.25 (bs, lH, NH), 4.28 (t, 2H, CH2-O-), 3.87 (t, 2H, CH2-N), 3.58 - (m, 1~, CaH), 3.09 (m, 2H, CH2-N), 1.20-1.90 (m, 10H, 5xCH2).
Purification: methanol Rf (silica) = 0.23 (methanol) ~ I S ~ t 6 5 9 5 6 W095/35297 PCT/CA95/0~356 _le 21 N-9-purinyl-7-pentyloxycarbonyl-L-arginine -Compound #21 Cl N ~ ~N H2~d/C N ~ N
N IN I E~o~col N IN 1l (CH2)5-O-CNH-CHCOOH (CH2)s-O-CNH-ICHCOOH
( ICH2)3 (Cl H2)3 NH NH
~C~ HN~ NH

lH NMR ~ (DMSO in ppm): 9.13 (s, lH, purine), 8.94 (s, lH, purine), 8.66 (s, lH, purine), 8.25 (bs, lH, NH), 7.44 (bs, 3H, guanidine), 6.82 (bs, lH, NH), 4.28 (t, 2H, CH2-O), 3.88 (t, 2H, CH2-N), 3.72 (m, lH, CaH), 3.07 (m, 2H, CH2-N), 1.19-1.95 (m, lOH, 5xCH2).
13C NMR (~ CD30D in ppm): 179.47, 159.21, 158.95, 153.79, 153.41, 140.07, 135.42, 66.06, 57.37, 45.38, 42.66, 31.88, 30.93, 30.11, 26.67, 24.65.
Purification- methanol R~ (silica) = 0.23 (methanol) le 22 Synthesis of N,N-Dimethylaminopurinyl Pentoxycarbonyl L-Valyl L-Prolyl L-Leucine -~ _ ~ #22 N

N~ NX~ ~_ NN

J o o ~OH

{
.? ~(~, t ~ 2165956 W095/3529'7 PCT/CA95/00356 lHNMR (CD30D, 400 MHz,~ in ppm) 8.22 (lH, s, purine); 8.05 (lH, s, purine); 4.60 (lH, t, C~_)j 4.3-3.6 (8H, n, N-C_2, C~H2, CH2-O, 2 x CaH)i 3.51 (6H, s, b, N-(CH3)2); 2.2-1 2 (14H, m, -(CH2)3-, 2 x C~H2, C~H, cY_2, C~H); 1.0-0.8 (12H, m, 2 x C~-CH3, 2 x C~-CH3.
m.p. = 168C
R~ silica (40~ methanol-ethyl acetate) = 0.40 _le 23 Synthesis of N,N-Dimethylaminopurinyl Pentoxycarbonyl L-Isoleucyl L-Prolyl L-IsoIeucine- ~ olln~ #23 N

N~CN
J O O _ ~OH

lHNMR (CD30D, 400 MHz, ~ in ppm); 8.21 (lH, s, purine); 8.03 (lH, s, purine)i 4.62 (1 H, t, CaH); 4.22 (4H, m); 4.03 (2H, m);
3.89 (lH, m, CaH); 3.67 (lH, d, CaH); 3.50 (6H, s, b, N-(CH3)2); 2.1-1.0 (16H, m, -(CH2)3-, C~H2 , 2 x C~H, 3 x C~H2);
0.95 (6H, d, 2 x C~CH3); 0.87 (6 H, t, 2 x C~-CH3).
m.p. (softens 83-86C) = 93C
RL silica (40% methanol-ethyl acetate) = 0.35 ~- ?~ 21 65956 W095/35297 PCT/CA95i~.?
_le 24 Synthesis of N-(6-Cyclopropylaminopurin-9-yl)-5-pentanol - Com~ound #24 Y y NH NH
N~ 5% HCI N~N
N IN'N MeOH N Nl (CH2)5--OSi--(C6H5) (CH2)5--OH
u lH NMR (~ CDCl3 in ppm): 8.46 (s, lH, purine), 7.77 (s, lH, purine), 6.42 (bs, lH, NH), 4.22 (t, 2H, CH2), 3.09 (bs, lH, OH), 1.94 (m, 2H, CH2), 1.63 (m, 2H, CH2), 1.45 (m, 2H, C_2), 0.94 (m, 2H, CH2), 0 69 (m, 2H, CH2).
Colorless oily material R~ = 0.3 10~ methanol/ethyl acetate Mass spectrum: H+ = 262 (HRMS) _le 25Synthesis of N-(6-cyclopropylAm;nopurin-9-yl)-7-pentyloxycarbonyl-D-arginine - C __~.d #25 Y Y
NH NH
N~N TEIF N~N
J~ ~ + COCI2 + D-A~ginine + NaHC03 H o ~ 1 ~

(CH2)5--OH (CHa)50 NHCHCOOH
( ICH2)3 HN,~;C~NH

1H NMR (~ DMSO in ppm): 8.22 (s, lH, purine), 8.14 (s, lH, purine), (bs, 4H, guanidine), 6.28 (d, lH, NH), 4.13 (t, 2H, CH2), 3.87 (m, 2H, CH2), 3.62 (m, lH, CaH), 3.02 (m, 2H, CH2), 1.2-1.8 (m, llH, 5xCH2 and CH), 0.69 (m, 2H, CH2), 0.67 (m, 2H, CH2 ) -13C NMR (~, DMSO in ppm): 175.53, 158.20, 157.63, 155.79, 152.62, 150.91, 141.03, 119.92, 63.69, 55.38, 43.12, 41.14, 30.10, 29.44, 28.49, 25.49, 24.38, 22.91, 6.78.
m.p. softens 147C melts 151C.
Rf = 0.34 (MeOH) Mass spectrum: H = 462 (HRMS) _le 26 Synthesis of N-(6-cyclopropylaminopurin-9-yl)-7-pentyloxycarbonyl-L-arginine - Com~ound #26 NE~ NH
N ~ N THF N ~ N
b J~ ~ + cocl~ +L-Arginine +NaHC03 HO ~ b J~ ~

(CH2)5- OH (CH2)5OCNHfH-COOH
(Cl H2)3 "C~

lH NMR (~ DMSO in ppm): 8.23 (s, lH, purine), 8.14 (s, lH, purine), 7.51 (bs, 4H, guanidine), 6.31 (d, lH, NH), 4.16 (t, 2H, CH2), 3.87 (t, 2H, C_2), 3.62 (m, lH, CaH), 3.02 (m, 2H, CH2), 1.2-1.85 (m, llH, 5xCH2 and CH), 0.72 (m, 2H, CH2), 0.68 (m, 2H, CH2).
13C NMR (~, CD30D in ppm): 179.41, 159.19, 158.95, 157.66, 154.09, 151.00, 143.00, 121.10, 66.12, 57.34, 45.33, 42.67, 31.90, 31.25, 30.14, 26.63, 25.11, 24.61, 8.14.
m.p. 144-146C
Rf = O . 35 (MeOH) Mass spectrum: M+ = 462 (HRMS).

WO 9S/35297 ~ r PCT/CA95~75 _le 27 Synthesis of N-(6-Azetidinepurin-9-yl)-5-pentanol - r __~.d #27 N N
~N THF N~J

(CH2)50~ Bu (CH2)5--OH
(C6H5)2 S lH NMR (~, CDCl3 in ppm): 8.25 (s, lH, purine), 7.66 (s, lH, purine), 4.44 (m, 4H, CH2), 4.10 (t, 2H, CH2), 3.55 (t, 2H, CH2), 3.21 (bs, lH, OH), 2.48 (m, 2H, CH2), 1.84 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.38 (m, 2H, CH2).
13C NMR (~, CDCl3 in ppm): 155.11, 153.42, 150.39, 140.37, 120.38, 62.79, 44.05, 32.57, 30.45, 30.22, 23.45, 18.23, 18.12.
m.p.: 104-106C
Rf = 0.33 (10~ MeOH/AcOEt) Mass spectrum: M = 262 (HRMS).

_le 28 Synthesis of N-(6-Azetidinepurin-9-yl)-7-pentyloxycarbonyl-D-arginine - C _ ~d #28 N N
N~N THF N~N
+COCI2+D-~&~e+ NaHCO3 N N O

(CH2)5--OH (CH2)50CNHfHCOOH
(Cl H2)3 NH
~C~

* W095/35297 6 PCT/CA95/00356 lH NMR (~, CD30D in ppm): 7.96 (s, lH, purine), 7.89 (s, lH, purine), 4.27 (m, 4H, 2xCH2), 4.02 (t, 2H, CH2), 3.79 (m, 3H, CH2 and CaH), 2.99 (m, 2H, CH2), 2.32 (m, 2H, CH2), 1.17-1.71 (m, 10H, 5xCH2).
13C NMR (~, CD30D in ppm): 181.56, 159.38, 159.14, 156.16, 153.89, 151.22, 143.21, 120.92, 66.30, 57.15, 48.71 45.24, 42.66, 31.51, 31.26, 30.15, 26.96, 24.64, 18.87.
m.p.: 190-192C
Rf: 0.25 (methanol) Mass spectrum: M' = 462 (HRMS) _le 29Synthesis of N-(6-Azetidinepurin-9-yl)-7-pentyloxycarbonyl-L-arginine - r ~.d #29 <> ~>
N N

~J + COC~2 + L-Arginine + NaHCO3 ~ J

(CH2)5- OH (CH2)5 OCNHfHCOOH
( ICH2)3 NIH
HN,~,C~NH

lH NMR (~, CD30D in ppm): 7.93 (s, lH, purine), 7.88 (s, lH, purine), 4.27 (m, 4H, 2xCH2), 4.01 (t, 2H, CH2), 3.79 (m, 3H, CH2 and CaH), 2.97 (m, 2H, CH2), 2.32 (m, 2H, CH2), 1.15-1.74 (m, 10H, 5xCH2).
13C NMR (~, CD30D in ppm): 179.44, 159.18, 158.95, 156.16, 153.89, 151.21, 143.20, 120.93, 66.12, 57.33, 48.69, 45.26, 42.65, 31.88, 31.33, 31.23, 30.16, 26.63, 24.61, 18.50.
m.p.: (softens at 175C) melts at 187C
Rf = 0.27 (methanol) Mass spectrum: M+ = 462 (HRMS) W095/35297 ~ ~ ~ ~ PCT/CA95/00356 r _ 1 e 30 Synthesis of trans-(N-6-chloropurin-9-yl)-4-methyl-cyclohexyl-methanol - C ~ d #30 cl N~ \>

H NMR (CDCl3, 400MHz, ~ in ppm); 8.73 (lH, s, purine); 8.05 (lH, s, purine); 4.12 (2H, d, N-CH2); 3.43 (2H, d, O-CH~); 1.89 (lH, m, CH)i 1.84-1.64 (4H, m, CH2-cyclohexane); 1.56 (lH, br s, OH); 1 45 (lH, m, CH)i 1.14-0.85 (4H, m, CH2-cyclohexane).

m.p. (softens 176C) = 178C
R = 0.4 (ethyl acetate) _le 31 Synthesis of trans-(N-6-dimethylaminopurin-9-yl)-4-methyl-cyclohexyl-methanol - ~c _ ~.d #31 N~ N
11 ~
N N ~OH

H NMR (CDCl3, 300MHz, ~ in ppm); ; 8.29 (lH, s, purine); 7.63 (lH, s, purine); 3.97 (2H, d, N-C_2); 3.49 ( 6H , br s, N-(CH3)2); 3.38 (2H, d, O-C_2); 2.46 (lH, br s, OH); 1.84 (lH, br m, C_-cyclohexane)i 1.71 (4H, m, 2 x CH2-cyclohexane);1.40 (lH, m, CH-cyclohexane); 0.90 (4H, m, CH2-cyclohexane).

13C NMR (CDCl3, 400MHz, ~ in ppm); 154.9, 152.3, 150.6, 138.7, 120.1, 68.2, 49.7, 40.2, 38.5, 38.2, 29.9, 28.6.
m.p. = 151-153C

R~ = 0.44 (10~ methanol-ethyl acetate) _le 32 Synthesis of trans-(N-6-dimethylaminopurin-9-yl)-4-methyl-cyclohexyl-methyloxycarbonyl-D-arginine - ~ __~.d #32 HNq~NH2 N HN~

N~N O NH~

lH ~R (DMSO-d6, 400MHz, ~ in ppm); 9.4 (lH, br s, COOH); 8.19 (lH, s, purine); 8.11 (lH, s, purine); 8.0-7.2 (4H, br, guanidine); 6.28 (lH, d, NH); 3.98 (2H, d, N-CH2); 3.69 (2H, d, O-CH2); 3.61 (lH, m, CaH); 3.43 (6H, br s, N-CH3)2); 3.00 (2H, br, C~H2); 1.9-0.8 (14H, m, C~H2, C~H2, 2 x CH-cyclohexane, 4 x CH2-cyclohexane).
13C NMR (DMSO-d6, 400MHz, ~ in ppm); 174.8, 156.8, 155.0, 153.8, 151.2, 150.0, 139.6, 118.7, 78.7, 67.9, 54.6, 48.2, 48.1, 37.1, 36.5, 29.3, 28.8, 27.8, 24.6.
m.p. (softens 157C) = 164-166C
R~ = 0.35 (methanol) _le 33 Synthesis of trans-(N-6-hydroxypurin-9-yl)-4-methyl-cyclohexyl-methanol -C _ou,~ #33 OH

~\\ N>~ =

lH NMR (CD30D, 400MHz, ~ in ppm); 8.05 (2H, s, purine)i 4.10 (2H, d, N-CH2); 3.35 (2H, d, O-CH2); 2.0-0.9 (10 H, m, 2 x CH-cyclohexane, 4 x CH2-cyclohexane) 13C NMR (CD30D, 400MHz, ~ in ppm); 156.7, 148.1, 144.1, 140.2, 122.7, 66.1, 48.7, 39.2, 37.5, 28.7, 27.6.
m.p. > 200C
Rf = 0.35 (20% methanol-ethyl acetate) ~ _le 34 Synthesis of trans-(N-6-methoxypurin-9-yl)-4-methyl-cyclohexyl-methanol - C __u..d #34 o/

N~

~OH

H NMR (CDC13, 300MHz, ~ in ppm); 8.52 (lH, s, purine); 7.84 (lH, s, purine); 4.17 (3H, s, O-CH3); 4.12 (2H, d, N-CH2); 3.43 (2H, d, O-CH2); 1.89 (lH, m, CH); 1.84-1.64 (4H, m, CH2-cyclohexane); 1.56 (lH, br s, OH); 1.45 (lH, m, CH); 1.14-0.85 (4H, m, CH2-cyclohexane).
m.p. (softens 159C) = 162C
Rf = 0.25 (ethyl acetate) _le 35 Synthesis of cis-(N-6-dimethylaminopurin-9-yl)-4-methyl-cyclohexyl-methanol - C _ ~-d~ 35 WO95l35Z97 Jr ~iC~ 2 1 6 5 9 5 6 PCT/CA95/03356 H3C~N,CH3 ~C ~

~OH

lH N~ (CDCl3, 300MHz, ~ in ppm); ; 8.31 (lH, s, purine); 7.66 (lH, s, purine)i 4.08 (2H, d, N-CH2); 3.55 (2H, d, O-C_2); 3.50 ( 6H , br s, N-(CH3)2); 3.28 (lH, br s, OH); 2.12 (lH, m, CH);
1.67 ~lH, m, CH); 1.5-1.3 (8H, m, CH2-cyclohexane).
13C NMR (CDCl3, 300MHz, ~ in ppm); 155.4, 152.7, 151.1, 139.1, 120.5, 65.9, 47.7, 39.1, 38.3, 36.3, 26.6, 25.4.
m.p. 153-156C
R = 0.3 (10% methanol-ethyl acetate) _~e 36 Synthesis of cis-(N-6-dimethylaminopurin-9-yl)-4-methyl-cyclohexyl-methyloxycarbonyl-D-arginine - C ~ d #36 N HNq,~NH2 ~ O J~NH~O~

lH NMR (DMSO-d6, 400MHz, ~ in ppm); 9.28 (lH, br s, COOH); 8.19 (lH, s, purine); 8.13 (lh, s, purine); 8.0-7.2 (4H, br, guanidine); 6.34 (lH, d, NH); 4.10 (2H, d, N-C_2); 3.85 (2H, d, O-CH2); 3.65 (lH, m, CaH); 3.44 (6H, br s, N-(CH3)2); 3.02 (2H, 2l 6~q56 W095/35297 ~ a ~ s PCTICA95/00356 m, C~H2); 2.09 (lH, m, CH); 1.8-1.2 (14H, m, C~H2, C~H2, 2 x CH-cyclohexane, 4 x CH2-cyclohexane).
13C NMR (DMSO-d6, 400MHz, ~ in ppm); 175.2, 157.7, 155.2, 154.1, 151.8, 150.2, 139 9, 119.3, 66.4, 55.3, 46.1, 48.0, 34 9, 34.7, 29.9, 25.5, 25.1, 24.4.
m.p. (softens 153C) = 168-170C
Rf = 0.35 (methanol) _le 37 Synthesis o~ N-(6-dimethylaminopurin-9-yl) 7-pentoxycarbonyl-D-citrulline - C __~.d #37 O~NH2 \N/ HN~

N ~ NH~/

lH NMR (DMSO - d6, 400 MHz, ~ in ppm); 8.19 (lH, s, purine);
8.15 (lH, s, purine); 6.21 (lH, d, NH); 6.11 (lH, s, NCO-NH);
5.42 (2H, s, N_2) 4.14 (2H, t, N-C 2); 3.86 (2H, m, O-CH2); 3.56 (lH, m, CaH); 3.35 (6H, br s, N-(CH3)2); 2.87 (2H, m, C~_2);
1.9-1.2 (lOH, m, (CH2)3~ C~H2~ C~H2) 13C NMR (CD30D, 400 MHz, ~ in ppm); 173.6, 158.4, 155.0, 153.8, 151.2, 149.7, 139.3, 118.7, 62.8, 54.8, 42.3, 37.4, 29.8, 28.5, 27.6, 25.7, 22Ø
m.p. (softens 172-176C) = 178-181C
Rf = 0.20 (40% methanol-ethyl acetate) _le 38 Synthesis of N-(6-methylaziridinepurin-9-yl)-5-pentanol - Compound #38 WO 9513529'7 PCT/CA95/00356 CH3~ CH3~7 Cl N
N~ N N~ N
N N THF ~NJ~N
(CH2)s--OH (CH2)s--OH

lH NMR (~, CDCl3 in ppm): 8.54 (s, lH, purine), 7.90 (s, lH, purine), 4.22 (t, 2H, CH2), 3.61 (t, 2H, C_2), 2.78 (m, lH, CH), 2.65 (d, lH, CH2), 2.40 (d, lH, CH2), 2.39 (bs, lH, OH), 1.94 (m, 2H, CH2), 1.59 (m, 2H, CH2), 1.50 (d, 3H, CH3), 1.40 (m, 2H, CH2 ) -13C NMR (~, CDC13 in ppm): 163.30, 152.99, 151.99, 142.80, 126.11, 62.81, 44.48, 35.97, 34.89, 32.51, 30.38, 23.50, 18.48.
Low melting point.
Rf = 0.4 (20% MeOH/AcOEt) Mass spectrum: M' = 262 Exam~le 39 Synthesis of N-(6-methylaziridine purine-9-yl)-7-pentyloxycarbonyl-D-arginine - ~c ~ #39 ~7 HN~NH2 HN

~CN O NH~

lH NMR (CD30D in ppm): 8.29 (s, lH, purine), 8.14 (s, lH, purine), 4.10 (t, 2H, CH2), 3.79 ~m, 3H, lxCH2 and CaH), 2.97 (m, 2H, CH2), 2.62 (m, lH, CH), 2.45 (d, lH, CH2), 2.19 (d, lH, CH2), 1.2-1.76 (m, 13H, 5xCH2 and lxCH3).

.j 2 1 6 5 9 5 6 m.p.: (softens at 190C) melts at 200C
Rf: O . 4 (methanol) Mass spectrum: M = 462 _le 40 N,N-(6-Dimethylaminopurinyl-9-yl)-7-thioethoxy-ethoxycarbonyl-D-arginine - Com~ound #40 Cl N
N ~ \> ~ ~ S "~"

N N

~ o ~ N ~ COOH ~ ~ ~ OH

NH
N ~ NH

WO 95/35297 ` `~ 2 1 ~ 5 9 5 6 PCTICA95/00356 r _le 41 meta-(N-6-Dimethylaminopurinyl-9-yl) methyl-benzyloxycarbonyl-D-arginine - C~ _lo~.d #41 HO--~OH

Cl N~C \> + HO ~f OSitBuPh2 N . N

N~ > 3 N ~CN~>

~--OH ~--~OSitBuPh2 N
N ~N~ NH2 ~ H COOH

Cu~ u~

W095/35297 :~ ~ ~ ~ PCT/CA931~C~56 _le 42 5-(N-6-Dimethylaminopurinyl-9-yl)-3-pentynyl-1-oxycarbonyl-D-arginine - C ~ d #40 OH ~ MeO2C OSitBuPh2 -- OSitBuPh2/\./

N~cN~
N HO ~ ~OSitBUPh2 \~

N

N~CN\>

N ~ OSitBuPh2 ~--0 HN~

5 Example 43 Synthesis of N-[6-(1-methyl-2-acetoxy)-ethylaminopurin-9-yl]-5-pentanol - ~ _ ~.d #43 o~

NH

~cN>

~ W09535~97 ~ S 2 ~ 6 5 9 5 6 PCT/CA95/0~356 lH NMR (~, CDCl3 in ppm): 8.36 (s, lH, purine), 7.76 (s, lH, purine), 6.58 (bs, lH, NH), 5.18 (m, lH, OH), 4.22 (t, 2H, CH2), 3.92 (bs, lH, CH), 3.63 (t, 2H, CH2), 2.05 (s, 3H, CH3), 1.3-1.9 (m, 4xCH2, lxCH3).
173.04, 156,6, 155.8, 154.2, 142.8, 120.5, 71.66, 68.61, 63.10, 45.43, 33.54, 31.42, 24.54, 21.69, 18.25.
Low melting point Rf = 0.5 15~ MeOH/AcOEt Mass spectrum: M~ = 322 Example 44 Synthesi~ of N-[6-(1-methyl-2-acetoxy), ethylaminopurin-9-yl]-7-pentyloxy-carbonyl-D-arginine - ~c _ ~.d #44 ~
HN~ NH

N ~ HN~

~0 NH~b/
o ~H NMR (~, CD30D in ppm): mixture of isomers, 8.05 (s, lH, purine), 7.89 (s, lH, purine), 4.92 (m, lH, CH), 4.03 (t, 2H, C_2) ~ 3.78 (m, 3H, CH2 and caH), 3.46 (d, 2H, CH2), 2.99 (m, 2H, CH2), 1.8 (s, 3H, CH3), 1.1-1.79 (m, 13H, 5xCH2 and lxCH3).
3C NMR (~, CD30D in ppm): mixture of isomers, 179.50, 159.19, 158.97, 156.88, 154.21, 154.14, 142.85, 142.75, 120.95, 68.13, 66.91, 66.11, 57.36, 45.32, 42.65, 31.89, 31.73, 31.25, 30.15, 30.04, 26.67, 24.61, 21.56, 18.09.
m.p.: (softens at 177C) melts at 185C
Rf: 0.35 (methanol) Mass spectrum: M~ = 522.

WO 9S/35297 ~ 2 1 6 5 9 5 6 PCT/CA95/00356 ~
_l e 45 Synthesis of N-(2,6-Dichloropurin-9-yl)-5-pentanol - C _GU~1~ #45 C~ /~N OH

lH NMR (~, CDCl3 in ppm): 8.11 (s, lH, purine), 4.29 (t, 2H, CH2), 3.66 (t, 2H, CH2), 2.00 (m, 2H, CH2), 1.64 (m, 2H, CH2), 1.48 (m, 2H, C_ 2), 1.3 (t, lH, OH).
13C NMR (â~ CDCl3 in ppm): 163.3, 150.2 149.3, 148.01, 128.00, l0 63.20, 44.80, 29.70, 26.00, 22.4.
m.p.: 133-135C
Rf: 0.4 596 methanol/ethyl acetate Mass spectrum: M+ = 260 (HRMS).

15 _1 e 46 Synthesis of N-(2,6-Dichloropurin-9-yl)-7-pentyloxycarbonyl-D-arginine - ~~ __u.~d #46 cl Cl ~ ~OH

20 lH NMR (~, DMSO in ppm): 9.33 (s, lH, COOH), 8.75 (s, lH, purine), 7.3-7.8 (bs, 4H, guanidine), 6.28 (d, lH, NH), 4.23 (t, 2H, CH2), 3.86 (t, 2H, CH2), 3.61 (m, lH, CaH), 3.015 (m, 2H, CH2), 1.2-1.9 (m, 10H, 5xCH2).
m.p.: Softens at 136C melts at 147C

2 ~ 6 5 9 5 6 Rf: 0.46 methanol Mass spectrum: Mf = 476 r _l Q 47 Synthesis of M-(2,6-Dichloropurin-9-yl)-7-S pentyloxycarbonyl-L-arginine - C~ d #47 CIl~N O NllJ~/

1H NMR (~, CD30D in ppm): 8.38 (s, lH, purine), 4.12 (t, 2H, CH2), 3.80 (m, 3H, CH2 and CaH), 2.97 (m, 2H, CH2), 1.2-1.8 (m, lOH, 5xCH2).
m.p.: Softens at 137C, melts at 147C
Rf: 0.45 (methanol) Mass spectrum: M = 476 Example 48 N-(2-Amino, 6-N, N-Dimethyl~m; nopurin-9-yl)-5-pentanol - Com~ound #48 N

~ ~H

W095/35297 PCT/CA95/~5 HNMR (~ in CDC13 ppm): 7.46 (s, lH, purine), 4.70 (bs, 2H, N_2 ), 4.04 (t, 2H, CH2), 3 65 (t, 2H, CH2), 3.46 (bs, 6H, 2 x CH3), 1.95 (m, 2H, CH2), 1.65 (m, 2H, CH2), 1.42 (m, 2H, CH2) 13CNMR (~ in CD30D ppm):
158.27, 154.08, 150.74, 136.12, 112.96, 60.28, 41.97, 36.50, 30.71, 28.38, 21.64.
m.p.: 139-141C
Rf: 0.55 (15% Methanol/Ethyl acetate) Mass spectrum: M~ = 265.
_le 49 Synthesis of N-(6-dime'hylamino-8-methylthiopurin-9-yl) 5-pentanol - C __~.d ~49 H3C~N,CH3 N~N~ CH3 IN fH

H NMR (CDCl3, 400MHz, ~ in ppm); 8.24 (lH, s, purine)i 4.08 (2H, t, N-CH2); 3.61(2H, t, O-C_2); 3.49 (6H, br s, N-(CH3)2);
2.70 (3H, s, S-CH3); 1.81 (2H, p, CH2); 1.67 (lH, br s, OH);
1.59 (2H, p, CH2); 1.39 (2H, p, CH2).
13C NMR (CDCl3, 400MHz, ~ in ppm); 152.30, 151.69, 150.29, 146.47, 119.39, 61.58, 41.77, 37.45, 31.28, 28.08, 21.81, 13.43.

~ ~ i ; ~r ; ~ ~ 1 6 5 9 5 6 Exam~le 50 Synthesis of N-(6-dimethylamino-8-methylthiopurin-9-yl) 7-pentoxycarbonyl-D-arginine - Com~ound #50 H3C~ N - CH3 N ~ N CH3 o '~ ~

HN

lH NMR (DMSO-d6, 400MHz, ~ in ppm); 8.13 (lH, s, purine); 8.0-7-2 (4H, br, guanidine)i 6.32 (lH, d, NH)i 4.01 (2H, t, N-CH2);
3.86 (2H, t, O-CH2); 3.65 (lH, m, Ca_); 3.41 (6H, br s, N-(CH3)2); 3.02 (2H, br, C~H2); 1.8-1.2 (lOH, m, C~H2, C~H2, -(CH2 ) - ) .
13C NMR (CDCl3, 400MHz, ~ in ppm);175.09, 156.83, 155.01, 152.01, 151.87, 150.46, 146.70, 118.84, 62.82, 54.61, 41.66, 39.91, 37.32, 29.26, 27.80, 27.69, 24.62, 22.05, 13.38.

_le 51 Synthesis of N-(6-methoxypurin-9-yl) 5-pentanol - C __ ~ #51 ~ ~0 2 ~ 6 5 9 5 6 W095/35297 ~ PCT/CA9S/00356 H NMR (CDCl3, 400MHz, ~ in ppm); 8.43 (lH, s, purine); 7.86 (lH, s, purine); 4.18 (2H, t, N-CH2); 4.09 (3H, s, O-CH3); 3.55 (2H, t, O-CH2); 3.09 (lH, br s, OH); 1.86 (2H, m, CH2); 1.53 (2H, m, CH2); 1.37 (2H, m, CH2).
13C NMR tCDCl3, 400MHz, ~ in ppm); 160.0, 150.9, 141.2, 120.4, 60.9, 53.2, 43.1, 30.9, 28.8, 22Ø
m.p.= 150C
Rf = 0.30 (15~ methanol-ethyl acetate) _le 52 Synthesis o~ N-(6-methoxypurin-9-yl) 7-pentoxycarbonyl-D-arginine - C~ d #52 H2N~f ;NH
HN~

NH~

lH NMR (DMSO-d6, 300MHz, ~ in ppm); 8.51 (lH, s, purine); 8.39 (lH, s, purine); 8.0-7.3 (4H, br, guanidine); 6.29 (lH, d, NH);
4.22 (2H, t, N-CH2); 4.08 (3H, s, O-CH3); 3.86 (2H, t, O-CH2);
3.62 (lH, m, CaH); 3.02 (2H, br, C~_2); 1.8-1.2 (lOH, m, C~H2, C
'YH2, (CH2)3) 13C NMR (CDCl3, 400MHz, ~ in ppm); 205.9, 175.8, 160.6, 157.7, 155.8, 152.4, 151.8, 120.9, 63.7, 55.4, 54.2, 43.6, 39.1, 30.1, 29.3, 28.5, 25.5, 22.9.
m.p. (softens 132C) = 148C
R~ = 0.35 (40~ methanol-ethyl acetate) W095l35297 PCTICA95100356 _le 53Synthesis of N-(2-chloro-6-methoxypurin-9-yl)-7-pentyloxycarbonyl-D-arginine - compound #53 o,CH3 CIl~N J HN COOH

NH

S lHNMR (~, DMSO in ppm) 9.41 ~bs, lH, COOH), 8.42 (s, lH, purine), 7.3-7.8 (bd, 4H, guanidine), 6.28 (d, lH, NH), 4.08 (s, 3H, CH3~, 3.87 (t, 2H, CH2), 4.18 (t, 2H, CH2), 4.08 (s, 3H, CH3), 3.87 (t, 2H, CH2), 4.08 ~s, 3H, CH3), 3.87 (t, 2H, CH2), 3.61 (m, lH, CaH), 3.04 (m, 2H, CH2), 1.22-1.87 (m, 10H, 5 X CH2).
m.p.: Softens at 128C, melts at 141C
Rf: 0.45 (Methanol) Mass spectrum: M+ = 471.

15 r _~e 54Synthesis of N-(6-dimethylaminopurin-9-yl) 7-pentoxycarbonyl-D-ornithine - C _ ' #54 \N/ H2N~
J

~CN~ OJ~NH~ ~

W095/35297 ~ C~ 2 i 6 5 9 5 6 PCT~CAg5~00356 ~
H NMR (DMSO-d6, 400MHz, ~ in ppm); 8.20 (lH, s, purine); 8.12 (lH, s, purine); 6.21 (lH, d, NH); 4.10 (2H, t, N-CH2); 3.87 (2H, t, O-CH2); 3.59 (lH, m, C~_); 3.4 (br, N-(CH3)2, N_2), 2.70 (2H, m, C~H2); 1.9-1.2 (lOH, m, (CH2)3~ C~H2~ C~H2)-13C NMR (CD30D, 400MHz, ~ in ppm);176.3, 156.2, 153.8, 150.7, 148.8, 138.9, 118.5, 63.5, 54.2, 42.5, 38.0, 37.0, 28.6, 28.3, 27.2, 22.4, 21.7.
m.p. (softens 185C) = 189-190C
Rf = 0.20 (methanol) _le 55 Synthesis of N-(6-dimethylaminopurin-9-yl) 7-pentoxycarbonyl-L-ornithine - Com~ouna #55 ~C J Hz Spectral properties were identical with compound #54.

_le 56 Synthesis of N-(6-dimethylaminopurin-9-yl) 7-pentoxycarbonyl-L-valine - C ~ d 56 \N/

~CN ! ~ ~1 lH NMR (DMSO - d6, 400 MHz, ~ in ppm); 8.19 (lH, s, purine);
8.16 (lH, s, purine); 6.30 (lH, d, NH); 4.13 (2H, t, N-CH2);

3.87 (2H, m, O-CH2); 3.64 (lH, m, CaH); 3.4 (br s, N-(CH3)2);
1.80 (2H, p; CH2,); 1.25 (2H, p, CH2,); 0.79 (3H, d, C~H3); 0.75 (3H, d, C~_3).
3C NMR (CD30D, 400 MHz, ~ in ppm); 174.6, 156.7, 153.7, 150.5, 148.9, 138.6, 118.6, 63.4, 59.2, 42.3, 36.729.6, 28.3, 27.3, 21.7, 17.5, 15.9.
m.p. (softens 140C) = 172-176C
Rf = 0.20 (30% methanol-ethyl acetate) _~e 57 Synthesis of N-(6-dimethylaminopurin-9-yl) 7-pentoxycarbonyl-D-valine - Compound #57 I5 Spectral properties were identical with compound #56.
_7e 58 Synthesis o~ N(N,N-dimethylaminopurin-9-yl)-7-pentyloxycarbonylethylamine hydrochloride -Compound #58 ~ ~ N ~ ~NH3 Cl 1HNMR (~ in DMSO ppm): 8.40 (s, lH, purine), 8.43 (s, lH, purine), 8.04 (bs, 3H, N_3), 4.24 (t, 2H, CH2), 4.02 (t, 2H, CH2), 2.99 (m, 2H, CH2 ), 2.67 (t, 2H, CH2 ), 1.83 (m, 2H, CH2), 1.62 (m, 2H,CH2), 1.28 (m, 2H, CH2).

WO95/35297 s= ? ~ 2 1 6 5 9 5 6 PCT/CA95/00~56 Rf: 0.3 10~ Methanol~Ethylacetate Mass spectrum: M~ = 321 r le 59 Synthesis N-(6-Mercaptopurin-9-yl)-pentanol -S Com~ound #59 SH

~N~

lHNMR (~ DMSO in ppm): 8.30 (s, lH, purine~, 8.18 (s, lH, C2-purine), 4.34 (t, lH, OH), 4.15 (t, 2H, C_2-0), 3.34 (t, 2H, CH2-N), 1.82 (m, 2H, CH2), 1.42 (m, 2H, CH2), 1.24 (m, 2H, CH2).
Rf: 0.57 30% Methanol/Ethylacetate Mass spectrum: M+ = 239 15 - _le 60 Synthesis of N-(6-Methylthiopurin-9-yl)-pentanol - C ~ d #60 '~ ~
T f .~
lHNMR (~ CDC13 in ppm): 8.74 (s, lH, purine), 7.95 (s, lH, purine), 4.27 (t, 2H, CH2-N), 3.65 (t, 2H, C_2-O), 2.74 (s, 3H, SCH3), 1.94 (m, 2H, CH2), 1.60 (m, 2H, C_2), 1.43 (m, 2H, CH2).
CNMR (~ CD30D in ppm): 163.23, 153.42, 149.96, 145.89, 132.48, 63.08, 45.60, 33.62, 31.24, 24.54, 12.29.
m.p.: 95-97C

r~ ~ IhC~ 2 t 6 5 9 5 6 W095/35297 ` PCTICA95/00356 Rf: 0.22 (Ethylacetate) Mass spectrum: M+ = 253 _le 61 Synthesis of N-(6-chloropurin-9-yl) 4-butanol -Compound ~61 Cl N ~

H NMR (CDCl3, 400 MHz, ~ in ppm): 8.66 (lH, s, purine), 8.16 (lH, s, purine), 4.33 (2H, t, N-CH2), 3.67 (2H, t, O-CH2), 3.04 (lH, br s, OH), 2.01 (2H, p, CH2), 1.55 (2H, p, CH2).
m.p. = 97C

_le 62 Synthesis of N-(6-dimethylaminopurin-9-yl) 4-butanol - Compound #62 H3C~N,CH3 N ~

H NMR (CDCl3, 400 MHz, ~ in ppm): 8.24 (lH, s, purine), 7.68 (lH, s, purine), 4.33 (2H, t, N-CH2), 3.89 (lH, br s OH), 3.64 (2H, t, O-CH2), 3.46 (6H, br, N-(CH3)2), 1.92 (2H, p, CH2), 1.53 (2H, p, CH2).
m.p. = 78C

Example 63 Synthesis of N-(6-dimethylaminopurin-9-yl)-6-butoxycarbonyl-D-arginine - C _ ~ #63 ~ ? ~ ~ ~ r 2 1 65 956 W095/3S297 PCT/CA95~ 5 H3C~N~cH3 N> O ~ NH COOH
O `~ (D-Arginine) HN

H NMR (DMSO-d6, 400 MHz, ~ in ppm); 8.20 (lH, s, purine), 8.16 (lH, s, purine), 8.1-7.3 (4H, br, guanidine), 6.40 (lH, d, NH), 4.16 (2H, t, N-CH2), 3.91 (2H, t, O-CH2), 3.65 (lH, M, C~H), 3.4 (6H, br, N-(CH3~2), 3.02 (2H, m, C~H), 1.9-1.3 (8H, m, C~H, CY_, -(CH2) 2-) ~
m.p. (softens 85C) = 140-142C

_le 64 Synthesis of N-(6-dimethylaminopurin-9-yl)-6-butoxycarbonyl-L-arginine - C _I~u--d #64 H3C~N,CH3 ~CN O~NH COOH
O ~ (L-Arginine) HN

Spectral properties were identical with compound #63 .
m.p. (softens 85C) = 139-142C

_le 65 Synthesis of N-(6-chloropurin-9-yl)-6-hexanol -Compound #65 ~ ~ r~ ~ ~ t ~; 2 1 6 5 9 5 6 W095/352~7 PCT/CA95/00356 Cl =OH

lH NMR (~, CDCl3 in ppm): 8.69 (s, lH, purine), 8.11 (s, lH, purine), 4.27 (t, 2H, CH2), 3.58 (t, 2H, CH2), 2.21 (bs, lH, OH), 1.91 (m, 2H, CH2), 1.43 (m, 2H, CH2), 1.35 (m, 4H, 2xCH2).
3C NMR (~, CDCl3 in ppm): 152.45, 151.60, 145.69, 132.16, 112.00, 63.00, 44.96, 32.86, 30.39, 26.87, 25.67 m.p. = 84-86C
Rf = 0.5 10~ (methanol/ethyl acetate) Mass spectrum: Mf = 255 Example 66 Synthesis of N-(6-N,N-dimethylaminopurin-9-yl)-6-hexanol - Com~ound #66 H3C~N,CH3 ~N

lH NMR (~, CDCl3 in ppm): 8.35 (s, lH, purine), 7.71 (s, lH, purine), 4.17 (t, 2H, CH2), 3.61 (t, 2H, CH2), 3.53 (bs, 6H, 2xCH3), 1.89 (m, 2H, CH2), 1.71 (bs, lH, OH), 1.55 (m, 2H, CH2), 1.45 ~m, 4H, 2xCH2).
3C NMR (~, CDCl3 in ppm): 154.44, 152.90, 150.95, 138.72, 120.53, 63.04, 44.08, 39.28, 32.95, 30.61, 26.80, 25.64.
m.p. 75-77C
Rf = 0.48 10% methanol~ethyl acetate Mass spectrum: M' = 264 _le 67 Synthesis of N-(6-N,N-dimethylaminopurin-9-yl)-8-hexyloxycarbonyl-D-aryinine - Cc _ ~-d #67 W095l35297 PCT/CA95100356 H3C~N,CH3 ~N ,~,O~NH~COOH

NH
HN~--NH2 lH NMR (~, CD30D in ppm): 7.98 (s, lH, purine), 7.~1 (s, lH, purine), 3.98 (t, 2H, CH2), 3.78 (m, 3H, CH2 and C~H), 3.27 (bs, 6H, 2xCH3), 2.96 (t, 2H, CH2), 1.1-1.78 (m, 12H, 6xCH2).

_le 68 Synthesis of N-6-N,N-dimethylaminopurine-9-yl)-8-hexyloxycarbonyl-L-arginine - C __uI-d #68 H3C~N,CH3 ~N ~O~NH~COOH

NH

lH NMR (~, CD30D in ppm): 8.00 (s, lH, purine), 7.82 (s, lH, purine), 4.00 (t, 2H, CH2), 3.80 (m, 3H, CH2 and CaH), 3.29 (bs, 6H, 2xCH3), 2.97 (t, 2H, CH2), 1.13-1.72 (m, 12H, 6xCH2).

Exam~le 69 Synthesis of cis-(N-6-hydroxypurin-9-yl)-4-methyl-cyclohexyl-methanol - C _-u--d #69 .- f~ 21 65956 7 ~ PCT/CA95/00356 H

~CN~

H NMR (CD30D, 400 MHz, ~ in ppm): 8.08 (lH, s, purine), 8.07 (lH, s, ~urine), 4.21 (2H, d, N-CH2), 3.49 (2H, d, O-CH2), 2.16 (lH, m, CH), 1.7-1.2 (9H, m, CH2-cyclohexane, CH).
m.p. >200C
Rf = O.3 (2096 methanol-ethyl acetate) _l e 70 Synthesis of cis-(N-6-hydroxypurin-9-yl)-4-methyl-cyclohexyl-methyloxycarbonyl-D-arginine - com~ouna #70 OH
N~cN~ o NH

O~NH~

15 lH NMR (CD30D, 400 MHz, ~ in ppm): 8.10 (lH, s, purine), 8.09 (lH, s, purine), 4.24 (2H, d, N-CH2), 3.65 (lH, m, C~H), 3.52 (2H, d, O-CH2), 2.95 (2H, m, C~H2), 2.2-1.2 (14H, m, 2xCH-cyclohexane, 4xCH2-cyclohexane, C~H2, C~H2).

20 _1 e 71 Synthesis of trans-(N-6-hydroxypurin-9-yl)-4-methyl-cyclohexyl-methyloxycarbonyl-D-arginine -- C _ _ ~ #71 2~ 65~56 WO 95/35297 ~ PCT/CA9~ 56 OH HNq~NH

N~CN OJ~NH ~OH

H NMR (CD30D, 300 MHz, ~ in ppm): 8.09 (2H, s, purine), 4.12 (2H, d, N-CH2), 3.68 (lH, m, CaH), 3.36 (2H, d, O-CH2), 3.01 (2H, m, C~H2), 2.0-0.9 (14H, m, 2xCH-cyclohexane, 4xCH2-cyclohexane, C~H2, CrH2).
m.p. >200C
R~ = 0.2 (methanol) 10 _le 72 Synthesis of N-(6-N,N dimethylaminopurin-9-yl)-5-pentylamine hydrochloride salt - C _~ d #72 H, Cl 15 lH NMR (â, DMSO in ppm): 8.20 (s, lH, purine), 8.16 (s, lH, purine), 7.84 (bs, 3H, NH3), 4.14 (t, 2H, CH2), 3.44 (bs, 6H, 2xCH3), 2.73 (t, 2H, CH2), 1.81 (m, 2H, CH2), 1.56 (m, 2H, CH2), 1.25 (m, 2H, CH2).

20 _le 73 Synthesis of N-(6-methylaziridinepurin-9-yl)-7-pentyloxycarbonyl-L-arginine - C~ #73 WO 95/35297 ~ r r ql ~ 2 1 6 5 9 5 ~ PCT/CA95l00356 ~ .
Nq~N

~N> _~, Spectral data of compound ~73 was comparable to that reported for compound #39 .

_l e 74 (2S,4S)-2-(N,N-Dimethylaminopurin-9-yl)-4-hydroxymethyl-1,3-dioxolane - Com~ound #74 N N
N~C \> nBu4NF N~CN,~
4~ OSi~3UPh2 4~ OH
O O

lH NMR (CDCl3): ~ 8.32 (s, lH, purine), 7.75 (s, lH, purine), 5.33 (dd, lH, J = 2.0, 6.6, H-2-dioxolane), 5.33 (bs, lH, OH), 4.45 (dd, lH, J = 6.6, 14.3, CH2-purine), 4.20 (dd, lH, J = 2.0, 14.3, CH2-purine), 4.20 (m, lH, H-4-dioxolane), 4.05 (d, 2H, J =
7.2, H-5), 3.78 (d, lH, J = 13.0, CH2-OH), 3.53 (bs, 6H, 15 (CH3)2N), 3.40 (d, lH, J = 13.0, CH2-OH).

}~xam~le 75 (lS,3R) and (lR,3S)-l-(N-6-Dimethylaminopurin-9-yl)methyl-3-cyclopentane methanol C _ ~u--d #75 W095/35297 ~ 2 1 6 5 9 5 6 PCTICA95/00356 N N

N~ ~ N\> nBU4NF N ~ \>
~ ~ OSitBUP~ OH

lH NMR (CDC13, 300 MHz): ~ 8.32 (s, lH, purine), 7.71 (s, lH, purine), 4.18 (dd, lH, J= 8.6, 13.7), 4.06 (dd, lH, J= 6.7, 13.7), 3.61-3.53 (m, 8H), 3.00 (bs, lH, OH), 2.48 (m,lH), 2.17 (m, lH), 1.88-1.68 '~Lll, 3H), 1.53 (m, lH), 1.43 (m, lH), 1.08 (m, lH).

_le 76 (lS,3R) and (lR,3S)-l-(N-6-Dimethylaminopurin-9-yl)methyl-3-(methyloxycarbonyl-D-arginine)cyclopentane - Compound #76 N N IH
N ~ N> l)COC12,THF N ~ \> ~N ~ N 2 N N 2)D-~ginine N N o ~
+/) ~ OH (/) ~ O N COOH

f~ 2 1 6 5 9 5 6 WO 95/35297 - ~ - PCT/CA95/00356 r _~ e 77 N,N-(6-Dimethylaminopurin-9-yl)-7-ethylamlnoethanol - Compound #77 N N
N~N,~ H2, Pd/C 10% N~C \>
~N ,OH --N~OH
CBZ H

S lHNMR (CDCl3, 400 MHz): ~ 8.34 (s, lH, purine), 7.79 (s, lH, purine), 4.29 (t, 2H, J= 5.8, -CH2-), 3.62 (m, 2H, -CH2-), 3.54 (bs, 6H, (CH3)2N), 3.11 (t, 2H, J=5.8, -CH2-), 2.81 (t, 2H, J=
5.2, -CH2-), 2.05 (bs, 2H, NH and OH).

_le 78 N,N-(6-Dimethylaminopurin-9-yl)-7-ethylaminoethoxycarbonyl-D-arginine Com~ound #78 N N
N~N,~ H2, Pd/C 10~ N~N~
N l~N~O~N~,COOH MeOH N N~ ~o~N COOH

,N NH~ (~NJ~NH2 H H

lH NMR (CDCl3, 300 MHz): ~ 9.36 (m, lH), 8.19 (s, lH, purine), 8.11 (s, lH, purine), 6.38 (d, lH, J=7.0, NH carbamate), 4.17 (t, 2H, J= 6.1, -CH2-), 3.91-3.87 (m, 2H, -CH2-), 3.65 (m, lH, CH-COOH), 3.43 (bs, 6H, (CH3)2N), 3.03-3.01 (m, 2H, CH2-NHC(NH)NH2), 2.90 (t, 2H, J= 6.1, -CH2-), 2.68 (t, 2H, J= 5.6, -20 CH2-), 1.64-1.44 (m, 4H, CH2-CH2-CH2NHC(NH)NH2).
.

-le 79 N,N-(6-Dimethylaminopurin-9-yl)-7-ethylaminoethoxycarbonyl-L-arginine -Compound #79 N N
N~N~ H2, Pd/C 10% N~CN> H
N l~N~O~N~COOH MeOH ~N~O~N~,COOH
CBZ ~ NH ~ NH
IN NH~ N NH2 H H

Spectral properties were identical with compound #78.

e 80 5-(N-6-Dimethylaminopurin-9-yl)-3-pentyn-1-ol -Compound #80 N N
N ~cN\>nBu4NF N~cN~
N N~ F, 0C N N~

. OSitBuPh~ ~--OH

lH NMR (CDC13): ~ 8.34 ~s, lH, H-2 purine), 7.89 (s, lH, H-8 purine), 4.91 (m, 2H, CH2-N), 3.74 (t, 2H, J = 6.2, CH2-OH), 3.52 (bs, 6H, (CH3)2N), 2.87 (bs, lH, OH), 2.50 (m, 2H, CH2-CH2OH).

r ¦ ~ 1 6 5 ~ 5 6 _~e 81 5-(N-6-Dimethylaminopurin-9-yl)-3-pentynyl-1-oxycarbonyl-~-aryinine - Com~ound #81 N N
1)COCI2,THF N ~ N~ N ~ NH2 N N~ 2)L-~ginine N N O ~ NH

~ OH ~ O IN COOH

Spectral properties were identical with compound #40.

_~e 82 N,N-(6-Dimethyl ~m; n opurin-9-yl~-7-thioethoxy-ethanol - Compouna #82 N N
> ~u4NF N ~ \>

~ S " ~_~OSi~uPh2 ~ S~_--OH

1H NMR (CDCl3): ~ 8.30 (s, lH, purine), 7.75 (s, lH, purine), 4.41 (t, 2H, J = 6.5, CH2 linker), 4.11 (bs, lH, OH), 3.73 (t, 2H, J = 6.5, C_ 2 linker), 3.51 (bs, 6H, (CH3)2N), 2.99 (t, 2H, J
= 6.5, CH2 linker), 2.68 (t, 2H, J = 6.5, CH2 linker).

1 6 5 9 5.6 r _le 83 N,N-(6-Dimethylaminopurin-9-yl)-7-thioethoxy-ethoxycarbonyl-L-aryinine - Compound #83 N N
N~N~ 1) COCl2, THF N~N~
~N N 2) L-arginine ~ N
¦~--`S ~ OH N ~ S ~ O ~ N ~ COOH
~ ~H
IN NH~

Spectral properties were identical with compound #40.

_le 84 (2S,4S)and (2R,4R)-2-(N,N-Dimethylaminopurin-9-yl)-4-(methoxycarbonyl-D-arginine)-1,3-oxathiolane - C _ ' #84 N N H
N ~ N~ 1)COC~,THP N ~ N~ ~N ~ NH~
N N 2)D-~ginine N N O ~

(+/-) ~ ~ OH (+/~) ~S ~ N~ COOH
H

1H NMR (CDCl3, 300 MHz): ~ 8.49 (s, lH, purine), 8.47 (s, lH, purine), 6.60 (bs, lH, NH c~rh~m~te), 6.22 (m, lH, H-2-oxathiolane), 4.26-4.03 (m, 3H), 3.63-3.00 (m, llH), 2.78-2.69 (m, 2H, H-5-oxathiolane), 1.53-1.40 (m, 4H, CH2-CH2-CH2NH-C(NH)NH2).

le 85 N,N-(6-Dimethylaminopurin-9-yl)-7-ethoxy-ethoxyethanol - Compound #85 N N
N~CN> nBu~N N~ \>

¦~ ~OSitBuP~2 l~o--~

lH NMR (CDCl3): ~ 8.29 (s, lH, purine), 7.80 (s, lH, purine), 4.33 (t, 2H, CH2), 3.82 (t, 2H, CH2), 3.68 (t, 2H, CH2), 3.55 (t, 2H, CH2), 3.50 (m, 6H, N(CH3)2).

Exam~le 86 M,N-(6-Dimethylaminopurin-9-yl)-7-ethoxy-ethoxycarbonyl-D-arginine - Compound #86 N N
N~N~ I) COCI2,1HF N~N~
N N 2) D-argilune N N
-- ~OH l ~O~N~COOH

NH
~NH NH2 lH NMR (DMSO-d6): ~ 8.25 (s, lH, purine), 8.14 (s, lH, purine), 6.5 (bd, lH, NH carbamate), 4.37 (t, 2H, CH2 linker), 4.03 (m, 2H, CH2 linker), 3.81 (m, 2H, CH2 linker), 3.72 (m, lH, CH-COOH), 3.60 (m, 2H, CH2 1 ;nk~r), 3.55-3.89 (m, 6H, N(CH3)2), 3.05 (m, 2H, CH2-NH-C(NH)NH2), 1.78-1.39 (m, 4H, CH2-CH2-CH2NH-C(NH)NH2).

W095l35297 ~ r`~ 2 1 6 5 9 5 6 PCT/CA95100356 Example 87 N,N-(6-Dimethylaminopurin-9-yl)-7-ethoxY-ethoxycarbonyl-L-arginine - C _-u~ld #87 N N
N ~ N~ 1)COCl2,THF N ~ N~ H
~N N 2)L-~ginine ~N N
l o~ I--O~O~N~,COOH

~NJ~

Spectral properties,were identical with compound #86.

_le 88 N-(6-Dimethylamino-8-bromopurin-9-yl)5-pentanol - C __ A #88 N N

N~N Acetate buffer, 0 5M Na+ N~N\~gr N N pH 4.5 N N

OH ~OH

lH NMR (CDCl3) 400 MHz): ~ 8.27 (s, lH, purine), 4.18 (t, 2H, J=7.2, CH2), 3.64 (t, 2H, J=6.3, CH2), 3.48 (bs, 6H, N(CH3)2), 2.02 (bs, lH, OH), 1.85 (quint, 2H, J=7.2, C_ 2) ~ 1. 63 (quint, 2H, J=6.3, CH2), 1.46 (m, 2H, CH2).

~ W095/35297 2 1 6 5 9 5 6 PCT/CA9S/OQ356 BIOLOGI~T. DATA

IN VITRO S~ -N1NI~:

S ExPI~ple 89: Mitogenic Proliferation on Spleen Cell Suspension Mitogenic lectin (mitogen) is a protein which binds and cross-links specific cells surface carbohydrate n determ;n~ntS~ and will polyclonally stimulate lymphoid cells. Lymphocyte activation by either antigens or mitogens results in intracellular changes and the subsequent development into a lymphoblast. Mitogenic stimulation of lymphocytes in vi tro is believed to mimic the series of events which occur in vivo following their stimulation by specific antigens. PHA, ConA, and PWM, LPS
mitogens can be used as a measurement of T cell and B cell activity, respectively.

Briefly, spleen mononuclear leukocytes from C57BL/6 mice were incubated in the presence or absence of mitogens with or without tested drugs. After 72 hours or 5 days, 3H
thymidine incorporation was recorded as an indication of cell trans~ormation and proliferation.
100 ~1 of a suspension of 2Xl06 cells/ml (2x105 cells/well) were incubated in presence of PHA or ConA or PWM or LPS at the following concentrations:

PHA = O.01% final concentration - O.001%
ConA = 2 ~g/ml - 1 ~g/ml PWM = O.2x - O.02x LPS = 5 ~g/ml - 2 ~g/ml 21 ~5956 WO9S/35297 ~ S PCTICA95100356 Cells were incubated in presence or absence of drugs for 72 hours. 0.5 or 1 ~Ci of tritiated thymidine (3H) was added per well the last 18 or 6 hours of incubation respectively. Cells were harvested and counted on a Beta S counter.

(Mitogenic Proliferation) Compound No. T (ConA)(M) B(LPS)(M) ST-789 0 1.5-3x(10-8-10 5) Xl 2 4 (1o-8 10-5) 2x(10 6 #2 2 4 ( 1o-8 10-5 ) o 2_6xlo-l2_lo-6) 2-2.5x(10 8-10 ) 2-6X(lo-l2_l0-8) 0 2-3~5X(lo-l2_lo-6) Example 90: Cytotoxic T Lymphocytes (CTL) and Mixed Lymphocyte Reaction (MLR) Assays Mixed lymphocyte reaction is an in vi tro counterpart of the allograft rejection. Briefly, T cell response was obtained when cells taken from two inbred strains from two outbred individuals of any species were mixed in vi tro in culture. To have a unidirectional response, the proliferation of either cell type may be blocked with X-irradiation or mitomycin C treatment.

After 4 days incubation, 3H thymidine uptake and cytotoxicity assay (CTL) were performed.

~ WO95/35~97 ~ ~ 3 t ~ 2 1 6 5 9 5 6 PCT/CA9~/0~
3 C57Bl/6 mice and 3 DBA/2 mice wre killed and lymphocytes prepared using lympholyte M. The cell concentration was adjusted to 10x106 cell/ml for each lysis. DBA/2 cells were irradiated with 3000 Rads. 1 ml o the C57 cells + lml of the DBA/2 cells + 1 ml of the drug at 3 different concentrations were incubated together for 5 days.
Positive control was IL-2 at 15 ng/ml final. After 5 days, the CTL and MLR tests were carried out.

MLR
The cells were resuspended and 100 ~l of cell suspension were deposited in each of the 96 wells in the plate, 50 ~1 of thymidine at 20 ~Ci/ml was added for 6 hrs. The cells were then collected and counted using a beta counter.

-P815 target cells were labelled with Cr5l. After labelling, the cells were resuspended to 5x104 cells/ml.
Effector cells were adjusted to 2.5x106 cells/ml, and then diluted 1:2 and 1:4 to obtain the necessary effector to target ratios:

50:1 (2. SX106 cells/ml: 5x104 cell/ml) 25:1 (1.25x106 cells/ml: 5x104 cells/ml) 2s 12.5:1 (0.625x105 cells/ml: 5x104 cells/ml) 100~1 of target cells + 100~1 Of T cells were incubated for 4 hrs and then 100~1 of supernatant was counted using a gamma counter.

W095135297 ~ t ~ ~ 2 t ~ 5 9 5 6 PCT/CA95J~56 (CTL and MLR Assays) Compound No. CTL (M) MhR (M) ST 689 ++++ (10-7 M)P 1.6x (10-5 M)P
ST 789 ++ (10-7) 2-3x(10-9-10-5) #III +++ (10-7 M) 1.5-2.5x (10-9-10-5 M) *V +++ (10-7 M) 1.5-2x (10-7-10-5 M) #1 ++++ (109) 1.5-4x(10 9-10 5) #2 0 1.5-2x(10 7-1o 5) ~3 + (10 5) 1.5x(10-9-10-5) #3a +++ (10-9-10-7 M) 1.5-2.9x (10-9-10-5 M) #5 + (10 7) 1. 5-2x(10 7-1o 5) #5a ++ (10-7-10-5 M) 1.5-2x (10-9-10-6 M) #6 +++ (10 9) 1.5-3x(10 9-10 5) #7 +++ (10-9) 1.5-2x(10 9-10 6) #7a ++++ (10-9 M)P 2x (10-9-10-5 M)P
#8 ++++ (10-9) 1.5-2x(10-9-10 5) #11 ++ (10 7) 2-2.5x(10 7-1o 5) #19 ++ (10-7 M)P P
#20 ++ (10-5) #51 ++ (10-7 M) 1.5-2x (10-9-10-6 M) #59 ++ (10-9 M) 2-2.4x (10-9-10-5 M) #60 + (10-7 M) 1.7-2x (10-9-10-5 M) For CTL Activity, the data expressed is as a % increase com.~ared to IL-2.
S IL-2 is 100~. 0 represents less than 20%, + represents 20-40%, ++
represents 40-60~, and +++ represent 60-80%, and ++++ represents 80+. P =
Pr~l; m; ~Ary result ~ WO 95/35297 ., ;~ r ~ 2 1 6 5 9 5 6 PCT/CA95/00356 IN VI~rO/EX VIVO S~:K~ N1N~

Example 91: Immunophenotyping After in vitro drugs analysis, the drugs were evaluated on - whole blood for drug stability and toxicity. Furthermore, in vivo /ex vivo analysis was performed on normal and cyclophosphamide immunosuppressed ~n;m~ls plus 5Fu-treated ~m~l S . Cell immunophenotyping was performed on mouse-treated blood and spleen. The following cell surface antigens were analyzed:
CD3 (all T cells), C~4 (T helper/inducer, binds class II-restricted T cells), CD8a (cytotoxic T cells, CTL
adhesion), CDlla (T, B, NK, some stem cells, CTL adhesion anti LFA-1~), MAC-1 (monocyte/macrophage), NK (natural killer cells), Ly5 (B cells), CD45 (all leukocytes, protein tyrosine phosphates), and TCR (T cell receptor).

C57BL/6 mice (6-8 weeks old) were injected daily for 4 consecutive days, sacrificed at day 5 and immunophenotyping was performed on blood and spleen cells.

The cells were washed twice in PBS, resuspended in 1 ml of RPMI 2% FBS, and incubated for 45 min. on ice with 2s monoclonal antibody. The cells were washed once, fixed with 1~ paraformaldehyde, then analyzed using XL Coulter~
counter. Results are presented in Table 3a and 3b.

WO9S/35297 ~ t~ 6 5 9 5 6 PCT1CA95/00356 -~
TABLE 3a Tmm11nophenotyping On Blood Cells Of Compound #l Treated-Mice (N-l0) Cellmarker Control 25mg/kg 50 mg/kg CD8+ mean 6.66 10.11 8.65 CD45+ STD 2.09 2.69 1.39 p 0.005 0.02 NK+ mean 6.01 5.90 8.14 CD3- STD 0.98 1.39 1.35 p 0.5 0.005 NK+ mean 3.43 5.84 3.25 CD3+ STD 0.76 2.08 0.57 p 0.02 0.289 mean 9.60 13.71 9.68 CDl lb+ STD 2.79 2.68 3.59 p 0.015 0.4 TABLE 3b Immunophenotyping On Spleen Cells Of Compound #l Treated-Mice (N~l0) Cell marker Control 25mg/kg 50 mg/kg mean 38.93 39.78 45.09 TCR+ STD 3.83 7.61 7.34 p 0.421 0.035 mean 55.49 54.08 50.35 Ly5 STD 3.44 7.30 6.72 p 0.37 0.034 ~ W095/35297 ;~ 2 1 6 5 9 5~ PCT/CA95/00356 TAB~E 4 Tmm7-nophenotyping On 8100d Cells Of Mice Treated With Compound #1 In Combination With 5Cyclophosphamide (N~4) CyClo-~Jhaa~JhumideCY + cpd #l CY + cpd #l Cell marker 100 mg/kg25 mg/kg 50 mg/kg mean 15.05 13.25 20.8 CD8+ STD 3.89 0.07 0.85 CD45+ p 033 Spleen: no effect TABLE 5a I unophenotyping On Blood Cells Of Nice Treated With Compound #1 In Combination With 5 Fluorouracil (N~4) 5FU 5FU +cpd#l 5FU+cpd#l Cell marker (80 mg/kg) 25 mg/kg 50 mg/kg mean 6.66 10.11 8.65 CD8+ STD 2.09 2.69 1.39 CD45+ p 0.005 0.022 mean 3.24 3.58 4.12 NK+ STD 0.66 1.01 0.74 p 0.38 0.01 }~ ~ C 2 t ~5~56 WO95/3S297 ~ ~ ~ PCTICA95/00356 TAB~E 5b T nophenotyping On Spleen Cells Of Mice ~reated With Compound #l In Combination With 5 Fluorouracil (N~4) 5 FU ~ FU + cpd #l5 FU + cpd #l Cell marker (80 mg/kg) 25 mg/kg 50 mg/kg CD4+ mean l O.0 13.19 12.06 CD45- STD 1.98 3.19 2.27 p 0.01 5 0.04 mean 4.22 3.32 3.17 NK+ STD 0.5 0.45 0.36 p 0.000~ 0.0001 A~ .IJn ASSESSMEN~ PROTOCOL

The compounds were tested for tumor growth control using the following procedures.

Example 92: Effect of compound ~l on growth of breast carcinoma in combination with cyclophosphz~m; ~ .

Balb/C Mice (n-5/Gr) were used along with DA-3 m~mmAry carcinoma cell line. The mice were treated from -2 to 13 days. ~n;m~1s were monitored for tumor takes/tumor size and body weights for three weeks from Day 0 until Day 21 D0 was the day of tumor cell inoculation and D21 was the day of experiment t~rm;n~tion.

Parameters of effect were measured by inhibition of tumor outgrowth and growth rate [tumors measured along the ~ W095/35297 ~ ?~ 2165956 PCT/C~95/00356 longest axis (length) and the perpendicular shortest axis (width) and the tumor volumes (T.V. + S.E.) was calculated by the formula T.V.=length (cm) x (width cm)2 /2.]
assessment of body weight loss.
- The statistical significance of difference between tumor takes and tumor sizes of control-untreated and drug-treated groups is estimated using the Chi-square and Student's t tests respectively with significance determined at p<0.05.

The mice were divided into the following 5 groups:

Gr.1 - Normal Saline (0.2 ml/mouse i.p. starting at D2) 15 Gr.2 - CY (100 mg/kg single bolus i.v. at DO) Gr.3 - Compound #1 (25 mg/kg i.p. starting at D2) Gr.4 - Compound #1 (50 mg/kg i.p. starting at D2) Gr.5 - CY (100 mg/kg i.v. at DO + compound #1 50 mg/kg i.p. starting at D2) Results are presented in Table 6 and Figures 1 and 2.

WO 95/35297 ~ ~-t~ t ~ 2 1 6 5 9 5 6 PCT/CA95/00356 Effect of compound #1 treatment on Tumor Outgrowth Group/Day 4 6 8 10 Gr.1: saline 5/5~ 515 515 515 Gr.2: CY ~ 100mg/kg 5/5 515 5/5 5/5 Gr.3: #1 ~ 25mg/kg 2/5t 215t 315 415 Gr 4: #1 ~ 50mg/kg 315 315 3/5 3/5 Gr.5: CY ~ 100mg/kg + #1 ~ 50mg/kg 4/5 5/5 5/5 5/5 * Tumor takes= # tumor-bearing mice/total # of mice S t p<0.05 by Chi-square test Example 93: Evaluation of Compound #1 in combination with cyclophosphamide (cytoxan) (CTX, 20 mg/kg) against DA- 3 - - ~y carcinoma.

Combination of compound #1 (25 and 50 mg/kg i.p. daily) plus CTX (20 mg/kg i.v. single bolus) was evaluated against day 4 established DA-3 tumors.

Results showed no significant effect of combination treatment of compound ~1 (at 25 mg/kg) plus CTX. However, a significant but transient effect was observed with CTX
plus compound #1 at 50 mg/kg from day 9 until day 18 (Figure 3). The decay of the positive anti-tumor effect is possibly due to the generation of T-supressor cells at the later stage of tumor growth. No significant body weight loss was observed (Figure 4).

i' 2165956 W095/35297 PCT/CA9~/0~356 Example 94: Evaluation of Compound #1 in combination with cyclophosphamide (cytoxan) (CTX, 28 mg/kg) against 3A-3 mammary carcinoma.
In another experiment, the CTX treatment was prolonged.
S Bal.b/c mice were injected s.c. with 5 x 105 DA-3 tumor cells at day 0. At day 4 when established tumors appeared, tumor-bearing ~n;m~l S were randomized (n=11/gr.) and injected with CTX (at 28 mg/kg) i.v. bolus injections at days 4, 11, and 18. Treatment with compound #1 was initiated using standard treatment regimen of daily i.p.
injections at 50 mg/kg starting from day 2 until day 28.

Results of this experiment (Table 7) show a highly statistically significant (p,0.001-p<0.005) anti-tumor effect of the compound ~1 (BCH-1393) + CTX combination treatment from day 11 until day 30 of tumor growth. No significant body weight loss was observed (Table 8).

Example 95: Evaluation of compound #1 in combination with 5F~ against colon adenocarcinoma.

C57BL/6 mice 6-8 weeks old (n=7-9/gr) were injected with 3x105 MC38 colon adenocarcinoma cells s.c. on day 0. On day 7, tumor-bearing mice were r~n~om;zed and injected with 5FU at 20 mg/kg either alone or in com.bination with levamisole at 20 mg/kg i.p. or with compound #1 at 25 and 50 mg/kg i.p. over a four week period. During this period, ~n;m~ls were treated for 5 consecutive days, untreated for 2 days, and treated again for 5 consecutive days per week for 4 weeks.

~ ~?~?~ t 65956 W095/3S297 PCT/CA9~l~C~
Results of this experiment show a significant dose-dependent anti-tumor effect following compound ~1 (at 50 and 25 mg/kg) + 5FU (20 mg/kg) compared to control untreated group (Figure 5). The anti-tumor effect of 5FU +
Compound #1 (at 50 mg/kg) was markedly better than that of 5FU + Levamisole. A moderate anti-tumor response was observed following treatment with 5FU (20 mg/kg) alone or with 5FU (20 mg/kg) plus Levamisole (20 mg/kg). This may be due to the fact that 20 mg/kg represents a suboptimal dose of 5FU for MC38 colon adenocarcinoma.

Example 96: In vivo toxicity of Compound #1 The objective o~ this study was to find the toxic dose of compound ~1 after repeated intravenous injections in 1S Fisher male and female rats.

Groups of 3 male rats, and 3 female rats were injected daily i.v. for 5 consecutive days. A first group received 500 mg/kg, a second group 250 mg/kg, and a third group 125 mg/kg. In addition, one male and one female were injected with 1000 mg/kg. An untreated group ( male and female) was included in the experiment. For all doses a constant volume of 0.1 ml/lOOg was used. Injections were started on day 0 and continued until day 4 (5 days). During treatment, weight changes were recorded daily and the rats were observed for at least 1 hour post-injection for signs of drug effect. On day 8, the rats were euthanatized and a macroscopic e~m;n~tion of the internal organs was performed.

Both rats (1 male and 1 female) injected with 1000 mg/kg i.v. showed severe colonic convulsions and died within 10 5 C' ~ t 2 1 65 956 woss/35297 PCT/CA95/00356 minutes. With 500 mg/kg, all rats were observed to have twitches of the torso area, tremors o the ~orepaws and jumping episods. These signs lasted less than 1 hour and were comparable after each of the five injections. The growth curves of the An;m~l were not affected when compared to controls. With the two lower doses (250 mg/kg and 125 mg/kg), no abnormal signs were observed at any time during dosing and the growth curves were normal (Figures 6 and 7). No drug induced changes were noted on necropsy of these ~n;m~l S .

Compound ~1 is well tolerated when injected i.v. in Fisher rats. A dose of 250 mg/kg injected for 5 consecutive days produced no signs of toxicity. The compound caused colonic convulsions and was lethal at the dose of 1000 mg/kg. A
dose of 500 mg/kg produced some short lasting abnormal signs but no lethality of effects on the growth of the ~n;m~1 ~.

CONCL~SIONS

From the data, in vitro, the compounds of the invention, in particular compound #1, appears to activate T cells (including CTL's) and B cells.
In vivo, the compound of the invention, in particular compound ~1, increases the number of CTL's.

The compounds of the present invention, in particular compound #1, appear to be well tolerated.

~ ,~ 2 1 6 5 9 ~ 6 Compound #l appears to inhibit tumor outgrowth in combination with cyclophosphamide against mouse m~mm~ry carcinoma in vivo .

Compound #l appears to inhibit tumor outgrowth in combination with 5FU against mouse colon adenocarcinoma ln vivo.

W095/35297 2 1 ~
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Claims (49)

WE CLAIM:

1. A compound of formula I:

or pharmaceutically acceptable derivatives thereof, wherein R1 is selected from the group consisting of hydrogen;
C1-16 alkyl; halogen; substituted or unsubstituted thiol; unsubstituted or substituted amino; and OR8 wherein R8 is selected from the group consisting of hydrogen, C1-16 alkyl, C1-8 acyl, and C7-18 aryl;
R2 and R3 are independently selected from the group consisting of hydrogen; C1-4 alkyl; amino; substituted or unsubstituted thiol; and halogen; and R4 is selected from the group consisting of a linear or cyclic carbon chain of the formula (CH0-2) 0-20 -X12 optionally interrupted with one or more heteroatom, and optionally substituted with one or more =O, or =S, and wherein X12, is selected from the group consisting of hydroxy, an aminoalkyl group, an amino acid, or a peptide of 2-8 amino acids, with the proviso that, when R1 is NH2, and R4 is pentyloxy carbonyl-L-arginine, then R2 is not hydrogen, and when R1 is OH, and R4 is pentyloxycarbonyl-L-arginine, then R2 is not NH2.

2. The compound according to claim 1, wherein R4 is (CH0-2) 1-8-X12, wherein X12 is OH.

3. The compound according to claim 1, wherein R4 is (CH2)-L -O-CO-X12, wherein h is a linear or cyclic carbon chain optionally interrupted with one or more O, S, or NH .

4. The compound according to claim 1, wherein X12 is (CH2)-NH2 wherein m is an integer between 1 and 6.

5. The compound according to claim 4, wherein n is 2.

6. The compound according to claim 3, wherein X12 is a naturally occuring amino acid in the D- or L-configuration.

7. The compound according to claim 6, wherein said amino acid is selected from the group consisting of:
arginine, glycine, alanine, glutamic acid, valine, ornithine, or citrulline, or conservative substitutions thereof.

8. The compound according to claim 7, wherein said amino acid is D-arginine.

9. The compound according to claim 7, wherein said amino acid is L-arginine.

10. The compound according to claim 3, wherein X12 is selected from a peptide of 2 to 8 amino acids.

11. The compound according to claim 10, wherein said peptide is Val-Pro-Leu, or Ile-Pro-Ile, or conservative substitutions thereof.

12. The compound according to claim 3, wherein L is selected from: -(CH2)n-, -(CH2)m-H-(CH2)m-, and (CH2)m-CC-(CH2)m-, wherein H is O, S, or NH, n is an integer between 1 and 6, and m is an integer between 1 and 3.

13. The compound according to claim 3, wherein L is selected from: phenyl, cyclohexyl, dioxolanyl, oxathiolanyl, and cyclopentyl.

14. The compound according to claim 1, wherein when R1 is C1-16 alkyl, R1 is an aromatic or non aromatic ring optionally interrupted with one or more heteroatom, and optionally substituted with one or more heteroatom, hydroxy, halogen, C1-16 alkyl, C1-16 acyl, C6-12 aryl, nitro, or substituted or unsubstituted amino.

15. The compound according to claim 1, wherein R1 is selected from the group consisting of: hydrogen, halogen, C1-6 alkyl, unsubstituted or substitued amino, OH, and OC1-6 alkyl, SH, or SC1-6 alkyl.

16. The compound according to claim 15, wherein said halogen is chloro.

17. The compound according to claim 15, wherein said unsusbtituted or substituted amino is represented by formula NR5R6 wherein R5 and R6 are independently selected from the group consisting of hydrogen, C1-4 alkyl, C1-4 alkoxy, C1-4 acyl, substituted or unsubstituted amino, and C6-10 aryl.

18. The compound according to claim 17, wherein R1 is selected from the group consisting of:
-N(CH3)2, -NHNH2, -NHCH3, -NH2, -N(NH2)CH3, -NH-CH(CH3)CH2-O-(CO)CH3, , or , wherein R20 is H or methyl.

19. The compound according to claim 15, wherein R1 is OCH3, or SCH3.

20. The compound according to claim 1, wherein R2 and R3 are independently selected from the group consisting of:
Cl, Br, I, and F.

21. The compound according to claim 20, wherein R2 and R3 are independently Cl, or Br.

22. The compound according to claim 21, wherein R2 is Cl.

23. The compound according to claim 21, wherein R3 is Br.

24. The compound according to claim 1, wherein R2 is NH2.

25. The compound according to claim 1, wherein R3 is SH, or SCH3.

26. The compound according to claim 1, wherein R1 is N(CH3)2; R2 and R3 are hydrogen; and R4 is pentyloxycarbonyl-D-arginine, or pharmaceutically acceptable derivatives thereof.

27. The compound according to claim 1 selected from the group consisting of:
Compound #IIIN-(6-Chloropurin-9-yl)-5-pentanol Compound #V N-(6-N,N-Dimethylaminopurin-9-yl)-pentanol Compound #1 N,N-Dimethylaminopurinyl Pentoxycarbonyl D-Arginine Compound #2 N,N-Dimethylaminopurinyl Pentoxycarbonyl L-Arginine Compound #3 N-Monomethylaminopurinyl Pentoxycarbonyl D-Arginine Compound #3a N-(6-N-Methyl-Aminopurin-9-yl)-pentanol Compound #4 N-Monomethylaminopurinyl Pentoxycarbonyl L-Arginine Compound #5 Aminopurinyl Pentoxycarbonyl D-Arginine Compound #5a N-(6-Aminopurin-9-Yl) 5-Pentanol Compound #6 Aminopurinyl Pentoxycarbonyl L-Arginine Compound #7 Hydrazinopurinyl Pentoxycarbonyl D-Arginine Compound #7a N-(6-Hydrazinopurin-9-yl) 5-Pentanol Compound #8 Hydrazinopurinyl Pentoxycarbonyl L-Arginine;
Compound #9 Chloropurinyl Pentoxycarbonyl D-Arginine;
Compound #10 Chloropurinyl Pentoxycarbonyl L-Arginine;
Compound #11 Hydroxypurinyl Pentoxycarbonyl D-Arginine;

Compound #12 Mercaptopurinyl Pentoxycarbonyl D-Arginine;
Compound #13 Mercaptopurinyl Pentoxycarbonyl L-Arginine;
Compound #14 N,N-Dimethylaminopurinyl Pentoxycarbonyl Glycine;
Compound #15 N,N-(6-Dimethylaminopurin-9-yl)-7'-ethoxy-ethoxycarbonyl-D-arginine;
Compound #16 (2S,4S)-2-(N,N-dimethylaminopurin-9-yl)-4-(methyloxycarbonyl-D-arginine)-1,3-dioxolane;
Compound #17 N-(6-Dimethylamino-8-bromopurinyl-Pentoxycarbonyl L-Arginine;
Compound #18 N-(6-dimethylamino-8-bromopurin-9-yl)7-pentoxycarbonyl-D-arginine;
Compound #19 N-9-purinyl-5-pentanol;
Compound #20 N-9-purinyl-7-pentyloxycarbonyl-D-arglnlne;
Compound #21 N-9-purinyl-7-pentyloxycarbonyl-L-arglnlne;
Compound #22 N,N-Dimethylaminopurinyl Pentoxycarbonyl L-Valyl L-Prolyl L-Leucine;
Compound #23 N,N-Dimethylaminopurinyl Pentoxycarbonyl L-Isoleucyl L-Prolyl L-Isoleucine;
Compound #24 N-(6-Cyclopropylaminopurin-9-yl)-5-pentanol;
Compound #25 N-(6-cyclopropylaminopurin-9-yl)-7-pentyloxycarbonyl-D-arginine;
Compound #26 N-(6-cyclopropylaminopurin-9-yl)-7-pentyloxycarbonyl-L-arginine;
Compound #27 N-(6-Azetidinepurin-9-yl)-5-pentanol;
Compound #28 N-(6-Azetidinepurin-9-yl)-7-pentyloxycarbonyl-D-arginine;
Compound #29 N-(6-Azetidinepurin-9-yl)-7-pentyloxycarbonyl-L-arginine;

Compound #30 trans-(M-6-chloropurin-9-yl)-4-methyl-cyclohexyl-methanol;
Compound #31 trans-(N-6-dimethylaminopurin-9-yl)-4-methyl-cyclohexyl-methanol;
Compound #32 trans-(N-6-dimethylaminopurin-9-yl)-4-methyl-cyclohexyl-methyloxycarbonyl-D-arginine;
Compound #33 trans-(N-6-hydroxypurin-9-yl)-4-methyl-cyclohexyl-methanol;
Compound #34 trans-(N-6-methoxypurin-9-yl)-4-methyl-cyclohexyl-methanol;
Compound #35 cis-(N-6-dimethylaminopurin-9-yl)-4-methyl-cyclohexyl-methanol;
Compound #36 cis-(N-6-dimethylaminopurin-9-yl)-4-methyl-cyclohexyl-methyloxycarbonyl-D-arginine;
Compound #37 N-(6-dimethylaminopurin-9-yl) 7-pentoxycarbonyl-D-citrulline;
Compound #38 N-(6-methylaziridinepurin-9-yl)-5-pentanol;
Compound #39 racemic N-(6-methylaziridine purine-9-yl)-7-pentyloxycarbonyl-D-arginine;
Compound #40 N,N-(6-Dimethylaminopurinyl-9-yl)-7-thioethoxy-ethoxycarbonyl-D-arginine;
Compound #41 Meta-(N-6-dimethylaminopurinyl-9-yl) methyl-benzyloxycarbonyl-D-arginine;
Compound #42 5-(N-6-Dimethylaminopurinyl-9-yl)-3-pentynyl-1-oxycarbonylD-arginine;
Compound #43 Racemic N-[6-(1-methyl-2-acetoxy)-ethylaminopurin-9-yl]-5-pentanol;
Compound #44 Racemic N-[6-(1-methyl-2-acetoxy), ethylaminopurin-9-yl]-7-pentyloxy-carbonyl-D-arginine;
Compound #45 N-(2,6-Dichloropurin-9-yl)-5-pentanol;
Compound #46 N-(2,6-Dichloropurin-9-yl)-7-pentyloxycarbonyl-D-arginine;

Compound #47 N-(2,6-Dichloropurin-9-yl)-7-pentyloxycarbonyl-L-arginine;
Compound #48 M-(2-Amino, 6-N, N-Dimethylaminopurin-9-yl)-5-pentanol;
Com.pound $49 N-(6-dimethyl amino-8-methylthiopurin-9-yl) 5-pentanol;
Compound #50 N-(6-dimethylamino-8-methylthiopurin-9-yl) 7-pentoxycarbonyl-D-arginine;
Compound #51 M-(6-methoxypurin-9-yl) 5-pentanol;
Compound #52 N-(6-methoxypurin-9-yl) 7-pentoxycarbonyl-D-arginine;
Compound #53 N-(2-chloro-6-methoxypurin-9-yl)-7-pentyloxycarbonyl-D-arginine;
Compound #54 N-(6-dimethylaminopurin-9-yl) 7-pentoxycarbonyl-D-ornithine;
Compound #55 N-(6-dimethylaminopurin-9-yl) 7-pentoxycarbonyl-L-ornithine;
Compound #56 N-(6-dimethylaminopurin-9-yl) 7-pentoxycarbonyl-L-valine;
Compound #57 N-(6-dimethylamino-9-yl) 7-pentoxycarbonyl-D-valine;
Compound #58 N(N,N-dimethylaminopurin-9-yl)-7-pentyloxycarbonylethylamine hydrochloride;
Compound #59 N-(6-Mercaptopurin-9-yl)-pentanol;
Compound #60 N-(6,-N-Methylthiopurin-9-yl)-pentanol;
Compound #61 N-(6-chloropurin-9-yl) 4-butanol;
Compound #62 N-(6-dimethylaminopurin-9-yl) 4-butanol;
Compound #63 N-(6-dimethylaminopurin-9-yl)-6-butoxycarbonyl-D-arginine;
Compound #64 N-(6-dimethylaminopurin-9-yl)-6-butoxycarbonyl-L-arginine;
Compound #65 N-(6-chloropurin-9-yl)-6-hexanol;
Compound #66 N-(6-N,N-dimethylaminopurin-9-yl)-6-hexanol;

Compound #67 N-(6-N,N-dimethylaminopurin-9-yl)-8-hexyloxycarbonyl-D-arginine;
Compound #68 N(6-N,N-dimethylaminopurine-9-yl)-8-hexyloxycarbonyl-L-arginine;
Compound #69 cis-(N-6-hydroxypurin-9-yl)-4-methyl-cyclohexyl-methanol;
Compound #70 cis-(N-6-hydroxypurin-9-yl)-4-methyl-cyclohexyl-methyloxycarbonyl-D-arginine;
Compound #71 trans-(N-6-hydroxypurin-9-yl)-4-methyl-cyclohexyl-methyloxycarbonyl-D-arginine;
Compound #72 N-(6-N,N dimethylaminopurin-9-yl)-5-pentylamine hydrochloride salt;
Compound #73 N-(6-methylaziridinepurin-9-yl)-7-pentyloxycarbonyl-L-arginine;
Compound #74 (2S,4S)-2-(N,N-Dimethylaminopurin-9-yl)-4-hydroxymethyl-1,3-dioxolane;
Compound #75 (1S,3R) and (1R,3S)-1-(N-6-Dimethylaminopurin-9-yl)methyl-3-cyclopentane methanol;
Compound #76 (1S,3R) and (1R,3S)-1-(N-6-Dimethylaminopurin-9-yl)methyl-3-(methyloxycarbonyl-D-arginine)cyclopentane;
Compound #77 N,N-(6-Dimethylaminopurin-9-yl)-7-ethylaminoethanol;
Compound #78 N,N-(6-Dimethylaminopurin-9-yl)-7-ethylaminoethoxycarbonyl-D-arginine;
Compound #79 N,N-(6-Dimethylaminopurin-9-yl)-7-ethylaminoethoxycarbonyl-L-arginine;
Compound #80 5-(N-6-Dimethylaminopurin-9-yl)-3-pentyn-1-ol;
Compound #81 5-(N-6-Dimethylaminopurin-9-yl)-3-pentynyl-1-oxycarbonyl-L-arginine;
Compound #82 N,N-(6-Dimethylaminopurin-9-yl)-7-thioethoxy-ethanol;

Compound #83 N,N-(6-Dimethylaminopurin-9-yl)-7-thioethoxy-ethoxycarbonyl-L-arginine;
Compound #84 (2S,4S)and (2R,4R)-2-(N,N-Dimethylaminopurin-9-yl)-4-(methoxycarbonyl-D-arginine)-1,3-oxathiolane;
Compound #85 N,N-(6-Dimethylaminopurin-9-yl)-7-ethoxy-ethoxyethanol;
Compound #86 N,N-(6-Dimethylaminopurin-9-yl)-7-ethoxy-ethoxycarbonyl-D-arginine;
Compound #87 N,N-(6-Dimethylaminopurin-9-yl)-7-ethoxy-ethoxycarbonyl-L-arginine; and Compound #88 N-(6-Dimethylamino-8-bromopurin-9-yl)-5-pentanol.

28.The compound according to claim 27 selected from the group consisting of:
Compound #IIIN-(6-Chloropurin-9-yl)-5-pentanol Compound #V N-(6-N,N-Dimethylaminopurin-9-yl)-per.tanol Compound #1 N,N-Dimethylaminopurinyl Pentoxycarbonyl D-Arginine Compound #2 N,N-Dimethylaminopurinyl Pentoxycarbonyl L-Arginine Compound #3 N-Monomethylaminopurinyl Pentoxycarbonyl D-Arginine Compound #3a N-(6-N-Methyl-Aminopurin-9-yl)-pentanol Compound #5 Aminopurinyl Pentoxycarbonyl D-Arginine Compound #5a N-(6-Aminopurin-9-Yl) 5-Pentanol Compound #6 Aminopurinyl Pentoxycarbonyl L-Arginine Compound #7 Hydrazinopurinyl Pentoxycarbonyl D-Arginine Compound #7a N-(6-Hydrazinopurin-9-yl) 5-Pentanol Compound #8 Hydrazinopurinyl Pentoxycarbonyl L-Arginine;

Compound #11 Hydroxypurinyl Pentoxycarbonyl D-Arginine;
Compound #19 N-9-purinyl-5-pentanol;
Compound #20 N-9-purinyl-7-pentyloxycarbonyl-D-arginine;
Compound #51 N-(6-methoxypurin-9-yl) 5-pentanol;
Compound #59 N-(6-Mercaptopurin-9-yl)-pentanol; and Compound #60 N-(6,-N-Methylthiopurin-9-yl)-pentanol.

29.The compound according to claim 28 being compound #1 -N2-(6-dimethylaminopurin-9-yl) 7-pentyloxycarbonyl-D-arginine.

30.A pharmaceutical composition contA;ning a compound according to claim 1, 27, or 29, wherein said compound is present in admixture with a pharmaceutically acceptable carrier.

31.A pharmaceutical composition according to claim 30 wherein compound is present in admixture with another therapeutically active agent.

32.A pharmaceutical composition according to claim 31 wherein said therapeutically active agent is cyclophosphamide.

33.A method for the treatment of immune deficiencies or control of cancer growth comprising the step of administering to a mammal a pharmaceutically acceptable amount of a compound according to claim 1, 27, or 29.

34.A method for increasing the number of cytotoxic T
lymphocytes in a mammal, including a human, comprising the step of administering a pharmaceutically acceptable amount of a compound according to claim 1, 27, or 29.

35.A method for the control of cancer growth in a mammal, including human, comprising the step of administering to a mammal a pharmaceutically acceptable amount of a compound according to claim 29.

36.A method for the control of mammary carcinoma in a mammal, including human, comprising the step of administering to a mammal a pharmaceutically acceptable amount of a compound according to claim 29, in combination with cyclophosphamide.

37.A pharmaceutical composition for the treatment of cancer comprising a compound according to claim 1, 27 or 29, in combination with 5-fluorouracil.

38.The pharmaceutical composition according to claim 37 wherein said cancer is colon carcinoma.

39.A method for the treatment of cancer in a mammal, including a human, comprising the step of administering a pharmaceutically acceptable amount of compound according to claim 1, 27, or 29, in combination with 5-fluorouracil.

40.The method according to claim 39, wherein said cancer is colon carcinoma.

41.The method according to claim 39, wherein said compound is administered in an amount ranging from about 1 to about 100 mg/kg.

42.The method according to claim 41, wherein said compound is administered in an amount ranging from about 2 to about 20 mg/kg.

43.The method according to claim 42, wherein said compound is administered at about 2.5 mg/kg.

44.The method according to claim 39, wherein said 5-fluorouracil is administered in an amount ranging from about 1 to about 50 mg/kg.

45.The method according to claim 44, wherein said 5-fluorouracil is administered in an amount ranging from about 5 to about 20 mg/kg.

46.The method according to claim 45, wherein said 5FU is administered at about 12 mg/kg.

47.The method according to claim 39, wherein said combination is administered sequentially.

48.The method according to claim 39, wherein said combination is administered simultaneously.

49.The method according to claim 39, wherein said combination is administered as a single formulation combining 5-FU and said compound.