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. Author manuscript; available in PMC: 2013 Jan 1.
Published in final edited form as: Eur J Med Chem. 2011 Nov 4;47C:167–174. doi: 10.1016/j.ejmech.2011.10.039

Synthesis and Evaluation of the Substrate Activity of C-6 Substituted Purine Ribosides with E. coli Purine Nucleoside Phosphorylase: Palladium Mediated Cross-Coupling of Organozinc Halides with 6-Chloropurine Nucleosides [1]

Abdalla E A Hassan 1,2, Reham A I Abou-Elkhair 1,2, James M Riordan 1, Paula W Allan 1, William B Parker 1, Rashmi Khare 1, William R Waud 1, John A Montgomery 1, John A Secrist III 1,*
PMCID: PMC3259247  NIHMSID: NIHMS340693  PMID: 22112758

Abstract

A series of C-6 alkyl, cycloalkyl, and aryl-9-(β-d-ribofuranosyl)purines were synthesized and their substrate activities with Escherichia coli purine nucleoside phosphorylase (E. coli PNP) were evaluated. (Ph3P)4Pd-mediated cross-coupling reactions of 6-chloro-9-(2,3,5-tri-O-acetyl-β-d-ribofuranosyl)-purine (6) with primary alkyl (Me, Et, n-Pr, n-Bu, isoBu) zinc halides followed by treatment with NH3/MeOH gave the corresponding 6-alkyl-9-(β-d-ribofuranosyl) purine derivatives 7–11, respectively, in good yields. Reactions of 6 with cycloalkyl(propyl, butyl, pentyl)zinc halides and aryl (phenyl, 2-thienyl)zinc halides gave under similar conditions the corresponding 6-cyclopropyl, cyclobutyl, cyclopentyl, phenyl, and thienyl -9-(β-d-ribofuranosyl)purine derivatives 12–16, respectively in high yields. E. coli PNP showed a high tolerance to the steric and hydrophobic environment at the 6-position of the synthesized purine ribonucleosides. Significant cytotoxic activity was observed for 8, 12, 15, and 16. Evaluation of 12 and 16 against human tumor xenografts in mice did not demonstrate any selective antitumor activity. In addition, 6-methyl-9-(β-d-arabinofuranosyl)purine (18) was prepared and evaluated.

Keywords: purine nucleoside phosphorylase, organozinc halides, cross-coupling reactions, 6-alkyl, cycloalkyl/aryl/heterocyclylpurine ribonucleosides

Introduction

Suicide gene therapy of cancer is an approach that is being evaluated as a potential treatment for solid tumors. We have developed a cancer gene therapy strategy that is based on the activation of a non-toxic purine nucleoside analog (prodrug) to a highly toxic purine analog by E. coli PNP selectively expressed in tumor cells [1–5]. E. coli PNP differs from human PNP in its ability to accept not only 6-oxopurine nucleosides, but also 6-aminopurine and certain adenine nucleoside analogs as substrates (Figure 1). This property has been used to cleave non-toxic adenine nucleoside analogs such as 9-(2-deoxy-β-D-ribofuranosyl)-6-methylpurine (MeP-dR, 1) and 2-fluoro-2′-deoxyadenosine (F-dAdo, 3), to the very toxic adenine analogs, 6-methylpurine (MeP, 2) and 2-fluoroadenine (F-Ade, 4) [6–8].

Figure 1.

Figure 1

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One of our goals has been to get more information about the E. coli PNP substrate structural requirements. We have reported on the correlation between various modifications at the sugar moiety of adenine nucleoside analogs and the substrate activity with E. coli PNP [5]. Crystal structures of a number of complexes of E. coli PNP with various compounds of varied substrate activities such as adenosine, MeP-dR, F-dAdo, and 2-fluoro-9-(β-D-arabinofuranosyl) adenine (F-araA, 5) showed unique positioning of MeP-dR at the active site [9]. The nucleoside base moiety of MeP-dR was shown to be fitted into a hydrophobic pocket at the active site, resulting in a 2.6 Å shift of the sugar moiety of MeP-dR from the phosphate binding site when compared with adenosine. Although the binding of MeP-dR was significantly different from that of the natural substrate, it was still an excellent substrate for this enzyme [5]. It has been postulated that in the mechanism of the phosphorolysis reaction catalyzed by E. coli PNP, the glycosidic bond breaking occurs ahead of the phosphate bond formation and that the transition state has a considerable oxocarbenium character that is stabilized and subsequently attacked by one of the phosphate oxygens [10]. These observations suggested that an increase of the hydrophobic interaction at the C6 position would have a positive impact on the cleavage activity. If it is the case, an enhancement of the cleavage activity of certain poor substrates such as arabinofuranosyladenine analogs might be observed. Herein, we report on the synthesis of selected C-6 alkyl and arylpurine nucleosides, their cleavage activity by E. coli PNP, and their evaluations both in vitro and in vivo.

Results and Discussions

Chemistry

Transition metal catalyzed cross-couplings of organometallics [11] (such as arylmagnesium halides [12], alkyl/arylzinc halides [13], alkyl/aryltin [14], trialkylaluminium [15], alkylcuprates [16] reagents and arylboronic acids [17] with 6-halopurines and 6-halopurine nucleoside analogs) have been effectively used for C-C bond formations at the C-6 position of purine nucleosides. We have previously reported on the application of the palladium-mediated cross-coupling of methylzinc bromide with N9-protected-6-chloropurine and suitably protected 6-chloro (9-β-d-ribo- and deoxyribofuranosyl) purines for the synthesis of MeP and the corresponding nucleosides [18]. The mildness of the reaction conditions as well as the stability, safety, and the ease of the preparation of the organozinc reagents prompted us to utilize the same chemistry for the introduction of different carbon substituents at the C-6 position. Treatment of 6-chloro-9-(2,3,5-tri-O-acetyl-β-d-ribofuranosyl)purine (6) [19,20] with MeZnBr, EtZnBr, n-PrZnBr, n-BuZnBr, and isoBuZnBr in THF in the presence of ca. 0.05 equivalents of (Ph3P)4Pd at 55 °C, followed by treatment with NH3/MeOH gave the corresponding 6-alkyl-9-(β-d-ribofuranosyl) purines (7–11, Scheme 1, Table 1) [18] in good yields. Treatment of 6 with sec-alkylzinc halides under the same conditions, however gave mainly the corresponding 6-primary alkyl derivatives with the 6-sec-alkyl derivatives as minor products, which were not isolated. Cyclopropyl [22], cyclobutyl, and cyclopentylzinc halides were cross-coupled with 6 in the presence of (Ph3P)4Pd efficiently to give, after deprotection of the sugar hydroxyl groups, the corresponding 6-cycloalkylpurine ribonucleosides 12–14 in good yields (Table 1). Phenyl and 2-thienylzinc bromides were also cross-coupled with 6 under similar conditions to give after removal of the acetyl groups by NH3/MeOH treatment, 6-phenyl and 6-(2-thienyl)-9-(β-dribofuranosyl) purine derivatives (15) [12a,17c,27] and (16) [27,28,30], respectively, in good yields.

Scheme 1.

Scheme 1

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aReaction conditions: a) RZnX, (Ph3P)4Pd, THF, 55 °C; b) NH3, MeOH

Table 1.

Pd(PPh3)4 Catalyzed Cross-Coupling of 6 with organozinc halides followed by treatment with NH3/MeOH

Entry RZnX Product, R Yield %
1 MeZnBr 7, CH3 95
2 EtZnBr 8, CH2CH3 82
3 n-PrZnBr 9, CH2CH2CH3 86
4 n-BuZnBr 10, CH2(CH2)2CH3 89
5 isoBuZnBr graphic file with name nihms340693t1.jpg 88
6 cycloPrZnBr graphic file with name nihms340693t2.jpg 92
7 cycloBuZnBr graphic file with name nihms340693t3.jpg 78
8 cyclopentylZnBr graphic file with name nihms340693t4.jpg 60
9 PhZnBr graphic file with name nihms340693t5.jpg 78
10 2-ThienylZnBr graphic file with name nihms340693t6.jpg 80
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6-Methyl-9-(β-d-arabinofuranosyl)purine (18) was prepared by (Ph3P)4Pd catalyzed cross-coupling of 6-chloro- 9-(2,3,5-tri-O-acetyl-β-d-arabinofuranosyl)purine 17 [23] with MeZnBr under similar conditions. The arabinofuranosyl-6-chloropurine derivative 17 was prepared from 9-(β-d-arabinofuranosyl)-hypoxanthine in two steps with minor modification of the literature procedure [20,23]. Cross-coupling of 17 with CH3ZnBr followed by removal of the acetyl groups under the standard conditions furnished 18 in high yield (Scheme 2). NOE studies showed a distinct glycosidic torsional angle preference for the arabinofuranosyl purine derivatives 7 and 18 compared with the corresponding ribofuranosyl derivatives. Irradiation at the H-8 proton of 18 resulted in 2% and 5% NOE enhancements at the H-1′ and H-3′ signals, respectively. Irradiation at H-3′ gave enhancements of 4–5% and 2% of the signals at H-8 and at H-4′, respectively. These results are very similar to those reported for 9-(β-d-arabinofuranosyl) adenine [24] and suggest a more syn-conformational preference for 18.

Scheme 2.

Scheme 2

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aReaction conditions: a) CH3ZnBr, (Ph3P)4Pd, THF, 55 °C; b) NH3, MeOH

Biology

Substrate characteristics with E. coli PNP

The rate of cleavage of 100 µM adenosine (the natural substrate) and its analog, MeP-R were 398,000 and 84,000 nmoles/mg/h, respectively. The cleavage activities of 6-ethyl, n-propyl, n-butyl, isobutyl, cyclopropyl, cyclobutyl, and cyclopentylpurine ribonucleosides (8–14) by E. coli PNP were as good as the 6-methyl derivative (7) (Table 2). Surprisingly, a considerable cleavage activity was also observed with 6-phenyl and 6-thienyl ribonucleosides 15 and 16. These results show the tolerance of E. coli PNP to the steric and hydrophobic effects at the C-6 position of the purine nucleosides and reflect a good accommodation of these hydrophobic substituents in the enzyme’s active site. On the other hand, the observed poor substrate activity of 6-methyl-9-(β-D-arabinofuranosyl)purine (18) reflects the impact of the conformation around the glycosidic bond as a determinant factor in placing the molecule in the proper position at the active site. It is of interest that arabinofuranosyl-MeP (18) was much worse as a substrate than F-araA (5).

Table 2.

Substrate activities of 6-substituted purine nucleoside analogs with E. coli PNP

Substrate C6-substituent Specific activity
(nmoles/mg/hr)		 (N)
Adenosine* amino 398,000 5
MeP-dR* methyl 461,000 10
F-araA* amino 1,300 4
7 methyl 84,000 8
8 ethyl 69,000 3
9 n-propyl 72,000 3
10 n-butyl 86,000 3
11 isobutyl 79,000 3
12 cyclopropyl 51,000 3
13 cyclobutyl 63,000 3
14 cyclopentyl 42,000 3
15 phenyl 21,000 3
16 thienyl 9,900 2
18 methyl 14 2
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Purified E. coli PNP (obtained from Dr. Steve Ealick, Cornell University, Ithaca, NY) was incubated at 25 °C with 100 µM of each nucleoside analog in the presence of 100 mM HEPES (pH 7.4), 50 mM phosphate (pH 7.4), 4% glycerol, 0.2 mM dithiothreitol, and an appropriate amount of enzyme to give a linear reaction. Samples were collected at various times after addition of substrate and the substrates and products were determined by monitoring UV absorbance as they eluted from a 150 × 4.6 mm, 5µm BDS hypersil C-18 column (Keystone Scientific Inc. Bellfonte, PA) using a 30-minute linear gradient of 5% to 50% acetonitrile in 50 mM NH3H2PO4 buffer pH 4.5 at a flow rate of 1 ml/minute. Each number is the average of at least 2 separate measurements (N).

*

From reference 5

Cytotoxicity

Because compound 7 is known to be very toxic to human cells, the 6-substituted purine nucleoside analogs were evaluated in a standard assay for cytotoxicity against CEM cells (Table 3). All of the compounds tested, except 11 and 18, were able to inhibit the growth of the CEM cells, but at greater concentrations than those required for compound 7. Because phosphorylation of compound 7 by adenosine kinase is required for its cytotoxicity, it is likely that this same activation is needed for the other compounds in Table 3. Thus, these results suggest that these compounds may be relatively poor substrates for this enzyme or that the phosphorylated metabolites are less active. Three of the most potent inhibitors (12,15,16) were also tested against a panel of solid tumor cell lines (Table 4), where they were generally quite active.

Table 3.

Inhibition of CEM cell growth by 6-substituted purine nucleoside analogs

Substrate C6-substituent IC50 (µM) (N)
7 methyl 0.02 2
8 ethyl 0.72 3
9 n-propyl 6.8 3
10 n-butyl 49 2
11 isobutyl >130 2
12 cyclopropyl 1 3
13 cyclobutyl 7 2
14 cyclopentyl 16 2
15 phenyl 0.62 3
16 thienyl 0.09 3
18 methyl >130 2
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CCRF-CEM cells (American Type Culture Collection) were incubated at 37 °C with various concentrations of the 6-substituted purine nucleoside analogs. Cell numbers were determined 72 hours after the addition of compound using a Coulter Counter and the amount of compound that resulted in 50% inhibition of cell growth was determined (IC50). Each number is the average of 2 or 3 separate measurements (N).

Table 4.

Inhibition of the growth of various solid tumor cell lines

Cell line IC50 of 6-substituted purine nucleoside analog (µM)
12 15 16
SNB7 (CNS) 33 31 10
DLD-1 (colon) 170 6 0.9
NCI-H23 (lung) 1 1.2 0.6
ZR-75-1 (mammary) 23 1.2 0.3
LOXIMVI (melanoma) >200 6.2 0.3
PC-3 (prostate) 67 1.2 0.9
CAKI-1 (renal) >200 31 30
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The above cells were plated in 96-well microtiter plates and incubated with various concentrations of compound number 12, 15, or 16 at 37 °C. Cell viability was measured after 72 hours of continuous incubation with compound using the sulforhodamine B assay (absorbance read at 570 nm), and the concentration of compound that inhibited cell growth by 50% was determined. The results shown are the result of one experiment.

Because of the sensitivity of the NCI-H23 tumor cell line to these compounds, the in vivo efficacy of compounds 12 and 16 were determined. NCI-H23 tumors were grown on the flanks of nude mice. When the tumors reached approximately 200 mg, the animals were treated with 33, 50, or 75 mg/kg of compound 12 or 16 (given ip daily for 9 consecutive days). Neither compound at any dose had any effect of tumor growth. Seventy-five mg/kg of compound 12 killed 5 of 6 mice, whereas with compound 16 this dose caused a 14% decrease in weight. Therefore, at maximally tolerated doses neither compound exhibited selective antitumor activity in mice.

Experimental

1H NMR and 13C NMR spectra were recorded on a Nicolet NT 300 NB spectrometer operating at 300.635 MHz (1H) or 75.6 MHz (13C). Chemical shifts are expressed in parts per million from tetramethylsilane. The hydrogen-decoupled 13C-NMR spectra were assigned by comparison of the JCH values obtained from hydrogen-coupled 13C-NMR spectra. When necessary, selective hydrogen decoupling was performed in order to confirm the assignments. Ultraviolet absorption spectra were determined on Perkin-Elmer Lambda 19 spectrometer by dissolving each compound in methanol or water and diluting appropriately with 0.1 N HCl, pH 7 buffer, or 0.1 N NaOH. Values are in nanometers, and numbers in parentheses are extinction coefficients (ε × 10−3). Mass spectra were recorded on a Varian/MAT 311A double-focusing mass spectrometer in the fast atom bombardment (FAB) mode (glycerol matrix). CHN elemental analysis was carried out on Perkin-Elmer 2400 elemental analyzer. HPLC analysis was carried out on a Hewlett-Packard 1100 series liquid chromatograph with a Phenomenex Sphenclone 5 µM ODS (1) column (4.6 mm × 25 cm) with UV monitoring (254 nm). All flash column chromatography used 230–400 mesh silica gel from E. Merck. TLC was done on Analtech pre-coated (250 µm) silica gel (GF) plates.

6-Ethyl-9-(β-d-ribofuranosyl)purine (8)[25]

A solution of EtZnBr (1.2 mmoL) was generated by dropwise addition of ZnBr2 (1.13M, 1.1 mL, 1.2. mmol) in THF to 2M solution of EtMgBr (1.1 mmol, 0.3 mL) in THF (6 mL) for 1h at −78 °C. The solution was allowed to warm gradually to room temperature and then (Ph3P)4Pd (27 mg, 0.02 mmol) in THF (2 mL) was added to the mixture. A solution of compound 6 (0.197 g, 0.477 mmol) in dry THF (4 mL) was added and the mixture was heated under argon for 1h at 55 °C. The mixture was then cooled down to room temperature and quenched with saturated solution of NH4Cl. The solvent was concentrated under reduced pressure and the residue was partitioned between CHCl3 and H2O. The residue obtained by evaporation of the dried organic phase was dissolved in MeOH saturated with NH3 (15 mL) and kept overnight at room temperature. The solvent was evaporated and the residue was purified by a flash silica gel chromatography (elution with 5% EtOH in CHCl3) to give (0.11 g, 82%) 8 as a white solid, m.p. 98–100 °C, 1:1 H2O-EtOH (lit. [25] 104–106 °C): HPLC [99.5%; RT, 10.64 min, 0.01M NH4H2PO4: MeOH, 20 min linear gradient]; MS m/z 281 (M+1)+, UV λmax pH 1, 265.3 (7.4); pH 7, 260.6 (8.0); pH 13, 261.2 (8.1); 1H NMR (Me2SO-d6) δ 8.84 (1H, s, H-2, 1JC,H = 204.4 Hz), 8.76 (1H, s, H-8, 1JC,H = 214.3 Hz), 6.02 (1H, d, H-1′, J1′,2′ = 5.8 Hz), 5.54 (1H, d, 2′-OH, J = 5.9 Hz), 5.26 (1H, d, 3′-OH, J = 4.9 Hz), 5.14 (1H, t, 5′-OH, J = 5.6 Hz), 4.64 (1H, ddd, H-2′, J2′3′ = 4.7 Hz), 4.19 (1H, ddd, H-3′, J3′4′ = 3.4 Hz), 3.98 (1H, ddd, H-4′), 3.70 (1H, ddd, H-5′a, J4′,5′a = 3.7 Hz, J5′a,5′b = 12.1 Hz), 3.64 (1H, ddd, H-5′b, J4′,5′b = 4.4 Hz), 3.12 (2H, q, 6-CH2CH3), 1.35 (3H, t, 6-CH3); 13C NMR (Me2SO-d6) δ 162.64 (C-6), 151.78 (C-2), 150.22 (C-4), 143.97 (C-8), 132.19 (C-5), 87.59 (C-1′), 85.67 (C-4′), 73.58 (C-2′), 70.33 (C-3′), 61.30 (C-5′), 25.70 (6-CH2CH3), 12.20 (6-CH3); Anal. Calcd. for C12H16N4O4; C, 51.42; H, 5.75; N, 19.99. Found C, 51.22; H, 5.65; N 20.89.

6-n-Propyl-9-(β-d-ribofuranosyl)purine (9) [25]

A solution of (Ph3P)4Pd (25 mg, 0.02 mmol) in THF (1 mL) was added to a solution of n-PrZnCl [generated as above from a 2M solution n-PrMgCl (0.53 mL) and 1.13M solution ZnBr2 (1 mL) at −78 °C to r.t., for 1h] in THF (5 mL) at room temperature. A solution of 6 (0.175 g, 0.424 mmol) in THF (2 mL) was added and the mixture was heated for 5 h at 55 °C. The mixture was then cooled down to room temperature and quenched with saturated solution of NH4Cl. The solvent was concentrated under reduced pressure and the residue was partitioned between CHCl3 and H2O. The residue obtained by evaporation of the dried organic phase was dissolved in MeOH saturated with NH3 (10 mL) and stirred for 2 h at room temperature. The solvent was evaporated and the residue was purified by a flash silica gel chromatography (elution with 5% EtOH in CHCl3) to give (0.107 g, 86%) 9 as a pale yellow waxy solid, which was recrystallized from EtOH, m.p. 104–106 °C (lit. [25] foam): HPLC [99%; RT 12.64 min; 0.01 M NH4H2PO4: MeOH; 20 min linear gradient from 10–90%]; MS m/z 295 (M+1)+, UV λmax pH 1, 266.0 (8.0); pH 7, 261.2 (8.3); pH 13, 261.6 (8.2); 1H NMR (Me2SO-d6) δ 8.83 (1H, s, H-2), 8.76 (1H, s, H-8), 6.02 (1H, d, H-1′, J1′2′ = 5.9 Hz), 5.54 (1H, d, 2′-OH, J =6.0 Hz), 5.26 (1H, d, 3′-OH, J = 4.9 Hz), 5.14 (1H, t, 5′-OH, J = 5.6 Hz), 4.65 (1H, ddd, H-2′, J1′2′ = 4.7 Hz), 4.19 (1H, ddd, H-3′, J3′,4′ = 3.5 Hz), 3.98 (1H, ddd, H-4′), 3.70 (1H, ddd, H-5′a, J4′,5a′ = 3.7 Hz, J5′a,5′b = 12.1 Hz), 3.59 (1H, ddd, H-5′b, J4′,5′b = 4.1 Hz), 3.10 (2H, t, 6-CH2CH2CH3), 1.85 (2H, m, 6-CH2CH2CH3), 0.94 (3H, t, 6-CH2CH2CH3); 13C NMR (Me2SO-d6) δ 161.55 (C-6), 151.72 (C-2), 150.22 (C-4), 144.00 (C-8), 132.64 (C-5), 87.54 (C-1′), 85.68 (C-4′), 73.54 (C-2′), 70.34 (C-3′), 61.30 (C-5′), 34.29 (6-CH2CH2CH3), 20.96 (6-CH2CH2CH3), 13.78 (6-CH2CH2CH3). Anal. Calcd. for C13H18N4O4·0. 5 H2O: C, 51.48; H, 6.31; N, 18.47. Found: C, 51.82; H, 6.25; N, 18.37.

6-n-Butyl-9-(β-d-ribofuranosyl)purine (10) [25]

A solution of (Ph3P)4Pd (23 mg, 0.02 mmol) in THF (1 mL) was added to a solution of n-BuZnCl [generated as above from a 2M solution n-BuMgCl (0.5 mL) and 1.13M solution ZnBr2 (1 mL) at −78 °C to r.t., for 1h] in THF (5 mL) at room temperature. A solution of 6 (0.166 g, 0.4 mmol) in THF (2 mL) was added and the mixture was heated for 2 h at 55 °C. The mixture was then cooled down to room temperature and quenched with saturated solution of NH4Cl. The solvent was concentrated under reduced pressure and the residue was partitioned between CHCl3 and H2O. The residue obtained by evaporation of the dried organic phase was dissolved in MeOH saturated with NH3 (10 mL) and kept for 3 h at room temperature. The solvent was evaporated and the residue was purified by a flash silica gel chromatography (elution with 5% EtOH in CHCl3) to give (0.11 g, 89%) 10 as a pale yellow waxy solid, which was recrystallized from H2O-EtOH, m.p. 98–100 °C (lit. [25] foam): HPLC [99%; RT 12.64 min; 0.01M NH4H2PO4: MeOH; 20 min linear gradient from 10–90%]; MS m/z 309 (M+1)+, UV λmax pH 1, 267.0 (8.1); pH 7, 260.9 (8.4); pH 13, 261.1 (8.7); 1H NMR (Me2SO-d6) δ 8.83 (1H, s, H-2), 8.75 (1H, s, H-8), 6.03 (1H, d, H-1′, J1′,2′ = 5.9 Hz), 5.55 (1H, d, 2′-OH, J = 5.9 Hz), 5.27 (1H, d, 3′-OH, J = 4.8 Hz), 5.13 (1H, t, 5′-OH, J = 5.6 Hz), 4.64 (1H, ddd, H-2′, J2′,3′ = 5.0 Hz), 4.18 (1H, ddd, H-3′, J3′,4′ = 3.4 Hz), 4.00 (1H, ddd, H-4′), 3.69 (1H, ddd, H-5′a, J4′,5′a = 3.5 Hz, J5′a,5′b = 12.1 Hz), 3.59 (1H, ddd, H-5′b, J4′,5′b = 4.1 Hz), 3.12 (2H, t, 6-CH2CH2CH2CH3), 1.81 (2H, m, 6-CH2CH2CH2CH3), 1.33 (2H, m, 6-CH2CH2CH2CH3), 0.91 (3H, t, 6-CH2CH2CH2CH3); 13C NMR (Me2SO-d6) δ 161.76 (C-6), 151.72 (C-2), 150.21 (C-4), 143.98 (C-8), 132.56 (C-5), 87.53 (C-1′), 85.68 (C-4′), 73.54 (C-2′), 70.33 (C-3′), 61.30 (C-5′), 31.94 (6-CH2CH2CH2CH3), 29.74 (6-CH2CH2CH2CH3), 21.87 (6-CH2CH2CH2CH3), 13.64 (6-CH2CH2CH2CH3); Anal. Calcd. for C14H20N4O4: C, 54.53; H, 6.54; N, 18.17. Found: C, 54.17; H, 6.35; N, 18.04.

6-Isobutyl-9-(β-d-ribofuranosyl)purine (11)

A solution of (Ph3P)4Pd (73 mg, 0.06 mmol) in THF (2 mL) was added to a 0.5-M solution of isoBuZnCl (10 mL) in THF (5 mL) at room temperature. A solution of 6 (0.520 g, 1.26 mmol) in THF (5 mL) was added and the mixture was heated for 2 h at 55 °C. The mixture was then cooled down to room temperature and quenched with saturated solution of NH4Cl. The solvent was concentrated under reduced pressure and the residue was partitioned between CHCl3 and H2O. The residue obtained by evaporation of the dried organic phase was dissolved in MeOH saturated with NH3 (10 mL) and kept for 4 h at room temperature. The solvent was evaporated and the residue was purified by a flash silica gel chromatography (elution with 6% EtOH in CHCl3) to give (0.34 g, 88%) of a white solid: MS m/z 309 (M + 1)+; UV λmax pH 1, 266.8 (8.8); pH 7, 261.6 (9.2); pH 13, 261.7 (9.1); 1H NMR (Me2SO-d6) δ 8.84 (1H, s, H-2), 8.75 (1H, s, H-8), 6.02 (1H, d, H-1′, J1′,2′ = 5.8 Hz), 5.52 (1H, d, 2′-OH, J = 6.0 Hz), 5.24 (1H, d, 3′-OH, J = 4.8 Hz), 5.12 (1H, t, 5′-OH, J = 5.2 Hz), 4.67 (1H, ddd, H-2′, J2′,3′ =5.0 Hz), 4.19 (1H, ddd, H-3′, J3′,4′ = 3.4 Hz), 3.98 (1H, ddd, H-4′), 3.69 (1H, ddd, H-5′a, J4′,5′a = 4.0 Hz, J5′a,5′b = 11.9 Hz), 3.57 (1H, ddd, H-5′b, J4′,5′b = 4,1 Hz), 2.97 (2H, d, 6-CH2CH(CH3)2), 2.34 (1H, m, 6-CH2CH(CH3)2), 0.93 and 0.90 (3H, d, 6-CH2CH(CH3)2); 13C NMR (Me2SO-d6) δ 161.02 (C-6), 151.69 (C-2), 150.28 (C-4), 144.08 (C-8), 133.01 (C-5), 87.50 (C-1′), 85.72 (C-4′), 73.54 (C-2′), 70.37 (C-3′), 61.33 (C-5′), 41.34 (6-CH2CH(CH3)2), 27.69 (6-CH2CH(CH3)2), 22.49 and 22.47 (6-CH2CH(CH3)2); Anal. Calcd. for C14H20N4O4: C, 54.54; H, 6.54; N, 18.17. Found: C, 54.58; H, 6.32; N, 18.06.

6-Cyclopropyl-9-(β-d-ribofuranosyl)purine (12)

A mixture of magnesium turnings (74 mg, 3.0 mmol) and cyclopropyl bromide (0.24 mL, 3.02 mmol) in anhydrous THF (6 mL) was heated for 1 h at 60 °C until complete dissolution of the magnesium. The solution was cooled to room temperature and was treated with 1.13M solution ZnBr2 (2.7 mL, 3.0 mmol) and the resulting white suspension was stirred for 1h at room temperature. (Ph3P)4Pd (50 mg, 0.04 mmol) in THF (1 mL) was added to the mixture, followed by the addition of 6 (0.22 g, 0.53 mmol) in THF (2 mL) and the mixture was heated for 4 h at 45 °C. The mixture was then cooled down to room temperature and quenched with saturated solution of NH4Cl. The solvent was concentrated under reduced pressure and the residue was partitioned between CHCl3 and H2O. The residue obtained by evaporation of the dried organic phase was dissolved in MeOH saturated with NH3 (10 mL) and kept for 2 h at room temperature. The solvent was evaporated and the residue was purified by a flash silica gel chromatography (elution with 7% EtOH in CHCl3 to give (0.148 g, 92%) as a pale yellow solid which was crystallized from MeOH-heptane, m.p. 154–156 °C (lit. [26] 158–160 °C); HPLC [99.8%; RT 12.62 min; 0.01M NH4H2PO4: MeOH; 20 min linear gradient from 10–90%]; MS m/z 293.1 (M+1)+; UV λmax pH 1, 276.5 (12.5); pH 7, 265.3 (12.4); pH 13, 265.6 (12.7); 1H NMR (Me2SO -d6) δ. 8.74 (1H, s, H-2), 8.72 (1H, s, H-8), 6.00 (1H, d, H-1′, J1′,2′ = 5.9 Hz), 5.52 (1H, d, 2′-OH, J = 5.9 Hz), 5.25 (1H, d, 3′-OH, J = 5.1 Hz), 5.15 (1H, dd, 5′-OH, J5′a′,5′-OH = 5.0 Hz, J5′b′,5′-OH = 6.1Hz), 4.61 (1H, ddd, H-2′, J2′,3′ = 4.9 Hz), 4.17 (1H, ddd, H-3′, J3′,4′ = 3.5 Hz), 3.99 (1H, ddd, H-4′), 3.70 (1H, ddd, H-5′a, J4′,5′a = 4.0 Hz, J5′,5′b = 12.1 Hz), 3.58 (1H, dd, H-5′b, J4′5′b = 4.1 Hz), 2.70 (1H, m, 6-cycloPr-CH), 1.27-1.23 (4H, m, 6-cycloPr-CH2-CH2); 13C NMR (Me2SO-d6) δ 162.88 (C-6), 151.85 (C-2), 149.54 (C-4), 143.73 (C-8), 132.14 (C-5), 87.59 (C-1′), 85.62 (C-4′), 73.58 (C-2′), 70.27 (C-3′), 61.26 (C-5′), 12.69 (6-cycloPr-CH), 10.94 (6-cycloPr-CHCH2CH2); Anal. Calcd. for C13H16N4O4: C, 53.42; H, 5.52; N, 19.18. Found: C, 53.30; H, 5.23; N 19.20.

6-Cyclobutyl-9-(β-d-ribofuranosyl)purine (13)

A mixture of magnesium turnings (45 mg, 1.8 mmol) and cyclobutyl bromide (0.25 mg, 1.85 mmol) in anhydrous THF (5 mL) was heated for 3 h at 60 °C until complete dissolution of the magnesium. The solution was cooled to −78 °C and treated ZnBr2 (1.13M, 1.6 mL, 1.8 mmol) in THF and the resulting white suspension was warmed gradually to room temperature and was stirred further for 1h at room temperature. (Ph3P)4Pd (27 mg) in THF (1 mL) was added, followed by addition of 3 (0.15 g, 0.36 mmol) in THF (2 mL) and the mixture was heated for 4 h at 55 °C. The mixture was then cooled down to room temperature and quenched with saturated solution of NH4Cl. The solvent was concentrated under reduced pressure and the residue was partitioned between CHCl3 and H2O. The residue obtained by evaporation of the dried organic phase was dissolved in MeOH saturated with NH3 (10 mL) and kept overnight at room temperature. The solvent was evaporated and the residue was purified by a flash silica gel chromatography (elution with 5% MeOH in CHCl3) to give (88 mg, 78%) as a pale yellow solid which was crystallized from EtOH in hexanes, m.p. 82–84 °C; HPLC [98%; RT 13.70 min; 0.01M NH4H2PO4: MeOH; 20 min linear gradient from 10–90%]; MS m/z 307 (M+1)+; UV λmax pH 1, 270.2 (9.9); pH 7, 263.1 (10.0); pH 13, 263.3 (10.0); 1H NMR (Me2SO-d6) δ 8.89 (1H, s, H-2), 8.74 (1H, s, H-8), 6.03 (1H, d, H-1′, J1′,2′ = 5.7 Hz), 5.52 (1H, d, 2′-OH, J = 5.2 Hz), 5.26 (1H, d, 3′-OH, J = 4.8 Hz), 5.14 (1H, t, 5′-OH, J = 5.6 Hz), 4.64 (1H, ddd, H-2′, J2′,3′ = 4.9 Hz), 4.29-4.18 (2H, m, 6-cycloBu; CHCH2CH2CH2 and H-3′), 4.01 (1H, ddd, H-4′, J3′,4′ = 3.2 Hz), 3.72 (1H, ddd, H-5′a, J4′,5′a = 3.5 Hz, J5′a,5′b = 12.1 Hz), 3.60 (1H, dd, H-5′b, J4′,5′b = 4.1 Hz), 2.60-2.47 (2H, m, 6-cycloBu CHCHaCHbCH2), 2.41-2.31 (2H, m, 6-cycloBu CHCHaCHbCH2), 2.20-1.93 (2H, m, HCHaCHbCH2); 13C NMR (Me2SO-d6) δ 163.14 (C-6), 151.87 (C-2), 150.33 (C-4), 143.90 (C-8), 131.51 (C-5), 87.64 (C-1′), 85.68 (C-4′), 73.64 (C-2′), 70.33 (C-3′), 61.31 (C-5′), 36.77 (6-cycloBu CHCH2CH2CH2), 26.96 (6-cycloBu CHCH2CH2CH2), 26.88 (6-cycloBu CHCH2CH2CH2), 18.17 (6-cycloBu CHCH2CH2CH2); Anal. Calcd. for C14H18N4O4 ∙0.5 H2O : C, 53.33; H, 6.07; N, 17.77. Found: C, 53.00; H, 5.95; N, 17.53.

6-Cyclopentyl-9-(β-d-ribofuranosyl)purine (14)

A solution of (Ph3P)4Pd (46 mg, 0.04 mmol) in THF (1.5 mL) was added to a solution of cyclopentyl ZnCl [generated as above from a 2M Et2O solution cyclopentyl MgCl (0.5 mL) and 1.13M THF solution ZnBr2 (1 mL) at −78 °C to r.t., for 1h] in THF (5 mL) at room temperature. A solution of 3 (0.224 g, 0.543 mmol) in THF (3 mL) was added and the mixture was heated for 45 min. at 55 °C. The mixture was then cooled down to room temperature and quenched with saturated solution of NH4Cl. The solvent was concentrated under reduced pressure and the residue was partitioned between CHCl3 and H2O. The residue obtained by evaporation of the dried organic phase was dissolved in MeOH saturated with NH3 (10 mL) and kept overnight at room temperature. The solvent was evaporated and the residue was purified by a flash silica gel chromatography (elution with 7% EtOH in CHCl3) to give (0.1 g, 60%) 14 as a pale yellow foam: HPLC [99%; RT 12.64 min; 0.01M NH4H2PO4: MeOH; 20 min linear gradient from 10–90%]; MS m/z 321.2 (M+1)+; UV λmax pH 1, 238.2 (6.4); pH 7, 262.2 (6.4); pH 13, 262.2 (6.4); 1H NMR (Me2SO-d6) δ 8.83 (1H, s, H-2), 8.74 (1H, s, H-8), 6.01 (1H, d, H-1′, J1′,2′. = 5.7 Hz), 5.53 (1H, d, 2′-OH, J = 5.6 Hz), 5.25 (1H, d, 3′-OH, J = 4.6 Hz), 5.13 (1H, t, 5′-OH, J = 5.5 Hz), 4.64 (1H,ddd, H-2′, J2′,3′ = 4.4 Hz), 4.18 (1H, ddd, H-3′, J3′,4′ = 3.1 Hz), 4.02 (1H, ddd, H-4′), 3.79 (1H, m, 6-CHCH2CH2CH2CH2), 3.68 (1H, ddd, H-5′a, J4′,5′a = 3.6 Hz, J5′a,5′b = 12.0 Hz), 3.56 (1H, dd, H-5′b, J4′,5′b = 4.6 Hz), 2.07-1.69 (8H, m, 6-CHCH2CH2CH2CH2), 13C NMR (Me2SO-d6) δ 165.59 (C-6), 152.36 (C-2), 150.76 (C-4), 144.36 (C-8), 132.57 (C-5), 87.59 (C-1′), 85.69 (C-4′), 73.57 (C-2′), 70.37 (C-3′), 61.33 (C-5′), 42.01 (6-CHCH2CH2CH2CH2), 32.09 and 32.06 (6-CHCH2CH2CH2CH2), 25.81 (6-CHCH2CH2CH2CH2); Anal. Calcd. for C15H20N4O4 ∙0.4 H2O : C, 54.96; H, 6.40; N, 17.17. Found: C, 55.11; H, 6.34; N, 16.84.

6-Phenyl-9-(β-d-ribofuranosyl)purine (1512) [a,17c,27]

A solution of PhZnBr (16.94 mmoL) was generated by dropwise addition of 1.13M solution of ZnBr2 (15 mL) in THF to 3M solution of PhMgBr (19.64 mmol, 5.64 mL) in THF (75 mL) at −0 °C for 1h. After the solution was allowed to warm to room temperature, a solution of (Ph3P)4Pd (0.5 g, 0.4 mmoL) in THF (10 mL) was added to it. A solution of compound 6 (3.55 g, 8.6 mmol) in dry THF (10 mL) was then added and the mixture was heated under argon for 4 h at 55 °C. The mixture was then cooled down to room temperature and quenched with saturated solution of NH4Cl. The mixture was then cooled down to room temperature and quenched with saturated solution of NH4Cl. The solvent was concentrated under reduced pressure and the residue was partitioned between CHCl3 and H2O. The residue obtained by evaporation of the dried organic phase was dissolved in MeOH saturated with NH3 (30 mL) and kept overnight at room temperature. The solvent was evaporated and the residue was purified by a flash silica gel chromatography (elution with 6% MeOH in CHCl3) to give (2.2 g, 78%) of 15 as a white solid with was crystallized from EtOH/toluene, m.p. 224–226 °C (lit. [17c] 228–230 °C): MS m/z 329 (M+1)+; UV λmax pH 1, 303.0 (18.3); pH 7, 288.4 (18.7); pH 13, 288.4 (18.4); 1H NMR (Me2SO-d6) δ 9.03 (1H, s, H-2), 8.92 (1H, s, H-8), 8.83-8.80 (2H, m, 6-Ph), 7.65-7.58 (3H, m, 6-Ph), 6.11 (1H, d, H-1′, J1′,2′ = 5.6 Hz), 5.60 (1H, d, 2′-OH, J = 5.9 Hz), 5.28 (1H, d, 3′-OH, J = 4.9 Hz), 5.16 (1H, t, 5′-OH, J = 5.5 Hz), 4.67 (1H, ddd, H-2′, J2′,3′ = 4.8 Hz), 4.23 (1H, ddd, H-3′, J3′,4′ = 3.9 Hz), 4.00 (1H, ddd, H-4′), 3.75 (1H, ddd, H-5′a, J4′,5′a = 3.6 Hz, J5′a,5′b = 11.9 Hz), 3.63 (1H, ddd, H-5′b, J4′,5′b = 4.1 Hz); 13C NMR (Me2SO -d6) δ 157.17(C-6 or C-4), 152.93 (C-4 or C-6), 151.85 (C-2), 144.86 (C-8), 135.19 (ipso C-6Ph), 131.08 (para C-6Ph), 130.82 (C-5), 129.33 and 128.63 (meta and ortho C-6Ph), 87.64 (C-1′), 85.63 (C-4′), 73.72 (C-2′), 70.21 (C-3′), 61.17 (C-5′); Anal. Calcd. for C16H16N4O4 ∙ 0.2 H2O: C, 57.85; H, 4.98; N, 16.94. Found: C, 57.78; H, 4.81; N, 16.99.

6-(2-Thienyl)-9-(β-d-ribofuranosyl)]purine (16) [27,28,30]

A mixture of magnesium turnings (466 mg, 19.2 mmol) and 2-thienyl bromide (1.8 mL, 19.2 mmol) in anhydrous THF (5 mL) was stirred under argon for 3 h at 37 °C. The resulting red color solution was cooled to 0 °C and treated with 1M THF solution of ZnBr2 (19.2 mL) and the thick suspension was stirred further for 1h at room temperature. (Ph3P)4Pd (277 mg, 0.24 mmol) in THF (5 mL) was added followed by the addition of a solution of 6 (2 g, 4.85 mmol) in THF (20 mL) and the mixture was heated for 2 h at 45 °C. The mixture was then cooled down to room temperature and quenched with saturated solution of NH4Cl. The solvent was concentrated under reduced pressure and the residue was partitioned between CHCl3 and H2O. The residue obtained by evaporation of the dried organic phase was dissolved in MeOH saturated with NH3 (20 mL) and kept overnight at room temperature. The solvent was evaporated and the residue was purified by a flash silica gel chromatography (elution with 5% MeOH in CHCl3) to give (1.3 g, 89%) as a yellow solid; MS m/z 307 (M+1)+; UV λmax pH 1, 338.8 (19.3); pH 7, 325.2 (24.1); pH 13, 324.8 (24.7); 1H NMR (Me2SO-d6) δ 8.91 (1H, s, H-2), 8.87 (1H, s, H-8), 8.84 (1H, dd, 6-C-S-CHCHCH, J = 1.1 Hz, J = 3.7 Hz), 7.94 (1H, dd, 6-C-S-CHCHCH, J = 1.1, J = 5.0 Hz), 7.36 (1H, dd, 6-C-S-CHCHCH, J = 5.0, J = 3.7 Hz), 6.06 (1H, d, H-1′, J1′2′ = 5.5 Hz), 5.57 (1H, d, 2′-OH, J = 5.9 Hz), 5.05 (1H, d, 3′-OH, J = 5.6 Hz), 5.15 (1H, t, 5′-OH, J = 5.5 Hz), 4.64 (1H, ddd, H-2′, J2′,3′ = 5.1 Hz), 4.21 (1H, ddd, H-3′, J3′,4′ = 3.2 Hz), 4.00 (1H, dd, H-4′), 3.71 (1H, ddd, H-5′a, J4′,5′a = 3.7, J5′a,5′b = 11.9 Hz), 3.60 (1H, dd, H-5′b, J4′,5′b = 4.6 Hz); Anal. Calcd. for C14H14N4O4S ∙ 0.3 H2O: C, 49.49; H, 4.33; N, 16.49. Found: C, 49.40; H, 4.03; N, 16.27.

6-Chloro-9-(tri-O-acetyl-β-d-arabinofuranosyl)purine (17) [23,30]

To a solution of 23 (1 g, 2.66 mmol) in anhydrous CHCl3 (25 mL) was added N,N-dimethylformamide (0.2 mL) and SOCl2 (4.5 mL, 30 mmol) dropwise over 10 min. The mixture was heated for 4 h at reflux temperature, then cooled down to room temperature and the solvent was evaporated. The residue was dissolved in EtOAc (50 mL) and neutralized with cold aqueous NaHCO3 solution. The organic phase was washed with H2O, dried over (MgSO4) and evaporated. The residue was purified by a flash silica gel chromatography (elution with; 1% MeOH in CHCl3) to give (1.05 g, 96%) of 17 as a colorless foam: MS m/z 413 (M+1)+; UV λmax pH 1, 263.2; pH 7, 263.2; pH 13, 261.6; 1H NMR (CDCl3) δ 8.78 (1H, s, H-2), 8.34 (1H, s, H-8), 6.66 (1H, d, H-1′, J1′,2′ = 4.6 Hz), 5.55 (1H, dd, H-2′, J2′,3′ = 3.1 Hz), 5.47 (1H, dd, H-3′, J3′,4′ = 4.5 Hz), 4.51 (1H, dd, H-5′a, J4′.5′a = 5.8 Hz, J5′a,5′b = 12.0 Hz), 4.49 (1H, dd, H-5′ b, J4′,5′ b = 4.4 Hz), 4.41 (1H, dt, H-4′), 2.19 (3H, s, Ac), 2.15 (3H, s, Ac), 1.90 (3H, s, Ac).

6-Methyl-9-(β-d-arabinofuranosyl)purine (18)

A solution of (Ph3P)4Pd (36 mg, 0.03 mmol) in THF (1 mL) was added to a solution of CH3ZnBr (1 mmol, generated as above) in THF (5 mL) at room temperature. A solution of 17 (0.167 g, 0.39 mmol) in THF (3 mL) was added at room temperature and the mixture was stirred for 5 h at 55 °C. After an aqueous work up, the residue obtained by evaporation of the dried organic phase was dissolved in MeOH saturated with NH3 (10 mL) and stirred for 3 h at room temperature. The solvent was evaporated and the residue was purified by silica gel chromatography (elution with 6% EtOH in CHCl3) to give (83 mg, 78%) of 18 as a colorless solid that was crystallized from hot ethanol, m.p. 220–222 °C; MS m/z 267.1 (M+1)+; UV λmax pH 1, 263.6 (7.4); pH 7, 260.3 (8.0); pH 13, 261.0 (8.3); 1H NMR (Me2SO-d6) δ 8.77 (1H, s, H-2), 8.56 (1H, s, H-8), 6.38 (1H, d, H-1′, J1′,2′ = 5.1 Hz), 5.66 (1H, br s, 2′-OH), 5.58 (1H, d, 3′-OH, J = 4.4 Hz), 5.11 (1H, br t, 5′-OH), 4.27 (1H, m, H-2′, J2′,3′ = 5.2 Hz), 4.20 (1H,ddd, H-3′, J3′,4′ = 5.2 Hz), 3.82 (1H, ddd, H-4′), 3.72-3.63 (2H, m, H-5′a,b), 2.72 (1H, s, 6-CH3); NOE: Irradiation at H-1′ an enhancements of 2%, 1–2% and 10% were observed at H-8, at H-4′ and H-2′, respectively. Irradiation at H-3′ gave enhancements of 4–5% and 2% of the signals at H-8 and at H-4′, respectively. 13C NMR (Me2SO-d6) δ 157.69 (C-6), 151.51 (C-2, 1JCH = 203.3 Hz), 150.09 (C-4), 144.55 (C-8, 1JCH = 215.5 Hz), 132.11 (C-5), 84.20 (C-4′), 83.71 (C-1′, 1JCH = 164.8 Hz), 75.64 (C-2′), 74.67 (C-3′), 60.65 (C-5′), 19.01 (6-CH3); Anal. Calcd. for C11H14N4O4; C 49.62, H 5.30, N 21.04; found C 49.45, H 5.15, N 21.00.

Acknowledgements

This investigation was supported by a National Cooperative Drug Discovery Grant (U19CA67763) from the National Cancer Institute. We thank M.D. Richardson, and J.C. Bearden of the Molecular Spectroscopy Laboratory of Southern Research Institute for analytical and spectral data and S. Campbell for HPLC analyses. We are grateful to M. Kirk, University of Alabama at Birmingham Comprehensive Cancer Center Shared Mass Spectrometry Facility, for supplying some of the mass spectral data. Special thanks are due to Dr. Omar Moukha-chafiq for technical assistance.

Abbreviations

E. coli PNP

Escherichia coli purine nucleoside phosphorylase

MeP-dR

9-(2-deoxy-β-d-ribofuranosyl)-6-methylpurine

F-dAdo

2-fluoro-2′-deoxyadenosine

F-araA

9-(β-d-arabinofuranosyl)-2-fluoroadenine

MeP

6-methylpurine

F-Ade

2-fluoroadenine

Footnotes

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References

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