6-Oxo and 6-Thio Purine Analogs as Antimycobacterial Agents

Abstract

6–Oxo and 6–thio purine analogs were prepared based on the initial activity screening of a small, diverse purine library against Mycobacterium tuberculosis (Mtb). Certain analogs showed moderate to good inhibitory activity. N⁹-substitution apparently enhances the antimycobacterial activity in the purine series described herein. Several 2-amino and 2-chloro purine analogs were also synthesized that showed moderate inhibitory activity against Mtb.

1. Introduction

In spite of the availability of highly active anti-tubercular agents, tuberculosis has remained one of the primary causes of human death and suffering worldwide. It is estimated that approximately one third of the world’s population is infected with the bacteria that causes tuberculosis, 2–3 million people die worldwide each year from the disease, and an additional 8 million people become ill with tuberculosis annually.¹ Mycobacterium tuberculosis (Mtb) is a facultative intracellular pathogen that persists primarily within macrophages in the human host, and these cells are involved in propagation of infection.² Intracellularly sequestered bacilli are considered more resistant to treatment and clearance due to limited access of drugs and the immune system to bacteria within macrophages, necessitating chronic treatment with high therapeutic doses for effective control and treatment of the disease.³ Additionally, AIDS patients and others with compromised immune systems are susceptible to other opportunistic mycobacteria including M. avium and M. kansaii, resulting in further morbidity and a high mortality.⁴ It is not surprising that drug resistance, from single drug resistant (SDR) up to totally drug resistant (TDR) strains,⁵ is now becoming commonplace considering the fact that virtually the same drug regimens have been in place and poorly deployed worldwide for over half a century. Treatment of highly resistant forms of Mtb is both difficult and expensive, and for these more intractable forms, few treatment options are available. Although newer drugs are now in clinical trials, these issues critically underscore the need for continued emphasis on the discovery of newer drugs with novel mechanisms of action.⁶

Phenotypic screening of diverse drug-like compound libraries against Mtb has been more recently implemented in order to respond to this need and discover compounds that are active against whole Mtb bacilli,⁷ potentially circumventing issues with antibacterial drug discovery using specific target based screens.⁸

Through similar screens of the Southern Research proprietary library, it has been found that several 9–benzylpurines with a variety of substitutions in the 2–, 6– and/or 8–positions exhibit inhibitory activity against Mtb.⁹ High inhibitory activity was found for 9–benzylpurines containing a phenylethynyl–, trans-styryl or aryl substituent in the 6-position, and generally chlorine in the 2–position tends to increase activity (compounds 1–4, Fig.–1). Several 6–arylpurines carrying a variety of substituents in the 9–position were prepared by Stille coupling between appropriately substituted 6–chloropurines and aryl(tributyl)tin, and the compounds were screened for antibacterial activity against Mtb H₃₇Rv.¹⁰ One of the derivatives, 9–benzyl–2–chloro–6–(2–furyl)purine (2), showed a MIC value of 0.78 µg/mL and also showed relatively low cytotoxicity against several singly drug-resistant strains. A series of 9–sulfonylated/sulfenylated–6–mercapto purines (3 and 4) has been prepared by reaction of 6–mercaptopurine with sulfonyl/sulfenyl halides.¹¹ These compounds constitute a new class of potent antimycobacterial agents, possessing excellent MIC values against Mtb H₃₇Rv, as well as appreciable activity against M. avium. A few compounds in this series have exhibited activity against several drug resistant strains of Mtb (e.g. compound 4). Currently, beyond the broad phenotypic activity, no target(s) has/have been identified.

Figure–1.

We have also synthesized a small library of 6–thioalkyl/aryl/benzyl purine analogs to generate a modest structure–activity relationship (SAR). All the purine derivatives synthesized were screened for their activity against two strains of Mtb (H₃₇Rv & H₃₇Ra) and three strains of M. avium (MAC NJ211, NJ168, NJ3404). Most of these derivatives were inactive against M. avium. In particular, (6–decylsulfanyl–purin–9–yl)–acetic acid ethyl ester 5 and the dodecyl derivative 6, and (6–decylsulfanyl–purin–9–yl)–acetic acid tert–butyl ester 7 exhibited MIC values of 1.56, 0.78 and 3.13 µg/mL respectively against the Mtb H₃₇Rv strain (Fig.–2).¹² Concurrent with the determination of MICs, analogs 5–7 were also tested for cytotoxicity (CC₅₀) in a cell proliferation assay for VERO cells at concentrations less than or equal to 62.5 µg/mL or 10 times the MIC for Mtb H₃₇Rv: selectivity is assigned by calculating the selectivity index (SI) ratio CC₅₀/MIC.

Figure–2.

Very little is known about the mechanism of action of these purine analogs. Purine salvage pathways are predicted to be present from the genome sequence of Mtb, and the metabolism in mycobacteria is similar to that in humans and other organisms.¹³ Information about the substrate preferences of the mycobacterial enzymes involved with purine metabolism is still unknown. The Mtb deoD gene encodes a presumptive purine nucleoside phosphorylase (PNP) and the gene was cloned, expressed, purified, and found to exhibit PNP activity.¹⁴ Modest biochemical work has been pursued on purine nucleosides with antimycobacterial activity, especially 2–methyladenosine that showed potent activity (99% inhibition, MIC = 3 µg/mL, IC₅₀ (VERO Cells = 1000 µg/mL, SI >1000).¹⁵ 2–Methyladenosine has demonstrated selective activity against Mtb, suggesting differences in the substrate preferences between mycobacterial and human adenosine kinases that might be exploited to develop novel nucleoside–based drugs for the treatment of mycobacterial diseases. Beyond the purine salvage pathways, ATP binding proteins and kinases are also being interrogated as new drug targets in Mtb.¹⁶

Based upon our previously synthesized purine series,¹² we planned to generate more diversified 6–substituted mercaptopurines analogs with substitutions at C–2 position along with 6–oxo substituted series for antimycobacterial screening. Compounds were prepared from a diversity perspective in order to further probe specific active scaffolds, but also to explore other purines such as the 2-amino and 2-chloropurine scaffolds.

2. Results and Discussion

2.1. Chemistry

In the first target set, synthesis of several new 6–oxoaryl/benzyl/aryl purines and their 2–amino or 2–chloro purine analogs (8–32) was carried out as shown in Scheme–1 by reacting a suitable 6–chloropurine analog with alcohols in the presence of sodium metal. Neutralization with acetic acid followed by the standard workup and column chromatographic purification on silica gel G produced pure products. A total of six analogs of 6–oxopurine, ten analogs of 2–amino–6–oxopurine and nine analogs of 2–chloro–6–oxopurine were prepared in the initial phase to determine their antibacterial activity. A total of nine analogs have shown >50% inhibitory activity against Mtb H₃₇Rv and are discussed herein (Table–1).

Scheme–1.

Table–1.

Antimycobacterial activity against Mtb and MAC of substituted purines.

Comp. No.	% Inhib. Mtb H37Rv	MIC90 Mtb H37Rv	MIC99 Mtb H37Ra	MIC99 MAC NJ211	CC50 VERO cells	Selectivity Index (SI) Mtb H37Rv
8	23	>6.25	>12.8	>12.8	n.d.	-
9	96	<6.25	>12.8	>12.8	n.d.	-
10	56	>6.25	>12.8	>12.8	n.d.	-
12	6	>6.25	>12.8	>12.8	n.d.	-
13	21	>6.25	>12.8	>12.8	n.d.	-
14	23	>6.25	>12.8	>12.8	n.d.	-
15	91	3.13	>12.8	>12.8	n.d.	-
16	94	0.78	>6.4	>6.4	>10	>12.8
18	20	>6.25	>12.8	>12.8	n.d.	-
21	93	>6.25	>12.8	>12.8	n.d.	-
23	24	>6.25	>12.8	>12.8	n.d.	-
24	24	>6.25	>12.8	>12.8	n.d.	-
25	23	>6.25	>12.8	>12.8	n.d.	-
26	55	>6.25	>12.8	>12.8	n.d.	-
27	59	>6.25	>12.8	>12.8	n.d.	-
28	8	>6.25	>12.8	>12.8	n.d.	-
32	35	>6.25	>12.8	>12.8	n.d.	-
33	100	1.56	>12.8(P)	>12.8	>62.5	>40.1
34	98	0.78	32	>128	>10	>12.8
35	14	>6.25	>12.8	>12.8	n.d.	-
36	100	6.25	16	>32	>10	>1.6
44	7	>6.25	>12.8	>12.8	n.d.	-
47	16	>6.25	>12.8	>12.8	n.d.	-
48	5	>6.25	>12.8	>12.8	n.d.	-
51	35	>6.25	>12.8	>12.8	n.d.	-
52	49	>6.25	>12.8	>12.8	n.d.	-
53	82	>6.25	>12.8	>12.8	n.d.	-
55	97	6.25	>12.8	>12.8	12.32	2.0
56	84	>6.25	>12.8	>12.8	n.d.	-
57	81	>6.25	>12.8	>12.8	n.d.	-
58	93	6.25	>12.8	>12.8	n.d.	-
59	96	6.25	>12.8	>12.8	n.d.	-
60	97	1.56	>12.8	>12.8	n.d.	-
61	74	>6.25	>12.8	>12.8	n.d.	-
62	89	>6.25	>12.8	>12.8	n.d.	-
63	86	>6.25	>12.8	>12.8	n.d.	-
64	90	>6.25	>12.8	>12.8	n.d.	-
65	92	6.25	>12.8	>12.8	8.04	1.3
66	98	6.25	>12.8	>12.8	n.d.	-
67	96	6.25	>12.8	>12.8	n.d.	-
68	92	>6.25	>12.8	>12.8	n.d.	-
69	85	>6.25	>12.8	>12.8	n.d.	-

% Inhibition was performed at 6.25 µg/mL concentration; Compounds not listed here showed no inihibition at 6.25 µg/mL; MIC’s are in µg/mL; CC₅₀ was determined in µg/mL; SI (Mtb H₃₇Rv) = CC₅₀/MIC₉₀.

As previously described by us,¹² substitution at the N⁹–position enhances antimycobacterial activity, and we have synthesized and screened four analogs 33–36 starting from active 6–oxopurine analogs 9, 10, 15 and 16 (Scheme–2). The synthesis of these analogs was carried out by reacting the appropriate commercially available purine analog with ethylbromoacetate and K₂CO₃ in anhydrous DMSO at room temperature.

Scheme–2.

Further analoging of compounds 34 and 35 was achieved by treating these with different D– and L–aminoacids. These compounds can utilize bacterial dipeptide transporters to enhance transport via membrane amino acids beyond the fact that adding amino acids at the end increases diversity for probing activity. It is sometimes the case that adding specific chirality can improve activity and only one stereoisomer shows activity. This result is, however, a gross generality as that is not always the case and sometimes both enantiomers are inactive or can show similar activity. Compounds 40–50 (Scheme–3) which contain D– or L– amino acid chains were synthesized from commercially available starting material, 2–amino–6–chloro–9H–purine–9–acetic acid (37). The 6–decyloxy derivative 38 was synthesized by reacting 37 with 1–decanol in the presence of sodium metal. The 6–decylthio derivative 39 was synthesized from 37 by the reaction with 1–decylthiol and (CH₃)₃COK as previously described.¹² Analoging of 38 and 39 was achieved by treating with different D– or L–amino acids using the coupling reagent benzotriazole–1–yl–oxy–trispyrrolidino-phosphonium hexafluorophosphate (PyBOP) in the presence of base Et₃N.

Scheme–3.

Alternatively, for diversity point of view, compounds 51–69 were prepared from easily accessible starting materials. 6–decylmercaptopurine analogs 51–54 were prepared starting from 6–chloropurine analogs by the reaction with 1–decylthiol and (CH₃)₃COK under reflux as previously described (Scheme–4A).^12,17 However, 6–decylmercapto purine analogs 55–69 were prepared from their corresponding 6-purinethiones by treatment with 1–chlorodecane/anhydrous K₂CO₃ as reported earlier (Scheme–4B).¹⁸

Scheme–4.

7–Deaza compound 70 was synthesized in three steps starting from commercially available 6–chloro–7–deazapurine 71 as shown in Scheme–5. The compound 71 was first converted to 6–thiomethyl–7–deaza purine 72 in excellent yield. Compound 72 was then reacted with ethylbromoacetate/K₂CO₃ followed by ester hydrolysis using 1N NaOH gave compound 73. Finally, coupling of 73 with 3,4–diemthylaniline, HATU and DIEA produced the desired product 70.

Scheme–5.

Reagents and conditions: a) NaSCH₃, MeOH, rt, overnight, 90%; b) (i) BrCH₂CO₂Et, K₂CO₃, DMF, rt, overnight, (ii) 1N NaOH, MeOH, rt, overnight, 82% in two steps; c) 3,4-dimethylaniline, HATU, DIEA, DMF, rt, 6h, 54%.

2.2. Anti-mycobacterial Activity

All new compounds were screened for their in vitro activity against strains Mtb H₃₇Ra and H₃₇Rv as well as one strain of M. avium (NJ211) and are described below. Compounds demonstrating at least 90% inhibition against H₃₇Rv strain at 6.25 µg/mL were re–tested to determine the MIC₉₀ in a BACTEC assay. Active compounds were then screened for cytotoxicity against mammalian VERO cells to determine their selectivity towards bacteria.

2.2.1. In vitro cell studies

The minimum inhibitory concentration (MIC₉₉, the lowest concentration that completely inhibits growth) of all compounds for Mtb H₃₇Ra were determined using a colorimetric (Alamar blue) microdilution broth assay reported previously.¹⁹ All the compounds were also screened against Mtb H₃₇Rv to determine the MIC₉₀ (the minimum concentration that inhibits 90% of growth). Out of 59 purine analogs synthesized, 26 analogs gave >50% inhibition against H₃₇Rv and are shown in Table–1 (compounds not incuded in Table–1 showed no inhibition against Mtb H₃₇Rv).

The antimycobacterial purine analogs 16, 33, 34, 36, 55 and 65 were examined for toxicity against mammalian cells (VERO cells),²⁰ and their cytotoxicity (CC₅₀) values are reported in Table–1. Their selectivity is assigned by calculating the selectivity index (SI) ratio CC₅₀/MIC. One of the more promising inhibitors of Mtb growth, compound 33, gave a SI > 40.

A 2–thiomethylpurine analog 74 (PubChem ID 726045, Fig.–3) commercially available from ChemBridge showed very potent activity (IC₉₀ < 0.1 µg/mL) with a selectivity index (SI) >150 against Mtb H₃₇Rv (PubChem Assay ID 1949). Unfortunately, this analog as well as the general class show only relatively poor bioavailablity and antitubercular activity in vivo.¹² Purines are potential substrates for a number of metabolic enzymes in vivo that include adenosine deaminase and xanthine oxidase that can alter biological properties and activity; a typical alteration that can modify metabolism involves preparation of 7–deaza analogs that can potentially circumvent metabolism and retain potent biological activity. Purine analogs modified in the five–membered ring have been synthesized and examined for antibacterial activity against Mtb H₃₇Rv in vitro.²¹ The 9–deaza analogs were only found to be weak inhibitors, but the 8–aza–, 7–deaza– and 8–aza–7–deazapurine analogs studied displayed excellent antimycobacterial activities, some even substantially better than the parent purine. Hence, the 7–deazapurine analog 70 of purine 74 was prepared and screened against Mtb. Interestingly, this modest alteration led to complete abolition of the whole cell antitubercular activity (IC₉₀ > 100 µM) in our case for compound 70. It is also notable that 70 (PubChem CID 44144249) showed modest inhibition (IC₉₀ = 7.31 µM) of Mtb H₃₇Rv in the MLSCN screen (Assay ID 485340) when screened in the presence of 6.5 µM (non–lethal, subinhibitory dose to bacteria) of the β–lactam antibiotic Meropenem. These data suggest that damage to the cell wall may allow uptake of 70 and activity against the/a molecular target. Speculatively, the purine analog 74 may be actively uptaken and inhibit a target or targets, but the 7–deaza analog 70 may lose the ability to penetrate the mycobacterial cell wall until the peptidoglycan is compromised by the β-lactam. This hypothesis, however, remains unproven.

Figure–3.

The two active purines 5 and 6 synthesized previously¹² were examined against a panel of Mtb strains resistant to currently used anti-TB drugs; CIP (ciprofloxacin), INH (isoniazid), KM (kanamycin), EMB (ethambutol), RMP (rifampin), PAS (p–aminosalicylic acid), and TAC (thiacetazone) (Table 2). All compounds examined retained their activity against all the drug-resistant strains applied in this study.

Table–2.

MIC of compounds 5 and 6 with H₃₇Rv, Erdman and single-drug resistant strains of Mtb in the Alamar blue assay.

	MIC90 (µg/mL) against drug resistance strains in
Compd	Alamar blue assay
	H37RvErdman		CIP	INH	KM	EMB	RMP	PAS	TAC
5	6.25	12.5	12.5	12.5	6.25	6.25	6.25	-	-
6	3.13	3.13	1.56	1.56	-	3.13	3.13	3.13	3.13

^*MIC of 5 and 6 were observed 1.56 and 0.78 µg/mL respectively against H₃₇Rv in Bactec assay. CIP (ciprofloxacin), INH (isoniazid), KM (kanamycin), EMB (ethambutol), RMP (rifampin), PAS (p-aminosalicylic acid), and TAC (thiacetazone)

2.2.2. In vivo animal studies²²

Concurrently with the SAR studies, both compounds 5 and 6 were selected for further evaluation in vivo in order to determine cytotoxicity and efficacy in a murine Mtb model to evaluate these scaffolds as further candidates for antitubercular drug discovery. The lead compounds of the series, compounds 6 and 16, were initially evaluated for in vivo toxicity using an acute toxicity mouse model. These compounds did not show any lethality at 100, 300 and 500 mg/kg. In the short term GKO mouse model, 6 showed poor efficacy in the lungs by reducing the bacterial load by 0.46 Log₁₀ CFU (mean CFU = 8.16) whereas the compound did not show any significant activity in the spleen (0.2 Log₁₀ CFU reduction, mean CFU = 6.83). For compound 16, there was no in vivo activity observed in the lungs or spleens in the GKO mouse model [−0.141 Log₁₀ CFU (mean CFU = 8.82) in lungs, and −0.05 Log₁₀ CFU (Mean CFU = 7.11)].

2.3. Conclusions

In the 6–oxo series, compound 9, 15 and 16 showed good activity against Mtb H₃₇Rv and possess a decyl/dodecyl chain at the C–6 position of purine. Based on their activity, compounds 29–34 possessing N⁹ substitution were synthesized and screened. Out of these four compounds, however, compound 35 significantly lost antimycobacterial activity over the comparator compound, 15. Structural diversity using D– and L–amino acids in compounds 34 and 35 did not increase the activity significantly. The diversity analogs 51–69 showed significant high % inhibition but also produced high MIC₉₀ values in the Mtb H₃₇Rv screen. In conclusion, small sets of diverse 6–substituted purine analogs were synthesized and screened against Mtb strains. Certain purine analogs synthesized showed interesting antimycobacterial activity against Mtb H₃₇Rv. At this point, however, no definitive structure–activity relationship has been determined.

3. Experimental

3.1. Synthesis

Purifications by column chromatography were done with silica gel (Merck, 60 A, 230–400 mesh). Target compounds were dried in vacuo at 25 °C and 78 °C over P₂O₅. Analytical results indicated by element symbols were within ±0.4% of the theoretical values, unless otherwise noted. In such noted cases, a high–resolution mass spectrum was obtained and the result listed. Where solvents are indicated in the formula, their presence was confirmed by ¹H NMR. The ¹H NMR spectra were determined with a Nicolet NMC 300 NB spectrometer using TMS as internal reference and chemical shifts (δ) are in ppm. ¹H NMR spectra were recorded in DMSO–d₆ as a solvent. NOE’s were performed on certain intermediates and target compounds to confirm structures. Mass spectra were recorded on a Varian MAT 311A mass spectrometer in the fast-atom-bombardment (FAB) mode. High-resolution mass spectra were obtained on a Bruker BIOTOF II spectrometer. Microanalyses were performed on a Perkin-Elmer 2400 CHN analyzer. All starting materials and reagents were purchased from commercial sources and used as such. Chemical yields have not been optimized and represent the result of a single preparation. All target compounds were stored under argon at −20 °C until used for screening. General synthetic procedures are described as below.

3.1.1. General Procedure for Synthesis of 6–Oxyalkyl/aryl/benzyl Purines (8–32)

To a solution of Na (1.36 equiv.) in the appropriate alcohol (20 equiv.) was added the appropriately substituted purine under an argon atmosphere. The reaction mixture was stirred at reflux for 2 to 6 h and then allowed to stir at room temperature overnight under argon. Deionized water (10–20 mL) was added and the reaction mixture was neutralized by addition of acetic acid. The neutralized solution was then extracted with diethyl ether. The ethereal layers were pooled, dried over Na₂SO₄, concentrated, and dried in vacuum over P₂O₅ at 25 °C. In most cases, the crude material was purified by column chromatography (CC) over silica gel G.

6–[(Isopropyl)oxy]purine (8)

CHCl₃–MeOH (99:1) with 0.5% NH₄OH was used as eluent for column chromatography (CC), off–white solid, yield 45%. ¹H NMR: δ 13.37 (1H, br s, NH), 8.46 (1H, s, H–2), 8.34 (1H, s, H–8), 5.58 (1H, m, OCH), 1.40 (6H, d, J = 6.2 Hz, 2xCH₃). FABMS m/z: 179 (M+H)⁺. ESI–MS: Found 179.0932, calcd. for C₈H₁₀N₄O 179.0927. Anal. (C₈H₁₀N₄O) Found: C, 54.24; H, 5.77; N, 30.35. Calcd. C, 53.92; H, 5.66; N, 31.44.

6–(Decyloxy)purine (9)

CHCl₃ was used as eluent for CC, off–white solid, yield 51%. ¹H NMR: δ 13.50 (1H, br s, NH), 8.47 (1H, s, ¹J_CH = 203.0 Hz, H–2), 8.36 (1H, s, ¹J_CH = 210.9 Hz, H–8), 4.52 (2H, t, J = 6.6 Hz, OCH₂), 1.80 (2H, m, CH₂), 1.34 (14H, m, 7xCH₂), 0.85 (3H, m, CH₃). FABMS m/z: 277 (M+H)⁺. Anal. (C₁₅H₂₄N₄O) Found: C, 64.86; H, 9.01; N, 19.90. Calcd. C, 65.19; H, 8.75; N, 20.27.

6–(Dodecyloxy)purine (10)

CHCl₃–MeOH (98:2) was used for CC, off–white solid, yield 63%. ¹H NMR: δ 13.40 (1H, br s, NH), 8.47 (1H, s, H–2), 8.36 (1H, s, H–8), 4.52 (2H, dd, J = 6.6, 6.7 Hz, OCH₂), 1.79 (2H, m, CH₂), 1.29 (18H, m, 9xCH₂), 0.85 (3H, m, CH₃). FABMS m/z: 305 (M+H)⁺. Anal. (C₁₇H₂₈N₄O) Found: C, 67.26; H, 9.02; N, 18.02. Calcd. C, 67.07; H, 9.27; N, 18.40.

6–[(4–Chlorophenyl)oxy]purine (11)

CHCl₃–MeOH (98:2) was used for CC, off–white solid, yield 84%. ¹H NMR: δ 13.61 (1H, br s, NH), 8.53 (1H, s, H–2); 8.44 (1H, s, H–8), 7.54 (2H, d, J = 8.9 Hz, H–3', H–5'), 7.36 (2H, d, J = 8.9 Hz, H–2', H–6'). FABMS m/z: 247 (M+H)⁺. Anal. (C₁₁H₇N₄ClO) Found: C, 53.51; H, 2.83; N 22.51. Calcd. C, 53.56; H, 2.86; N, 22.71.

6–[(Cyclohexylmethyl)oxy]purine (12).²³

CHCl₃–MeOH (98:2) was used for CC, off–white solid, yield 32%. ¹H NMR: δ 13.39 (1H, br s, NH), 8.43 (1H, s, H–2); 8.33 (1H, s, H–8), 4.31 (2H, d, J = 6.3 Hz, OCH₂), 1.50 (11H, m, C₆H₁₁). FABMS m/z: 233 (M+H)⁺. Anal. (C₁₂H₁₆N₄O· 0.1H₂O) Found: C, 61.59; H, 7.01; N, 23.60. Calcd. C, 61.57; H, 6.98; N, 23.93.

6–[(3,4–Dichlorobenzyl)oxy]purine (13)

CHCl₃–MeOH (99:1) with 0.5% NH₄OH was used for CC, off–white solid, yield 35%. ¹H NMR: δ 13.51 (1H, br s, NH), 8.52 (1H, s, H–2); 8.42 (1H, s, H–8), 7.82 (1H, d, J = 1.8 Hz, H–2'), 7.69 (1H, d, J = 8.2 Hz, H–5'), 7.53 (1H, dd, J = 1.8, 2.1 Hz, H–6'), 5.62 (2H, s, OCH₂). FABMS m/z: 295 (M+H)⁺. Anal. (C₁₂H₈N₄Cl₂O· 0.45CH₃OH) Found: C, 48.63; H, 2.94; N, 17.73. Calcd. C, 48.31; H, 3.19; N, 18.10.

2–Amino–6–[(isopropyl)oxy]purine (14)

Off–white solid, yield 59%. ¹H NMR: δ 7.78 (1H, s, H–8), 6.14 (2H, br s, NH₂), 5.48 (1H, ddd, J = 6.2, 6.3, 6.3 Hz, OCH), 1.34 (6H, d, J = 6.2 Hz, 2xCH₃). FABMS m/z: 194 (M+H)⁺. Anal. (C₈H₁₁N₅O) Found: C, 49.91; H, 5.83; N, 35.86. Calcd. C, 49.73; H, 5.74; N, 36.25.

2–Amino–6–(decyloxy)purine (15)

CHCl₃–MeOH (20:1) was used for CC, off–white solid, yield 62%. ¹H NMR: δ 12.4 (1H, br s, NH), 7.81 (1H, s, H–8), 6.19 (2H, br s, NH₂), 4.37 (2H, dd, J = 6.6, 6.7 Hz, OCH₂), 1.74 (2H, m, CH₂), 1.33 (14H, m, 7xCH₂), 0.85 (3H, s, CH₃). FABMS m/z: 292 (M+H)⁺. Anal. (C₁₅H₂₅N₅O) Found: C, 61.73; H, 8.58; N, 23.81. Calcd. C, 61.83; H, 8.65; N, 24.03.

2–Amino–6–(dodecyloxy)purine (16)

CHCl₃–MeOH (20:1) was used for CC, off–white solid, yield 59%. ¹H NMR: δ 12.4 (1H, br s, NH), 7.80 (1H, s, H-8), 6.19 (2H, br s, NH₂), 4.37 (2H, dd, J = 6.6, 6.7 Hz, OCH₂), 1.73 (2H, m, CH₂), 1.30 (18H, m, 9xCH₂), 0.85 (3H, m, CH₃). FABMS m/z: 320 (M+H)⁺. Anal. (C₁₇H₂₉N₅O· 0.5H₂O) Found: C, 61.92; H, 8.97; N, 21.24. Calcd. C, 62.17; H, 9.21; N, 21.32.

2–Amino–6-(benzyloxy)purine (17)

CHCl₃–MeOH (20:1) was used for CC, off–white solid, yield 45%. ¹H NMR: δ 12.6 (1H, br s, NH), 7.95 (1H, s, H-8), 7.44 (2H, m, H–2', H–6'), 7.24 (3H, m, H–3', H–4', H–5'), 6.25 (2H, br s, NH₂). FABMS m/z: 228 (M+H)⁺. Anal. (C₁₁H₉N₅O· 0.2CH₃OH); Found: C, 57.74; H, 4.36; N, 29.83. Calcd. C, 57.58; H, 4.23; N, 29.98.

2–Amino–6–[(4–chlorophenyl)oxy]purine (18)

CHCl₃–MeOH (20:1) was used for CC, off–white solid, yield 83%. ¹H NMR: δ 12.6 (1H, br s, NH), 7.94 (1H, s, H–8), 7.49 (2H, m, H–2', H–6'), 7.29 (2H, m, H–3', H–5'), 6.30 (2H, br s, NH₂). FABMS m/z: 262 (M+H)⁺. Anal. (C₁₁H₈N₅ClO· 0.4H₂O) Found: C, 49.12; H, 3.66; N, 25.76. Calcd. C, 49.14; H, 3.30; N, 26.05.

2–Amino–6–[(4–methylphenyl)oxy]purine (19)

CHCl₃–MeOH (20:1) was used for CC, yellow solid, yield 83%. ¹H NMR: δ 12.5 (1H, br s, NH), 7.93 (1H, br s, H–8), 7.22 (2H, d, J = 8.2 Hz, H–3', H–5'), 7.10 (2H, d, J = 8.4 Hz, H–2', H–6'), 6.22 (2H, br s, NH₂), 2.32 (3H, s, CH₃). FABMS m/z: 242 (M+H)⁺. Anal. (C₁₂H₁₁N₅O· 0.4H₂O) Found: C, 58.03; H, 4.91; N, 28.26. Calcd. C, 58.01; H, 4.79; N, 28.19.

2–Amino–6–[(3–methylphenyl)oxy]purine (20)

CHCl₃–MeOH (20:1) was used for CC, light brown solid, yield 85%. ¹H NMR: δ 12.56 (1H, br s, NH), 7.94 (1H, br s, H–8), 7.31 (1H, dd, J = 7.9, 8.2 Hz, H–5'), 7.03 (3H, m, H–2', H–4', H–6'), 6.26 (2H, br s, NH₂), 2.33 (3H, s, CH₃). FABMS m/z: 242 (M+H)⁺. Anal. (C₁₂H₁₁N₅O· 0.45H₂O) Found: C, 58.03; H, 4.93; N, 27.82. Calcd. C, 57.80; H, 4.81; N, 28.09.

2–Amino–6–[(cyclohexylmethyl)oxy]purine (21).²³

CHCl₃–MeOH (20:1) was used for CC, off–white solid, yield 66%. ¹H NMR: δ 12.38 (1H, br s, NH), 7.79 (1H, s, H–8), 6.21 (2H, br s, NH₂), 4.20 (2H, d, J = 6.2 Hz, OCH₂), 1.50 (11H, m, C₆H₁₁). FABMS m/z: 248 (M+H)⁺. Anal. (C₁₂H₁₇N₅O· 0.3CH₃OH) Found: C, 57.25; H, 7.03; N, 27.09. Calcd. C, 57.50; H, 7.14; N, 27.26.

2–Amino–6–(benzyloxy)purine (22).²⁴

CHCl₃–MeOH (9:1) was used for CC, off–white solid, yield 41%. ¹H NMR: δ 12.44 (1H, br s, NH), 7.83 (1H, br s, H–8), 7.50 (2H, m, H–2', H–6'), 7.38 (3H, m, H–3', H–4', H–5'), 6.29 (2H, br s, NH₂), 5.48 (2H, s, OCH₂). FABMS m/z: 242 (M+H)⁺. Anal. (C₁₂H₁₁N₅O·0.4CH₃OH) Found: C, 58.84; H, 4.89; N, 27.27. Calcd. C, 58.62; H, 5.00; N, 27.56.

2–Amino–6–[(3,4–dichlorobenzyl)oxy]purine (23)

CHCl₃–MeOH (99:1) with 0.5% NH₄OH was used for CC, off–white solid, yield 57%. ¹H NMR: δ 12.5 (1H, br s, NH), 7.85 (1H, br s, H–8), 7.79 (1H, d, J = 1.9 Hz, H–2'), 7.67 (1H, d, J = 8.4 Hz, H–5’), 7.51 (1H, dd, J = 2.1, 2.1 Hz, H–6'), 6.32 (2H, br s, NH₂), 5.48 (2H, s, OCH₂). FABMS m/z: 310 (M+H)⁺. Anal. (C₁₂H₉Cl₂N₅O) Found: C, 46.48; H, 3.19; N, 22.34. Calcd. C, 46.47; H, 2.92; N, 22.58.

2–Chloro–6–(methoxy)purine (24)

CHCl₃–MeOH (98:2) was used for CC, off–white solid, yield 49%. ¹H NMR: δ 13.67 (1H, br s, NH), 8.41 (1H, br s, H–8), 4.10 (3H, s, CH₃). FABMS m/z: 185 (M+H)⁺. ESI–MS: Found 185.0221, calcd. for C₆H₅ClN₄O 185.0225. Anal. (C₆H₅ClN₄O) Found: C, 37.36; H, 2.51; N, 28.77. Calcd. C, 37.23; H, 3.12; N, 28.94.

2–Chloro–6–[(isopropyl)oxy]purine (25)

CHCl₃–MeOH (99:1) with 0.5% NH₄OH was used for CC, white solid, yield 62%. ¹H NMR: δ 8.40 (1H, s, H–8), 5.52 (1H, septet, J = 6.2 Hz, OCH), 1.40 (6H, d, J = 6.2 Hz, 2xCH₃). FABMS m/z: 213 (M+H)⁺. Anal. (C₈H₉ClN₄O) Found: C, 45.15; H, 4.34; N, 26.31. Calcd. C, 45.19; H, 4.27; N, 26.35.

2–Chloro–6–(decyloxy)purine (26)

Cyclohexane–EtOAc (5:1) was used for CC, white solid, yield 62%. ¹H NMR: δ 8.41 (1H, s, H–8), 4.51 (2H, t, OCH₂), 1.81 (2H, m, CH₂), 1.21 (14H, m, 7xCH₂), 0.86 (3H, m, CH₃). FABMS m/z: 311 (M+H)⁺. Anal. (C₁₅H₂₃ClN₄O) Found: C, 57.85; H, 7.41; N, 17.98. Calcd. C, 57.96; H, 7.46; N, 18.02.

2–Chloro–6–(dodecyloxy)purine (27)

Cyclohexane–EtOAc (5:1) was used for CC, white solid, yield 71%. ¹H NMR: δ 13.62 (1H, br s, NH), 8.41 (1H, s, H–8), 4.51 (2H, t, J = 6.6 Hz, OCH₂), 1.79 (2H, m, CH₂), 1.28 (18H, m, 9xCH₂), 0.85 (3H, m, CH₃). FABMS m/z: 339 (M+H)⁺. Anal. (C₁₇H₂₇ClN₄O) Found: C, 60.28; H, 8.09; N, 16.17. Calcd. C, 60.25; H, 8.03; N, 16.53.

2–Chloro–6–[(4–chlorophenyl)oxy]purine (28)

CHCl₃–MeOH (98:2) was used for CC, off–white solid, yield 56%. ¹H NMR: δ 13.86 (1H, br s, NH), 8.58 (1H, s, H–8), 7.57 (2H, m, H–2', H–6'), 7.40 (2H, m, H–3', H–5'). FABMS m/z: 281 (M+H)⁺. Anal. (C₁₁H₆Cl₂N₄O) Found: C, 47.34; H, 2.32; N, 19.80. Calcd. C, 47.00; H, 2.15; N, 19.93.

2–Chloro–6–[(4–methylphenyl)oxy]purine (29)

CHCl₃–MeOH (98:2) was used for CC, off–white solid, yield 91%. ¹H NMR: δ 13.82 (1H, br s, NH), 8.55 (1H, s, H–8), 7.29 (2H, d, J = 8.2 Hz, H–3', H–5'), 7.20 (2H, dd, J = 2.3, 8.6 Hz, H–2', H–6'), 2.36 (3H, s, CH₃). FABMS m/z: 261 (M+H)⁺. Anal. (C₁₂H₉ClN₄O) Found: C, 55.15; H, 3.56; N, 21.36. Calcd. C, 55.29; H, 3.48; N, 21.49.

2–Chloro–6–[(3–methylphenyl)oxy]purine (30)

CHCl₃–MeOH (98:2) was used for CC, off–white solid, yield 58%. ¹H NMR: δ 13.8 (1H, br s, NH), 8.56 (1H, s, H–8), 7.38 (1H, dd, J = 7.9, 8.1 Hz, H–5'), 7.14 (3H, m, H–2', H–4', H–6'), 2.36 (3H, s, CH₃). FABMS m/z: 261 (M+H)⁺. Anal. (C₁₂H₉ClN₄O) Found: C, 55.47; H, 3.74; N, 21.14. Calcd. C, 55.29; H, 3.48; N, 21.49.

2–Chloro–6–[(cyclohexylmethyl)oxy]purine (31)

CHCl₃–MeOH (98:2) used for CC, off–white solid, yield 76%. ¹H NMR: δ 13.64 (1H, br s, NH), 8.43 (1H, s, H–8), 4.33 (2H, d, J = 6.3 Hz, OCH₂), 1.5 (11H, m, C₆H₁₁). FABMS m/z: 267 (M+H)⁺. Anal. (C₁₂H₁₅ClN₄O) Found: C, 53.91; H, 5.70; N, 20.94. Calcd. C, 54.04; H, 5.67; N, 21.01.

2–Chloro–6–[(3,4–dichlorobenzyl)oxy]purine (32)

Cyclohexane–EtOAc (5:1) was used for CC, off–white solid, yield 84%. ¹H NMR: δ 13.73 (1H, br s, NH), 8.47 (1H, s, H–8), 7.85 (1H, d, J = 1.8 Hz, H–2’), 7.71 (1H, d, J = 8.2 Hz, H–5’), 7.50 (1H, ddd, J = 1.9, 2.1, 8.2 Hz, H–6'), 5.59 (2H, s, OCH₂). FABMS m/z: 329 (M+H)⁺. Anal. (C₁₂H₇Cl₃N₄O· 0.15C₄H₈O₂) Found: C, 44.32; H, 2.49; N, 16.17. Calcd. C, 44.15; H, 2.41; N, 16.34.

3.1.2. General Procedure for Synthesis of N⁹–substituted Purines (33–36)

To a suspension of the appropriate substituted purine, dry K₂CO₃ (1 equiv), and anhydrous DMSO, was added ethylbromoacetate (1.04 equiv) dropwise at 0 °C under an argon atmosphere. The reaction mixture was allowed to stir at 0 °C for 4 to 6 h and then allowed to stir at room temperature overnight under argon. Deionized water (10–20 mL) was added and the reaction mixture was extracted with diethyl ether. The ethereal layers were pooled, dried over Na₂SO₄, concentrated, and dried in vacuo over P₂O₅ at 25 °C. The crude material was purified by column chromatography (CC) over silica gel G (230–400 mesh).

9–Purine acetic acid, 6–(decyloxy) ethyl ester (33)

CHCl₃ (100%) was used for CC, off–white solid, yield 42%. ¹H NMR: δ 8.50 (1H, s, H–2), 8.35 (1H, s, H–8), 5.18 (2H, s, NCH₂), 4.54 (2H, dd, J = 6.6, 6.7 Hz, OCH₂), 4.17 (2H, ddd, J = 7.0, 7.1, 7.2 Hz, OCH₂), 1.80 (2H, m, CH₂), 1.40 (2H, m, CH₂), 1.22 (15H, m, 6xCH₂, CH₃), 0.85 (3H, m, CH₃). FABMS m/z: 363 (M+H)⁺. Anal. (C₁₉H₃₀N₄O₃) Found: C, 62.72; H, 8.11; N, 15.16. Calcd. C, 62.96; H, 8.34; N, 15.46.

9–Purine acetic acid, 6–(dodecyloxy) ethyl ester (34)

CHCl₃ (100%) was used for CC, off-white solid, yield 77%. ¹H NMR: δ 8.50 (1H, s, H–2 or H–8), 8.35 (1H, s, H–2 or H–8), 5.18 (2H, s, NCH₂), 4.54 (2H, dd, J = 6.5, 6.8 Hz, OCH₂), 4.17 (2H, ddd, J = 7.0, 7.1, 7.1 Hz, OCH₂), 1.80 (2H, m, CH₂), 1.3 (21H, m, 9xCH₂, CH₃), 0.85 (3H, m, CH₃). FABMS m/z: 391 (M+H)⁺. ESI–MS: Found 391.2700, calcd. for C₂₁H₃₄N₄O₃ 391.2704. Anal. (C₂₁H₃₄N₄O₃· 0.5H₂O) Found: C, 63.16; H, 8.46; N, 12.98. Calcd. C, 63.13; H, 8.83; N, 14.02.

9–Purine acetic acid, 2-amino–6–(decyloxy) ethyl ester (35)

CHCl₃ (100%) was used for CC, off–white solid, yield 51%. ¹H NMR: δ 7.82 (1H, s, H–8), 6.43 (2H, br s, NH₂), 4.91 (2H, s, NCH₂), 4.39 (2H, dd, J = 6.6, 6.7 Hz, OCH₂), 4.15 (2H, ddd, J = 7.1, 7.1, 7.1 Hz, OCH₂), 1.76 (2H, m, CH₂), 1.24 (17H, m, 7xCH₂, CH₃), 0.85 (3H, m, CH₃). FABMS m/z: 378 (M+H)⁺. ESI–MS: Found 378.2491, calcd. for C₁₉H₃₁N₅O₃ 378.2500.Anal. (C₁₉H₃₁N₅O₃· 0.3H₂O) Found: C, 59.39; H, 7.81; N, 17.95. Calcd. C, 59.60; H, 8.32; N, 18.29.

9–Purine acetic acid, 2–amino–6–(dodecyloxy) ethyl ester (36)

CHCl₃ (100%) was used for CC, off–white solid, yield 31%. ¹H NMR: δ 7.82 (1H, s, H–8), 6.43 (2H, br s, NH₂), 4.91 (2H, s, NCH₂), 4.39 (2H, dd, J = 6.6, 6.8 Hz, OCH₂), 4.15 (2H, ddd, J = 7.0, 7.1, 7.1 Hz, OCH₂), 1.74 (2H, m, CH₂), 1.24 (21H, m, 5xCH₂, CH₃), 0.85 (3H, m, CH₃). FABMS m/z: 406 (M+H)⁺. Anal. (C₂₁H₃₅N₅O₃) Found: C, 61.82; H, 8.42; N, 16.99. Calcd. C, 62.20; H, 8.70; N, 17.27.

3.1.3. General Procedure for Synthesis of 6–Decyloxy (38) and 6–Decylthio (39) Substituted Purine Intermediates

The starting material, 2–amino–6–chloro–9H–purine–9–acetic acid (37), for the synthesis of 38 and 39 was purchased commercially and used as such. The 6–decyloxy derivative 38 was synthesized in the same manner as described above for compound 8. The 6-decylthio derivative 39 was synthesized by the method previously described by us.¹²

2–Amino–6–decyloxy–9H–purine–9–acetic acid (38)

CHCl₃–MeOH (3:1) was used for CC, off–white solid, yield 65%. ¹H NMR: δ 7.69 (1H, s, H–8), 6.20 (2H, br s, NH₂), 4.38 (2H, t, J = 6.6 Hz, OCH₂), 4.28 (2H, s, NCH₂), 1.74 (2H, m, CH₂), 1.37 (14H, m, 7xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: Found 350.2204 (M+H)⁺, calcd. for C₁₇H₂₇N₅O₃ 350.2187.

2–Amino–6–decylthio–9H–purine–9–acetic acid (39)

CHCl₃–MeOH (7:1) was used for CC, off–white solid, yield 64%, ¹H NMR: δ 7.81 (1H, s, H–8), 6.31 (2H, br s, NH₂), 4.40 (2H, br s, NCH₂), 3.26 (2H, t, J = 7.1 Hz, SCH₂), 1.65 (2H, m, CH₂), 1.37 (14H, m, 7xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: Found 366.1959 (M+H)⁺, calcd. for C₁₇H₂₇N₅O₂S 366.1958. Anal. (C₁₇H₂₇N₅O₂S ·2.25H₂O) Found: C, 50.68; H, 6.54; N, 16.86. calcd. C, 50.29; H, 7.82; N, 16.86.

3.1.4. General Procedure for Synthesis of 6–Decyloxy or 6–Decylthio–9–L–Phe/or Ala– substituted Purine Derivatives (39-50)

To a solution of the appropriate intermediate (38 or 39) in anhydrous pyridine (5 mL) under argon, was added the appropriate amino acid ester HCl (1.5 equiv.) followed by triethylamine (1.5 equiv.). The resulting solution was chilled slightly and PyBOP (1.5 equiv.) was added. The reaction solution was allowed to stir under argon overnight at room temperature. Deionized water (20 mL) was added and extracted with CHCl₃ (2 × 350 mL). The combined organic layers were washed with 1N HCl (50 mL), d. H₂O (50 mL), dried over Na₂SO₄, concentrated, re-dissolved in toluene and co-evaporated to remove excess pyridine. The resulting residue was dried in vacuo over P₂O₅ at 25 °C. The crude material was then purified by column chromatography (CC) over silica gel G (230–400 mesh).

L–Alanine, N–[2–(2–amino–6–decyloxy–9H–purin–9–yl)–1–oxoethyl]–, methyl ester (40)

CHCl₃–MeOH (95:5) was used for CC, off–white solid, yield 57%. ¹H NMR: δ 8.74 (1H, d, J = 7.0 Hz, NHCH), 7.76 (1H, s, H–8), 6.36 (2H, br s, NH₂), 4.74 (2H, br s, NCH₂), 4.39 (2H, t, J = 6.6 Hz, OCH₂), 4.29 (1H, m, NHCH), 3.63 (3H, m, CO₂CH₃), 1.74 (2H, m, CH₂), 1.25 (17H, m, CH₃, 7xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: Found 435.2721 (M+H)⁺, calcd. for C₂₁H₃₄N₆O₄ 435.2714. Anal. (C₂₁H₃₄N₆O₄) Found: C, 53.87; H, 7.23; N, 16.49.

L–Phenylalanine, N–[2–(2–amino–6–decyloxy–9H–purin–9–yl)–1–oxoethyl]–, methyl ester (41)

CHCl₃–MeOH (99:1) was used for CC, off–white solid, yield 51%. ¹H NMR: δ 8.76 (1H, d, J = 7.5 Hz, NHCH), 7.69 (1H, s, H–8), 7.25 (5H, m, Ar), 6.33 (2H, br s, NH₂), 4.71 (2H, dd, NCH₂), 4.48 (1H, m, NHCH), 4.38 (2H, t, J = 6.5 Hz, OCH₂), 3.59 (3H, s, CO₂CH₃), 2.98 (2H, m, CH₂Ph), 1.73 (2H, m, CH₂), 1.25 (14H, m, 7xCH₂), 0.85 (3H, m, CH₃). FABMS m/z: 511 (M+H)⁺. Anal. (C₂₇H₃₈N₆O₄) Found: C, 63.41; H, 7.23; N, 16.44. Calcd. C, 63.51; H, 7.50; N, 16.46.

L–Phenylalanine, N–[2–(2–amino–6–decyloxy–9H–purin–9–yl)–1–oxoethyl]–, ethyl ester (42)

CHCl₃–MeOH (99:1) was used for CC, off–white solid, yield 59%. ¹H NMR: δ 8.76 (1H, d, J = 7.5 Hz, NHCH), 7.70 (1H, s, H–8), 7.25 (5H, m, Ar), 6.33 (2H, br s, NH₂), 4.72 (2H, dd, NCH₂), 4.48 (1H, m, NHCH), 4.39 (2H, t, J = 6.4 Hz, OCH₂), 4.03 (2H, dd, CO₂CH₂), 2.98 (2H, m, CH₂Ph), 1.74 (2H, m, CH₂), 1.24 (14H, m, 7xCH₂), 1.07 (3H, t, J = 7.0 Hz, CH₃), 0.85 (3H, m, CH₃). FABMS m/z: 525 (M+H)⁺. Anal. (C₂₈H₄₀N₆O₄·0.8H₂O) Found: C, 62.63; H, 7.38; N, 15.30. Calcd. C, 62.39; H, 7.78; N, 15.59. 20982

L–Alanine, N–[2–(2–amino–6–decylthio–9H–purin–9–yl)–1–oxoethyl]–, methyl ester (43)

Petroleum ether–EtOAc (1:4) was used for CC, off–white solid, yield 66%. ¹H NMR: δ 8.76 (1H, d, J = 7.0 Hz, NHCH), 7.84 (1H, s, H–8), 6.44 (2H, br s, NH₂), 4.75 (2H, s, NCH₂), 4.31 (1H, m, NHCH), 3.63 (3H, s, CO₂CH₃), 3.26 (2H, t, J = 7.0 Hz, SCH₂), 1.64 (2H, m, CH₂), 1.40 (2H, m, CH₂), 1.29 (15H, m, CH₃, 6xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: Found 451.2487 (M+H)⁺, calcd. For C₂₁H₃₄N₆O₃S 451.2486. Anal. (C₂₁H₃₄N₆O₃S) Found: C, 56.98; H, 7.64; N, 17.34. Calcd. C, 55.98; H, 7.61; N, 18.65.

D–Alanine, N–[2–(2–amino–6–decylthio–9H–purin–9–yl)–1–oxoethyl]–, methyl ester (44)

CHCl₃–MeOH (98:2) was used for CC, off–white solid, yield 57% yield. ¹H NMR: δ 8.75 (1H, d, J = 6.9 Hz, NH), 7.84 (1H, s, H–8), 6.45 (2H, s, NH₂), 4.76 (2H, m, NCH₂), 4.31 (1H, m, CH), 3.63 (3H, s, CH₃), 3.27 (2H, m, SCH₂), 1.64 (2H, m, CH₂), 1.37 (2H, m, CH₂), 1.31 (3H, d, J = 7.2 Hz, CH₃), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: found 451.2481 (M+H)⁺, calcd. 451.2485 for C₂₁H₃₄N₆O₃S. Anal. (C₂₁H₃₄N₆O₃S) Found: C, 56.98; H, 7.64; N, 17.34. Calcd. C, 55.98; H, 7.61; N, 18.65.

L–Phenylalanine, N–[2–(2–amino–6–decylthio–9H–purin–9–yl)–1–oxoethyl]–, methyl ester (45)

CHCl₃–MeOH (99:1) was used for CC, off–white solid, yield 69%. ¹H NMR: δ 8.77 (1H, d, J = 7.5 Hz, NHCH), 7.77 (1H, s, H–8), 7.22 (5H, m, Ar), 6.40 (2H, br s, NH₂), 4.72 (2H, dd, NCH₂), 4.48 (1H, m, NHCH), 3.59 (3H, s, CO₂CH₃), 3.26 (2H, m, SCH₂), 2.99 (2H, m, CH₂Ph), 1.65 (2H, m, CH₂), 1.24 (14H, m, 7xCH₂), 0.85 (3H, m, CH₃). FABMS m/z: 527 (M+H)⁺. Anal. (C₂₇H₃₈N₆O₃S) Found: C, 61.39; H, 7.23; N, 15.76. Calcd. C, 61.57; H, 7.26; N, 15.96.

D–Phenylalanine, N–[2–(2–amino–6–decylthio–9H–purin–9–yl)–1–oxoethyl]–, methyl ester (46)

CHCl₃–MeOH (98:2) was used for CC, off-white solid, yield 87%. ¹H NMR: δ 8.79 (1H, d, J = 7.5 Hz, NHCH), 7.77 (1H, s, H–8), 7.22 (5H, m, Ar), 6.42 (2H, br s, NH₂), 4.73 (2H, m, NCH₂), 4.48 (1H, m, NHCH), 3.59 (3H, s, CO₂CH₃), 3.26 (2H, t, J = 7.1 Hz, SCH₂), 3.03 (1H, dd, J = 5.9, 13.6 Hz, CH₂Ph), 2.93 (1H, dd, J = 8.4, 13.6 Hz, CH₂Ph), 1.65 (2H, m, CH₂), 1.39 (2H, m, CH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). FABMS m/z: 527 (M+H)⁺. Anal. (C₂₇H₃₈N₆O₃S) Found: C, 61.51; H, 7.24; N, 15.88. Calcd. C, 61.57; H, 7.26; N, 15.96.

L–Phenylalanine, N–[2– (2–amino–6–decylthio–9H–purin–9–yl)–1–oxoethyl]–, ethyl ester (47)

Petroleum ether–EtOAc (1:2) was used for CC, off–white solid, yield 60%. ¹H NMR: δ 8.78 (1H, d, J = 7.5 Hz, NHCH), 7.78 (1H, s, H–8), 7.23 (5H, m, Ar), 6.41 (2H, br s, NH₂), 4.75 (1H, d, NCH₂), 4.72 (1H, d, NCH₂), 4.48 (1H, m, NHCH), 4.02 (2H, m, OCH₂CH₃), 3.26 (2H, m, SCH₂), 2.98 (2H, m, CH₂Ph), 1.65 (2H, m, CH₂), 1.29 (14H, m, 7xCH₂), 1.07 (3H, t, J = 7.0 Hz, CO₂CH₂CH₃), 0.85 (3H, m, CH₃). ESI–MS m/z: Found 541.2976 (M+H)⁺, calcd. For C₂₈H₄₀N₆O₃S 541.2955. Anal. (C₂₈H₄₀N₆O₃S ·0.5H₂O) Found: C, 62.80; H, 7.72; N, 15.03. Calcd. C, 61.18; H, 7.52; N, 15.29.

L-Serine, N–[2–(2–amino–6–decylthio–9H–purin–9–yl)–1–oxoethyl]–, methyl ester (48)

CHCl₃-MeOH (9:1) was used for CC, off-white solid, yield 53%. ¹HNMR: δ 8.74 (1H, d, J = 7.8 Hz, NHCH), 7.84 (1H, s, H-8), 6.44 (2H, br s, NH₂), 5.16 (1H, t, J = 5.4 Hz, OH), 4.81 (2H, m, NCH₂), 4.38 (1H, m, NHCH), 3.73 (1H, m, CH₂), 3.64 (3H, s, CH₃), 3.62 (1H, m, CH₂), 3.26 (2H, t, J = 7.1 Hz, SCH₂), 1.65 (2H, m, CH₂), 1.40 (2H, m, CH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). FABMS m/z: 467 (M+H)⁺. Anal. (C₂₁H₃₄N₄O₄S ·0.15H₂O) Found: C, 53.80; H, 7.10; N, 17.51. Calcd. C, 53.75; H, 7.37; N, 17.91.

D–Serine, N–[2–(2–amino–6–decylthio–9H–purin–9–yl)–1–oxoethyl]–, methyl ester (49)

CHCl₃–MeOH (9:1) was used for CC, off–white solid, yield 59%. ¹HNMR: δ 8.74 (1H, d, J = 7.8 Hz, NHCH), 7.84 (1H, s, H–8), 6.44 (2H, br s, NH₂), 5.17 (1H, t, J = 5.4 Hz, OH), 4.80 (2H, m, NCH₂), 4.38 (1H, m, NHCH), 3.73 (1H, m, CH₂), 3.64 (3H, s, CH₃), 3.62 (1H, m, CH₂), 3.26 (2H, m, SCH₂), 1.65 (2H, m, CH₂), 1.40 (2H, m. CH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). FABMS m/z: 467 (M+H)⁺. Anal. (C₂₁H₃₄N₆O₄S) Found: C, 54.04; H, 7.09; N, 18.01. Calcd. C, 54.06; H, 7.34; N, 18.00.

L–Serine, N–[2–(2–amino–6–decylthio–9H–purin–9–yl)–1–oxoethyl]–, ethyl ester (50)

CHCl₃–MeOH (95:5) was used for CC, off–white solid, yield 83%. ¹HNMR: δ 8.70 (1H, d, J = 7.8 Hz, NHCH), 7.84 (1H, s, H–8), 6.44 (2H, br s, NH₂), 5.15 (1H, t, J = 5.6 Hz, OH), 4.80 (2H, m, NCH₂), 4.35 (1H, m, NHCH), 4.10 (2H, dd, J = 7.1, 14.2 Hz, OCH₂), 3.73 (1H, m, CH₂OH), 3.63 (1H, m, CH₂OH), 3.26 (2H, t, J = 7.1 Hz, SCH₂), 1.18 (3H, t, J = 7.1 Hz, CH₃), 1.65 (2H, m, CH₂), 1.40 (2H, m, CH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). FABMS m/z: 481 (M+H)⁺. Anal. (C₂₂H₃₆N₆O₄S) Found: C, 55.39; H, 7.43; N, 17.25. Calcd. C, 54.98; H, 7.55; N, 17.49.

3.1.5. General procedure for synthesis of purine analogs 51–54

The 6-decylmercapto purine analogs 51–54 were prepared starting from 6-chloropurine analogs by treating overnight with 1-decanethiol in presence of (CH₃)₃CO⁻K⁺ in isopropanol (3 mL) under reflux condition. The reactions were cooled to room temperature, 2 mL of deionized water were added and the mixture acidified with acetic acid (0.5 mL). Solid was collected by filtration and washed with water followed by petroleum ether. The resulting material was dried under vacuum at 78 °C overnight and analyzed.

N–(6–(Decylthio)–9H–purin–2–yl)acetamide (51)

Starting from N–(6–chloro–6,9–dihydro–1H–purin–2–yl)acetamide¹⁷ (100 mg, 0.47 mmol), 106 mg (Yield 64%). ¹H NMR: δ 10.33 (1H, s, NH), 8.25 (1H, s, H–8), 3.33 (2H, m, SCH₂), 2.20 (3H, s, COCH₃), 1.69 (2H, m, CH₂), 1.42 (2H, m, CH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: 350 (M+H)⁺. Anal. (C₁₇H₂₇N₅OS); Found: C, 58.26; H, 7.97; N, 19.87. Calcd. C, 58.42; H, 7.79; N, 20.04.

3–(2–Amino–6–(decylthio)–9H–purin–9–yl)propane–1,2–diol (52)

Starting from 3–(2–amino–6–chloro–1H–purin–9(6H)–yl)propane–1,2–diol¹⁷ (100 mg, 0.41 mmol), 82 mg (Yield 52%). ¹H NMR: δ 7.82 (1H, s, H–8), 6.44 (2H, br s, NH₂), 5.07 (1H, d, J = 5.1 Hz, CHOH), 4.16 (1H, dd, J = 3.3, 13.5 Hz, NCH₂), 3.85 (1H, dd, J = 7.9, 13.5 Hz, NCH₂), 3.77 (1H, m, CHOH) 3.37 (2H, m, CH₂OH), 3.26 (2H, dd, J = 6.9, 7.2 Hz, SCH₂), 1.65 (2H, m, CH₂), 1.38 (2H, m, CH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: 382 (M+H)⁺. Anal. (C₁₈H₃₁N₅O₂S); Found: C, 56.59; H, 8.04; N, 18.39. Calcd. C, 56.66; H, 8.19; N, 18.36.

9–Benzyl–6–(decylthio)–9H–purin–2–amine (53)

Starting from 9–benzyl–6–chloro–6,9–dihydro–1H–purin–2–amine¹⁷ (100 mg, 0.38 mmol), 135 mg (Yield 88%). ¹H NMR: δ 8.02 (1H, s, H–8), 7.31 (3H, m, Ar), 7.22 (2H, m, Ar), 6.48 (2H, br s, NH₂), 5.25 (2H, s, NCH₂), 3.26 (2H, dd, J = 6.9, 7.2 Hz, SCH₂), 1.65 (2H, m, CH₂), 1.38 (2H, m, CH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: 398 (M+H)⁺. Anal. (C₂₂H₃₁N₅S); Found: C, 66.50 H, 7.89; N, 17.42. Calcd. C, 66.46; H, 7.86; N, 17.61.

2–((2,6–Bis(decylthio)–9H–purin–9–yl)methoxy)ethanol (54)

Starting from 2–((2,6–dichloro–1H–purin–9(6H)–yl)methoxy)ethanol¹⁷ (100 mg, 0.38 mmol), 40 mg (Yield 26%). ¹H NMR: δ 8.43 (1H, s, H–8), 5.57 (2H, s, NCH₂), 4.66 (1H, t, J = 5.3 Hz, OH), 3.51 (2H, m, OCH₂), 3.45 (2H, m, CH₂OH), 3.27 (2H, dd, J = 4.5, 7.8 Hz, SCH₂), 3.18 (2H, dd, J = 7.2, 7.5 Hz, SCH₂), 1.70 (4H, m, 2xCH₂), 1.41 (4H, m, 2xCH₂), 1.24 (24H, m, 12xCH₂), 0.84 (6H, m, 2xCH₃). ESI–MS m/z: 539 (M+H)⁺. Anal. (C₂₈H₅₀N₄O₂S₂); Found: C, 62.53; H, 9.46; N, 10.22. Calcd. C, 62.41; H, 9.35; N, 10.40.

3.1.6. General procedure for synthesis of purine analogs 55–69

The 6–decylmercapto purine analogs 55–69 were prepared starting from 6-purinethiones which were treated overnight with 1–chlorodecane/anhydrous K₂CO₃ in DMAc (3 mL) at room temperature. Deionized water (2 mL) was added and the mixture was acidified with acetic acid (0.2 mL). Solid was collected by filtration and washed with water followed by petroleum ether. The material was dried under vacuum at 78 °C overnight and analyzed.

6–(Decylthio)–9–ethyl–9H–purin–2–amine (55)

Starting from 2–amino–9–ethyl–1H–purine–6(9H)–thione²⁵ (100 mg, 0.51 mmol), 80 mg (Yield 47%). ¹H NMR: δ 7.94 (1H, s, H–8), 6.42 (2H, br s, NH₂), 4.03 (2H, dd, J = 7.2, 14.4 Hz, NCH₂), 3.26 (2H, t, J = 7.2 Hz, SCH₂), 1.64 (2H, m, CH₂), 1.34 (3H, t, J = 7.2 Hz, CH₃), 1.40 (2H, m, CH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: 336 (M+H)⁺. Anal. (C₁₇H₂₉N₅S. 0.1H₂O); Found: C, 60.47; H, 8.75; N, 20.54. Calcd. C, 60.53; H, 8.73; N, 20.76.

6–(Decylthio)–2–ethyl–9H–purine (56)

Starting from 2–ethyl–1H–purine–6(9H)–thione²⁶ (100 mg, 0.55 mmol), 56 mg (Yield 31%). ¹H NMR: δ 13.2 (1H, br s, NH), 8.31 (1H, s, H–8), 3.33 (2H, dd, J = 6.9, 7.2 Hz, SCH₂), 2.89 (2H, dd, J = 7.5, 15 Hz, CH₂), 1.70 (2H, m, CH₂), 1.41 (2H, m, CH₂), 1.31 (3H, t, J = 7.5 Hz, CH₃), 1.23 (12H, m, 6xCH₂), 0.84 (3H, m, CH₃). ESI–MS m/z: 321 (M+H)⁺. Anal. (C₁₇H₂₈N₄S.0.2H₂O); Found: C, 63.14; H, 8.70; N, 17.29. Calcd. C, 63.00; H, 8.83; N, 17.28.

9–Cycloheptyl–6–(decylthio)–9H–purine (57)

Starting from 9–cycloheptyl–1H–purine–6(9H)–thione²⁷ (100 mg, 0.40 mmol), 124 mg (Yield 79%). ¹H NMR: δ 8.68 (1H, s, H–2), 8.54 (1H, s, H–8), 4.64 (1H, m, NCH), 3.32 (2H, dd, J = 7.2, 7.5 Hz, SCH₂), 2.08 (6H, m, 3xCH₂), 1.65 (8H, m, 4xCH₂), 1.41 (2H, m, CH₂), 1.23 (12H, m, 6xCH₂), 0.84 (3H, m, CH₃). ESI–MS m/z: 389 (M+H)⁺. Anal. (C₂₂H₃₆N₄S.0.1H₂O); Found: C, 67.77; H, 9.37; N, 14.34. Calcd. C, 67.68; H, 9.35; N, 14.35.

9–Allyl–6–(decylthio)–9H–purine (58)

Starting from 9–allyl–1H–purine–6(9H)–thione (100 mg, 0.52 mmol), 72 mg (Yield 42%). ¹H NMR: δ 8.70 (1H, s, H–2), 8.44 (1H, s, H–8), 6.08 (1H, m, CH), 5.21 (1H, m, CH_2a), 5.06 (1H, m, CH_2b), 4.89 (2H, m, NCH₂), 3.35 (2H, dd, J = 7.2, 7.5 Hz, SCH₂), 1.70 (2H, m, CH₂), 1.41 (2H, m, CH₂), 1.23 (12H, m, 6xCH₂), 0.84 (3H, m, CH₃). ESI–MS m/z: 333 (M+H)⁺. Anal. (C₁₈H₂₈N₄S.0.1H₂O); Found: C, 64.74; H, 8.55; N, 16.72. Calcd. C, 64.67; H, 8.50; N, 16.76.

2–(6–(Decylthio)–9H–purin–9–yl)ethyl acetate (59)

Starting from 2–(6–thioxo–1H–purin–9(6H)–yl)ethyl acetate (100 mg, 0.42 mmol), 146 mg (Yield 92%). ¹H NMR: δ 8.71 (1H, s, H–2), 8.47 (1H, s, H–8), 4.50 (2H, m, NCH₂), 4.41 (2H, m, OCH₂), 3.33 (2H, dd, J = 7.2, 7.5 Hz, SCH₂), 2.93 (3H, s, CH₃), 1.70 (2H, m, CH₂), 1.41 (2H, m, CH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: 379 (M+H)⁺. Anal. (C₁₉H₃₀N₄O₂S.0.15H₂O); Found: C, 59.69; H, 7.88; N, 14.61. Calcd. C, 59.86; H, 8.01; N, 14.69.

2–(6–(Decylthio)–1H–purin–9(6H)–yl)acetonitrile (60)

Starting from 2–(6–thioxo–1H–purin–9(6H)–yl)acetonitrile (100 mg, 0.52 mmol), 75 mg (Yield 43%). ¹H NMR: δ 8.79 (1H, s, H–2), 8.51 (1H, s, H–8), 5.55 (2H, m, NCH₂), 3.36 (2H, dd, J = 7.2, 7.5 Hz, SCH₂), 1.71 (2H, m, CH₂), 1.41 (2H, m, CH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: 332 (M+H)⁺. Anal. (C₁₇H₂₅N₅S); Found: C, 61.48; H, 7.36; N, 20.99. Calcd. C, 61.60; H, 7.60; N, 21.13.

(E)–N–Benzylidene–6–(decylthio)–9H–purin–9–amine(61)

Starting from (E)–9–(benzylideneamino)–1H–purine–6(9H)–thione²⁸ (100 mg, 0.39 mmol), 82 mg (Yield 53%). ¹H NMR: δ 9.78 (1H, s, CH), 8.90 (1H, s, H–2), 8.82 (1H, s, H–8), 7.95 (2H, m, Ar), 7.60 (3H, m, Ar), 3.37 (2H, dd, J = 7.2, 7.5 Hz, SCH₂), 1.71 (2H, m, CH₂), 1.41 (2H, m, CH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: 396 (M+H)⁺. Anal. (C₂₂H₂₉N₅S.0.1H₂O); Found: C, 66.52; H, 7.07; N, 17.62. Calcd. C, 66.50; H, 7.36; N, 17.62.

6–(Decylthio)–9–ethyl–9H–purine (62)

Starting from 9–ethyl–1H–purine–6(9H)–thione²⁹ (100 mg, 0.55 mmol), 160 mg (Yield 90%). ¹H NMR: δ 8.70 (1H, s, H–2), 8.48 (1H, s, H–8), 4.27 (2H, dd, J = 7.2, 14.4 Hz, NCH₂), 3.34 (2H, dd, J = 7.2, 7.5 Hz, SCH₂), 1.70 (2H, m, CH₂), 1.43 (3H, t, J = 7.2 Hz, CH₃), 1.40 (2H, m, CH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: 321 (M+H)⁺. Anal. (C₁₇H₂₈N₄S); Found: C, 63.72; H, 8.94; N, 17.44. Calcd. C, 63.71; H, 8.81; N, 17.48.

9–Benzyl–6–(decylthio)–9H–purine (63)

Starting from 9–benzyl–1H–purine–6(9H)–thione¹⁷ (100 mg, 0.41 mmol), 59 mg (Yield 37%). ¹H NMR: δ 8.71 (1H, s, H–2), 8.59 (1H, s, H–8), 7.33 (5H, m, Ar), 5.47 (2H, s, NCH₂), 3.35 (2H, m, SCH₂), 1.70 (2H, m, CH₂), 1.40 (2H, m, CH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: 383 (M+H)⁺. Anal. (C₂₂H₃₀N₄S); Found: C, 69.05; H, 7.83; N, 14.66. Calcd. C, 69.07; H, 7.90; N, 14.65.

2–(6–(Decylthio)–9H–purin–9–yl)cyclohexanol (64)

Starting from 9–(2–hydroxycyclohexyl)–1H–purine–6(9H)–thione^18d (100 mg, 0.40 mmol), 80 mg (Yield 51%). ¹H NMR: δ 8.68 (1H, s, H–2), 8.42 (1H, s, H–8), 4.99 (1H, d, J = 4.2 Hz, OH), 4.55 (1H, m, NCH), 3.97 (1H, br s, CHOH), 3.34 (2H, m, SCH₂), 2.30 (1H, m, CH₂), 1.74 (6H, m, CH₂), 1.42 (5H, m, CH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: 391 (M+H)⁺. Anal. (C₂₁H₃₄N₄OS); Found: C, 64.45; H, 8.83; N, 14.34. Calcd. C, 64.58; H, 8.77; N, 14.34.

9–Cyclopentyl–6–(decylthio)–9H–purine (65)

Starting from 9–cyclopentyl–1H–purine–6(9H)–thione^18a (100 mg, 0.48 mmol), 134 mg (Yield 82%). ¹H NMR: δ 8.69 (1H, s, H–2), 8.52 (1H, s, H–8), 4.93 (1H, m, NCH), 3.33 (2H, m, SCH₂), 2.16 (2H, m, CH₂), 2.03 (2H, m, CH₂), 1.89 (2H, m, CH₂), 1.70 (2H, m, CH₂), 1.40 (2H, m, CH₂), 1.23 (12H, m, 6xCH₂), 0.84 (3H, m, CH₃). ESI–MS m/z: 361 (M+H)⁺. Anal. (C₃₀H₃₂N₄S.0.1H₂O); Found: C, 66.06; H, 9.10; N, 15.89. Calcd. C, 66.29; H, 8.96; N, 15.46.

9–Butyl–6–(decylthio)–9H–purine (66)

Starting from 9–butyl–1H–purine–6(9H)–thione^18a (100 mg, 0.48 mmol), 124 mg (Yield 74%). ¹H NMR: δ 8.69 (1H, s, H–2), 8.47 (1H, s, H–8), 4.24 (2H, dd, J = 6.9, 7.2 Hz, NCH₂), 3.34 (2H, t, J = 7.2 Hz, SCH₂), 1.81 (2H, m, CH₂), 1.69 (2H, m, CH₂), 1.39 (2H, m, CH₂), 1.23 (14H, m, 7xCH₂), 0.89 (3H, t, J = 7.2 Hz, CH₃), 0.84 (3H, m, CH₃). ESI–MS m/z: 349 (M+H)⁺. Anal. (C₁₉H₃₂N₄S.0.2H₂O); Found: C, 64.88; H, 9.28; N, 15.89. Calcd. C, 64.80; H, 9.27; N, 15.90.

2–(6–(Decylthio)–9H–purin–9–yl)cyclopentanol (67)

Starting from 9–(2–hydroxycyclopentyl)–1H–purine–6(9H)–thione^18c (100 mg, 0.42 mmol), 94 mg (Yield 59%). ¹H NMR: δ 8.68 (1H, s, H–2), 8.45 (1H, s, H–8), 4.92 (1H, d, J = 4.2 Hz, OH), 4.78 (1H, m, NCH), 4.18 (1H, m, CHOH), 3.34 (2H, m, SCH₂), 2.29 (2H, m, CH₂), 2.11 (2H, m, CH₂), 1.94 (2H, m, CH₂), 1.70 (2H, m, CH₂), 1.40 (2H, m, CH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: 377 (M+H)⁺. Anal. (C₂₀H₃₂N₄OS); Found: C, 63.88; H, 8.56; N, 14.75. Calcd. C, 63.79; H, 8.57; N, 14.88.

N–(6–(Decylthio)–9H–purin–9–yl)acetamide (68)

Starting from N–(6–thioxo–1H–purin–9(6H)–yl)acetamide^18b (100 mg, 0.48 mmol), 40 mg (Yield 24%). ¹H NMR: δ 8.69 (1H, s, H–2), 8.48 (1H, s, H–8), 3.35 (2H, m, SCH₂), 2.10 (3H, s, CH₃), 1.71 (2H, m, CH₂), 1.40 (2H, m, CH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: 350 (M+H)⁺. Anal. (C₁₇H₂₇N₅OS.0.1H₂O); Found: C, 58.01; H, 8.03; N, 19.80. Calcd. C, 58.12; H, 7.80; N, 19.93.

9–Cyclohexyl–6–(decylthio)–9H–purine (69)

Starting from 9–cyclohexyl–1H–purine–6(9H)–thione^18a (100 mg, 0.43 mmol), 93 mg (Yield 58%). ¹H NMR: δ 8.68 (1H, s, H–2), 8.54 (1H, s, H–8), 4.45 (1H, m, NCH), 3.33 (2H, m, SCH₂), 1.91 (8H, m, 4xCH₂), 1.69 (2H, m, CH₂), 1.41 (4H, m, 2xCH₂), 1.24 (12H, m, 6xCH₂), 0.85 (3H, m, CH₃). ESI–MS m/z: 375 (M+H)⁺. Anal. (C₂₁H₃₄N₄S.0.1H₂O); Found: C, 66.97; H, 9.19; N, 14.85. Calcd. C, 67.01; H, 9.16; N, 14.89.

3.1.7. N-(3,4-Dimethylphenyl)-2-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)acetamide (70)

Compound 73 (110 mg, 0.58 mmol) was dissolved in dry DMF (5 mL) under argon and 3,4-dimethylaniline (106 mg, 0.87 mmol) was added followed by the addition of HATU (265 mg, 0.70 mmol) and DIEA (0.16 mL, 0.16 mmol). The reaction mixture was stirred at room temperature overnight. The resulting solid was filtered and washed with ether to give pure 70 (86 mg, Yield 54%) after drying overnight under vacuum over P₂O₅ at 78 °C. mp: 203–205 °C. ¹H NMR: δ 10.26 (1H, s, CONH), 8.60 (1H, s), 7.54 (1H, d, J = 3.6 Hz), 7.35 (1H, s), 7.27 (1H, d, J = 2.0, 11.0 Hz), 7.05 (1H, d, J = 8.1 Hz), 6.54 (1H, d, J = 3.3 Hz), 5.11 (2H, s), 2.66 (3H, s), 2.17 (3H, s), 2.16 (3H, s). ESI–MS: m/z calc. for C₁₇H₁₈N₄OS (M+H)⁺: 327.1274, found: 327.1278.

3.1.8. 4-(Methylthio)-7H-pyrrolo[2,3-d]pyrimidine (72)

To a methanol (15 mL) solution of commercially available 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (400 mg, 2.60 mmol) was added NaSCH₃ (272 mg, 3.90 mmol), and the reaction mixture was stirred overnight at room temperature. The mixture was concentrated and the crude material was purified by column chromatography using 5% CHCl₃–MeOH to yield pure product 68 (386 mg, Yield 90%). ¹H NMR: δ 12.08 (1H, br s, NH), 8.58 (1H, s, H–2), 7.45 (1H, d, J = 3.4 Hz, H–8), 6.47 (1H, d, J = 3.5 Hz, H–7), 2.64 (3H, s, SCH₃). ESI–MS: m/z calc. for C₇H₇N₃S (M+H)⁺: 166.0433, found: 166.0432.

3.1.9. 2-(4-(Methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)acetic acid (73)

Compound 74 (100 mg, 0.61 mmol) was dissolved in dry DMF (3 mL) under argon and K₂CO₃ (100 mg, 0.73 mmol) was added followed by ethylbromoacetate (0.061 mL, 0.73 mmol). The reaction mixture was stirred overnight at room temperature. Solvent was removed, and the residue was dissolved in CHCl₃ (25 mL) and washed with water (2×15 mL). The chloroform layers were dried over Na₂SO₄ and concentrated. The crude material was purified by column chromatography over silica gel G (230–400 mesh) using 10% CHCl₃–MeOH as the eluant to yield the pure ethyl ester of 73 (120 mg). This material was then dissolved in dry MeOH (3 mL) and 1N NaOH (1 mL) was added. The reaction mixture was stirred overnight at room temperature. The resulting solid was filtered, washed with water and dried at 78 °C under vacuum to give pure 73 (110 mg, Yield 82% in 2 steps). ¹H NMR: δ 8.55 (1H, s, H–2), 7.40 (1H, d, J = 3.5 Hz, H–8), 6.41 (1H, d, J = 3.5 Hz, H–7), 4.56 (1H, s, NCH₂), 2.65 (3H, s, SCH₃). ESI–MS: m/z calc. for C₉H₉N₃O₂S (M+H)⁺: 224.0488, found: 224.0490.

3.2. Biology

3.2.1. Activity against Mtb H₃₇Ra and MAC strain

All compounds were tested for their inhibitory activity against Mtb H₃₇Ra (ATCC 25177) and MAC NJ211 strains. The screening was performed at 1.28 and 12.8 µg/mL in Middlebrook 7H9 broth supplemented with 0.2% glycerol and ADC enrichment using a colorimetric (Alamar blue) microdilution broth assay.³⁰ The active compounds (≤12.8 µg/mL) were retested using two-fold dilutions to obtain the actual MIC₉₉. The MIC₉₉ was recorded as the lowest drug concentration that inhibited the growth completely.

3.2.2. Activity against H₃₇Rv.²⁰

The primary screen was conducted at 6.25 µg/mL against Mtb. H₃₇Rv (ATCC 27294) using radiometric BACTEC assay in a 12B medium by Tuberculosis Antimicrobial Coordinating Facility (TAACF). Compounds demonstrating at least 90% inhibition at 6.25 µg/mL were re-tested to determine the MIC₉₀, defined as the lowest concentrating inhibiting growth by 90% or higher.

3.2.3. Cytotoxicity against VERO cells and Selectivity Index (SI).²⁰

Concurrent with the determination of MIC₉₀’s, compounds 16, 33, 34, 36, 55 and 65 were tested for cytotoxicity (CC₅₀) in VERO cells using Promega non-radioactive cell proliferation assay kits by TAACF. The selectivity index is defined as the ratio of the measured CC₅₀ in VERO cells to the MIC₉₀ (against Mtb H₃₇Rv).