AU717160B2 - Novel substituted purinyl derivatives with immunomodulating activity - Google Patents

Description

1too.

*.06 64S *000

AUSTRALIA

Patents Act 1990 BioChem Pharma Inc.

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Novel substituted purinyl derivatives with immunomodulating activity The following statement is a full description of this invention including the best method of performing it known to us:-

V.

5

S.

S. S

S*

Pharma47 PCT NOVEL SUBSTITUTED PURINYL DERIVATIVES WITH IMMUNOMODULATING ACTIVITY 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.

Background of the Invention 15 The primary function of the immune 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 autoimmune disorders.

Modulation of the immune system through pharmaceutically *induced stimulation or suppression offers an important 25 approach to the control of disease. Compounds which nonspecifically stimulate the immune system are of potentially significant medicinal importance and have been the object of a lengthy research effort. Often, the research results show that immunomodulating compounds are either weak immunostimulants, and hence not very effective, or potent immunostimulants and, therefore, effective but toxic by virtue of this potent immunostimulating activity.

Date: June 16, 1995 1 9 Time: 11:38 AM Pharma47 PCT 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 humans 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.

15 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 administration. However, these results are preliminary. With many nucleoside compounds, toxicity is an important issue which must also be closely analyzed.

A particular class of nucleoside immunostimulants has .25 arisen from inosine and other similar hypoxanthinecontaining compounds. A well know example is isoprinosine, an inosine-containing complex. Isoprinosine has been thoroughly studied as an immunomodulator and referred to as a "gold standard" by C.D. Simone et al. in Thymus, 19, 51-55, 1992. Some rationale for the activity of hypoxanthine- (inosine) containing compounds arises from the observation that a lack of adenosine deaminase, the enzyme which converts adenosine to inosine, results in severe combined immunodeficiency disease (SCID).

Date: June 16, 1995 2 Time: 11:38AM Pharma 47 PCT 1- Although very nontoxic, isoprinosine is not an effective immunomodulator, 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, 49-54, 1991 (suppl. 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, 15 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, S1-S112 (1992). L-Arginine was selected because it is known to play a role in immune activation and is present at the terminus of many immunomodulatory peptides such as tuftsin, substance P, thymopentin, and splenopentin. ST 789 is further described in European 25 Patent Application #91830284, publication #464,009, published January 2, 1992. Analogues of ST 789 are also described in the European publication where oligopeptides composed of naturally occurring L-amino acids replace Larginine. However, the purine base portion of the molecule remains hypoxanthine.

While no immunological comparison was made with isoprinosine, a similar pattern emerged. The compounds are nontoxic but, at best, moderate immunostimulants. For Date: June 16,1995 3 Time: 11:38AM Pharma47 PCT example, there was no indication that ST 789, or analogues thereof, could stimulate 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 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 terminus dipeptide Lleucyl L-methionine. The purine base remains 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 15 been reported for compounds including a purine base other than hypoxanthine. Further, because of the requirement for physiologically active amino acids in mammalian 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 adenine 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 deaminase, 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 mammals in Date: June 16, 1995 4 Time: 11:38AM Pharma 47 PCT the presence of xanthine oxidase enzyme. However, allopurinol was noted to be immunosuppressive and ST 689 was not significantly more immunostimulatory than ST 789 in most of the immunology assays reported in the '151 patent.

Levamisole is another immunoregulator 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, 24 262-4].

The prior art indicates that there is a need for compounds which have the ability to stimulate a number of immune S 15 cell subsets and thereby possess significant immunomodulating 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 immunostimulatory capability both in vitro and in vivo.

25 Specifically, there is provided a compound which possesses activity in increasing the amount of cytotoxic T cells in vitro 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.

Date: June 16, 1995 Time: 11:38AM 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 R

(I)

or pharmaceutically acceptable derivatives thereof, wherein 10 RI is substituted amino represented by formula NR 5

R

6 5 6 wherein R 5 and R 6 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, with the proviso that R 5 and R 6 are not both S 15 hydrogen or amino; R 5 and R 6 can also be connected to the nitrogen atom to form a saturated C3-6 heterocyclic ring optionally substituted with C 1 -4 *alkyl;

R

2 and R 3 are independently selected from the group consisting of hydrogen; C1- 4 alkyl; amino; substituted or unsubstituted thiol; and halogen; and

R

4 is represented by the formula R12-X 12 wherein R 12 is a saturated or unsaturated linear hydrocarbon chain of 5-20 carbons optionally containing one or more interruptions within the chain by a heteroatom, and optionally substituted with one or more or and X 12 is selected from the group consisting of an aminoalkyl group, a known amino acid bound by its a-amino group or a peptide of 2-8 amino acids.

If Pharma 47 PCT The following definitions are used herein.

The term "alkyl" as employed herein includes both straight and branched chain radicals, for example methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4trimethylpentyl, 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.

15 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 0, S, SO, S02, and N, or the ring may be unsubstituted or substituted with, for example, halogen, 20 alkyl, acyl, hydroxy, aryl, and amino, said heteroatom and substituent may also be substituted with, for example, alkyl, acyl, aryl, aralkyl.

The term "acyl" as used herein refers to carbonyl groups 25 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 containing 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 Date: June 16, 1995 7 Time: 11:38 AM Pharma47 PCT naphthyl may be for example C1- 4 alkyl, halogen, C 1 -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, C1- 8 alkyl, C 1 -8 acyl, C6- 12 aryl, hydroxy, and hydrogen.

The term "amino acid" as employed herein includes and 15 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 P-alaline.

A list of non natural amino acids may be found in "The Peptides", vol 5, 1983, Academic Press, Chapter 6 by D.C.

Roberts and F. Vellaccio.

The term "linear 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 example, a carbon chain which either precedes or follows the ring.

The term "pharmaceutically acceptable derivative" as employed herein, includes any pharmaceutically acceptable salt, ester, or salt of such ester, of a compound of formula I or any other compound which, upon administration to the recipient, is capable of providing (directly or Date: June 16,1995 8 Time: 11:38AM indirectly) a compound of formula I or an active metabolite or residue thereof.

Brief Description of Drawings Figure 1 illustrates the variations in tumor growth for mice treated with cyclophosphamide, or compound or both.

Figure 2 illustrates the body weight variations for mice treated with the same regimen as in Figure 1 Figure 3 illustrates the variations in tumor volume for 15 mice treated with Cytoxan, or compound #1 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 #1.

Figure 6 illustrates the growth curves of male Fisher rats 25 treated with compound #1 at high doses.

Figure 7 illustrates the growth curves of female Fisher rats treated with compound #1 at high doses.

Description of the Invention In one aspect of the invention, there is provided a compound of formula wherein R, is (CH 2 L -O-CO-X 12 wherein L is selected from the group consisting of

-(CH

2

(CH

2

(CH

2 and (CH 2

(CH

2 )m wherein Q RA is O, S, or NH, n is an integer between 3 and 6, and m is \1 integer between 1 and 3.

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

More preferably, X 12 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, alanine, 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.

15 In an alternative embodiment of the invention, X 1 may be a peptide of 2 to 8 amino acids.

Preferably, such a peptide can be Val-Pro-Leu, or Ile-Pro- Preferably, R 4 is (CH 2 L -O-CO-X 12 wherein L can be selected from the group consisting of: phenyl, cyclohexyl, a dioxolanyl, oxathiolanyl, and cyclopentyl.

oo 25 More preferably, Ri can be OH, OCH 3 SH or SCH 3 Preferably, R, can be selected from the group consisting of: N (CH 3 21

-NHNH

2

-NHCH

3 -N (NH 2

CH

3 -NH-CH (CH 3

CH

2 (CO) C11 3 NlI ,or **a V. V V

C

V.

V V V V

C

V

V

0

V

*0*e V S V 0

V.

V

V

,wherein R 20 is H or methyl.

Even more preferably, R, can be: -N(CH 3 2 15 Even more preferably, R, can be: -NHNH 2 Even more preferably, R, can be: -NHCH 3 ,and Even more preferably, R, can be: -N(NH 2

)CH

3 Most preferably, R, can be -N(CH 3 2 20 In a further alternative embodiment of the invention, R 2 and R, can be independently selected from the group consisting of: Cl, Br, I, and F.

Preferably, R 2 and R 3 can be independently Cl or Br.

More preferably, R 2 can be H, Cl, or NHl 2 More preferably, R 3 can be H, Br, or Sf1 or SCH 3 Preferred. compounds of the present invention are selected from: Compound #III N-5- (6-N-Chloropurin-9-yl) pentanol Compound #V N-5- N-Dimethylaminopurin- 9-yl) -pentanol Compound #1 N-5- N-Dimethylaminopurin- 9-yl) -pentoxycarbonyl-D-Arginine t0 Compound #2 N-5- N-Dimethylaminopurin- 9-yl) -pentoxycarbonyl-L-Arginine Compound #3 N-5- (6-N-Methylaminopurin-9yl) -pentoxycarbonyl-D-Arginine Compound #3a N-5- (6-N-Methylaminopurin-9- :9 15 yl)-pentanol 00,Compound #4 N-5- (6-N-Methylaminopurin-9yl) -pentoxycarbonyl-L-Arginine Compound #5 N-5- (6-N-Aminopurin-9-yl) pentoxycarbonyl-D-Arginine Compound #t5a N-5-(6--N-Aminopurin-9-yl)- "lee pentanol Compound #6 N-5- (6-N-Aminopurin-9-yl) goes pentoxycarbonyl-L-Arginine Compound #7 N-5- (6-N-Hydrazinopurin-9-yl) 25 pentoxycarbonyl-D-Arginine Compound #7a N-5- (6-N-Hydrazinopurin-9-yl) pentanol Compound #8 N-5- (6-N-Hydrazinopurin-9-yl) pentoxycarbonyl-L-Arginine; Compound #9 N-5-(6-N-Chloropurin-9-yl)pentoxycarbonyl--D--Arginine; Compound #10 N-5- (6-N-Chloropurin-9-yl) pentoxycarbonyl-L-Arginine; Compound #11 N-5- (6-N-Hydroxypurin-9-yl) pentoxycarbonyl-D-Arginine; Compound #12 N-5- (6-N-Mercaptopurin-9-yl) pentoxycarbony1-D-Arginine; Compound #13 N-5- (6-N-Mercaptopurin-9-yl)pentoxycarbonyl-L-Arginine; Compound #t14 N-5-(6-N,N-Dimethylaminopurin- 9-yl) -pentoxycarbonyl-Glycine; Compound #15 N-5- (6-N,N-Dimetliylaminopurin-9-yl) ethoxy-ethoxycarbonyl-D-Arginine; Compound #16 (2S, 4S) (6-N,N- Dime thylaminopurin- 9-yl) (inethyloxycarbonyl-D- Arginine) 3-dioxolane; Compound #17 N-5- N-Dimethylamino-8bromopurin- 9-yl) -pentoxycarbonyl-L-Arginine; Compound #18 N-5- N-Dimethylamino-8- 15 bromopurin-9-yl) -pentoxycarbonyl-D-Arginine; Compound #19 N-5- (Purin-9-yl) -pentanol; Compound #20 N-5- (Purin-9-yl) t) pentoxycarbonyl-D-Arginine; :Compound #21 N-5- (Purin-9-yl) pentoxycarbonyl-L-Arginine; Compound #22 N-5- N-*Dimethylaminopurin- 9-yl) -pentoxycarbonyl-L-Valyl-L-Prolyl- L-Leucine; Compound #23 N-5- N-Dimethylaminopurin- 25 9-yi) -pentoxycarbonyl-L- Isoleucyl-L-Prolyl-L- Isoleucine; *Compound #24 N-5-(6-N- Cyclopropylaminopurin-9-yl) -pentanol; Compound #25 Cyclopropylaminopurin-9-yl) -pentoxycarbonyl-D- Arginine; Compound #26 N5(6-N Cyclopropylaminopurin-9-yl) -pentoxycarbonyl-L- Arginine; Compound #27 N-5- (6-N-Azetidinepurin-9-yl) 4 -T R4 pentanol; Compound #28 N-5- (6-N-Azetidinepurin--9-yl) penptoxycarbonyl-D-Arginine; Compound 429 N-5- (6-N-Azetidinepurin-9-yl) pentoxycarbonyl-L-Arginine; Compound #30 trans-(6-N-Chloropurin--9-yl)- 4-methyl-cyclohexyl-methanol; Compound #31 trans-(6-N,N- Dimethylaminopurin-9-yl) -4-methyl-cyclohexylmethanol; Compound #32 trans- N- Dimethylaminopurin-9-yl) -4-methyl-cyclohexylmethyloxycarbonyl-D-Arginine; Compound #33 trans- (6-N-Hydroxypurin-9-yl) 4-me thyl-cyclohexyl-methanol; Compound #34 trans-(6-N-Methoxypurin-9-yl)- 4-me thyl-cyclohexyl-methanol; Compound #35 cis- N-Dimethylaminopurin-9-yl) -4methyl-cycloliexyl-methanol; Compound #36 cis- N-Dimethylaminopurin- 9-yl) -4-methyl-cyclohexyl-methyloxycarbonyl-Darginine; Compound #37 N-5- N-Dimethylaminopurin- 9-yi) -pentoxycarbonyl-D-Citrulline; Compound #38 N-5- (6-N-Methylaziridinepurin- 9-yl)-pentanol; Compound #39 N-5- (6-N-Methylaziridinepurin- 9-71) -pentoxycarbonyl-D-Arginine; Compound 440 N-5- N-Dimethylaminopuri-n- 9-yl) -7-thioethoxy-ethoxycarbonyi-D-Arginine; Compound #41 Meta- N-Dimethylaminopurin-9-yl) methyl -benzyloxycarbonyl -D-Arginine; Compound #42 N-5- N-Dimethylaminopurin- 9-yl) -3-pentynyl-1-oxycarbonyl-D-Argiiine; Compound #43 N-5- (1-methyl-2-acetoxy) ethylaminopurin-9-yl] -pentanol; Compound #44 N-5- (1-Iethyl-2-acetoxy) ethylaminopurin-9-yl] -pentyloxycarbonyl-D-Arginine; Compound #45 N-5- 6-Dichloropurin-9-yl) pentanol; Compound 46N-5- 6-Dichloropurin-9-yl) pentoxycarbonyl-D--Arginine; Compound #4 N-5- 6-Dichloropurin-9-yl) pentoxycarbonyl-L-Arginine; Compound #48 N-5-(2-Amino-6-N, N- Dimethylaminopurin-9-yl) -pentanol; Compound #49 N-5- N-Dimethylamino-8methylthiopurin-9-yl) -pentanol; Compound #t50 N-5- N-Dimethylamino-8methyl thiopurin-9-yl) -pentoxycarbonyl-D-Arginine; Compound #51 N-5-(6-N-Methoxypurin-9-yl)-pentanol; ::Compound #52 N-5- (6-N-Methoxypurin-9-yl) *pentoxycarbonyl-D-Arginine; :Compound #t53 N-5- (2-chloro-6-methoxypurin- 9-yl) -pentoxycarbonyl-D-Arginine; Compound #54 N-5- N-Dimethyiaminopurin- 9-yl) -pentoxycarbonyl-D-Ornithine; Compound #55 N-5- N-Dimethylaminopurin- 9 -yl) -pentoxycarbonyl-L-Ornithine; 9***Compound #56 N-5- (6-N,N-Dimethylaminopurin- 25 9-yl)-pentoxycarbonyl-L-Valine; Compound #57 N-5- N-Dimethylaminopurin- 9-yl) -pentoxycarbonyl-D-Valine; Compound #58 N-5- N-Dimethylaminopurin- 9-yl) -pentoxycarbonylethylamine hydrochloride; Compound #59 N-5-(6-N-Mercaptopurin-9-yl)pentanol; Compound #60 N-5- (6-N-Methylthiopurin-9yl) -pentanol; Compound #61 N-4- (6-N-Chloropurin-9-yl)but ano 1; Compound #62 N-4-(6-N,N-Dimethylaminopurin- 9-.yl) -butanol; Compound #63 N-4- N-Dimethylaminopurin- 9-yl) -butoxycarbonyl-D-Argirnine; Compound #64 N-4-(6-N,N-Dimethylaminopurin- 9-yl) -butoxycarbonyl-L-Arginine; Compound #65 N-6- (6-N-Chloropurin-9-yl) hexanol; Compound #66 N-6- N-Dimethylaminopurin-9-yl) hexanol; Compound #67 N-6- N-Dimethylaminopurin- 9-yl) -hexyloxycarbonyl-D-Arginine; Compound #68 N-6-(6-N,N- Dimethylaminopurlne-9-yl) -hexyloxycarbonyl-L- 15 Arginine; Compound #69 cis- (6-N-Hydroxypurin-9-yl) -4methyl-cyclohexyl-methanol; Compound #70 cis- (6-N-Hydroxypurin-9-yl) -4inethyl-cyclohexyl-methyloxycarbonyl-D-Arginine; Compound #71 trans-(6-N-Hydroxypurin-9-yl)- 4-methyl-cyclohlexyl-methyloxycarbonyl-D-Arginine; Compound #72 N-5- (6-N,N-Dimethylaminopurin- 9-yl) -pentylamine hydrochloride salt; Compound #73 14-5- (6-N4-Me Lhylaziridiniepurin- 9-yl)-pentyloxycarbonyl-L-Arginine; Compound #74 (2S, 4S) N- Dimethylaminopurin-9-yl) -4-hyclroxymethyl-1, 3dioxolane; Compound #75 (1S,3R) and (lR,3S)-1-(6-N,N- Dimethylaminopurin-9-yl) -methyl-3-cyclopentane methanol; Compound #76 (lS,3R) and (1R,3S)-1-(6-N,N- Dimethylaminopurin-9-yl) -methiyl-3- (methyloxycarbonyl-D-Arginine) -cyclopentane; Compound #77 N-Dime thylaminopurin-9- -7-ethylaminoethanol; Compound #78 (6-N,N-Dimethylaminopurin-9yl) -7-ethylaminoethioxycarbonyl-D-Arginine; Compound #f79 N-Dimethylaminopurin-9yl) -7-ethylaminoethoxycarbonyl-L-Arginine; Compound 480 N-5- N-Dimethyiaminopurin- 9-yl) -3-pentyn-1-ol; Compound #81 N-5- N-Dimethylaminopurin- 9-yl) -3-pentynyl-l-oxycarbonyl-L-Arginine; Compound #82 N-Dimethylaminopurin-9yl) -7-thioethoxy-ethanol; Compound #83 N-Dimethyiaminopurin-9yl) -7-thioethoxy-ethoxycarbonyl-L-Arginine; Compound #t84 (2S,4S)and (2R,4R)-2-(6-N,N- Dimethylaminopurin-9-yl) (methoxycarbonyl-D- Arginine) 3-oxathiolane; :Compound #85 N-Dimethylaminopurin-9yl) -7-ethoxy-ethoxyethanol; Compound #86 N-Dimethylaminopurin-9yl) -7-ethoxy-ethoxycarbonyl-D-Arginine; :Compound #87 (6-N,N-Dimethylaminopurin-9yl) -7-ethioxy-ethoxycarbonyl-L-Arginine; and Compound #88 N-5- N-Dimethylamino-8bromopurin-9-yl)-pentanol.

*More preferably,-the compound of the present invention is selected from: Compound #111 N-5- (6-N-Chloropurin-9-yl) pentanol Compound #V N-5- N-Dimethylaminopurin- 9 -yl) -pentanol Compound #1 N-5- N-Dimethylaminopurin- 9-yl) -pentoxycarbonyi-D-Arginine Compound #2 N-5- N-Dimethylaminopurin- 9 -yl) -pentoxycarbonyl-L-Arginine Compound #3 N-5- (6-N-Methylaminopurin-9yl) -pentoxycarbonyl-D-Arginine Compound #f3a N-5- (6-N-Methylaminopurin-9yl) -pentanol Compound #5 N-5- (6-N-Aminopurin-9-yl) pentoxycarbonyl-D-Arginine Compound #5a N-5- (6-N-Aminopurin-9-yl) pentanol Compound #6 N-5- (6-N-Aminopurin-9-yl) pentoxycarbonyl-L-Arginine Compound #7 N-5- (6-N-Hydrazinopurin-9-yl) pentoxycarbonyl-D-Arginine Compound #7a N-5-(6-N-Ilydrazinopurin-9-yl)pentanol :Compound #8 N-S- (6-N-Hydrazinopurin-9-yl) pentoxycarbonyl-L-Arginine; Compound #11 N-5- (6-N-Hydroxypurin-9-yl) pentoxycarbonyl-D-Arginine; Compound #19 N-5- (Purin-9-yl) -pentanol; Compound #20 N-S- (Purin-9-yl) pentoxycarbonyl-D-Arginine; Compound f#51 N-B- (6-N-Methoxypurin--9-yl) -pentanol; #59 N-5-(6-N-Mercaptopurin-9-yl)pentanol; and Compound #60 N-5- (6-N-Methylthiopurin-9yl) -pentanol.-- Most preferably, the compound of the present invention is N-Dimethylaminopurin-9-yl) -pentoxycarbonyl-Darginine .The following abbreviations and definitions are used PHA phytohemagglutinin ConA concanavalin A CY cyclophosphamide PWM pokeweed mitogen LPS lipopolysaccharide DEAD diethylazodicarboxylate PBS phosphate buffered saline TBDPSC1 tert-butyldiphenylsilyl chloride CTX Cytoxan The term "conservative substitution" as employed herein refers to modifications and substitutions of amino acids which are conservative ones, i.e. those having a minimal influence on the secondary structure and hydropathic 15 nature of the amino acid or peptide. These include substitutions such as those described by Dayhoff in the Atlas of Protein Sequence and Structure 5, 1978, and by Argos in EMBO 8, 779-785, 1989. For example, amino acids belonging to the following groups represent conservative changes: ala, pro, gly, glu, asp, gin, asn, ser, thr; cys, ser, tyr, thr; val, ile, leu, met, ala, se o *o r' Pharma47 PCT 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 immunostimulant 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

OS

20 in the D-configuration.

It has surprisingly been discovered that the compounds of the invention possess in vitro and in vivo activity to increase the number of cytotoxic T lymphocytes in the 25 mammal 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 nontoxic substitute to levamisole in the treatment of malignant melanoma.

When tested in mice against a control group, the compounds of the present invention significantly inhibit tumor Date: June 16, 1995 Time: 11:38AM Pharma47 PCT growth when used in combination with cyclophosphamide or particularly against mammary and colon carcinoma respectively.

The compounds of 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, 39-47, 1990, 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 carry out a modification of this synthetic procedure, as outlined in the following examples, wherein the purine ring is already constructed by use of 6chloropurine as a starting material. This avoids the need to build the purine ring and thereby provides a more efficient and higher yield preparation of the desired immunostimulant. This preferred synthetic pathway is outlined in Scheme 1.

In Scheme 1, R 4 which is (CHo- 2 1 8

O-CO-X

12 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 25 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.

Date: June 16,1995 21 Time: 11:38AM C-Pharma 47PT SCEME 1 Ri

PG-L

NaHITHF HO OH N N L% PG/ R N N R 3 IV 2 DEAD PPh 3

/THF

N N R

R

4

'PG

i(CH,) 4

NF/THF

AcOH *R 1 N fN 2 N N R.

N N oI o R 4

O

thcpeeneCfDEDCn Ph/THF t il opudII Compoun IV canbe pre amino acidg nw tehiqe0i h As Nel ,cnb de efr hsse ra lae ste usn ehiueIelkon nteat Date:~ Jue1, 952 Tie 11:38 AM Pharma 47 PCT Compound III is deprotected by using methodology well known to those skilled in the art, for example, with

(C

4

H

9 4 NF/THF and AcOH for a OTBDPS protecting group, to yield compound II. This compound is further optionally reacted with an amino acid or a peptide group of 1-8 amino acids in length, for example, in the presence of ClCOCI/THF and H20. The resultant compound is a compound of formula I.

Those skilled in the art will appreciate that compounds of formula 1 wherein R 4 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 of a mammal, preferably a human, comprising the step of administering a compound of formula I, a pharmaceutical composition, or a pharmaceutically 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.

Date: June 16, 1995 23 Time: 11:38AM Pharma 47 PCT It will be further appreciated that the amount of a compound of the invention required for use in treatment will vary not only with the particular compound selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of 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 of body weight per day.

Preferably, doses will range from about 1 to about 100 mg/kg/day. More preferably between about 2 to about mg/kg. Most preferably about 2.5 mg/kg. Still, most 15 preferably about 450 mg/m 2 e The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example as two, three, four or more sub- 20 doses per day.

The compound is conveniently administered in unit dosage e*.form; for example containing 10 to 1500 mg, conveniently to 1000 mg, most conveniently 50 to 700 mg of active 25 ingredient per unit dosage form.

Ideally the active ingredient should be administered 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 administered as a bolus.

Desirable blood levels may be maintained by a continuous infusion or by intermittent infusions.

Date: June16, 1995 24 Time: 11:38AM Pharma 47 PCT 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 or a pharmaceutically acceptable derivative thereof together with one or more pharmaceutically acceptable carriers thereof and, optionally, other therapeutic ingredients.

The carrier(s) must be "acceptable" 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) administration or in a form suitable for administration 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 of pharmacy. All methods include the step of bringing into association the active compound with liquid carriers or finely divided solid carriers or both and 25 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 transdermal patch. Such transdermal patches may contain penetration enhancers such as linalool, carvacrol, thymol, citral, menthol and tanethole. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may Date: June16,1995 Time: 11:38AM Pharma 47 PCT be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or colouring agents.

Pharmaceutical formulations suitable for oral administration 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, cr 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 administration 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 20 as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges or, gelatin or blister packs from which S" the powder may be administered with the aid of an inhalator or insufflator.

S. 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 combination 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 or a Date: June 16,1995 26 Time: 11:38AM Pharma 47 PCT physiologically acceptable derivative thereof together with another therapeutically active agent.

Such therapeutically active agents include cytotoxic agents used to treat tumors. Such cytotoxic agents include cyclophosphamide, or 5-fluorouracil Preferably, cyclophosphamide doses used in the treatment of tumors range from about 10 to 1000 mg/m 2 Morepreferably, from about 100 to about 500 mg/m 2 Most preferably, about 350 mg/m 2 /day.

Also preferably, 5-fluorouracil doses used in the treatment of tumors ranges from about 0.1 to about 250 15 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 of the tumor, etc..

The combinations referred to above may conveniently be S. 25 presented for use in the form of a pharmaceutical composition and thus pharmaceutical composition comprising a combination as defined above together with a pharmaceutically acceptable carrier thereof comprise a further aspect of the invention.

The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.

Date: June 16, 1995 27 Time: 11:38 AM Pharma47 PCT When the compound of formula 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 is used alone. Appropriate doses will be readily appreciatedby those skilled in the art.

In a further embodiment of the invention, there is provided a method of treatment of immune deficiencies or for the control of tumor growth comprising the step of administering 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 administering a pharmaceutically acceptable amount of a compound of the invention, in combination with 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.

Date: June16,1995 28 Time: 11:38AM

EXAMPLES

The compounds of formula I were synthesized and tested for immunological activity using the procedures outlined below.

Exa!Mple la Synthesis of N-5- (6-N,N-Dimethylaminopurin-9-yl) pentoxycarbonyl-D-Arginine Compound #1 C L be.

S

S. *S S C

S

S S

S

S

**SS

5*5*

S

S

.555 555555

C

5555

S

S

*5.S

S

S S

S.

S

TBDPSCI

NaN/THE' OTD- HO (CH, 2

OH

I1

DEAD

PPh./'rNF C1

OTBDPS

(C

4 9 4

NF/TNE'

iAcOH Ci-

N

OH

II ClCOC/Taluene D-Arginine! H 2 0 HNN(CH3) 2 [120 Compound #1 11H NN 2 Step 1: a) Synthesis of protected 1,5 pentanediol 36.3 mmole of pentanediol was dissolved in 75 ml dry tetrahydrofuran and stirred under argon flow. Sodium hydride (1.4 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 stirred at ambient temperature and under argon overnight. The suspension was then poured onto 100 ml ether. The etheral suspension was washed with potassium carbonate (100 ml), brine (100ml) and dried with magnesium sulfate. Removal of the solvent in vacuo gave 10.3 30.1 mmole of product in 98% yield which was used without further purification.

20 b) Coupling of Compound I with 6-chloropurine To a stirred solution of triphenylphosphine (4.7 g., I 17.9 mole), and 6-chloropurine 15.1 mmole) S: in 100 ml dry tetrahydrofuran, under argon flow, was 25 added diethylazodicarboxylate (DEAD, 2.8 ml, 17.9 mmole). After 10 minutes, compound I (4.7 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 removed 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 7.6 mmole, was obtained in 55% yield as a colorless oil.

Step 2: Removal of silyl protecting group 2.3g, 4.4 mmole of compound II was dissolved in 40 ml dry tetrahydrofuran and stirred under argon flow.

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 removed 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 minimal 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 1.0g., 4.2 mmole of 6-chloropurinyl pentanol, compound :III was dissolved in 75 ml dry tetrahydrofuran and *20 stirred under argon flow. Toluenic phosgene (4.4 ml., 5 8.3 mmole) was added and the reaction was monitored by TLC (developed in methanol) and continued until the intermediate chloroformate was the predominant product *eeo (6-10 hours). The solvent was removed in vacuo, and 25 the residue was taken up in 50 ml.dry tetrahydrofuran.

D-arginine (0.94 5.4 mmole), dissolved in 5 ml.

water, was added to the chloroformate suspension.

Another 5 ml. aliquot 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 The toluene was back extracted with water (60 ml.) and the combined aqueous portions were brought to slightly alkaline pH by the addition of 5% sodium bicarbonate. Water was removed in vacuo and the residue was dissolved in methanol (10 ml.).

After filtration, the methanolic solution was added dropwise 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 to the aqueous 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 removed 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 4 0 C 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- S. arginine, compound 0.79 1.8 mmole in 43% yield.

20 mp (softens 119 0 C) 1230 125°C Rf silica (methanol) 0.30 1 HNMR (DMSO d 6 300 MHz, 5 in ppm); 9.40 (1H, br, s, COOH); 8.20 (1H, s, purine); 8.15 (1H, s, purine); 7.3 (4H, b, guanidine); 6.33 (1H, d, NH); 4.13 (2H, S 25 t, N-CH 2 3.86 (2H,t, O-CH 2 3.63 (1H, m, CaH); 3.36 (6H, s, b, N-(CH 3 2 3.02 (2H, b, CaH); 1.8-1.2 m, C H, C'H,-(CH 2 3 MS (high-resolution FAB, glycerol); m/e, 450.25780; calculated for M+H

(C

19

H

32 0 4

N

9 450.25773.

Example lb Alternative Synthesis of N-5-(6-IN,N-Dimethyl aiinopurin-9--yl) pentoxycarbonyl-D-Arginine- Compound #1

CI

NN

OTBDPS

H 1 (C If), 1120 I C 4 FIT HF A c 0HI

N

N N V.

V

V

V V

V

V

V

V

V

V

V

V

V.

V V V

V.

V

V

I CCOC] /101uene D-Arginine/ 112 0

N

N N N rill Compound #1 NJJ Nl A modified synthesis of compound #1 was undertaken by reaction of protected 6-chioropurinyl pentanol, compound II (prepared as described in example la) with aqueous dimethylamine, followed by deprorection to yield compound V, and coupling with D-arqinine (the coupling reactLion is as described in example la) to 33 Step 1: to Rj, give the product. The spectral and chromatographic properties were identical to the product obtained from the synthesis described in example la.

A typical example of the reaction of protected 6chloropurinyl pentanol, compound II, with dimethylamine is as follows; to 0.13 0.26 mmole of compound II dissolved in 20 ml tetrahydrofuran was added dimethylamine (40% 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 94% yield.

Example 2 Synthesis of N-5-(6-N,N-Dimethylaminopurin-9-yl)- 9..

pentoxycarbonyl-L-Arginine Compound #2 S. 20 The L-enantiomer of compound #1,compound was synthesized as described above in example lb to give 50 mg. of product as a white solid.

mp (softens 118 0 C) 123° 125"C.

Spectral properties were identical with compound t# S* Example 3 Synthesis of N-5-(6-Methylaminopurin-9-yl)- S"o pentoxycarbonyl-D-Arginine Compound #3 9* Compound #3 was synthesized as described above in example lb, except that dimethylamine was replaced with methylamine aqueous solution) to give 6-methylamiinopurinyl pentanol compound #3a. This was then coupled with D-arginine, as described in example la to give 32 mg. of product.

mp (softens at 127'C) 133 0

C.

Rf silica (methanol) 0.20 iHNMR (DMSO-d6, 300 MHz, 6 in ppm); 9.30 (1H, b, COOH), 8.21 (1H, s, purine); 8.13 (1H, s, purine); 7.8-7.2 (4H, b, guanidine); 6.29 (1H, d, NH); 4.13 (211, t, N-CH2); 3.86 (2H, t, O-CH 2 3.61 (1H, m, 3.17 (1H, m, CH 3 3.02 (3H, b, HN-CH 3 2.97 (2H, b, CSH); 1.9- 1.2 (10H, m, C 1 HI, C'H, (CH 2 3 Example 4 Synthesis of N-5-(6-N-Methylaminopurin-9-yl)pentoxycarbonyl-L-Arginine Compound #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 123 0 C) 1320 134 0

C.

Rf silica (methanol) 0.20.

Spectral properties were identical with compound #3.

S

Example 5 Synthesis of N-5-(6-N-Aminopurin-9-yl)l pentoxycarbonyl-D-Arginine Compound 2 Compound #5 was synthesized as described above in example Ib except that protected 6-chloropurinyl pentanol, compound II, was reacted with ammonia gas instead of dimethylamine. The 6- Sm; aminopurinyl (adenine) product was thus deprotected to give 25 the alcohol compound #5a. This was then coupled with Darginine to give 260 mg. of product as a white solid. A typical example of the reaction of compound II with ammonia is as follows; 0.42g, 0.88 mmole of compound II was dissolved in ml absolute ethanol, and the solution was placed on an ice bath. Ammonia 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 120°C oil bath. Solvent was removed in vacuo, yielding 0.40 0.88 mmole of product in 95% yield.

This product was used without further purification.

Characteristics of compound TFp p(softens 143 0 C) 150 0

C.

Rf silica (methanol) 0.20 'HNMR (DMSO-dS, 300 MHz, 5 in ppm); 9.40 (1H, b, COOH); 8.14 (1H, s, purine); 8.13 (1H, s, purine); 8.0 7.0 (6H, In, guanidine,-NH 2 6.36 (1H, b, NH); 4.13 (2H, t, N-CH 2 3.87 (2H, t, O-CH2); 3.65 (1H, m, C 3.03 (2H, b, C'SH); 1.9-1.2 m, CPH, C'H, (CH 2 3 Example 6 Synthesis of N-5-(6-N-Aminopurin-9-yl)pentoxycarbonyl-L-Arginine Compound #6 The L-enantiomer of compound compound was synthesized as described above in example 5 to give 93 mg of product as a white solid.

mp (softens at 1430C 1530 155 0

C.

15 Rf silica (methanol) 0.22 Spectral properties were identical with compound o Example 7 Synthesis of N-5-(6-N-Hydrazinopurin-9-yl)pentoxycarbonyl-Arginine Compound #7 Compound #7 was synthesized as described above in example la, o except that dimethylamine was replaced with hydrazine from the corresponding alcohol compound #7a Thus, in a typical o: example, 50 mg, 0.11 mmole of 6-chloropurinyl pentoxycarbonyl 25 D-arginine, dissolved in 5 ml 95% ethanol, was reacted with o g hydrazine hydrate (12 l, 0.40 mmole) at ambient temperature overnight. The reaction was then slowly cooled to 0 C 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 130 0 C) 134 0

C

Rf silica (methanol) 0.27 'HNMR (DMSO-d 6 300 MHz, 5 in ppm); 9.20 (1H, b, COOH); 8.23 (1H, s, purine); 8.14 (1H, s, purine); 7.4 (3H, b, guanidine); 6.6 (2H, b, NHz); 6.43 (1H, d, NH); 4.14 (2H, t, N-CH2); 3.87 (2H, t, O-CH2); 3.64 (111, m, CH); 3.04 (2H, b, C 1.8-1.3 m, CPH, CH, (CH 2 3 Example 8 Synthesis of N-5-(6-N-Hydrazinopurin-9-yl)pentoxycarbonyl-L-Arginine Compound #8 The L-enantiomer of compound compound was synthesized as described in example 7 to give 40 mg of product as a white solid.

mp (softens at 130 0 C 134 0

C.

Rf silica (methanol) 0.27 Spectral properties were identical with compound #7.

Example 9 Synthesis of N-5-(6-N-Chloropurin-9-yl)- S 15 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.

o* mp(softens 137 0 C) 1450 148 0

C.

Rf silica (methanol) 0.35 1HNMR (DMSO-ds, 300 MHz, 5 in ppm); 9.15 (1H, b, COOH); 8.78 25 (1H, s, purine); 8.74 (1H, s, purine); 7.8-7.2 (4H, b, guanidine); 6.33 (1H, d, NH); 4.29 (2H, t, N-CH 2 3.88 (2H, t,

O-CH

2 3.64 (1H, m, 3.04 (2H, b, C 1.95-1.20 (10H, In, C H, CH, (CH 2 3 Example 10 Synthesis of N-5-(6-N-Chloropurin-9-yl)pentoxycarbonyl-L-Arginine Compound The L-enantiomer of compound compound #10, was synthesized as described above in example 9 to give 65 mg of product as a white solid.

mp (softens at 137 0 C) 143-146 0

C.

f silica (methanol) 0.26 -n 37 Spectral properties were identical with compound #9.

Example 11 Synthesis of N-5-(6-N-Hydroxypurin-9-yl)-D- Arginine Compound #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 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 hydrochloric acid), the solvent 15 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 163 0 C) 182 0

C.

Rf silica (methanol) 0.22 1 HNMR (DMSO-d6, 300 MHz, 5 in ppm); 9.28 (1H, b, COOH); 8.10 (1H, s, purine); 8.04 (1H, s, purine); 7.8-7.2 (4H, b, 25 guanidine); 6.38 (1H, d, NH); 4.32 (1H, b, OH); 4.13 (2H, t,

N-CH

2 3.87 (2H, t,O-CH 2 3.61 (1H, m, C II); 3.04 (2H, b,

C

6 1.8-1:1 (10H, m, CH, C'H, (CH2)3-) Example 12 Synthesis of Mercaptopurinyl Pentoxycarbonyl D- Arginine Compound #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 The reaction was then stored at 0 C overnight and the lting crystals were filtered and washed with cold absolute ethanol. The product was recrystallized from absolute ethanol to give a white solid, 48 mg, 0.11 mmole in 61% yield.

mp(softens 180 0 C) 200 0

C.

Rf silica (methanol) 0.50 'HNMR (DMSO-d6, 300 MHz, 6 in ppm); 9.15 (1H, b, COOH); 8.29 (1H, s, purine); 8.18 (1H, s, purine); 7.5-7.3 (4H, b, guanidine); 6.39 (1H, d, NH); 4.13 (2H, t, N-CH 2 3.87 (2H, t,

O-CH

2 3.65 (1H, in, C 3.04 (2H, b, C8H); 1.90-1.23 (10H, m, C H, C'H, (CH 2 3 Example 13 Synthesis of N-5-(6-N-Mercaptopurin-9-yl)pentoxycarbonyl-L-Arginine Compound #13 The L-enantiomer of compound #12, compound #13, was 15 synthesized as described in example 12 to give 42 mg of product as a white solid.

mp (softens at 180 0 C 200 0

C.

Rf silica (methanol) 0.50 Spectral properties were identical with compound #12 Example 14 Synthesis of N-5-(6-N,N-Dimethylaminopurin-9-yl)pentoxycarbonyl Glycine Compound #14 Compound #14 was synthesized as described above in example lb 25 except that the coupling reaction was undertaken on smaller '9 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 100 0 C) 126 0

C.

Rf silica (1:1 methanol-ethyl acetate) 0.35 1 HNMR (DMSO-d6, 300 MHz, 5 in ppm); 8.20 1H, purine); 8.16 1 H, purine); 6.05 (1H, t, NH); 4.14 (2H, t, N-CH 2 3.86 (2H, t, O-CH 2 3.33 (6H, s, b, N-(C11 3 2 3.18 (2H, d, C 'H 2 1.80 (2H, m, CH 2 1.55 (2H, m, CH 2 1.26 (2H, m, CH 2 Example 15 N-5-(6-N,N-Dimethylaminopurin-9-yl)-7'-ethoxyethoxycarbonyl-D-Arginine Compound Step 1: N,N-(6-Dimethylaminopurin-9-yl)-5-ethoxyethoxy-tbutyldiphenylsilane To a solution of alcohol (0.201 g, 1 eq) in anh. THF (2.9 ml), at room temperature, under argon, were added successively 6-chloropurine (90 mg, 0.58 mmol), Ph 3

P

(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 Hexanes/EtOAc) to give a mixture of (EtO 2

CNH)

2 and the coupled purine. To a solution of this mixture in THF (6 ml), at room temperature, was added 40% Me 2

NH/H

2 0 (0.70 ml, 10 eq). The solution was stirred at room temperature for 45 minutes and was then poured in sat. aq. NaHCO 3

/CH

2 Cl 2 The phases were ~separated and the aqueous phase was extracted with

CH

2 Cl 2 The combined organic extracts were dried over MgS0 4 the solids filtered and the solvents

S

25 evaporated. The residue was purified by flash chromatography (silica gel, 2:8 Hex/AcOEt) to give 0.16 g of the coupled dimethyl amino purine.

Step 2: (6-N,N-Dimethylaminopurin-9-yl)-7-ethoxyethanol compound 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 nBu 4 NF 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 immediately purified by flash chromatography (silica gel, 4:1 AcOEt/MeOH) to give 72 ing of the alcohol compound #85 as a clear oil.

HNMR (CDC1 3 5 8.29 11, purine), 7.80 111, purine), 4.33 2H, CH2), 3.82 2H, CH 2 3.68 (t, 2H, CHi 2 3.55 2H, CH 2 3.50 6H, N(CH 3 2 Step 3: N-5-(6N,N-Dimethylaminopurin-9-yl)-7'-ethoxyethoxycarbonyl-D-Arginine Compound 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 COC1 2 /Toluene 1.93 M (0.30 ml, 2 15 eq) and the solution was stirred at room temperature for 5 hours. The THF was evaporated in 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 H20). The flask containing the Darginine solution was rinsed with 0.5 ml 1120 and the reaction mixture was stirred at room temperature for hours. It was then extracted with toluene and the toluene phase was back-extracted with H20. The combined aqueous layers were brought to pH 7.5-8.0 (NaHCO 3 25 and the water was evaporated. The residue was purified by flash chromatography (silica gel, 100% MeOH) The fractions containing the compound were evaporated and the residue was dissolved in a minimum quantity of MeOH. Et20 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%).

H NMR (DMSO-d 6 5 8.25 1H, purine), 8.14 11H, purine), 6.5 (bd, 1H, NH), 4.37 2H, CHz linker), 4.03 211, CI1 2 linker), 3.81 2H, CHl linker),3.72 1 H 3.60 211, CH 2 linker), 3.55-3.89 (m, 6H, N (CH 3 2 3 .05 2H, CL"1 2 1. 78-1 .39 (mn, 4H. CPH2 C'H2 Example 16 (2S,4S)-2--(6-N,N-Diiethylaininlopurifl-9-y1)-4- (inethyloxycarbonyl-D-arqinine) 3-dioxolane Compound #16 0 O 0 OSitBuPh 2 00 i 2 0.110_r OSitBtPh 2 1-10 0* o 0 \-OD OSitl~uPh 2 0 cl

N

4

N

K~N

0 OSitBiPli 2 0

N

N

0 0 .cooI-I Compound #16 Step 1: (4S) 2-diniethyl-1, 3-dioxolane--4-tbutyldiphienyls ilylinethanol To a solution of (4s)-2,2-dimethyl-l,3-dioxolane-4methanol (ig, 7.57 mmols) in anh. CH 2 C1 2 7 6ml) at room temperature, under argon, were added successively imidazole (1.03g., 2eq) and t-BuPh 2 SiCl (1.95 ml, 1.1 eq). A white precipate formed immediately. This suspension was stirred at room temperature for 1 hour and then poured in sat. aq.

NaHCO 3 The phases were separated and the aqueous layer was extracted with CH 2 C12 The combined organic extracts were dried over MgSO 4 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/H 2 0 (15 ml), at room temperature, was added TFAA (0.5 ml, 2.4 eq) and the solution was heated at 50 0 C for 5 hours. It was 20 then poured in sat. aq. NaHCO 3

/CH

2 Cl 2 and the phases were separated. The aqueous layer was extracted with CH 2 C1 2 (2x) and the combined organic extracts were dried over MgSO 4 The solvents were evaporated and the residue was purified by flash chromatography (silica gel, 1:1 Hex/EtOAc) to give 0.62 g. of the diol as a clear oil.

Step 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.

NaHCO 3

/CII

2 C1 2 The phases were separated and the aqueous layer was extracted with CH 2 C1 2 The combined organic extracts were dried over MgSO 4 and S43 the solvents were evaporated. The residue was purified by flash chromatography (silica gel, 9:1 Hex/EtOAc) to give 0.49 g of a 5:1 (cis/trans) mixture of the dioxolanes.

Step 4: (2S,4S)-2-Hydroxymethyl-4-tbutyldiphenylsilyloxymethyl-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 pl, 0.1 eq).

The solution was stirred for 18 hours after which it was poured in sat. aq. NH 4 Cl/CH 2 Cl 2 The phases were separated and the aqueous phase was extracted with 15 CH 2 C1 2 The combined organic extracts were dried over MgSO 4 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.

Step 5: (2S,4S)-2-(N,N-dimethylaminopurin-9-yl)-4-tbutyldiphenylsilyloxymethyl-l,3-dioxolane 0 25 The compound was prepared using a similar method as in Example 15, step 1.

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

Purification: 10% MeoH/EtoAc 1 HNMR (CDCl 3 5 8.32 1H, purine), 7.75 1H, purine), 5.33 (dd, 1H, J=2.0, 6.6, H-2-dioxolane, 5.33 (bs, 1H, OH), 4.45 (dd, 1H, J=6.6, 14.3, C 2zpurine), 4.20 (dd, 18, J=2.0, 14.3, CH 2 -purine), 4.20 (mn, 1H, 11-4--dioxolane), 4.05 2H, J-7.2, H- 3.78 1H, J=13.0, CH 2 -OH) 3.53 (bs, 611, (C11 3 2 3.40 1H, J-13.0, C11 2 -011) S tep 7: (2S, 4S)-2-(6-N,N-Dinethylaininopurin-9-yl)-4- (methyloxycarbonyl-D-arginine) -1,3-dioxolane Compound #t16 The compound #16 was prepared using a similar method as in Example 15, step 3.

Purification: MeoH 100% 1 HNMR (DMSO-d,) 6 8.43 1H1, purine) 8.11 (s, 1H, purine), 6.6 (mn, 18, NH), 5.28, 1H, 11-2dioxolane), 4.39 2H, CH 2 -purine), 4.26 (mn, 1H, -4-dioxolane), 3.97-3.81 (mn, 3H, Cc'H, CH 2

-OCO-D-

arginine), 3.71 (mn, 2H, 11-5-dioxolane), 3.39 (bos, 611, (CHA) 2 N) 3 .07 (in, 2H, C aH2), 1.70-1.45 (mn, 4H, H 1

J

2

C'H

2 Example 17 Synthesis of N-5-(6-N,N-Diinethylamino-8bromopurin-9-yl) -pentoxycarbonyl L-Arginine 25 Compound #17 H

NH

N N 0 N N Br 'N H O 0 1H NMR (DMSO-d 6 400 MHz, 6in ppm): 9.5 (1H, s, b, COOH),8.19 (18, s, purine), 8.1-7.2 (4H, b, guanidine), 6.30 (18, d, NH), 4.11 (2H, t, N-CH 2 3.87 (2H, t, CH 2 3.64 (1H, in, CLH),3.39 (6H, s, b, N-(CH 3 3.02 (2H, m, C 1.8-1.2 (10H, In, Coli, CUH, -(CH 2 3

-)I

m.p. (softens 115-1180) 124-127'C.

Rf silica (70% methanol-ethyl acetate) 0.25 Example 18 Synthesis of N-5-(6-N,N -Diinethylamino-8bromopurin-9-yl)-7-pentoxycarbonyl-D-arginine Compound #18

HN

1. N~ N O Br ONH N N0

NH

IH NMR (DMSO d 6 400 MHz, 8 in ppm); 9.08 (1H, br s, COOH); 8.18 (1H, s, purine); 7.9-7.3 (4H, br s, guanidine); 6.34 (111, d, NH); 4.10 t, N-CR1 2 3.86 (211, in, 0-CR 2 3.55 (1H, m, C7-H); 3.35 (br s, N-(CR 3 3.03 (2H, m, CO'H 2 1.9-1.2 m, (CI1 2 3 CYH2, CP11 2 in.p. (softens 116 0 C) 122-125 0

C.

s Rf silica (70% methanol-ethyl acetate) 0.25 1 Example 19 N-5-(Purin-9-yl)-pentanol Compound #19

CI

N N N N (C11 2 5

-OI

I-I,/Pd/C Ethatiol 25 0 C/overnight N' -N C OI 1H NMR 5 (CDCl 3 in ppm): 9.12 1H, purine), 8.96 111, purine), 8.10 1N, purine), 4.30 2H, CH 2 3.63 Ct, 2H, CH 2 1.97 3H, CR 2 and OH), 1.62 2H, CR 2 1.47 2H,

CR

2 13C NMR (5 CDC1 3 in ppm): 153.09, 151.96, 149.15, 145.80, 134.60, 62.78, 44.38, 32.47, 30.27, 23.57.

10 Purification 5% MeOH/AcOEt Rf (silica) 0.29 MeON/AcOEt) @0 0 0 0 i i 0 0 000.

0 0000 0 0 0000 0* 0 Example 20 N-5-(Purin-9-yl)-pentoxycarbonyl-D-arginine Compound

CI

N N H 2 P/lVC Ethaniol N N 0 25C (C 11 2 -C -Nil -CI-COOII (CI 12)3 11

NI-I

I-IN

NHI

2 N N 0 I I I

(C

2 5 C -NII-CI-I-COOI (Cpi 2 3

NI-I

1IN NI1 2 1H NMR 5 (DMSO in ppm): 9.10 1i, purine), 8.93 1H, purine), 8.64 11, purine), 7.77 (bs, 4H, guanidine), 6.25 Cbs, 1H, NH), 4.28 21, CH 2 3.87 2H, CH 2 3.58 1H, CaH), 3.09 2H, CH 2 1.20-1.90 IOH, 5xCH 2 Purification: methanol Rf (silica) 0.23 (methanol) 47 O~FF8~i Example 22. N-5- (Purin-9-yl) -pentoxycarbonyl-L-Argini-ne Compound #21

CIC

NN NII I-1 2 /PCd/C Ethianol 2 5'C N N N4 N 0 (C1-19 5 0 CIIC OOH

NIL

fIN NHi 2 a.

.9 a. 9 0 9**a a *aa9 a a 0 Oa a 9* 9***aa 11H NMR 5 (DMSO in ppm): 9.13 1H, purine), 8.94 111, purine), 8.66 1Hi, purine), 8.25 Cbs, 1H, NH), 7.44 (bs, 3H, guanidine), 6.82 (bs, 1H, NH), 4.28 2H, CH 2 3.88 21-1, CH 2 3.72 (in, 1H-, CO H) 3.07 211, CH 2 1.19- 10 1.95 (in, 101l, 5xCI1 2 1CNMR (5 CD 3 0D 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 15 Rf (silica) 0.23 (methanol) Example 22 Synthesis of N-5- (6-N,N--Diinethylaminiopujrin-9yl) -pentoxycarbonyl-L-Valyl-L-Prolyl-L-Leucine Compound #22

OFFIGO

1 HNMR (CD 3 OD, 400 MHz,8 in ppm) 8.22 (1H, s, purine); 8.05 (1H, s, purine)-; 4.60 (1H, t, CI); 4.3-3.6 (8H, n, N-CH 2 C5H- 2

CH

2 -Of 2 x CaH); 3.51 (6H, s, b, N-(CH 3 2 2.2-1.2 (14H, m, (Cl 2 3 2 x CPH 2 CPH, CYH 2 COH) 1. 0-0.-8 (12H, m, 2 x CV-

CH

3 ,f 2 X CY-C1 3 m.p. 168 0

C

Rf silica (40% methanol-ethyl acetate) 0.40 Example 23 Synthesis of N-5- (6-N,N-Dimethylaminopurin-9yl) -pentoxycarbonyl-L-Tsoleucyl-L-Prolyl-L- Isoleucine- Compound #23 0 0 0 0 000 @0 0 jO 0 0000 0* *0 0 0 to0 00 000 0 0 OH 15 1 HNDIR (CD 3 OD, 400 Mfz, 5 in ppm); 8.21 (1H, s, purine); 8.03 (1H, s, purine); 4.62 (1 H, t, COIH); 4.22 (4H, in); 4.03 in); 3.89 (1l11, m, CUH); 3.67 (15, d, CcaS); 3.50 (6H, s, b, N- (C-13 2 1-1. 0 (165, mn, (CH 2 3 CPH2 2 x CPFI-, 3 x C7-1 2 0.95 (6H, d, 2 x CR25 3 0. 87 (6 H, t, 2 x CY-CH 3 rn.p. (softens 83-86 0 C) =93 0

C

Rf silica (40% methanol-ethyl acetate) 0.35 -4 49 Example 24 Synthesis of N-5- (6-N-Cyclopropylaminopurin-9yl)-pentanol Compound #24

Y

Nil K N

(CH

2 5 -OSi -(C1 15) tiBu 7 NHl

NN

(l) 5 1I 0 0*

S.

00 0 0 *0 00 0 0 S S 0 0

S

*000

S

S

*0*0*0 0 0*0S 0

S.

0 5 0 *5 0 1H NMR (5 CDC1 3 in ppm): 8.46 1H, purine), 7.77 111, purine), 6.42 Cbs, 1H, NH), 4.22 Ct, 2H, CR1 2 3.09 Cbs, 111, OH), 1.94 Cmn, 211, CH 2 1.63 Cmn, 2H, CH 2 1.45 Cmn, 2H, CH 2 0.94 (Cm, 2H, CH 2 0.69 Cin, 21-1, CR 2 10 Colorless oiiy material Rf= 0.3 10% methanol/ethyl acetate Mass spectrum: H'I= 262 CHRMS) Example 25 7 N1l N ~N N N

(C-

2 5 Synthesis of N-5-(C6-N-Cyclopropylaminopurin-9yl) -7-pentoxycarbonyl-D-Arginine- Compound 7 NI1I 11WT N

COCI

2 D-Arginine NaHCO, 112 10 L I N N 0 -01-1 (C11,),OCNI Id ICOOl I (CI-1 2 3 H N NI-I 2 iH NMR C5 DMS0 in ppm) 8.22 Cs, 111, purine) 8.14 Cs, 1Wi, purine), Cbs, 4H, guanidine), 6.28 111, NHi), 4.13 2H,

CR

2 3.87 Cmn, 2H, CH 2 3.62 Cmn, 1li, CO-H), 3.02 Cmn, 2H, CR 2 b 1.2-1.8 (im, 11H1, 5xCH 2 and CH), 0.69 (mn, 211, C11 2 0.67 (mn, 2H, C-f 2 1CNMR DMS0 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.

in.p. softens 147 0 C melts 151 0

C.

Rf 0.34 (MeOH) Mass spectrum: H' 462 (HRMS) Example 26 Synthesis of N-5-(6-N-Cyclopropylaininopurin-9yl)-pentoxycarbonyl-L-A7rgi-nine -Compound #26 NH- Nil N THE N N' S 1+ COC 2 L-Arginine+ NaHCO,

I"I

1 120 N N~ N 0

(CH

2 5 01-1(C11 2 ),OCNI IClI COOlf (CI 12)3 4'H NMR (8 DMS0 in ppm): 8.23 1H1, purine), 8.14 1H1, go****purine), 7.51 Cbs, 411, guanidine), 6.31 111, NH), 4.16 (t, 21Hf CH 2 3.87 2H, CH 2 3.62 (rn, 11I, CO1lfl, 3.02 (in, 211,

CH-

2 1.2-1.85 (in, 11H, 5XCH- 2 and CH), 0.72 (in, 2H, CH 2 0.68 (mn, 21-1, CH1 2 13 C NMR CD 3 0D 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. 144-146 0

C

Rf= 0.35 (MeOH) Mass spectrum;: M' 462 (HRMS) Dr;% 51

OFRI&

Example 27 Synthesis of N-5-(6-N-Azetidinepurin-9-yl)pentanol Compound #27 N N TII N; N N N BuNF- TIKN N N N N

(CH

2 5 Si u

(C-

2 5

-OH

(C

6

H

5 2 1H NMR CDC1 3 in ppm): 8.25 1H, purine), 7.66 1H, purine), 4.44 Cm, 4H, CH 2 4.10 2H, Cli 2 3.55 Ct, 2H,

CH

2 3.21 Cbs, 1H, OHf), 2.48 (in, 2H, CHI 2 1.84 2H, CH 2 1.54 Cm, 211, CH 2 1.38 Cm, 21-, CH 2 13 C NMR C5, CDC1 3 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.

104-106'C Rf 0.33 (10% MeGH/AcOEt) orsa Mass spectrum: M' 262 (HRMS) @000 Example 28 Synthesis of N-5-C6-N-Azetidinepurin-9-yl)pentoxycarbonyl-D-Arginine Compound #28 *see*: N N COC12 D-Arginirne NaiICO T N N N N 0

IICOO-

NII

1H NMR CD 3 0D in ppm): 7.96 111, purine), 7.89 Cs, 1H, purine) 4-.27 (in, 4H, 2xCH 2 4.02 2H, CH 2 3.79 (in, 3H, CU12 and CO%1), 2.99 Cmn, 2H, CH 2 2.32 Cmn, 211, CH 2 1.17-1.71 Cmn, 10OU, 5xCH 2 13 C NMR CD 3 0D 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. 190-192 0 c Rf: 0.25 (methanol) Mass spectrum: M+ 462 (HRMS) Example 29 Synthesis of N-5- C6-N-Azetidinepurin-9-yl) pentoxycarbonyl-L-Arginine Compound #29 0 0 o a.

N N NNTf F N- N I COC1 2 L-Arginine NaHCO 3 K N N N 0

II

(CH

2 3

NH

fUN

NIT

2 1 H NMR CD 3 0D in ppm): 7.93 Cs, 1H, purine), 7.88 Cs, 1H, purine), 4.27 Cmn, 4H, 2xCH1 2 4.01 2H, CH 2 3.79 Cmn, 3H,

CU

2 and CU 2.97 Cmn, 2H,-CH1 2 2.32 Cmn, 2H, CH 2 1.15-1.74 (Cm, 10H, 5xCH 2 13 C NMR CD 3 0D 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.

(softens at 175'C) melts at 187 0

C

Rf= 0.27 (methanol) Mass spectrum: M+ 462 (HPJYS) 53 b

-I

XIFIC*'

Example 30 Synthesis of trans-(6-N-Chloropurin-9-yl)-4riTiethyl-cyclohexyl-methanol Compound

CI

NN

N

N

0OH 1H1 NMR (CDC1 3 400MHz, 5 in ppm) 8 .73 (1H, s, purine) 8. (1H1, s, purine); 4.12 (2H, di, N-CH 2 3.43 (2H1, d, O-CH 2 1.89 (1H1, m, ClI); 1.84-1.64 (4H, m, CH 2 -cyclohexane); 1.56 (111, br s, OH); 1.45 (1H1, m, CHI); 1.14-0.85 (4H, m, CH 2 (softens 176 0 C) 178 0

C

Rf 4 (ethyl acetate) *Example 31 Synthesis of trans-(6-N,N-Dimethiylaminopurin- :9-yl) -4-methyl--cyclohiexyl-meth-anol- Compound #31

N

*00.

N

N

NN

IH *M CC3 0Mz npm; 82 1,s uie;76 13C NMRj (CDC1 3 30MHz, 5 in ppm); 158.29, 152.s, purine) 1876 120.1, 68.2, 49.7, 40.2, 38.5, 38.2, 29.9, 28.6.

N-45-,MP 151-153 0

C

Rf 0.44 (10% methanol-ethyl acetate) Example 32 Synthesis of trans-(6-N,N-Dimethylaniflopurin- 9-yl) -4 -methyl-cyclohiexyl-methyloxycarbonyl-D- Arginine Compound #32 NN INH N

NN

0 N N 0ONH OH 1H NMR (DMSO-d 6 400MHz, 5 in ppm); 9.4 (1H, br s, C0011); 8.19 (111, s, purine); 8.11 (1H, s, purine); 8.0-7.2 (4H, br, guanidine); 6.28 (1H, d, NH); 3.98 (211, d, N-OH 2 3.69 (2Hl, d, 0-CH 2 3.61 (111, mn, CO.H); 3.43 (6H, br s, N-OH1 3 2 3.00 (21f, br, C 1.9-0.8 (14H, mn, CPH 2

CYH

2 2 x CHcyclohexane, 4 x CH 2 -cyclohexane).

13C NMR (DMsO-d 6 400MHz, 5 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.

(softens 1570C) 164-1660C 9 to. Rf- 0.35 (methanol) Example 33 Synthesis of trans- (6-N-hydroxypurin-9-yl) -4methyl-cyclohexyl-methanol -Compound #33 1H NMR (CD 3 OD, 400MHz, 8 in ppm) 8. 05 (2H, s, purine) 4. (2H, d, N-CH 2 3. 35 (2H, d, 0-C1 2 2. 0-0. 9 (10 H, m, 2 x CH1cyclohexane, 4 x CH 2 -cyclohexane) 1CNMR (CD 3 OD, 400MHz, 5 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. 200 0

C

Rf 0.35 (20% methanol-ethyl acetate) 9* 9 9** 9**9 *9 9 9 9 99** 9 *99* 9 9 9*999* 9999 9 9 9 9 999.9.

Example 34 Synthesis of trans-(6-N-Methoxypurin-9-yl) -4methyl-cyclohexyl-methanol Compound #34 Oil 'H NMR (CDC1 3 300Mliz, 5 in ppm) 8 .52 (1H, s, purine) 7 .84 (111, s, purine); 4.17 (3H, s, O-CH 3 4.12 (2H, d, N-CH 2 3.43 (2H, d, 0-CH 2 1.89 (1H, m, CH); 1.84-1.64 (4H, m, CH 2 cyclohexane); 1.56 (111, br s, OH); 1.45 (1H, m, CH); 1.14-0.85 (4H, m, C1 2 -cyclohaexane).

m.p. (softens 159 0 C) =162 0

C

Rf=0.25 (ethyl acetate) Example 35 F1 Synthesis of cis- N-Dimethylaiinopurin-9yl) -4 -iethyl-cyclohexyl-methianol Compound# 56 H 3 C N. 'C 1 3

N

0OH 111 NMR (CDCl 3 300MHz, 5 in ppm); 8. 31 (1H, s, purine) 7. 66 (111, s, purine) 4. 08 di, N-CH1 2 3. 55 (211, d, O-CH1 2 3.50 6H br s, N-(CH 3 2 3.28 (1H1, br s, OH); 2.12 (1H, in, CH); 1.67 (1H, mn, CII); 1.5-1.3 (8H, m, CH 2 -cyclohexane).

1CNMR (CDCl 3 300MHz, 5 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.

10 mn.p. 153-1560C Rf 0.3 (10% mnethanol-ethyl acetate) **c Example 36 synthesis of cis- (6-N,N-Dimethylaminopurin-9yl) -4-mnethyl--cyclohexyli--nethyloxycarbonyl-D- Arginine Compound #36 HNyNH 2 IIN lHNMR (DMSO-d 6 400Mlz, 5 in ppmn); 9. 28 (111, br s, C0011); 8. 19 (1H, s, purine) 8.13 (1h, s, purine) 8 2 (411, br, guanidine); 6.34 (1H, d, NH); 4.10 (211, d, N-CU1 2 3.85 (2FI,d, 0-CU1 2 3.65 (11-1, in, CaOH); 3.44 (6H, br s, N-(CH 3 2 3.02 2H, m, C 5

H

2 2.09 (1H, m, CH); 1.8-1.2 (14H1, m, cpH 2

CYH

2 2 x CH-cyclohexane, 4 x CH 2 -cyclohexane).

1CNMR (DMSO-d 6 400MHz, 5 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 153 0 C) 168-170 0

C

Rf=0.35 (methanol) Example 37 Synthesis of N-5-(6-N,N-Dimethylaminopurin-9yl) 7-pentoxycarbonyl-D-Citrulline- Compound #37 0 NH 2

N

0

N

0 15 1H NMR (DMS0 d 6 400 MHz, 8 in ppm); 8.19 (1H, s, purine); 8.15 (1H, s, purine); 6.21 (1H, d, NH); 6.11 (1H, s, NCO-NH); 5.42 (2H, s, NH 2 4.14 (2H, t, N-CH 2 3.86 (2H, m, O-CH 2 3.56 (1H, mn, CaH); 3.35 (6H, br s, N-(CH 3 2 2.87 (211, m, C8 H12); 1.9-1.2 (1011, m, (C-1 2 3

C'YH

2

CPHF

2 1 3 C NMR (CD 3 OD, 400 MHz, 5 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, 22.0.

m.p. (softens 172-176'C) 178-181'C Rf =0.20 (40% methanol-ethyl acetate) Example 38, Synthesis of N-5-(6-N-Methylaziridinepurin-9yl)-pentanol Compound #38 58

CH

3 %t H lj 117 N NN N1 N

N

011 (U 2 5 01-I 1NMR CDC1 3 in ppm): 8.54 1H, purine), 7.90 1H1, purine), 4.22 211, CH 2 3.61 211, CH 2 2.78 Cmn, 11f, CII), 2.65 1H1, CH 2 2.40 1H1, CH 2 2.39 (bs, 111, OH), 1.94 Cmn, 211, CH 2 1.59 Cmn, 211, CH 2 1.50 311, CH 3 1.40 Cmn, 211, CH 2 1 NMR CDC1 3 in ppm): 163.30, 152.99, 151.99, 142.80, 10 126.11, 62.81, 44.48, 35.97, 34.89, 32.51, 30.38, 23.50, 18.48.

Low melting point.

Rf= 0.4 (20% MeOHI/AcOEt) Mass spectrum: M+ 262 Example 39 Synthesis of N-5-C6-N-Methiylaziridinepurine-9yl)-pentoxycarbonyl-D-Arginine Compound #39 N

N

N 0 0 N NH 2 Y

O

4 59 (softens at 190 0 C) melts at 200 0

C

Rf: 0.4 (methanol) Mass spectrum: M' 462 Example 40 N-5- N-Dimethylaminopurin-9-yl) -7-thioethoxyethoxycarbonyl-D-Arginine Compound 111N 1 Cl

N

'N N> 1

N

OSiti~uPh,

N

N N [N N

H

0 O N

COOH

NH

N NH 2 I'lN 3 N

N

N Oil1 NJ S TnI Example 41 Meta- (6-N,N-Dimethylaminopurin-9-yl) -methylbenzyloxycarbonyl-D-Arginine Compound #41 HO OH c I N-1 N H OSiLI~uPh 2 N

N

-12 I-,N

N

0 0 0 0000 00 0 0 0 0 0 0000 0 0 0000 0000 0 0 0000 000000 0 000.

0 0 0000 00 0 0 0 *00000

N

3 N~

N

OH OSitBuPh 2 yNH2 M-1 COO"l Compound #f41 Example 42 N-5- (6-N,N-Dimethylaminopurin-9-yl) 3 -pentynyl-1oxycarbonyl-D-arginine Compound #42

I

OH h

CN

KNNo OMtBu Ph 2 .OSitBuPi2 [eO 2 C 7N/OSitEtitPb 4 N

N

N N112 NHl ON COOH 11 N

N

N

5 Example 43 Synthesis of N-5- (1-methyl--2-acetoxy) ethylaminopurin-9-yl]-pentanol Compound #43 1 H NMR CDC1 3 in ppm) 8.36 1H, purine), 7.76 1H, purine), 6.58 (bs, 1H, NH), 5.18 (in, 1H, OH), 4.22 2H,

CH

2 3 .92 (bs, 1H, CH) 3 .63 2H, CH 2 2. 05 3H, CHA), 1. 3-1. 9 4xCH 2 1xCH 3 173.04, 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% MeOfR/AcOEt Mass spectrum: W~ 322 Example 44 Synthesis of N-5-[6-(1-methyl-2-acetoxy), ethylaminopurin-9-yl] -pentyloxy-carbonyl-D- Arginine -Compound #44 0 *HNy NH2

NH

NH

NN

N

OH

*N I 0 'H NMR CD 3 0D in ppm) mixture of isomers, 8.05 Cs, 1H, purine), 7.89 Cs, 1H, purine), 4.92 Cm, 1H, CH), 4.03 2H,

CR

2 3.78 3H, CH 2 and co'H) 3.46 2H, CR 2 2.99 2H,

CR

2 1.8 3H, CHA), 1.1-1.79 (in, 13H, 5xCH 2 and 1xCH 3 1 3 C NMR CD 3 0D 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.

ni.p. (softens at 177'C) melts at 185oC Rf: 0.35 (methanol) Mass spectrum: M+ 522.

63 -I r'1 Example 45 Synthesis of 6-Dichloropurin-9-yl) pentanol Compound

S

S

*SS*

S

I H NMR CDC1 3 in ppm): 8.11 1H, purine), 4.29 2H,

CH

2 3. 66 2 H, CHA), 2. 00 (in, 21H, CH 2 1. 64 (mn, 2 H, C11 2 1. 48 (in, 211, CHA)1 1. 3 1H, OH) 13C NMR CDC1 3 in ppm) :163.3, 150.2 149.3, 148.01, 128.00, 10 63.20, 44.80, 29.70, 26.00, 22.4.

m.p. 133-135'C

R

1 0.4 5% methanol/ethyl acetate Mass spectrum: M' 260 (HRMS) 15 Example 46 Synthesis of N-5- 6-Dichloropurin-9-yl) petitoxycarbonyl-D-Argiiine Compound #46 [INy NHI

NH,

1H1 NMR DM50 in ppm) 9.33 1H1, COOH), 8.75 1H, purine), 7.3-7.8 (bs, 4H, guanidine), 6.28 1H, NH), 4.23 2H1, CH2), 3.86 211, CH 2 3.61 (in, 1H, 3.015 (mn, 211, el1 2 1.2-1.9 (in, 10O1, 5xCH 2 INIs~mp.:Softens at 136 0 C melts at 147aC Rf: 0.46 methanol Mass spect-rum: M' 476 Example 47 Synthesis of N-5- 6-Dichloropurin-9-yl) -7pentoxycarbonyl-L-Arginine Compound #47 @0 0 000* 000 0000 66 00 of 1 H NMR CD 3 0D in ppm) 8.38 1H-, purine), 4.12 21-1, 10 Cl- 2 3.30 (in, 3H, CH- 2 and C~xH), 2.97 (in, 2H, CH- 2 1.2-1.8 (in, 10H, 5xCH- 2 Softens at 137 0 C, melts at 147 0

C

Rf: 0.45 (methanol) Mass spectrum: 476 Example 46 Nl-5- (2-Arino-6-N,N-Dimethylaminopurin-9-yl) pentanol. Compound #48

NI--

Oc 1'INMR (5 in CDC13 ppm) 7.46 Cs, 1H, purine) 4.70 (bs, 2H, NH 2 4.04 Ct, 2H, CH 2 3.65 2H, CH 2 3.46 (bs, 6H, 2 x eli 3 1.95 Cm, 211, C11 2 1.65 2H, C1H 2 1.42 (im, 2H, CH 2 1 3 CNMR (5 in CD 3 0D 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. 139-141 0

C

Rf: 0.55 (15% Methanol/Ethyl acetate) Mass spectrum: M± 265.

Example 49 Synthesis off N-5-C6-N,N-Dimethylamnino-8methylthiopurin-9-yl)-pentanol -Compound #49 0000 1. [IC CII1 00 N N N CH 3 4S I OO*N

OH

0* *00 201CNMR CCDC 3 40MHz, 5 in ppm); 8.2.30, s5169 purn50.08,

I

Examnple 50 Synthesis of N-5- (6-N,N-Diimethylamino-8methylthiopurin-9-yl) -pentoxycarbonyl-D- Arginine Compound N cH 3 0 H N /CO0O0H H N H N NH 2 o *0

A

0 4 4 '0 0 0**0

A

A

0*00*t 0 000* 4 4 0*

A

0)0 4 i 1 11 NMR (DMSO-d 6 400MHz, 5 in ppm); 8.13 (1H, s, purine); 7-2 (4H, br, guanidine); 6.32 (1H1, d, NH); 4.01 (211, t, N-

CH

2 3.86 (2H1, t, O-CH1 2 3.65 (1H, m, CaH); 3.41 (6H, br s, 10 N-(CHF 3 2 3.02 (211, br, C8H 2 1.8-1.2 (10H1, m, CPH 2 CYH1 2

(CH

2 13 C NMR (CDC1 3 400MHz, 8 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.

Examiple 51 Synthesis of N-5- (6-N-Methoxypurin-9-yl) pentanol Compound #51 0 1H1 NMR (CDC1 3 400MHz, 5 in ppm) 8 .43 (11H, s, purine) 7. 86 (111, s, purine) 4. 18 (2H, t, N-COH 2 4. 09 (311, s, 0-C0113) 3. 55 (211, t, O-CH 2 3. 09 (1H, br s, OH) 1. 86 (2H, m, OH 2 1.53 m, Cl- 2 1.37 (2H, m, eli 2 1 3 C NMR (CD01 3 400MHz, 5 in ppm); 160.0, 150.9, 141.2, 120.4, 60.9, 53.2, 43.1, 30.9, 28.8, 22.0.

1500C Rf= 0.30 (15% methanol-ethyl acetate) Examplea 52 Synthesis of N-5-(6-N-Methoxypurin-9-yl)pentoxycarbonyl-D-Arginine Compound #52 H 2 N NH

HN

N\ 0 KN N 0 NH O 0 1H- NMR (DMSO-d 6 300MHz, 5 in ppm); 8.51 (11H, s. purine); 8.39 (1H1, s, purine); 8.0-7.3 (411, br, guanidine); 6.29 (111, d, NHl); 4.22 (211, t, N-OH 2 4.08 (311, s. 0-C- 3 3.86 (2H, t, 0- C-1) 3.2SH ,CX .2 (H r 512 1 8 1 2 (0 ,m

CPH

2

O'YH

2 (012)3).

13C NMR (00013, 400MHz, 8 in ppm); 205.9, 175.8, 160.6, 157.7, *see*: 20 155.8, 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 132 0 C) =1480C Rf= 0.35 (40% methanol-ethyl acetate) Example 53 Synthesis of N-5- (2-chloro-6-rnethoxypurin-9yl)-pentoxycarbonyl-D-Arginine Compound #53 0. CH 3 N N CI N N 0 0 H N CO0O0H N VH HN1 NH 2 9 9 a a a.

a a a a 9* a a a a a a. 11HNMR DMSO in ppm) 9.41 Cbs, 1H, C0011), 8.42 1H1, purine), 7.3-7.8 (bd, 4H, guanidine), 6.23 1H, NH), 4.08 3H, Cl- 3 3.87 2H, Cl- 2 4.18 2H, CH- 2 4.08 3H, CH 3 3.87 2H, CH 2 4.08 3H, CH 3 3.87 211, CH 2 3.61 (in, 1H, C(XI), 3.04 10 (mn, 2H, CH- 2 1.22-1.87 Cm, 10H, 5 X CH 2 in.p. Softens at 128'C, melts at 141 0

C

Rf: 0.45 (Methanol) Mass spectrum: M+ 471.

15 Example 54 Synthesis of N-5- (6-N,N-Dimethylaninopurifl-9yl)-pentoxycarbonyl-D-Ornithine Compound #54 N 1H NMR (DMSO-d 6 400OMHz, 5 in ppm) 8. 20 (1H, s, purine) 8. 12 (1H, s, pu-rifle); 6.21 (111, d, NH); 4.10 (211, t, NT-CH 2 3.87 (2H1, t, O-CH 2 3.59 (1H, m, CaH); 3.4 (br, N-(CH 3 2

NH

2 2.70 (2H, ini, C8H 2 1.9-1.2 (1011, m, (CH 2 3

CPH

2 CYF1 2 1 3 C NMR (CD 3 OD, 400MHz, 5 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 185 0 C) 189-190 0

C

Rf= 0.20 (methanol) I0 Example 55 Synthesis of N-5- N-Dimethylaminopurin-9yl)-pentoxycarbonyl-L-ornithine Compound *t 9 .9 .9 9 *9 9 9 0 i*9* *9*9 9e9* 9*9* 9 9 9 99** 9 9 9 9 9.

a I--,H 2

N

N

N N 0 N N0 N H 0 Spectral properties were identical with compound #54.

Example 56 Synthesis of N-5- (6-N,N-Dimethylarninopurin--9yl)-pentoxycarbonyl-L-Valine Compound 56 1NM?. (DMSO d 6 400 MHz, 5 in ppm) 8. 19 (111, s, purine) 8.16 (1H, s, purine); 6.30 (1H, d, NH); 4.13 (2H, t, N-CH 2 3. 87 (211, in, O-CH 2 3. 64 (1H, m, CaH) 3. 4 (br s, N- C11 3 2 1. 80 (2H, CH1 2 1. 25 (211, p, elI 2 0. 79 (3H, d, CYH 3 0.75 (311, d, CYH- 3 1.3C NMR (CD 3 OD, 400 Mfz, 5 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 140 0 C) 172-176 0

C

R= 0.20 (30% methanol-ethyl acetate) Example 57 Synthesis of N-5- N-Dimethylarninopurin-9yl)-pentoxycarbonyl-D-Valine Compound #57 Y N 0H *2 a..

a

C.

a 'a a *aa.

a at, *a.a *aaa a a 15 Spectral properties were identical with compound #t56.

Example 58 Synthesis of N-5- N-Dimethylarninopurin-9yl) -pentoxycarbonylethylainine hydrochloride Compound #58 1 N 1 H1IMR (5 in DMSO ppmn): 8.40 1H1, purine), 8.43 1H, purine), 8.04 (bs, 3H1, NH 3 4.24 2H, CIA 2 4.02 2H, C2,2.99 (in, 211, CIA 2 2.67 2H1, CH 2 1.83 (in, 211,

CH

2 1.62 (in, 2H,CH 2 1.28 (in, 2H, CH 2 Rf: 0.3 10% Methanol/Ethylacetate Mass spect-rum: M+ 321 ExaIMple 59 Synthesis N-5- (6-N-Mercaptopurin-9-yl) pentanol Compound #59 q

S

S

S

.555

S

S

S

*5

S

lHNMR (8 DMS0 in ppm) 8. 30 1H-, purine) 8.18 1H, C 2 t) purine), 4.34 Ct, 1H, OH), 4.15 211, CH 2 3.34 211, CH1 2 1.82 (xn, 21-1, Cl1 2 1.42 (in, 211, C11 2 1.24 (in, 211,

CH

2 Rf: 0.57 30% Methanol/Ethylacetate Mass spectrum: M+ 239 15 ExaMple 60 Synthesis of N-5-(6-N--Methylthiopurin-9--yl)pentanol Compound IHNMR (5 CDC1 3 in ppm) 74 1H, purine) 7. 95 111, purine), 4.27 211, CH 2 3.65 211, CH 2 2.74 (s,3H, Sell 2 1.94 21-, CH 2 1.60 (in, 211, CH1 2 1.43 2H,CH 2 1 3 CNMR (8 CD 3 0D in ppm): 163.23, 153.42, 149.96, 145.89, 132.48, 63.08, 45.60, 33.62, 31.24, 24.54, 12.29.

-97C Rf: 0.22 (Ethylacetate) Mass spectrum: Examle 61 253 Synthesis of N-4- (6-N-Chloropurin-9-yl) butanol -Compound #61

CI

NN

KN N O 'H NMR (CDC1 3 400 MHz, 5 in ppm) 8.66 (1H, s, purine), 8.16 (1H, s, purine) 4. 33 (2H, t, N-CH2), 3. 67 (2H, t, O-CH 2 3. 04 (1H, br s, OH) 2. 01 (2H, p, CH 2 1. 55 (211, p, CH 2 m 97 0

C

S

S

S S *5S*

S

*5S5

S

S..

S

5.

S

Example 62 Synthesis of N-4- (6-N,N-Dimethylaminopurin-9yl)-butanol Compound #62 3 C N. l 'H NMR (CDC1 3 400 MHz, 5 in ppm) 8.24 (1H, s, purine), 7.68 (1H, s, purine), 4.33 (2H, t, N-CH 2 3.89 (1H, br s OH), 3.64 20 (2H, t, O-CH 2 3.46 (6H, br, N- (CH 3 2 1.92 (2H, p, CL] 2 1.53 (2H, p, CH 2 m.p. 78'C Example 63 Synthesis of N-4- N-Dimethylaminopurin-9yl) -butoxycarbonyl-D-Arginine Compound #63 H 3 C N N H C 3 N

N\

N N H (C 0OH N -A rg in in e) H N H N NH 2 'H NMR (DMSO-d6, 400 MHz, 8 in ppm) 8. 20 (1H, s, purine) 8. 16 (1H, s, purine), 8.1-7.3 (4H, br, guanidine), 6.40 (1Hl, d, NH) 4 .16 (2H, t, N-OH 2 3. 91 (2H, t, 0-CH2), 3. 65 (1H, M, O 5H), 3.4 (6H, br, N-(CH 3 2 3.02 (2H, in, O 6H), 1.9-1.3 (811, m, m.p. (softens 850C) 140-1420C 9.

9 9 9.

0* 9

S..

9 9

S

9* 9* 9 9 9 9 9**9 9 9 9

S

9 9 9. *59959 9 10 Example 64 Synthesis of N-4- N-Dimethylaminopurin-9yl)-butoxycarbonyl-L-Arginine Compound #64 HG N N C H 3 N 0~ NH1 CO0O0H N f Ir (L -A rgin ine) 0 H N H N NH 2 Spectral properties were identical with compound #63.

m.p. (softens 850C) 139-1420C Example 65 Synthesis of N-6- (6-N-Ohloropurin-9-yl) hexanol Compound

CI

N

N\

N 0 'H NMR CDC1 3 in ppm): 8.69 1H, purine), 8.11 1H, purine), 4.27 2H, CH2), 3.58 2H, CH12), 2.21 (bs, 1H, OH), 1.91 2H, CH 2 1.43 21, CH 2 1.35 4H, 2xCH 2 13C NMR CDC1 3 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. 84-86"C Rf 0.5 10% (methanol/ethyl acetate) Mass spectrum: M 255 Example 66 Synthesis of N-6-(6-N,N-dimethylaminopurin-9yl)-hexanol Compound #66

H

3 C CH 3 N

H

0 N

N

e** S 15 H NMR CDC 3 in ppm): 8.35 11, purine), 7.71 11, purine), 4.17 2H, CH2), 3.61 2H, CH 2 3.53 (bs, 6H, 0 2xCH 3 1.89 2H, CH2), 1.71 (bs, 1H, OH), 1.55 2H, CH2), 1. 45 4H, 2xCH1 2 C3 NMR CDC1 3 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. 75-770°C Rf 0.48 10% methanol/ethyl acetate Mass spectrum: M 264 Example 67 Synthesis of N-6-(6-N,N-Dimethylaminopurin-9yl)-hexyloxycarbonyl-D-Arginine Compound #67 H 3 c N CH 3

N

N H H1N

NH

2 1 H NMR CD 3 0D in ppmn): 7. 98 1H, purine) 7. 81 1H, purine), 3.98 211, CH 2 3.78 (in, 3H, CH- 2 and C aH), 3.27 (bs, 6H, 2xCH 3 2.96 2H, CH 2 1.1-1.78 (in, 12H, 6xCH 2 Example 68 Synthesis of N-6(6-N,N-Dimethiylaminopurine-9yl)-hexyloxycarbonyl-L-Arqinine Compound #68 99 9 9, 0 *9

S.

9

S..

6.

9 9 9 9 9 9 9994, 9 9 *999 0999 9 9 9 9999 99999* 9 9 9..

9 9999**

S

N 0,NH, COOH 0

NH

HN NIfl 2 H N4MR CD 3 ,OD in ppm) 8.00 1H, purine), 7.82 1H, purine) 4.00 2H, CH 2 3. 80 3H, CH 2 and C aH), 3.29 (bs, 6H, 2xCIII) 2. 97 211, CH 2 1. 13-1.72 (in, 12H, 6XCI 2 Example 69 Synthesis of cis- (6-N-FHydroxypurin-9-yl) -4inethyi-cyclohexyl-inethanol Compound #69 O1il 'H NMR (CD 3 OD, 400 MHz, 8 in ppm): 8.08 (1H, s, purine), 8.07 (1H, s, purine) 4. 21 (2H, d, N-CH2), 3. 49 (2H, d, O-CH2), 2. 16 (1H, m, CH), 1.7-1.2 (9H, m, CH,2-cyclohexane, CH).

in.p. >200 0

C

Rf= 0.3 (20% inethanol -ethyl acetate) Example 70 S

S@

S

S..

5 0 0 *5e0

S

S

*5*0

S

.5 Synthesis of cis-(6-N-Hydroxypurin-9-yl)-4mie thyl-cyclohexyl-metlhyloxycarbonyl-D-Arginine Compound 0OH N

N

15 'H NMR (CDOD- 400 MHz, 5 in ppm): 8.10 (1H, s, purine), 8.09 (1H, s, purine), 4.24 (2H, d, 3.65 (1H, mn, CxH), 3.52 (2H, d, O-CH,) 2. 95 (2H, in, C 5H2), 2.2-1.2 (14H, mn, 2xCHcyciohexane, 4xCH-cyclohexane, C H2, C'112) Example 71 Synthesis of trans- (6-N-Hlydroxypurin-9-yl) -4methyl-cyclohexyl-inetLhyiloxycarbonyl-D-Arginine Compound #71 NN Ni Ir 0 1H NMR (CDOD, 300 MHz, 6 in ppm): 8.09 (211, s, purine), 4.12 (2H, d, N-CH 2 3.68 (1H, m, COH), 3.36 (2H, d, O-CH 2 3.01 (2H, m, C 8

H

2 2.0-0.9 (14H, m, 2xCH-cyclohexane, 4xCH 2 cyclohexane,

CPH

2

C"H

2 rn.p. >200 0

C

Rf= 0.2 (methanol) Example 72 Synthesis off N-5- N-Dimethylamiinopurin-9yl)-pentylamine hydrochloride salt Compound #72 0 0 so*.

0

S..

00* 0 0 0 1H NMR DMSO in ppm): 8.20 1H, purine), 8.16 1l1, purine), 7.84 (bs, 3H, NH 3 4.14 2H, Cl-I 2 3.44 (bs, 6H, 2 xCHA). 2 73 2H, CHA)1 1. 81 (in, 2 H, Cl-i 2 1. 56 2 H, CH 2 1.25 (in, 2H, CH 2 Example 73 Synthesis of N-5- (6-N-Methylaziridinepurin-9yl) -pentyloxycarbonyl-L-Arginine Compound #73 "h1 7 N N

N

NN

KN N 0 0 -k N0 0 Spectral data of compound #73 was comparable to that reported for compound #39.

Example 74 *9 9 9 9.

9.

.9 *99 9.

*9 9 .9 9 9 9 9 9 9 99..

99.9 9 9 9.9 i 9 9 9999 9 9 9 9 9 9 99 999999 0

N>

(2S, 43)-2- (6-N,N-Dimethylaminopurin-9-yl) -4hiydroxymethyl-1,3-dioxolane -Compound #74 N 1iiBu 4

NFN

N

N

OsitBLuPh 2 01-\01 0 11H NMR (CDCl 3 5 8.32 1H, purine), 7.75 1H, purine), 5.33 (dd, 1H, J 2.0, 6.6, H-2-dioxolane), 5.33 (bs, 1H, OH), 4.45 (dd, 1H, J 6.6, 14.3, CH 2 -purine), 4.20 (dd, 1Hi, J 2.0, 14.3, CH 2 -purine), 4.20 (in, 1H, H-4-dioxolane), 4.05 (d, 2H, J 7.2, 3.78 1H, J 13.0, CH 2 3.53 (bs, 15 611, (CH 3 2 3.40 1H, J CH 2 -Ol)- Example 75 C1S,3R) and (lPR,3S)-l-(6-N,N- Dimetlhylaminopurin-9-yl) methyl-3--cyclopentane methanol -Compound 79

OM

N

N

N

N nB'1 4

NF

TfHF OS ItfuPh 2 N-

N

N

N

OH

NHDMR (CDC1 3 300 MHz): 8 8.32 111, purine), 7.71 1H, purine), 4.18 (dd, 1H, J= 8.6, 13.7), 4.06 (dcl, 1H, J= 6.7, 13.7), 3.61-3.53 (in, 8H), 3.00 (bs, 1H, OH), 2.48 2.17 (mn, 1H), 1.88-1.68 Cm, 3H), 1.53 (in, 1H), 1.43 (in, 1H), 1.08 1H).

Example 76 0 *0

C.

.00 0 0 0* 0 0 0 0 0 0 *00* *000 *0 *00* *000 0 *000 *000** 0 000.

0 0 *0 0 (1S,3R) and (lR,3S)-1-6-N,N- Dimethaylaminopurin-9-yl) methyl-3- Cmethyloxycarbonyl-D-Arginine) cyclopentane Compound #76

-N

NN

N

7 C 1) COCI 2

THIF

2) D-afginine 1N

N

l-NFI 2

NH

FF\

Example 77

N

N

N';

(6-Nq,N-Dirnethylaminopurin-9-yl) -7ethylaminoethanol Compound #77

N

H-1 2 Pd/C 10% McOH

N

NI\

N

N

0OH

H

ICBZ

'H1 NMR (CDC1,, 400 MHz) 5 8.34 1H, purine) 7.79 1H, purine) 4.29 2H, J= 5. 8, -C- 2 3. 62 (in, 2H, -CF1 2 3. 54 (bs, 6H, (CH 3 2 3.11 2H, J=5.8, -CH 2 2.81 2H-, J= 2, -CH 2 2. 05 (bs, 2H, NH and OH) 9* 9 9 9 9 99 9** 99 9 99 9 9 9 9 9 99 9 9 09 9 9999 9 9 99.9 9 9 *999 9 999.

9 9 9999 9 9 *9 9 99 999.9.

10 Example 78 N-Diinethylarninopurin-9-yl) -7etlhylaininoethoxycarbonyl-D-Arginine Compound #78 1 1 N N N CBZ 0 il N NHl 2 112: Pd/c 10% MeOll

N

N) N N COOHI 11 0 Nil N )Nil 2* 15 'H1 NMR (CDC1,, 300 MHz) :5 9. 36 (mn, 1H) 8. 19 ilI, purine) 8. 11 1H-, purine) 6. 38 1H, J=7 NH carbamate) 4. 17 2H, J= 6.1, 3.91-3.8-7 (mn, 21-1, 3.65 (mn, 111, CH-COOH) 3.43 (bs, 6H, (CHA)2N) 3.03-3.01 (mn, 2H, CH 2 NHC (NH) NH,) 2. 90 2H, J= 6. 1, -CH2-) 2. 68 2H, J= 5. 6, -CH2-) 1 1 .64-1 .44 (ri, 4H, CH2-CH2-CH 2 NHC (NH) NH2) Example 79 N-Dimethylaminopurin-9-yl) -7ethylaminoethoxycarbonyl-L-Arginine Compound #79 N 1 NeIN N N CBZ N0

NNH

2 1 1-12, Pd/C 10% MeOH I N II N

N

N COOH 110

NH-

N Nf 2 1-1 Spectral properties were identical with compound #78.

9 9 9e

B..

B B B B B. B B

B

B B B

B

B

*BB.

B

B

B B..B

B.

9 B B B

B.

Example 80 N-5-(6-N,N-D 1-al Coxnpo

N

N N T.

I- OSiluh 2 imethylaminopurin-9-yl) -3-pentynund 3u 4 Nr IF, OOC '-1N

N

KN

1H NMR (CDCl 3 5 8.34 1H, H-2 purine) 7.89 1H, H-8 purine), 4.91 (in, 211, CH 2 3.74 2H, J 6.2, CH 2 -OH1), 3.52 (bs, 6H, (CH1 3 2 2.87 (bs, 1H1, OH), 2.50 (in, 2H, CH 2 15 CH 2

OH).

82 2b~

-I

Example 81 N-5-C6-N,N-Dimethylaminopurin-9-yl)-3pentynyl-l-oxycarbonyl-L-Arginine Compound N N 1) COCI,THF 2) L-argiiiii

NY

N y NH 2

NII

0 0 N COOI

H

OH

Spectral properties were identical with compound Example 82 (6-N,N-Dimethylaminopurin-9-yl)-7-thioethoxyethanol Compound #82 00 0c 00 0 00 0r 0 0000 0 0 00 0 0 0 0 0 0 0 0 0000 00 0 00

IN

N N S ,-,,_,,0SI3Uhh iiBu 4

NF

TIIF

\N

-NN

N

N

N

S~~0 1 H NMR (CDCl 3 5 8.30 1H, purine), 7.75 1H, purine), 4.41 2H, J 6.5, CR 2 linker), 4.11 (bs, 1H, OHf), 3.73 (t, 2H, J 6.5, CI 2 linker), 3.51 (bs, 6H, (CH 3 2 2.99 2H, J CH 2 linker), 2.68 2H, J C- 2 linker).

83 FF\Cr

-I

C

Example 83

N

Ne ICN N

I

S

(6-NN-Dimethylaminopurin-9-yl)-7-thioethoxyethoxycarbonyl-L-Arginine Compound #83 1 1 N 1-1 1) COCI TIT N N 21 2) L-arginine I I N N S y 0 NI-1 N NI-1 2 1-1 Spectral properties were identical with compound Example 84 V.us 00 a Soo.

0 0: 0 0 0 *too **to 000000 *Gore: 9 06 (2S,4S)and (2R,4R)-2-(6-NN- Dimethylaniinopurin-9-yl)-4-(methoxycarbonyl-D- Arginine)-1,3-oxathiolane Compound #84

N

N

N

N

Oil

S

1) COCI 21THF 2) D-argiiiine N rN CNH 2 N N N11 I Y 0 0 WIC001-1 SJ I I I 1H NMR (CDC13, 300 MHz): 5 8.49 1H, purine) 8.47 Iff, purine), 6.60 (bs, Iff, NH carbamate), 6.22 1H, H-2oxathiolane), 4.26-4.03 3H), 3.63-3.00 11H), 2.78-2.69 15 (in, 2-H, H-5-oxathiolane), 1.53-1.40 4H, CH2-CH2-CH2NH- C(NH)NH2).

Example 85 (6-NN--Dirnethylaminopurin-9-yl)-7-ethoxyethoxyethanol Compound 84

N

L N 0 ~OSitBtPh 2 O1u 4

NF

TIIF

N

N

.N.0 1 11i NMR (CDC1 3 5 8.29 11H, purine) 7.80 1H, purine), 4. 33 211, CL!f 2 3. 82 211, CH 2 3. 68 2H, C-1 2 3. 2H, CR 2 3.50 6H, N(Cfi 3

S

a..

S

*5*e

S.

S S S S

S

a..

S

a.

C

S.

S

Example 86 N-Dimeth-ylaminopurin- 9-yl) -7-ethoxyethoxycarbonyl-D-Arginine Compound #86 N 1-1N N N\1) CO C ITIF NJN\\ LN N 2) D-arginineI N N 'C0011 NIl IJ NIl 1H NMR (DMSO-d 6 8.25 1H, purine) 8.14 Cs, 1H, purine) 6. 5 (bd, 1H, NH carbarnate) 4 .37 2H, C11 2 linker) 4.03 211, CR 2 linker), 3.81 2H1, CR 2 linker), 3. 72 (m, 1H, CH-COOH), 3.60 211, CR 2 linker), 3.55-3.89 611, N (CH 3 2 3.05 2Hf- CH 2 -NH-C(NH)N1 2 1.78-1.39 411l,

CH

2

-CH

2

-CH

2 NH-C (NH) NH 2

-II

Example 87 et

N

N

N

i-N, N-Dimethylaminopurin-9-y1)-7-ethoxy- :hoxycarbonyl-L-Arginine Compound #87 1) COCI 2

TI-IF

2) L-argimie N

N

_N N iCO 0

NI-I

Spectral properties were identical with compound #t86.

Example 88 9 *9 *9 9 9 9 9 9 N-5- C 6 -N,N-Dimethylamino-8-bromopurin-9-yl) pentanol Compound #88 1-1N N 1:CN N

N

I-,N'I,

NIN.

Acetate buffer, 0.5M N a+ p H

N

Br

NO

'H NMR (CDC1 3 400 Mliz): 8. 27 1H, purine) 4. 18 Ct, 211, J=7.2, 0112) 3. 64 Ct, 2H, J=6.3, CH 2 3. 48 Cbs, 6H, NCCH 3 2 ),r 2.02 Cbs, 1H, OH), 1.85 (quint, 2H, J=7.2, CH 2 1.63 Cquint, 211, J=6.3, CH 2 1.46 (in, 2H, OH 2 Pharma 47 PCT BIOLOGICAL

DATA

IN VITRO SCREENING: Example 89: Mitogenic Proliferation on Spleen Cell Suspension Mitogenic lectin (mitogen) is a protein which binds and cross-links specific cells surface carbohydrate in determinants, 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 vitro 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.

20 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, H thymidine incorporation was recorded as an indication of cell transformation and proliferation.

100 p1 of a suspension of 2X10 6 cells/ml (2xl0 5 cells/well) were incubated in presence of PHA or ConA or PWM or LPS at the following concentrations: PHA 0.01% final concentration 0.001% ConA 2 g/ml 1 jg/ml PWM 0.2x 0.02x LPS 5 .ig/ml 2 pg/ml Date: June 16, 1995 87 Time: 02:59 PM Pharma47PCT Cells were incubated in presence or absence of drugs for 72 hours. 0.5 or 1 ICi 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 counter.

TABLE 1 (Mitogenic Proliferation) Compound No. T (ConA)(M)

B(LPS)(M)

ST-789 0 1.5-3x(10 -10- 5 #1 2-4x(10-8-10 s 2x(10- 6 #2 2-4x(10- 8 -10 5 0 #3 2-6x1012-10- 6 2-2.5x(10- 8 -10- 6 #7 2-6x(10- 1 2 -10- 8 0 #9 2-3.5x(10- 12 -10 6 I. Example 90: Cytotoxic T Lymphocytes (CTL) and Mixed Lymphocyte Reaction (MLR) Assays *e o Mixed lymphocyte reaction is an in vitro 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 vitro in culture. To have a unidirectional response, the proliferation of either cell type may be blocked with Xirradiation or mitomycin C treatment.

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

Date: June 16, 1995 Time: 02:59 PM Pharma 47 PCT 3 C57B1/6 mice and 3 DBA/2 mice wre killed and lymphocytes prepared using lympholyte M. The cell concentration was adjusted to 10x10 6 cell/ml for each lysis. DBA/2 cells were irradiated with 3000 Rads. 1 ml of 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 gi of cell suspension were deposited in each of the 96 wells in the plate, 50 pl of thymidine at 20 Ci/ml was added for 6 hrs. The cells were then collected and counted using a beta counter.

CTL

P815 target cells were labelled with Cr 51 After labelling, the cells were resuspended to 5x10 4 cells/ml.

Effector cells were adjusted to 2.5x10 6 cells/ml, and then diluted 1:2 and 1:4 to obtain the necessary effector to target ratios: 50:1 (2.5x10 6 cells/ml: 5x10 4 cell/ml) 25:1 (1.25xl0 6 cells/ml: 5x10 4 cells/ml) 25 12.5:1 (0.625x10 6 cells/ml: 5x10 4 cells/ml) 100pi of target cells 100l Of T cells were incubated for 4 hrs and then 100pl of supernatant was counted using a gamma counter.

Date: June 16, 1995 89 Time: 02:59 PM Pharma 47 PCT TABLE 2 (CTL and MLR Assays) Compound No. CTL I MLR (M) ST 689 (10-7 M)P 1.6x (10- 5

M)

P

ST 789 (10 7 2-3x(10- 9 #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 1 0 9 1.5-4x(10 9 #2 0 1.5-2x(10- 7 -10- 5 #3 (10-s) 1.5x(10-9-10-s5) #3a (10-9-10- 7 M) 1.5-2.9x (10-9-10- 5

M)

#5 (10- 7 1.5-2x(10-7-10-5) #5a (10-7-10- 5 M) 1.5-2x (10-9-10- 6

M)

#6 (10- 9 1.5-3x(10-10- 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(10-7-10-5) #19 (10-7 M) P 0O (10s #51 (10-7 M) 1.5-2x (10-9-10- 6

M)

#59 (10-9 M) 2-2.4x (10-9-10- 5

M)

(10-7 M) 1.7-2x (10-9-10- 5

M)

For CTL Activity, the data expressed is as a increase compared to IL-2.

IL-2 is 100%. 0 represents less than 20%, represents 20-40%, represents 40-60%, and represent 60-80%, and represents 80+. P Preliminary result Date: June 16, 1995 Time: 02:59 PM Pharma 47 PCT IN VIVO/EX VIVO SCREENING 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 animals plus animals. Cell immunophenotyping was performed on mousetreated blood and spleen. The following cell surface antigens were analyzed: CD3 (all T cells), CD4 (T helper/inducer, binds class IIrestricted T cells), CD8a (cytotoxic T cells, CTL adhesion), CDlla B, NK, some stem cells, CTL adhesion 15 anti LFA-la), 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 20 consecutive days, sacrificed at day 5 and immunophenotyping was performed on blood and spleen cells.

*ee The cells were washed twice in PBS, resuspended in 1 ml of RPMI 2% FBS, and incubated for 45 min. on ice with 25 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.

Date: June 16. 1995 91 Time: 02:59 PM Pharma 47 P'CT 4 4.

*4 4 0 S 0 *0*0 4* S 0

S.

000 4* *5 S TABLE 3a Iwmunophenotyping On Blood Cells of Compound #1 Treated-Mice Cell marker Control 25 mg/kg 50 mg/kg CD8+ mean 6.66 10.11 8.65 0D45+ STD 2.09 2.69 1.39 p 0.05 0.005 NK+ mean 3.03 5.84 3.25 CD3+ STD 0.76 2.08 0.57 p 0.02 0.289 mean 9.60 13.71 9.68 CDIlb+ STID 2.79 2.68 3.59 p 0.015 0.4 TABLE 3b Izmunoplenotyping on Spleen Cells of Compound #1 Treated-Mice Cell marker Control 25 mg/kg 50 mg/kg mean 38.93 39.78 45.09 TCR+ STD 3.83 7.61 7.34 P 0.37 0.034 4* 4 4 04 S S *4 Date: June 16, 1995 Time: 02:59 PM 4

S.

S

6S

S

55 4 .5 S S 5.

CO S *5 S S p Pharma 47 PCT TABLE 4 Immunophenotyping On Blood Cells Of Mice Treated With Compound #1 In Combination With Cyclophosphamide (N=4) cyclophosphamide CY cpd #1 CY cpd #1 Cell marker 100 mg/kg 25 mg/kg 50 mg/kg mean 15.05 13.25 20.8 0D8+ STD 3.89 0.07 0.85 0D45+ p 0.33 0.05 Spleen: no effect TABLE Immulnophenotyping on Blood Cells of Mice Treated With Compound #1 In Combination With 5 Fluorouracil (N=4) 1 5FU 5 FU cpd #1 5 FU +cpd #1 Cell marker]j (80 mg/kg) 25 mg/kg 50 mg/kg mean 6.66 10.11 8.65 0D8+ STD 2.09 2.69 1.39 0D45+ 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

S.

S.

.5 5 a

S.

Date: June 16, 1995 Time: 02:59 PM Pharma 47 PCT TABLE Immunophenotyping On Spleen Cells Of Mice Treated With Compound #1 In Combination With 5 Fluorouracil (N=4) 5FU cpd #1 5FU cpd#1 Cell marker (80 mg/kg) 25 mg/kg 50 mg/kg CD4+ mean 10.0 13.19 12.06 STD 1.98 3.19 2.27 p 0.015 0.04 mean 4.22 3.32 3.17 NK+ STD 0.5 0.45 0.36 p 0.0005 0.0001

IL

1. ANTITUMOR ASSESSMENT PROTOCOL The compounds were tested for tumor growth control using the following procedures.

Example 92: e Effect of compound #1 on growth of breast carcinoma in combination with cyclophosphamide.

Balb/C Mice (n-5/Gr) were used along with DA-3 mammary carcinoma cell line. The mice were treated from -2 to 13 days. Animals were monitored for tumor takes/tumor size and body weights for three weeks from Day 0 until Day 21.

DO was the day of tumor cell inoculation and D21 was the day of experiment termination.

Parameters of effect were measured by inhibition of tumor outgrowth and growth rate [tumors measured along the Date: June 16, 1995 94 Time: 02:59 PM Pharma47PCT longest axis (length) and the perpendicular shortest axis (width) and the tumor volumes was calculated by the formula T.V.=length (cm) x (width cm) 2 assessment of body weight loss.

The statistical significance of difference between tumor takes and tumor sizes of control-untreated and drugtreated 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.l Normal Saline (0.2 ml/mouse i.p. starting at D2) 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) 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.

9.

.9 9* 9* Date: June 16, 1995 Time: 02:59 PM Pharma 47 PCT TABLE 6 Effect of compound #1 treatment on Tumor Outgrowth Group/Day 4 6 8 Gr.1: saline 5/5* 5/5 5/5 Gr.2: CY 100mg/kg 5/5 5/5 5/5 Gr.3: #1 25mg/kg 2 2/5' 3/5 Gr.4: #1 50mg/kg 3/5 3/5 3/5 CY 100mg/kg #1 50mg/kg 4/5 5/5 5/5 Tumor takes= tumor-bearing mice/total of mice 5 t p<0.05 by Chi-square test a a. Example 93: Evaluation of Compound #1 in combination with cyclophosphamide (cytoxan) (CTX, mg/kg) against DA-3 mammary 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 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).

Date: June 16, 1995 96 Time: 02:59 PM S- Pharma 47 PCT Example 94: Evaluation of Compound #1 in combination with cyclophosphamide (cytoxan) (CTX, 28 mg/kg) against DA-3 mammary carcinoma.

In another experiment, the CTX treatment was prolonged.

Balb/c mice were injected s.c. with 5 x 10 5 DA-3 tumor cells at day 0. At day 4 when established tumors appeared, tumor-bearing animals were randomized (n=ll/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 15 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).

9 Example 95: Evaluation of compound #1 in combination 20 with 5FU against colon adenocarcinoma.

C57BL/6 mice 6-8 weeks old (n=7-9/gr) were injected with 3x10 5 MC38 colon adenocarcinoma cells s.c. on day 0. On day 7, tumor-bearing mice were randomized and injected with 5FU at 20 mg/kg either alone or in combination with levamisole at 20 mg/kg i.p. or with compound #1 at 25 and mg/kg i.p. over a four week period. During this period, animals were treated for 5 consecutive days, untreated for 2 days, and treated again for 5 consecutive days per week for 4 weeks.

Date: June 16, 1995 97 Time: 02:59 PM SPharma47PCT Results of this experiment show a significant dosedependent-anti-tumor effect following compound #1 (at and 25 mg/kg) 5FU (20 mg/kg) compared to control untreated group (Figure The anti-tumor effect of 5FU Compound #1 (at 50 mg/kg) was markedly better than that of 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 of this study was to find the toxic dose of compound #1 after repeated intravenous injections in 15 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 20 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/100g 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 examination 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 Date: June 16, 1995 98 Time: 02:59 PM Pharma47PCT minutes. With 500 mg/kg, all rats were observed to have twitches of the torso area, tremors of the forepaws and jumping episods. These signs lasted less than 1 hour and were comparable after each of the five injections. The growth curves of the animal 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 No drug induced changes were noted on necropsy of these animals.

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 15 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 animals.

20 CONCLUSIONS From the data, in vitro, the compounds of the invention, in particular compound appears to activate T cells (including CTL's) and B cells.

In vivo, the compound of the invention, in particular compound increases the number of CTL's.

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

Date: June 16, 1995 99 Time: 02:59 PM Pharma 47 PCT Compound #1 appears to inhibit tumor outgrowth in combination with cyclophosphamide against mouse mammary carcinoma in vivo.

Compound #1 appears to inhibit tumor outgrowth in combination with 5FU against mouse colon adenocarcinoma in vivo.

C

a. r r u Date: June 16, 1995 Time: 02:59 PM *a .e ac e. S e a a* a .e a. a a a a. a. *a.a a. e.a a a.

.e a a 89Q Table 7 balb-c mice, 5.OxlQ sc DA-3 cells p #28, testing compound #1 with Cytoxan injections of cells April 17th, treatmnet with compound #1 started on April measurement and treatment with Cytoxan started on April 2lth, 1995, Data of tumor sizes including mice with tumor only DAY 4 7 9 1 4 1 6 2 3 2 8 3 Or #1 nin 1p WDays 3-28) 3.5 11.s 20.3 34.4 01. 00.5 77.3 88.8 127.3 100.7 1419 200.9 aer 1.37 2.11 3.22 3.70 7.30 0.00 7.51 10.39 13.92 22.70 21.0 21.5 mlce number n- I i/l I n-11/l1 n-il/l11 n-ll/11 n-li11l n-11111 "11l/11 n-11111l n-11/1i n-Il11lI n-Il1Il no-Il/li Or #2 0TX 28mg/kg 1p (Days 4, 11, 18) 4.2 10.5 18.4 30.7 50.0 5 4.5 53.4 88.0 83.4 100.3 130.0 11.0 Sam 1.45 2.37 3.81 4.83 7,93 0,88 8.43 8.80 8.48 8.81 12.42 13.08 p Volve 0.7017 0.7422 0.7392 0.5500 0.3110 0.1182 0.2337 0.4220 0.0603 0.0372 0.0618 0.0463 mice number n-iil l/li n -Il/il1 n- il/l1 n-I1111I1 n-111 l 1/Il n"111n111 n- li n11/11l n-ll/Il "11l/111 no-il/11 Or# 3 OCH-1393 50mg/kg 1v (days 3-281 2 .7 10.2 18.1 28.2 50.1 58.5 08.3 84.2 131.4 130.0 162.5 1 87.2 gemr 1.01 2.87 4.40 0.82 10.21 12.O 15.45 18.03 30.55 28.51 28.05 ;5.01 p value 0.0480 0.7141 0.0927 0.5044 0.358 0.4480 0.6310 0.7030 0.0174 0.4335 0.4087 0.63&3 mice number "1i1111 n-11I/11 n-1ilI n-Il/l11I n-11/1I "1111/i n-Il11Il naill In-ll/Il n-il/Il nv-Il/Il n-Il/Il Or#4 BCH-1393 50mg/kg IpINays3.28) CTX 28mg/kg 1v (days 4, 11, 18) 3.o a.5 12.8 20.2 128.5 41.7: 44.8 83.4 100 85.4 104.2 110.11 gemr 1.40 1.85 3.44 3.77 5.58 0.40 0.52 0,50 9.90 12.57 13.311 4.05 P value 0.6825 0.0083 0.11M 0.0001 0.0014 0.0031 0.0025 0.0102 0.0010 0.0050 0.0030 .01 mice number n-Illi n-li/il in-li/Il naIC/Il n-10C/1I n,-I0C/l no-IC/l n-1111 n-10IlnI1 Iln11Il1SOI Table 8 balb-c mice, 5.OxlO 5 sc DA-3 cells p #28, testing compound #1 with Cytoxan injections of cells April 17th, treatment with compound #1 started on April m~easuremnent and treatment with Cytoxan started on April 21th, 1995, L;ita of body weight (including every mice in the group) DAY__ 4 7 9 11 14 16 18 2123 2528 Or #1 sallne 1p (Days 3-201 58 2 19,7 18,5 18.0 18.2 10.8 18.5 Mi.0 18.7 18.0 18.$ 20.0 earn 0.25 0.34 0.32 0.30 0.31 0.31 0.27 0.27 0.20 0.28 0.26 0.31 mice number r-I1 -lI n-li /Il n- li/Il n- li/Il n-l/Il n-li/il1 n-il/li n-111 -11 il/li1 n-li/il n-li/Il n-li/il Or CTX 28mg/kg 1p (Days 4, 11,18 18. 9 2 19.8 19.8 19.8- 18.2 18.7 18.0 18.8 18.7 20.1 20.3 20.4- Gern 0.22 0.28 0.31 0.40 0131 0.28 0.27 0.25 0.28 0.27 0.20 8.38 p Volue 1.0000 0.8471 0.4468 0,0040 1.0000 0.8403 0.8208 0.0400 1.0000 0.51141 02721 0.431201 mile number n-Il/Iil n-Il/i1 n-Il/Il n-Il/li n-MIlI n-Il/1I n-li/11I ni-Il/il nai1/Il n-i/li n-li/Il1 n-IilI 3 BCH-1393 50mg/kg IV (day, 3-28) 18.0 19.1 18.3 18.8 18.8 18.5 18.8 18.3 18.3 18R.5 18.7 20.0 gam 0.27 0.33 0.38 0.33 0.33 0.35 0.37 0.32 0.34 0.35 0.30 0.41 P valve 0.1704 0.-2144 0.7362 0.1308 0.5701 0.5830 0.2023 0,4121 0.3254 0.6087 0.5530 1.000 mice numb., n-111/i1 n-1l/I1 n-Il/il n-I11/Il n-Il/Il n-Il/li n-W/li n-Il/Il n-Il/il n-li/il n-il/Il n-Il11li G4#4 BCH-1393 50mg/kg Ip(Doys 3-28) CTX 28mg/kg 1v (days.44,11, 181)- 18.7 19.2 19.1 18.0 10.0 18.5 10.3 18.8 181.8 18.5 19.4 18.3 earn 0.28 0.17 0.10 0.18 0.23 0.15 0.18 0.20 0,17 0.20 0.23 0.20 p value 0.2721 0.1904 0.3403 0.0854 0.1847 0.3203 0.4353 0.0500 0.0117 0.4582 0.2201 0.1060 mice number n-111111 n-Il/i i n-li/li n-i011Il n-IC/I n-IC/Il n-1/11 n-IC/Il n-10II n-IC/ilI n-i101il n-iC/Il

Claims (48)

1. A compound of formula I: R N N R 4 or a pharmaceutically acceptable derivative thereof, wherein R, is substituted amino represented by formula NR 5 "R wherein R 5 and R' are 10 independently selected from the group consisting of hydrogen, C 1 4 alkyl, C.4 alkoxy, C1-4 ayl, substituted or unsubstituted amino and C.-o aryl, with the proviso .that R and R6 are not both hydrogen or amino; R" and R 6 can also be connected to the nitrogen atom to form a saturated C. 3 6 heterocyclic ring 15 optionally substituted with C,4 alkyl; R2 and R 3 are independently selected from the group consisting of hydrogen; C.4 alkyl; amino; substituted or unsubstituted thiol; and halogen; and R 4 is represented by the formula R 1 -X 12 wherein R 12 is a saturated or 0 unsaturated linear hydrocarbon chain of 5-20 carbons optionally containing one or more interruptions within the chain by a heteroatom, and optionally substituted with one or more or and X 12 is selected from the group consisting of an aminoalkyl group, a known amino acid bound by its a-amino group or a peptide of 2-8 amino acids. 25 2. The compound according to claim 1, wherein R 4 is (CH2)-L-O-CO-X 2 wherein L is selected from the group consisting of (CH2)j-Q-(CH2)m, and (CH 2 2 wherein Q is O, S, or NH, n is an integer between 3 and 6, and m is an integer between 1 and 3.

3. The compound according to claim 1, wherein X' 2 is (CH2)m-NH 2 wherein in is an integer between 1 and 6.

4. The compound according to claim 3 wherein in is 2.

5. The compound according to claim 2, wherein X 1 2 is a naturally occuring L-alpha amino acid or its D-stereoisomer.

6. The compound according to claim 5, wherein said amino acid is selected from the group consisting of: arginine, glycine, alanine, glutamic acid, valine, ornithine, or citrulline or conservative substitutions thereof.

7. The compound according to claim 5, wherein said amino acid is selected from the group consisting of: arginine, glycine, alanine, glutamic acid, valine, ornithine, or citrulline.

8. The compound according to claim 6, wherein said amino acid is D- arginine.

9. The compound according to claim 6, wherein said amino acid is L- arginine.

10. The compound according to claim 2, wherein X 12 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, Ile-Pro-lle.

12. The compound according to claim 1, wherein R4 is (CH 2 wherein L is selected from the group consisting of phenyl, cyclohexyl, dioxolanyl, oxathiolanyl, and cyclopentyl. 25 13. The compound according to claim 1, wherein R 1 is selected from the group consisting of: 0. -N(CH3),, -NHNH 2 -NHCH 3 30 -N(NH2)CH 3 -NH-CH(CH 3 )CI- 2 -O-(CO)CH 3 N-- ,or NH wherein R 2 is H or methyl.

14. The compound according to claim 1, wherein R 2 and R3 are independently selected from the group consisting of Cl, Br, I, and F. The compound according to claim 14 wherein R2 and R3 are independently Cl or Br.

16.

17.

18.

19. 20 3 .0 3: 5 0 The compound according to claim 15 wherein R 2 is Cl. The compound according to claim 15 wherein R 3 is Br. The compound according to claim 1, wherein R 2 is NH 2 The compound according to claim 1, wherein R 3 is SH or SCI- 3 A compound selected fromn the group consisting of: Compound #111 N-5-(6-N-Chloropurin-9-yl)-pentanol Gomnpound #V N-5 -(6-N,N-Dimethylaininop urin-9-yl) pentanol Compound #1 N-S -(6-N,N-Dimethylaxninopurini-9-yl) pentoxycarbonyl-D-Argiiniiie Compound #2 N-5-(6-N,N-Dimethylaminopurini-9-yl)- pentoxyc arbonyl-L-Arginine Compound #3 N-S -(6-N-Me thylaminiopurini-9-yl) pen toxycarbonyl-D-Arginine Compound #3 a N-S -(6-N-Me thylaninopurin-9-yl) pentanol Compound #4 N-5-(6-N-Methylaininopurini-9-yl)- pentoxycarbonyl-L-Arginine Compound #5 N-5-(6-N-Aminopurin-9-yl)- pentoxycarbomiyl-D-Arginine Compound #5 a N-S -(6-N-Aminopurin-9-yJ -pentanol Compound #6 N-5-(6-N-Aminopurin-9-ylJ- pentoxycarbonyl-L-Arginine Compound #7 N-5-(6-N-Hydraziniopuriin-9-yl)- p entoxyc arbo nyl-D-Arg inine Compound #7a N-5-(6-N-Hydraziniopurini-9-yl)-pentaiioI Compound #8 N-5-(6-N-Hydrazinopurini-9-yl)- pe ntoxycarbonyl-L-Arginine; Compound #9 N-5-(6-N-Chloropurin-9-yl)- pen toxycarbonyl-D-Arginine; Compound #10 N-5 -(6-N-Chloropurin-9-yl)- pentoxycarbonyl-L-Arginine; Compound #11 N-5-(6-N-Hydroxypurin-9-yl) pentoxycarbonyl-D-Arginine; Compound #12 N-S -(6-N-Mercap topurin-9-yl)- pentoxycarboilyl-D-Arginine; Compound #13 N-5-(6-N-Mercap topurini-9-yl) pen toxycarbonyl-L-Arginine; Compound #14 N-5-(6-N,N-Dimethylamninopurin-9-yl)- penitoxycarbonyl-Glycine; Compound #15 N-5-(6-N,N-Dimethylaininopurin-9-yl)-7'-ethoxy- ethoxycarbonyl-D-Argininie; Compound #16 (2S,4S)-2-(6-N,N-Dimethylaminiopuriin-9- yl)-4-(methyloxycarbonyl-D-Argininie)-1, 3-dioxolane; Compound #17 N-5 N-Dime thylarnino-8- bromop urin-9-yl)-pentoxycarboniyl-L-Argininie; Comipound #18 N-5-(6-N,N-Dimetllylamino-8- bromop urin-9-ylJ -pentoxycarbonyl-D-Arginine; Compound #19 N-5 -(Purin-9-yl) -pen tanol; Compound #20 N-5-(Purin--9-yl)-pentoxycarbonyl-D- Arginine; Compound #21 N-5-(Purin-9-yl)-pentoxycarbonyl-L- Arginine; Compound #22 N-5 -(6-N,N-Dimethylaminopurin-9-yl) peiitoxycarbonlyl-L-Valyl-L-Prolyl- L-Le ucine; Compound #23 N-S -(6-N,N-Dimethylarniiopurin-9-yl) pentoxycarbonyl-L-Isoleucyl-L-Prolyl-L-Lsole ucine; pentanol; Compound #25 N-S -(6-N-Cyclopropylaminopuriin-9-yl)- pentoxycarbonyl-D-Arginine; #26 N-5-(6-N-Cyclopropylaminiopuriin-9-yl)- pentoxycarbonyl-L-Arginine; *Compound #27 N-5-(6-N-Azetidinepurin-9-yl) -pentanlol; Compound #28 N-S -(6-N-Azetidinepurin-9-yl)- pentoxycarbonvl-D-Arginine; Compound #29 N-5-(6-N-Azetidinepurin-9-yl)- pentoxycarbonyl-L-Arginine; Compound #30 trans -(6-N-Chlorop Lrin-9-yl) -4-niethyl- cyclohexyl-methanol; Compound #31 tranis-(6-N,N-Dimethylaminopurin-9-yl) 1~ 0 4-miethyl-cyclohexyl-inethanol; :Compound #32 trans-(6-N, N-Dim-e thylaminlopuriin-9-yl) 4-methyl-cyclohexyl-methyloxycarbonyl-D-Argiine; Compound #33 trans -(6-N-Hyd roxyp urin-9-yl) -4-me thyl- cyclohexyl-iie thanol; Compound #34 trans -N-Me thoxyp uri n-9-yl) -4-methyl- cyclohiexyl-ilnethailol; Compound #35 cis-(6-N,N-Dirnethlylamiinopurin-9-yl) -4-miethyl- cyclohexyl-ne thanol; Compound #36 cis-(6-N,N-Dimiethiylaininopurin-9-yl)-4- me thyl-cyc lollexyl-me thyloxycarb onyl-D- argi nine; Compound #37 N-S -(6-N,N-Dimie thylaininopurini-9-yl)- peiitoxycairbonyl-D-Citrullille; Comipound #38 N-S -(6-N-Metliylaziridiinepurin-9-yl) pentanol; Compound #39 N-5-(6-N-Methylaziridiinepurini-9-yl)- ,'~pelntoxycarboniyl-D-Arginine; Comhpound #40 N-S -(6-N,N-Dimnethlylaminopurin-9-yl)-7- thice thoxy-e thoxycarbonyl-D-Arginine; Compound #41 Meta-(6-N,N-Dimethylaminiopurin-9-yl) -methyl- benzyloxycarbonyl-D-Argininie; Compound #42 N-5 -(6-N,N-Dimethiylalniinopurin-9-yl)-3- pentyniyl-i-oxycarboniyl-D-Argiine; Compound #43 N-5-t6-(l i-e thyI-2-acetoxy)- e thylamiinopurin-9-yl] -penitan-ol; Conmpound #44 N-5 '-inethyl-2-ace toxy)- e lhylaminiopurin-9-ylJ -pentyloxycarbonyl-D-Argiinie; Compound #45 N-5-(2 ,6-Dichloropurin-9-yl) -pentanol; Compound #46 N-S ,6-Dicliloropurin-9-yl) pen toxycarboiiyl-D-Arginine; Compound #47 N-5-(2 ,6-Dichloropurin-9-yl)- pentoxycarbonyl-L-Arginine; Compound #48 N-5-(2-Amino-6-N, N- Dietlhylainiopurin-9-yl)-pentanol; .Compound #49 N-5-(6-N,N-Dimetllylanmino-8- 20 ethyltlliopurin-9-yl) -pentailol; #50 N-5-(6-N,N-Jiiniethiylaiiino-8- methyl thiopurini-9-yl) -pentoxycarbonyl-D-Argiinie; Compound #51 N-S-6NM tloyui--l-penitanol; pentoxycarbonyl-D-Arginine; .25 Compound #53 N-5-(2-chloro-6-m-iethoxypurini-9-yl)- *pentoxycarbonyl-D-Arginine;l) ~Compound #54 N-5-(6-N,N-Dimietllylaino i-9y) pentoxycarbonyl-D-Ornitlhine; Compound #55 N-5-(6-N,N-Dirnethylaminopuriin-9-yl)- peiitoxycarbonyl-L-Ornlitliine; :Compound #56 N-S N-Dim-e thylainopurin-9-yl) peintoxycarbonyl-L-Valine; Compound #57 N-5-(6-N,N-Diimethylaiiinopurin-9-yl)- pentoxycarbonyl-D-Valine; Compound #58 N-5-(6-N,N-Dim-ietliylaminiopurin-9-yl)- pentoxycarbonyle thylainine hydrochloride; Compound #59 N-5 -(0-N-Mercap top urin-9-yl) -pen Laanol; Compound //600 N-5-(6-N-M\/ethiyltllioptirin-9-yi) pentanol; Compound #61 N-4-(6-N-Ghloropurinl-9-yl)-butanol; Compound #62 N-4-(6-N,N-Diniethlylaminopurin-9-yl)- butanol; Compound #63 N-4-(6-N,N-Dimiethiylainiopurin-9-yl) b utoxycarbonyl-D-Arginine; Compound #64 N-Dime tlylaminopurini-9-yl) utoxycarbon-yl-L-Arginiine; Compound #65 N-6-(6-N-Clilorop uriin-9-yl) -hexaniol; Compound #66 N-6-(6-N,N-Dimiethylaininopurin-9-yl)-hexaniol; Compound #67 N-6-(6-N,N-Dimetllylaminopurini-9-yl)- hexyloxycarbonyl-D-Arginiine; Compound #68 N-6-(6-N,N-Dimethylarninopurine-9-yl)- hexyloxycarbonyl-L-Argininie; Comipound #69 cis-(6-N-Hydroxypuriin-9-yl)-4-methyl- cyclohexyl-me timanol; Compound #70 cis-(6-N-Hydroxypuriin-9-yl) -4-mnethyl- cyclohexyl-mie thyloxycarbonyl-D-Arginiine; Compound #71 trans -(6-N-Hydroxyp urin-9-yl) -4-ie thyl- cyclohexyl-ie thyloxycarbonyl-D-Argiinine; Compound #72 N-S N-Dimiethylamii nop urin-9-yl) pentylamine hiydrochloride salt; Compound #73 N- (6-N-Me tlylaziridinepurin-9-yl) pentyloxycarbonyl-L-Arginine; SCompound #74 (2S,4S)-2-(6-N,N-Diniethylamiinopurini- S 9-yl) -4-hydroxymethyl-1, 3-dioxolane; Compound #75 (1S,3R) and 20 Dime thylamninop urin-9 -yl) -me thyl- 3-cyc lope ntane methanol; Compound #76 (1S,3R) and .Dimiethylamiliopurini-9-yl) -iethyl- 3- (iethyloxycarbonyl-D- Arginine) -cyclopentane; Compound #77 (6-N,N-Dimiethlylaniniop urin-9-yl) -7- ethylaininoethanol; Cornp ound #78 (6-N,N-Dime thylaminlop urin-9-yl) -7- e thylaminoetloxycarbonyl-D-Argiinie; **g:Compound #79 (6-N,N-Diinethylanhiinopurini-9-yl) -7- ethylarninoe thloxycarbonyl-L-Arginine; 0 Compound #80 N-5-(6-N,N-Dine thylaininiopurin-9-yl)-3- pentyn-i-ol; Compound #81 N-5-(6-N,N-Diimethylainiopurin-9-yl)-3- 5penitvnyl-1-oxycarbonyl-L-Arginine; Compound #82 (6-N,N-Dime thylaminlop urin-9-yl) -7- thioe thoxy-ethanol; Compound #83 (6-N,N-Dimnetliylaminopurin-9-yl)-7- thioe thoxy-e thoxycarbonyl-L-Arginine; Compound #84 (2S,4S)and (2R,4R)-2-(6-N,N- Dime thlylaniinopurin-9-yl)-4-(methioxycarbonylDArginine) 3- oxathiolane: Compound #85 (6-N,N-Dime thylamino ptriin-9-yl) -7- ethoxy-e thoxyethanol; Compound #86 (6-N,N-Dime thylainiopurini-9-yl) -7- ethoxy-ethoxycarbonyl-D-Argininie; #87 (6-N,-Diinethylaininopurin-9-yl)-7- ethoxy-ethoxycarboniyl-L-Arginine; and Compound #88 bronlopurin--9-yl)-pentanol. N-S -(6-N,.N-Diinethylamino-8- 0 0 0 00 S. *0* *5 S S. S S. *S S S S *SS* *555 S S S *5*S S S. S. S S. 55S555

21. The compound according to claim 20 selected from the group consisting of: Compound #III N-5-(6-N-Chloropurini-9-yl)-peiitan-ol Compound #V N-5-(6-N,N-Dimethylarninopurini-9-yl)- pentanol Compound #1 N-S -(6-N,N-Dimethlylaminopuirini-9-yl)- 0 pentoxycarbonyl-D-Arginine Compound #2 N-5-(6-N,N-Dimietllylaminiopurin-9-yl)- pentoxycarbonyl-L-Arginine Compound #3 N-5 -(6-N-Me thylaminiopurini-9-yl) pen toxycarbonyl-D-Arginine Compound #3a N-S -(6-N-Methylaminiopur-in-9-yl) pentanol Compound #5 N-5 -(6-N-Aminop urin-9-yl) pentoxycarbonyl-D-Arginine Compound #5a N-5-(6-N-Aminopurini-9-yl)-pentanol 0 Compound #6 N-S -(6-N-Aniinopurin-9-yl)- pen toxycarbonyl-L-Argiine Compound #7 N-5-(6-N-Hydrazinopurin-9-yl)- pentoxycarbonyl-D-Arginine Comipound #7a N-S -(6-N-H-ydrazinopurin-9-yl)-pentanlol 5Compound #8 N-5-(6-N-Hydrazinopurini-9-yl)- pentoxycarbonyl-L-Arginine; Compound #11 N-S -(6-N-I-yd roxypurin-9-yl) pen toxycarboilyl-D-Arginine; Comipound #19 N-S -(Purin-9-yl)-pentatiol; J Compound #20 N-S -(Purin-9-yl) -pentoxycarboniyl-D- Arginime; Compound #51 N-S (6-N-Methoxyp urin-9-yl) -pen tanol; Compound #59 N-5 -N-Merc ap top urin-9-yl) -p entanol; and Compound #60 N-S -(6-N-Metliylthiopurin-9-yl) pentanol.--

22. The compound according to claim 21 being compound #1 N-S-(6-N,N- Dimiethylamiinop urin-9-yl) -p entoxycarboniyl-D-Arginine.

23. A pharmaceutical comnpositioni containing a compound according to either claim 1 or claim 20, wherein said compound is present in admixture with a pharmaceutically acceptable carrier.

24. A pharmaceutical composition according to claim 23 wherein compound is present in admixture with another the rapeutic ally active agent. A pharmaceutical composition according to claim 24 wherein said therapeutically active agent is cyclophosphamide.

26. A method for the treatment of immune deficiencies or inhibition of tumor growth comprising the step of administering to a mammal a pharmaceutically acceptable amount of a compound according to either claim 1 or claim

27. 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 either claim 1 or claim

28. A method for the inhibition of tumor growth in a mammal, including human, comprising the step of administering to a mammal a pharmaceutically acceptable amount of a compound according to claim 22.

29. A method for the control of mammary carcinoma in a mammal, 20 including human, comprising the step of administering to a nmarnnal a pharmaceutically acceptable amount of a compound according to claim 22, in combination with cyclophosphamide. A pharmaceutical composition for the treatment of cancer comprising a 25 compound according to either claim 1 or claim 20, in combination with

31. The pharmaceutical composition according to claim 30 wherein said cancer is colon carcinoma.

32. 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 either claim 1 or claim 20, in combination with

33. The method according to claim 32, wherein said cancer is colon carcinoma.

34. The method according to claim 32, wherein said compound is administered ili an amount ranging from 1 to 100 mg/kg. The method according to claim 34, wherein said compound is administered in an amount ranging from 2 to 20 mg/kg.

36. The method according to claim 35, wherein said compound is ST inistered at 2.5 mg/kg. 109

37. The method according to claim 32, administered in an amount ranging from

38. The method according to claim 37, administered in an amount ranging from

39. The method according to claim 38, at 12 mg/kg. The method according to claim 32, administered sequentially.

41. The method according to claim 32, administered simultaneously. wherein said 5-fluorouracil is 1 to 50 mg/kg. wherein said 5-fluorouracil is 5 to 20 mg/kg. wherein said 5FU is administered wherein said combination is wherein said combination is o0 :0. 20 25 e

42. The method according to claim 32, wherein said combination is administered as a single formulation combining 5-FU and said compound.

43. Use of a pharmaceutically acceptable amount of a compound according to either claim 1 or claim 20 for the treatment of immune deficiencies or inhibition of tumor growth in a mammal.

44. Use of a pharmaceutically acceptable amount of a compound according to either claim 1 or claim 20 for increasing the number of cytotoxic T lymphocytes in a mammal, including a human.

45. Use of a pharmaceutically acceptable amount of a compound according to claim 22 for the inhibition of tumor growth in a mammal, including human.

46. Use of a pharmaceutically acceptable amount of a compound according to claim 22, in combination with cyclophosphamide, for the control of mammary carcinoma in a mammal, including human.

47. Use of a pharmaceutically acceptable amount of compound according to either claim 1 or claim 20, in combination with 5-fluorouracil, for the treatment of cancer in a mannal, including a human.

48. The use according to claim 47, wherein said cancer is colon carcinoma.

49. The use according to claim 47, wherein said compound is administered in an amount ranging from 1 to 100 mg/kg.

50. The use according to claim 49, wherein said compound is administered T- /in an amount ranging from 2 to 20 mg/kg.

51. The use according to claim 50, wherein said compound is administered at 2.5 mg/kg.

52. The use according to claim 47, wherein said 5-fluorouracil is administered in an amount ranging from 1 to 50 mg/kg.

53. The use according to claim 52, wherein said 5-fluorouracil is administered in an amount ranging from 5 to 20 mg/kg.

54. The use according to claim 53, wherein said 5FU is administered at 12 mg/kg. The use according to claim 47, wherein said combination is administered sequentially.

56. The use according to claim 47, wherein said combination is administered simultaneously.

57. The use according to claim 47, wherein said combination is administered as a single formulation combining 5-FU and said compound. *0 *0 of 00 *9 0 *see 0 00 go 0 000 0 0 0 0 0 f 0 0 00 00 0 S. 0 DATED this 18th day of January 2000 Patent Attorneys for the Applicant: F.B. RICE CO.