Selective A₃ Adenosine Receptor Antagonists Derived from Nucleosides Containing a Bicyclo[3.1.0]hexane Ring System

Abstract

We have prepared 5′-modified derivatives of adenosine and a corresponding (N)-methanocarba nucleoside series containing a bicyclo[3.1.0]hexane ring system in place of the ribose moiety. The compounds were examined in binding assays at three subtypes of adenosine receptors (ARs) and in functional assays at the A₃ AR. The H-bonding ability of a group of 9-riboside derivatives containing a 5′-uronamide moiety was reduced by modification of the NH, however these derivatives did not display the desired activity as selective A₃ AR antagonists, as occurs with 5′-N,N-dimethyluronamides. However, truncated (N)-methanocarba analogues lacking a 4′-hydroxymethyl group were highly potent and selective antagonists of the human A₃ AR. The compounds were synthesized from D-ribose using a reductive free radical decarboxylation of a 5′-carboxy intermediate. A less efficient synthetic approach began with L-ribose, which was similar to the published synthesis of (N)-methanocarba A₃AR agonists. Compounds 33b – 39b (N⁶-3-halobenzyl and related arylalkyl derivatives) were potent A₃AR antagonists with binding K_i values of 0.7 − 1.4 nM. In a functional assay of [³⁵S]GTPγS binding, 33b (3-iodobenzyl) completely inhibited stimulation by NECA with a K_B of 8.9 nM. Thus, a highly potent and selective series of A₃AR antagonists has been described.

Introduction

There are four subtypes of receptors for adenosine 1, designated A₁, A_2A, A_2B, and A₃.¹ Selective antagonists of the A₃ adenosine receptor (AR) are of potential use for clinical targets, including the treatment of cancer,² glaucoma³ and inflammation.⁴ A number of competitive tricyclic⁵ and nucleoside⁶ antagonists of ARs have been reported recently. The structure of some of the antagonists may resemble adenosine, while other compounds possess a completely different structure. An important advantage of nucleoside-based A₃AR antagonists is their species-independent activity.⁷ These antagonists typically possess high affinity at the human, rat, and mouse A₃ARs and therefore may be suitable for evaluation in small animal models or for further development as drugs. For example, A₃AR antagonists derived from adenosine have been shown to lower intraocular pressure in a mouse model of glaucoma.⁷

We have previously shown that local modifications of the potent AR agonist Cl-IB-MECA 2 may result in a complete loss of agonist efficacy while retaining a strong binding inhibition constant (K_i).⁸ These modifications resulted in potent ARs antagonists of this type in compounds 3 and 4. The most probable reason for the drastic change in activity is a disruption of H-bonding at the 5′ position either through the introduction of an additional N-methyl substituent, or enforcement of a conformation of the amide group at this position that is unable to activate the receptor.⁹ Another approach to adenosine-derived A₃AR antagonists involves the complete removal of the substituent at position 4′. This approach has been successfully implemented in thionucleoside antagonists of the class represented by compound 6, which was derived from 5, the 4′-thio analogue of Cl-IB-MECA.¹⁰

Both the binding and selectivity of prototypical A₃AR agonists can be substantially increased by replacing the flexible ribose scaffold in the series of compounds related to 2 with a rigid bicyclo[3.1.0]hexane ring system. The resulting (N)-methanocarba adenosine agonists 7 − 9 have been found to possess high potency and enhanced selectivity for the A₃ receptor,¹¹ and agonists of this class are currently under development as anti-arthritis drug candidates.

This study reports on a new series of selective A₃AR antagonists of the (N)-methanocarba family. Design of these compounds involved the disruption of H-bonding at the 4′ position through removal of the N-methylcarboxamide function while retaining the conformationally restricted (N)-methanocarba scaffold bearing 6-(arylalkylamino)-2-chloropurine moieties, as in agonists 7 − 9.

Results

Chemical synthesis

Prior to probing the structure activity relationship (SAR) with the more synthetically challenging (N)-methanocarba system, we explored alternatives to the N,N-dimethyl group as a means of obtaining A₃AR antagonists in the ribose series similar to compound 3. The corresponding methyl ester 10b (Scheme 1A, prepared from the protected intermediate 10a)^10,12 was prepared using a previously reported synthetic approach. This ester group was directly aminolyzed using several hydroxylamino nucleophiles to provide 11 and 12. Alternately, the ester was hydrolyzed to yield the carboxylic acid 13. All of these derivatives were intended for testing at ARs, because they have altered H-bonding ability in relation to 1. Attempts to prepare an active ester from 13 as an intermediate for further amide synthesis were unsuccessful.

Scheme 1.

a) K₂CO₃, MeOH, H₂O; b) K₂CO₃, MeOH, H₂O, MeONH or MeONHMe; (c) two steps¹¹; (d) TFA, EtOH-water (e) R³H (amine), EtOH.

In the (N)-methanocarba series, the intermediate 2′,3′-isopropylidene protected derivatives 15 − 17 were prepared from the key intermediate 14 as described in the literature (Scheme 1B).^11,13 The key intermediate 14 was in turn prepared according to our recent modified procedure.^13b The isopropylidene group in 15 − 17 was conveniently removed by using aqueous TFA in EtOH to afford the requisite 4′-ester methanocarba derivatives 18 − 20 in good yields. Attempts to react the ester group of either 15 − 17 or 18 − 20 with dimethylamine to prepare dimethylamides similar to 3 in the (N)-methanocarba series, i.e. 21, were unsuccessful.

Our initial synthetic approach (Scheme 2) toward (N)-methanocarba derivatives lacking the 4′ substituent was based on our previously published synthesis of (N)-methanocarba agonists from L-ribose 22.¹³ It involved protection of a key alcohol in 14 as a benzyl ether followed by hydrolysis of the ester group in 24 with a subsequent free-radical reductive decarboxylation of 25 using the methodology of Barton,¹⁴ leading to conversion into the desired compounds by previously described methodology.¹³ The decarboxylation of 25 to yield 26 was performed using a one-pot modification,¹⁵ which involved reaction with a mixture of tributyltin hydride, 2,2′-dithiopyridine-1,1′-dioxide, tributylphosphine, and a radical initiator. We found that under these conditions preparation of benzyl ether 26 could be achieved, although the reaction yield was variable and difficult to reproduce. Another problem was the difficulty of deprotection of benzyl ether 26 by hydrogenation, which gave alcohol 27 in poor yield.

Scheme 2.

a) 7 steps¹³; b) CF₃SO₃H (cat.), DCM; c) BnCl, NaH, THF; d) NaOH/H₂O/MeOH, reflux; e) PBu₃, AIBN, Bu₃SnH, toluene; f) H₂/Pd, MeOH.

These synthetic problems prompted us to evaluate an alternative synthetic approach (Scheme 3). It involved the synthesis of alcohol 29 from inexpensive D-ribose using the same methodology.¹³ Alcohol 29 was protected with TBDPS-Cl followed by alkaline hydrolysis, thus providing acid 30.

Scheme 3.

a) 7 steps¹³; b) TBDPS-Cl, imidazole, DMF; c) NaOH, H₂O, MeOH, reflux; d) 2-mercaptopyridine N-oxide, DCC, toluene; e) (Me₃Si)₃SiH, AIBN, toluene; f) Bu₄NF, THF; g) 2,6-dichloropurine, PPh₃, DIAD, THF; h) RNH_2, EtOH; i) TFA/H₂O/MeOH.

Reductive decarboxylation of acid 30 was initially carried out through a sequential one-pot procedure involving a DCC-mediated coupling of the acid with 2-mercaptopyridine N-oxide at room temperature followed by reductive free radical decarboxylation with tributyltin hydride at 80 °C.^14a However, the resultant ether 31 was difficult to separate from toxic and foul-smelling tributyltin-containing byproducts. A modified approach for the reductive decarboxylation used nontoxic tris(trimethylsilyl)silane as a hydrogen donor¹⁶ and produced the silyl ether 31 in 40% yield. To the best of our knowledge, this is the first application of tris(trimethylsilyl)silane for reductive decarboxylation, and this reaction may possess a considerable preparative potential.

In contrast to the low-yielding hydrogenation of 26, silyl ether 31 was smoothly deprotected with TBAF. The resultant alcohol 27 was converted into a key dichloropurine derivative 32 through a Mitsonobu reaction (Scheme 3).¹³ The dichloropurine derivative 32 reacted with an excess of the corresponding primary amine to give the N⁶ substituted and 2′,3′-isopropylidene protected derivatives compounds 33a – 39a, followed by acid catalyzed deprotection to give the N⁶-3-halobenzyl and related arylmethyl derivatives 33b – 39b.

Pharmacological activity

The 3-chloro and 5-chloro-2-methoxy derivatives, 8 and 9, respectively, of compound 7 were used for comparison in the biological assays (Table 1). Binding assays were carried out using standard radioligands and membrane preparations from Chinese hamster ovary (CHO) cells (A₁ and A₃) or HEK293 cells (A_2A) stably expressing a hAR subtype (Table 1). Functional effects at the A₃AR were determined in assays of adenylate cyclase or guanine nucleotide binding.^22,23 Replacement of the amide NH of 2 with an isosteric, but non H-bond donating, ester oxygen in 10b resulted in a one-order of magnitude loss of affinity at the A₁ and A₃ARs, and no change at the A_2AAR. Insertion of an O between the NH and the methyl group in 11 reduced the A₃AR affinity by 62-fold. Both 10b and 11 lost nearly all efficacy as A₃AR agonists in relation to full agonist 2. This confirmed previous findings that the efficacy of adenosine derivatives in activating the A₃AR was highly dependent on structural factors in the region of the 4′ carbon. The N-methylated derivative 12 had no effect on the A₃AR affinity, while completely abolishing activation of the receptor. Unfortunately, compound 12 was not a suitable lead as an A₃AR antagonist, because it was only 4-fold selective in comparison to the A₁AR. However, this compound could be a useful pharmacological probe of mixed selectivity for the A₁AR and A₃AR. Compound 12 was tested in a functional assay at the A₁AR ([³⁵S]GTPγS binding in A₁AR-expressing CHO cells) and found to lack the ability to activate the receptor (data not shown). Therefore, compound 12 appears to be a mixed A₁/A₃ AR antagonist. Compound 13, derived by saponification of 12, was nearly inactive at the A₁ and A_2AARs and a very weak agonist at the A₃AR. Thus, a negative charge in the region of the 4′ carbon appears to be highly detrimental toward interaction with ARs.

Table.

Potency of a series of (N)-methanocarba adenosine derivatives at three subtypes of human ARs and the functional efficacy at the A₃AR.

	Structure			Affinity (Ki, nM) or % inhibitiona			%Efficacyb
Compound	R1	R3	R2	A1	A2A	A3	A3
Series A
2c	CH3NH	Cl	3-I-Phenyl-CH2	222 ± 22	5360 ± 2470	1.4 ± 0.3	100
3d,e	(CH3)2N	Cl	3-I-Phenyl-CH2	5870 ± 930	>10,000	29.0 ± 4.9	0
10b	CH3O	Cl	3-I-Phenyl-CH2	1600 ± 130	4770 ± 240	12.4 ± 2.4	11
11	CH3ONH	Cl	3-I-Phenyl-CH2	870 ± 180	1670 ± 350	86.2 ± 8.4	4
12	CH3O(CH3)N	Cl	3-I-Phenyl-CH2	252 ± 8	(52 ± 3%)	70.2 ± 6.8	0
13	HO	Cl	3-I-Phenyl-CH2	(18 ± 5%)	(4 ± 4%)	58 ± 3%	42
Series B
7	CH3NH	Cl	3-I-Phenyl-CH2	136 ± 22d	784 ± 97d	1.5 ± 0.2c	100c
8c	CH3NH	Cl	3-Cl-Phenyl-CH2	260 ± 60	2300 ± 100	0.29 ± 0.04	103 ± 7
9c	CH3NH	Cl	5-Cl-2-MeOPh-CH2	240 ± 50	1200 ± 100	1.5 ± 0.0	107 ± 15
18	C2H5O	Cl	3-Cl-Phenyl-CH2	4820 ± 680	(19%)	310 ± 66	54
19	C2H5O	Cl	5-Cl-2-MeOPh-CH2	1010 ± 120	(37%)	196 ± 33	39
20	C2H5O	SCH3	3-Cl-Phenyl-CH2	6630 ± 120	(20%)	1010 ± 130	0
Series C
33be	-	Cl	3-I-Phenyl-CH2	3040 ± 610	1080 ± 310	1.44 ± 0.60	1.0 ± 3.2f
34b	-	Cl	3-Cl-Phenyl-CH2	3070 ± 1500	4510 ± 910	1.06 ± 0.36	2.9 ± 3.7f
35be	-	Cl	3-Br-Phenyl-CH2	1760 ± 1010	1600 ± 480	0.73 ± 0.30	5.8 ± 0.8f
36b	-	Cl	1-Naphthyl-CH2	1120 ± 640	1530 ± 350	1.42 ± 0.12	3.1 ± 0.3f
37b	-	Cl	2,5-diMeO-Ph-CH2	3000 ± 1260	2620 ± 730	1.58 ± 0.56	4.6 ± 3.8f
38b	-	Cl	2-OH-5-MeO-Ph-CH2	1110 ± 300	6870 ± 1440	4.06 ± 0.35	0.4 ± 1.3f
39b	-	Cl	trans-2-Ph-cyclopropyl	1790 ± 1430	2010 ± 890	1.30 ± 0.39	9.7 ± 4.1f

^aAll experiments were done on CHO or HEK (A_2A only) cells stably expressing one of four subtypes of human ARs. The binding affinity for A₁, A_2A and A₃ARs was expressed as K_i values (n = 3−5) and was determined by using agonist radioligands ([³H]CCPA or [³H]RPIA; [³H]CGS21680; or [¹²⁵I]I-AB-MECA; respectively). A percent in parentheses refers to inhibition of radioligand binding at 10 μM.

^bUnless noted, the efficacy at the human A₃AR was determined by inhibition of forskolin-stimulated cyclic AMP production in AR-transfected CHO cells, as described in the text. At a concentration of 10 μM, in comparison to the maximal effect of a full agonist NECA at 10 μM. Data are expressed as mean ± standard error (n = 3).

^cValues from reference ¹¹.

^dValues from reference ⁸.

^e3, MRS3771; 33b, MRS5127; 35b, MRS5147.

^fA₃AR functional assay consisted of stimulation of [³⁵S]GTPγS binding at 10 μM, expressed as a percentage of the full effect induced by 10 μM NECA (100 ± 5%).

ND not determined.

The (N)-methanocarba agonists 7 − 9 were already reported to be potent and selective at the A₃AR and fully efficacious.¹¹ Replacement of the amide NH of 8 or 9 with an ester oxygen resulted in greatly reduced affinity at the three AR subtypes, and only partial activation of the A₃AR was observed. Thus, this modification is unlikely to provide an entry into selective A₃AR antagonists. Replacement of the 2-chloro of 18 with methylthio, which was tolerated in the ribose series of A₃AR agonists,^6b resulted in 20 and failed to increase the affinity. However, compound 20 completely lacked the ability to activate the A₃AR and thus appears to be a weak antagonist.

Compounds 33b – 35b (3-halobenzyl) in the (N)-methanocarba series were potent A₃ AR ligands with binding K_i values of 0.7 − 1.4 nM. Compound 35b (3-bromobenzyl analogue) proved to be the most potent A₃AR ligand of this series in binding with a K_i value of 0.73 nM, and it displayed high selectivity (2400-fold and 2190-fold in comparison to the A₁ and A_2AAR, respectively). The most A₃AR selective compound was the 3-chloro analogue 34b with 2900-fold and 4250-fold selectivity in comparison to the A₁ and A_2AAR, respectively. The SAR of substitution of the N⁶-benzyl group further showed that dimethoxy substitution (37b), fusion of the phenyl ring to a second ring (36b), and extension by one carbon (i.e., in the rotationally constrained 2-phenylcyclopropyl analogue, 39b) were all tolerated with nanomolar binding affinity at the A₃AR. Compound 38b, a demethylated analogue of 37b, was slightly less potent in binding to the A₃AR.

Compounds 33b – 39b were examined for the ability to stimulate [³⁵S]GTPγS binding in membranes of CHO cells expressing the human A₃AR. None of the truncated (N)-methanocarba derivatives induced substantial (>10%) activation of the A₃AR. In the same functional assay applied to detect antagonism, a representative analogue 33b completely inhibited stimulation by 1 μM NECA (5′-N-ethylcarboxamidoadenosine) with an IC₅₀ of 29.8 nM (Figure 1). Schild analysis of the right shifts by 33b of the response curves in the inhibition of adenylate cyclase by NECA provided a K_B value of 8.9 nM. Thus, truncated (N)-methanocarba derivative displayed A₃AR antagonist properties.

Figure 1.

Functional antagonism by the 3-iodobenzyl analogue 33b tested in an assay of guanine nucleotide binding ([³⁵S]GTPγS) in membranes of CHO cells expressing the human A₃AR.

A₃AR molecular modeling: Docking of nucleoside agonist and antagonists

The previous docking study showed that the binding site of a full agonist (Cl-IB-MECA 2) in the A₃AR was located in the upper transmembrane domain (TM) near EL2 (second extracellular loop) in Figure 2A.¹⁷ In the present study, the nucleoside antagonist 33b in Figure 2B was initially docked to the A₃AR model based on the structure with Cl-IB-MECA 2 bound, followed by minimization and molecular dynamics, and the complexes were compared. These two A₃AR selective ligands were exactly overlapped in the binding site with respect to the phenyl ring of the N⁶ substituent and the ribose ring, as depicted in Figure 2C. Common binding sites were conserved: 1) The NH of the side-chain of Q167 in EL2 formed H-bonds with the 2′-hydroxyl group; 2) The 3′-hydroxyl group interacted with the side chain of H272^7.43 and the backbone of S271^7.42. However, different additional interactions were evident for agonist 2 and antagonist 33b. In the agonist binding domains, additional interactions were present at the site of helical bending of TM6 (due to a Pro residue) in proximity to TM7. The additional interactions were shown in the interaction of the ribose 4′O with T94^3.36, the 5′-carbonyl group with S272^7.42, and the 5′-terminal methyl group with the hydrophobic side-chain of F239^6.44 in the conserved FxxxWxP motif of Class A GPCRs.

Figure 2.

Docking complexes of A₃AR ligands. (A) nucleoside agonist 2, Cl-IB-MECA in light pink color, (B) nucleoside antagonist 33b in cyan color, and (C) the superimposition of agonist 2 and antagonist 33b. Intermolecular H-bonding is indicated with a dotted line. Using Pymol program, all ligands are represented by stick models and the A₃AR are shown in ribbon model with different colors for each TM (TM1: purple, TM2: blue, TM3: light blue, TM4: green, TM5: yellow, TM6: orange, TM7: red).

The docking complex of compound 33b in Figure 2B indicated the importance for agonism of binding at the helical bending region of TM6 in proximity to TM7. Compound 33b, an (N)-methanocarba analogue of Cl-IB-MECA 2 lacking the 5′-group, bound to the A₃AR with a similar binding affinity to 2 but did not induce activation. The docking result showed a loss of the typical interaction of the 5′-position with T3.36, S7.42, and F6.44. The analysis of binding energy showed losses of 1.3 kcal/mol, 3.6 kcal/mol, and 2.8 kcal/mol related to binding in TM3, TM6 and TM7, respectively, compared to the agonist 2. However, the N⁶-(3-iodobenzyl) moiety formed the same hydrophobic interaction with F168 in EL2 with the similar binding energy in TM5 (0.04 kcal/mol energy difference). The EL2, which interacted with the common 2′-hydroxyl group, revealed a more favorable energy of interaction, by 0.5 kcal/mol, for the antagonist 33b. Since this docking result is based on the structure of the meta-I state of rhodopsin, in which a different rotamer of W6.48 was revealed following an anti-clockwise rotation from the extracellular perspective, the docking with the ground-state structure, the inverse-agonist preferred form, was more energetically favorable in the antagonist 33b-bound structure. However, the agonist 2 displayed a severe steric clash at W6.48 and failed to form an optimum interaction in the ground state.

As reported previously,^8,9 the flexibility and the additional H-bonding of the 5′-substituent correlated with putative conformational changes of the receptor associated with the movement of W6.48 upon activation. Selective A₃AR agonists, such as Cl-IB-MECA 2 and its 4′-thio analogue 5, have been successfully transformed into antagonists selective for the A₃AR by appending an additional N-methyl group on the 5′-uronamide position or by reducing the flexibility through cyclization of the 5′ substituent.⁸ The 5′-CO group of those antagonists was oriented perpendicular to the ribose ring and could form a H-bond with W6.48, thus blocking the shift of W6.48 side-chain during the conformational change. Thus, the 5′-cyclized uronamide MRS1292 and the 5′-N,N-dimethyluronamide 3 did not adopt the typical receptor-bound agonist conformation because of a locked conformation and a different energetically favored geometry, respectively.^8,17 The current mode of docking of a new nucleoside antagonist 33b also supports the view that the additional binding domains present in the full agonist 2 but absent in 33b are associated with receptor activation. Thus, nucleoside ligands have spatially distinct regions to fulfill separate roles in binding and activation processes.

Discussion

The truncation of the 5′-CONHCH₃ of A₃AR-selective agonists proved to be the most useful means of converting agonists into pure antagonists. This reduces both the H-bond donating and accepting abilities of the ribose or ribose-like ring. Other means of diminishing the H-bonding ability through methylation, etc. tended to reduce the potency and or selectivity, or allow residual efficacy to remain, which was evident in assays of adenylate cyclase.

The AR binding affinities of the five agonists (i.e., 5′-CONHCH₃ derivatives) reported by Tchilibon et al.¹¹ were compared to those of the corresponding to truncated compounds 33b – 35b, 37b, and 39b. The mean affinities (nM) at the human A_2A and A₃ARs were comparable in the two series (A_2AAR: 3800 for the agonists, 2400 for the truncated analogues; A₃AR: 1.22 for the agonists, 0.87 for the truncated analogues). However, the mean affinities (nM) at the human A₁AR were higher in the agonist series (610) in comparison to the truncated analogues (2500). Thus, the A₃AR selectivity in comparison to the A₁AR tended to be higher in the truncated series. The relatively close correspondence of the AR binding affinities in the agonist and truncated antagonist series is consistent with the mode of overlay of the representative ligands docked in the receptor, i.e. the riboside agonist 2 and the truncated (N)-methanocarba antagonist 33b. The moderate hydrophobicity of the truncated (N)-methanocarba analogues was similar to the corresponding agonist series (e.g., 7 − 9) and in a favorable range for in vivo administration. For example, the clog P value of the 3-bromo truncated derivative 35b is 1.96 in comparison to 2.17 for the 3-bromo agonist analogue.

In conclusion, a highly potent and selective series of A₃AR antagonists in the truncated (N)-methanocarba series has been described. These analogues, particularly the 3-bromobenzyl 35b and 3-chlorobenzyl 34b analogues, can now be examined in models of glaucoma and other diseases for which modulation of the A₃AR has been proposed to be useful.

Experimental Section

Chemical synthesis

Materials and instrumentation

D-ribose, L-ribose, and other reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO). Alcohol derivative 14 was prepared as reported.^{10 1}H NMR spectra were obtained with a Varian Gemini 300 spectrometer using CDCl₃ and CD₃OD as solvents. Chemical shifts are expressed in σ values (ppm) with tetramethylsilane (σ 0.00) for CDCl₃ and water (σ3.30) for CD₃OD. TLC analysis was carried out on aluminum sheets precoated with silica gel F₂₅₄ (0.2 mm) from Aldrich. HPLC mobile phases consisted of System A: linear gradient solvent system: CH₃CN/triethyl ammonium acetate from 5/95 to 60/40 in 20 min, flow rate 1.0 mL/min; System B: linear gradient solvent system: CH₃CN/tetrabutyl ammonium phosphate from 20/80 to 60/40 in 20 min, flow rate 1.0 mL/min. Low-resolution mass spectrometry was performed with a JEOL SX102 spectrometer with 6-kV Xe atoms following desorption from a glycerol matrix or on an Agilent LC/MS 1100 MSD, with a Waters (Milford, MA) Atlantis C18 column. High resolution mass spectroscopic (HRMS) measurements were performed on a proteomics optimized Q-TOF-2 (Micromass-Waters) using external calibration with polyalanine, unless noted. Observed mass accuracies are those expected based on known performance of the instrument as well as trends in masses of standard compounds observed at intervals during the series of measurements. Reported masses are observed masses uncorrected for this time-dependent drift in mass accuracy.

5-[2-Chloro-6-(3-iodo-benzylamino)-purin-9-yl]-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid methyl ester (10b)

The methyl ester 10a (0.062 g, 0.1 mmol) was dissolved in MeOH (5 mL), potassium carbonate (0.028 g, 0.2 mmol) was added, and the mixture was stirred at room temperature for 10 min. Acetic acid (0.2 mL) was added to neutralize the base, the reaction mixture was concentrated under reduced pressure and subjected to preparative thin layer chromatography by using chloroform/methanol (9:1) as solvent to afford the diol 10b as a colorless solid (0.023 g, 43%). 1H NMR (CDCl₃) δ 8.20 (s, 1H), 7.73 (s, 1H), 7.66 (d, 1H, 7.5 Hz), 7.29 (d, 1H, 6.5 Hz), 7.12 (t, 1H, 4.0, 2.4 Hz), 6.05 (d, 1H, 5.1 Hz), 4.70−4.82 (m, 2H),4.55−4.67 (m, 2H), 3.79 (s, 3H), HRMS calculated for C₁₈H₁₈ClIN₅O₅⁺ (M+H)⁺: Exact Mass: 546.0041; found, 546.0042. HPLC: RT 21.5 min (98%) in solvent system A, 10.5 min (99%) in system B.

5-[2-Chloro-6-(3-iodo-benzylamino)-purin-9-yl]-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid methoxy-amide (11)

The methyl ester 10a (0.031 g, 0.05 mmol) was dissolved in MeOH (5 mL), potassium carbonate (0.054 g, 0.4 mmol) was added, and the mixture was stirred at room temperature for 10 min. Methoxyamine hydrochloride (0.017 g, 0.2 mmol) was added and the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was treated with acetic acid (0.3 mL) and concentrated under reduced pressure. The residue was subjected to preparative thin layer chromatography by using chloroform/methanol (8:2) as solvent to afford the diol 11 as a colorless solid (0.004 g, 16%). ¹H NMR (CD₃OD) δ 8.26 (s, 1H), 7.79 (s, 1H), 7.61 (d, 1H, 6.5 Hz), 7.40 (d, 1H, 6.5 Hz), 7.10 (t, 1H, 4.2, 2.4 Hz), 5.98 (d, 1H, 6.6 Hz), 4.72−4.82 (m, 2H), 4.45−4.67 (m, 2H), 3.78 (s, 3H), HRMS calculated for C₁₈H₁₉ClIN₆O₅⁺ (M+H)⁺: 561.0156; found 561.0150. HPLC: RT 19.4 min (98%) in solvent system A, 11.1 min (98%) in system B.

5-[2-Chloro-6-(3-iodo-benzylamino)-purin-9-yl]-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid methoxy-methyl-amide (12)

The methyl ester 10a (0.031 g, 0.05 mmol) was dissolved in MeOH (5 mL), potassium carbonate (0.054 g, 0.4 mmol) was added, and the mixture was stirred at room temperature for 10 min. N,O-dimethylhydroxylamine hydrochloride (0.019 g, 0.2 mmol) was added and the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was treated with acetic acid (0.3 mL) and concentrated under reduced pressure. The residue was subjected to preparative thin layer chromatography by using chloroform/methanol (8:2) as solvent to afford the diol 12 as a colorless solid (0.004 g, 14%). ¹H NMR (CD₃OD) δ 8.29 (s, 1H), 7.74 (s, 1H), 7.52 (d, 1H, 5.5 Hz), 7.36 (d, 1H, 8.5 Hz), 7.14 (t, 1H, 4.1, 2.5 Hz), 6.04 (d, 1H, 6.4 Hz), 4.62−4.80 (m, 2H), 4.42−4.62 (m, 2H), 3.58 (s, 3H), 2.68 (s, 3H) HRMS calculated for C1₉H₂₁ClIN₆O₅⁺ (M+H)⁺: 575.0312; found, 575.0310. HPLC: RT 15.2 min (98%) in solvent system A, 18.5 min (99%) in system B.

5-[2-Chloro-6-(3-iodo-benzylamino)-purin-9-yl]-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid (13)

The methyl ester 10a (0.062 g, 0.1 mmol) was dissolved in EtOH (3 mL), lithium hydroxide (0.012 g, 0.5 mmol) was added, and the mixture was stirred at room temperature for 10 h. Acetic acid (0.2 mL) was added to neutralize the base, and the reaction mixture is concentrated under reduced pressure. The resulting residue was triturated with ethyl acetate (10 mL). The solid obtained was filtered and dried to afford acid 13 as a colorless solid (0.027 g, 53%). ¹H NMR (CD₃OD) δ 8.91 (s, 1H), 7.79 (s, 1H), 7.61 (d, 1H, 7.2 Hz), 7.39 (d, 1H, 7.2 Hz), 7.10 (t, 1H, 5.8 Hz), 6.15 (d, 1H, 6.9 Hz), 4.62−4.80 (m, 2H), 4.35−4.42 (m, 2H). HRMS calculated for C1₁₇H₁₆ClIN₅O₅⁺ (M+H)⁺: 531.9879; found, 531.9910. HPLC: RT 15.2 min (98%) in solvent system A, 18.0 min (95%) in system B.

(1′S, 2′R, 3′S, 4′S, 5′S)-4′-[2-Chloro-6-(4-chloro-benzylamino)-purin-9-yl]-2′,3′-dihydroxy-bicyclo[3.1.0]hexane-1-carboxylic acid ethyl ester (18)

Compound 15 (10 mg, 0.02 mmol) was treated with a solution of trifluoroacetic acid in ethanol (10% TFA, 1.0 mL) and H₂O (0.1 mL). The mixture was heated to 70°C for 3 h. The solution was then cooled to room temperature and the solvent was removed under vacuum. The residue obtained was subjected to preparative silica gel column chromatography (CHCl₃: MeOH, 85:15) to afford diol (18). (7 mg, 74%). ¹H NMR (CDCl₃) δ 7.70 (s, 1H), 7.83 (d, 1H, 8.0 Hz), 7.26 (d, 1H, 8.5 Hz), 7.14 (t, 1H, 4.2, 2.8 Hz), 5.40−5.48 (m, 1H), 4.95 (s, 2H), 4.1−4.4 (m, 3H), 3.61 (s, 1H), 2.20−2.12 (m, 1H), 1.67−1.63 (m, 1H), 1.26 (t, 3H, 8.5, 6.5 Hz), 0.97−0.93 (m, 1H). HRMS calculated for C₂₁H₂₂Cl₂N₅O₄⁺ (M+H)⁺: 478.1043; found 478.1042. HPLC: RT 22.1 min (98%) in solvent system A, 17.5 min (99%) in system B.

(1′S, 2′R, 3′S, 4′S, 5′S)-4′-[2-Chloro-6-(2-chloro-5-methoxy-benzylamino)-purin-9-yl]-2,3-dihydroxy-bicyclo[3.1.0]hexane-1-carboxylic acid ethyl ester (19)

This was prepared following the same procedure as for compound (18) starting from the isopropylidene derivative 16 to give 19 in 71% yield. ¹H NMR (CDCl₃) δ 7.63 (s, 1H), 7.28 (s, 1H), 7.12 (d, 1H, 2.4 Hz), 6.72(d, 1H, 8.7 Hz), 6.32 (bs, 1H), 5.27 (s, 1H), 4.72 (d, 2H, 27 Hz), 4.0−4.2 (m, 3H), 3.78 (s, 3H), 2.08−2.10 (m, 1H), 1.74−1.81 (m, 1H), 1.51−1.59 (m, 1H), 1.22 (t, 3H, 1.2, 6.9 Hz). HRMS calculated for C₂₂H₂₄Cl₂N₅O₅ (M+H)⁺: 508.1154; found 508.1163. HPLC: RT 22.6 min (99%) in solvent system A, 21.2 min (100%) in system B.

(1′S, 2′R, 3′S, 4′S, 5′S)-4′-[6-(4-Chloro-benzylamino)-2-methylsulfanyl-purin-9-yl]-2,3-dihydroxy-bicyclo[3.1.0]hexane-1-carboxylic acid ethyl ester (20)

This was prepared following the same procedure as for compound (18) starting from the isopropylidene derivative 17 to afford 20 in 68% yield. ¹H NMR (CDCl₃) δ 7.60 (s, 1H), 7.35 (s, 1H), 7.18−7.29 (m, 3H), 6.26 (bs, 1H), 5.31−5.36 (m, 1H), 4.78−4.85 (m, 2H), 4.21−4.29 (m, 3H), 2.52 (s, 3H), 2.20−2.39 (m, 1H), 1.64−1.71 (m, 1H), 1.51−1.59 (m, 1H), 1.26−1.31 (m, 4H). HRMS calculated for C₂₂H₂₅ClN₅O₄S⁺ (M+H)⁺: 490.1316; found 490.1299. HPLC: RT 23.45 min (97%) in solvent system A, 21.2 min (98%) in system B.

Ethyl (1S, 2R, 3S, 4S, 5S)-2,3-O-(isopropylidene)-4-O-benzyl-2,3,4-trihydroxybicyclo[3.1.0]hexanecarboxylate (24)

Alcohol 14 (240 mg, 1.00 mmol) was dissolved in 5.0 mL of a DMF:THF (4:1) solution. The solution was cooled to −70°C under nitrogen, and 60% NaH in mineral oil (45.0 mg, 1.10 mmol) was added. The reaction was stirred at −70°C for 5 min and then allowed to warm to 0°C. Benzyl bromide (130 μl, 1.10 mmol) was added to the mixture. The mixture was stirred for 5 min at 0°C, allowed to warm to room temperature, and then stirred under nitrogen gas for an additional 16 h. The solvent was removed under vacuum. The resulting orange oil was dissolved in 50 mL ethyl acetate, washed with 3x50 mL water, and dried over Na₂SO₄. The extract was concentrated to a pale yellow-orange oil and purified by column chromatography (silica gel, ethyl acetate:hexanes 9:1) to provide 24 (220 mg, 66%) as a colorless oil. ¹H NMR (CDCl₃): 7.42−7.27 (m, 5H), 5.26 (d, 1H, 6.7 Hz), 4.69 (s, 2H), 4.56 (t, 1H, 6.7 Hz), 4.25−4.05 (m, 4H), 2.31−2.29 (m, 1H), 1.85 (t, 1H, 4.5 Hz), 1.59 (s, 3H), 1.54−1.50 (m, 1H), 1.31 (s, 3H), 1.28−1.22 (m, 4H). HRMS calculated for C₁₉H₂₄NaO₅⁺ (M+Na)⁺: 355.1521; found, 355.1482.

(1S, 2R, 3S, 4S, 5S)-2,3-O-(Isopropylidene)-4-O-benzyl-2,3,4-trihydroxybicyclo[3.1.0]-hexanecarboxylic acid (25)

A mixture of 1.50 mL of 6N KOH(aq) and 24 (220 mg, 0.66 mmol) in a sealed vial was heated to 80°C and stirred for 6 h. The mixture was cooled to 0°C and diluted with 10 mL of ice water. The mixture was neutralized upon the dropwise addition of 0.50 mL glacial acidic acid. The neutralized solution was extracted with 3×10 mL ethyl acetate. The extracts were combined and dried over Na₂SO₄. The solvent was removed under vacuum and the resulting carboxylic acid 25 was used without further purification (200 mg, 99%). ¹H NMR (CDCl₃) 7.42−7.27 (m, 5H), 5.21 (d, 1H, 6.7 Hz), 4.69 (s, 2H), 4.56 (t, 1H, 6.7 Hz), 4.23 (t, 1H, 5.0 Hz) 2.31−2.40 (m, 1H), 1.91 (t, 1H, 4.5 Hz), 1.58 (s, 3H), 1.54−1.50 (m, 1H), 1.31 (s, 3H). HRMS calculated for C₁₈H₂₁O₇⁻ (M+HCO₂)⁻: 349.1287; found 349.1287.

(1R, 2R, 3S, 4S, 5S)-2,3-O-(Isopropylidene)-4-O-benzyl-2,3,4-trihydroxybicyclo-[3.1.0]hexane (26)

Carboxylic acid 25 (200 mg, 0.66 mmol) was dissolved in 15.0 mL of dry toluene. The solution was purged of oxygen by 3 cycles of freeze and thaw under vacuum. 2,2′-Dithiopyridine-1,1′-dioxide (199 mg, 0.79 mmol), Bu₃SnH (956 mg, 3.30 mmol), and Bu₃P (332 mg, 1.65 mmol) was added to the solution. The reaction was covered with foil and stirred at room temperature under nitrogen gas for 3 h. Over the course of 3 h, the reaction mixture turned slightly yellow. At the end of 3 h, AIBN (54.0 mg, 0.33 mmol) was added to the mixture. The reaction was then heated to 90°C for an additional hour and the yellow color disappeared. The reaction was cooled to room temperature. The reaction mixture was diluted with 30 mL H₂O and extracted with 3×20 mL ethyl acetate. The extract was dried over Na₂SO₄ and the solvent was removed under vacuum. The crude oil was purified with silica preparative TLC using 20% ethyl acetate/hexanes to give 26 (85.0 mg, 49%). ¹H NMR (CDCl₃) 7.42−7.27 (m, 5H), 4.82 (t, 1H, 6.0 Hz), 4.69 (s, 2H), 4.50 (t, 1H, 6.7 Hz), 4.30 (t, 1H, 5.0 Hz), 1.70−7.75 (m, 1H), 1.60 (s, 3H), 1.31−1.26 (m, 4H), 0.65−0.60 (m, 1H). HRMS calculated for C₁₆H₂₆NO₃⁺ (M+NH₄)⁺: 278.1756; found 278.1752.

(1R, 2R, 3S, 4S, 5S)-2,3-O-(Isopropylidene)-2,3,4-trihydroxybicyclo[3.1.0]hexane (27), Method A

Benzyl ether 26 (50.0 mg, 0.33 mmol) was dissolved in a mixture of 2.0 mL anhydrous methanol and 0.10 mL formic acid. The solution was stirred in an ice bath and palladium black (8.50 mg, 10% w/w) was added. The solution was carefully purged with H₂(g) and fitted with a balloon filled with H₂(g). The reaction mixture was heated to 50°C and stirred for 16 h. The H₂(g) was discharged and the reaction mixture filtered through a nylon syringe filter. The filter was washed with 10 mL of methanol and the filtrate was dried under vacuum. The resulting oil was purified on a silica preparative TLC plate with 30% ethyl acetate/hexanes as eluant to give 27 as a homogeneous product (15.0 mg, 27%). ¹H NMR (CDCl₃) 4.88 (t, 1H, 4.2 Hz), 4.53 (bs, 1H), 4.48 (t, 1H, 6.7 Hz), 4.30 (t, 1H, 5.0 Hz), 2.40 (bs, 1H), 1.87−1.83 (m, 1H), 1.67−1.63 (m, 1H), 1.55 (s, 3H), 1.29 (s, 3H), 0.99−0.95 (m, 1H), 0.68−0.60 (m, 1H). HRMS calculated for C₉H₁₄O₃ (M)⁺: 170.0943; found, 170.0942.

(1′R, 2′R, 3′S, 4′S, 5′S)-4′-[(2,6-Dichloropurine)-2′,3′-O-(isopropylidene)-2′,3′-dihydroxybicyclo[3.1.0]hexane (32)

A mixture of triphenyl phosphine (46 mg, 0.176 mmol) and 2,6-dichloropurine (33 mg, 0.176 mmol) in dry THF (2.0 mL) was treated with diisopropylazodicarboxylate (36 mg, 0.176 mmol). The mixture was stirred at room temperature for 20 min. The mixture was then added to a stirring solution of 27 (15 mg, 0.088 mmol) dissolved in dry THF (0.50 mL). The mixture was stirred for an additional 8 h at room temperature. The solvent was evaporated, and the crude oil was purified with silica preparative TLC in 30% ethyl acetate/hexanes solution to give 32 (15 mg, 50%) ¹H NMR (CDCl₃) 8.15 (s, 1H), 5.38 (t, 1H, 4.2 Hz), 5.06 (s, 1H), 4.67 (d, 1H, 6.7 Hz), 2.20−2.15 (m, 1H), 1.67−1.63 (m, 3H), 1.56 (s, 3H), 1.26 (s, 3H), 1.03−0.97 (m, 3H). HRMS calculated for C₁₄H₁₄Cl₂N₄O₂ (M)⁺: 340.0494; found, 340.0495.

(1R, 2S, 3R, 4R, 5R)-3,4-O-(Isopropylidene)-2-O-(tert-butyldiphenylsilyl)-2,3,4-trihydroxybicyclo[3.1.0]hexane-1-carboxylic acid (30)

tert-Butyldiphenylsilyl chloride (2.70 g, 10 mmol) and triethylamine (2.0 g, 20 mmol) were added to a solution of alcohol 29 (prepared from D-ribose 28 following the standard procedure,¹³ 1.22 g, 5 mmol) and imidazole (140 mg, 2 mmol) in DMF (3 mL) while stirring at room temperature. The solution was stirred at 60 °C for 16 h. The reaction mixture was cooled to room temperature and diluted with a 4:1 ethyl acetate – hexane mixture (50 mL), washed with water, dried, and solvent was evaporated. The residue was purified by flash chromatography (0 to 10% ethyl-acetate – hexane) to give ethyl (1R, 2S, 3R, 4R, 5R)-2,3-O-(isopropylidene)-4-O-(tert-butyldiphenylsilyl)-2,3,4-trihydroxybicyclo[3.1.0]hexane-1-carboxylate. The compound was dissolved in MeOH (5 mL), 2N aq. NaOH (5 mL) was added, and the reaction mixture was refluxed for 2 h. The reaction mixture was neutralized with NaH₂PO₄, and extracted with DCM. The combined DCM solutions were dried and evaporated, and the residue was purified by flash chromatography to give title compound 30 (1.65 g, 73%). ¹H NMR (CDCl₃), δ: 7.72 (d, 4H, J=7.8 Hz), 7.39 (m, 6H), 5.05 (d, 1H, J=6.3 Hz), 4.43 (t, 1H, J=6.0 Hz), 4.08 (t, 1H, J=6.6 Hz), 2.26 (m, 1H), 1.97 (s, 3H), 1.56 (s, 3H), 1.52 (m, 1H), 1.21 (s, 3H), 1.08 (s, 9H).

(1S, 2S, 3R, 4R, 5R)-3,4-O-(Isopropylidene)-2-O-(tert-butyldiphenylsilyl)-2,3,4-trihydroxybicyclo[3.1.0]hexane (31)

A 1M solution of DCC in oxygen-free toluene (0.96 mL) was added to a solution of acid 30 (363 mg, 0.80 mmol), 2-mercaptopyridine N-oxide (112 mg, 0.88 mmol), and AIBN (40 mg, 0.24 mmol) in dry oxygen-free toluene (4 mL). The reaction mixture was stirred for 4 h at 25 °C, tris(trimethylsilyl)silane (0.50 mL, 1.6 mmol) was added, and the reaction mixture was heated at 85 °C for 4 h. The reaction mixture was evaporated, and the residue was separated by flash chromatography (0 to 10% ethyl acetate – hexane mixture) to afford the title compound 31 (121 mg, 40%). ¹H NMR (CDCl₃), δ: 7.76 (d, 4H, J=7.8 Hz), 7.39 (m, 6H), 4.66 (t, 1H, J=6.0 Hz), 4.44 (t, 1H, J=6.6 Hz), 4.03 (t, 1H, J=6.6 Hz), 1.6 (m, 1H), 1.57 (s, 3H), 1.45 (m, 1H), 1.33 (s, 1H), 1.20 (s, 3H), 1.09 (s, 9H), 0.58 (m, 1H).

(1R, 2R, 3S, 4S, 5S)-2.3-O-(Isopropylidene)-2,3,4-trihydroxy-bicyclo[3.1.0]hexane (27), Method B

A 1M solution of tert-butylammonium fluoride in THF (1 mL) was added to a solution of silyl ether 31 (102 mg, 0.25 mmol) in THF (1 mL). The reaction mixture was left at 20 °C for 16 h and evaporated. The residue was diluted with ethyl acetate (20 mL) and washed with a small amount of brine. The ethyl acetate solution was dried and evaporated, and the residue was purified by flash chromatography to afford the title compound 27 (33 mg, 84%). ¹H NMR and MS are provided under Method A.

General procedure for preparation of compounds 33b – 39b

An amine (RNH₂ in Scheme 3, 0.5 mmol) was added to a solution of 32 (20 mg, 0.06 mmol) in DCM (0.1 mL). The reaction mixture was stirred at room temperature for 16 h. The solvent was removed under vacuum, and the residue was separated by flash chromatography (30 to 100% ethyl acetate-hexane) to afford the corresponding 6-alkylaminopurine derivative that was dissolved in a mixture of MeOH (4 mL), TFA (0.2 mL) and water (2 mL). The reaction mixture was stirred at 70 °C for 16 h, and then evaporated. The residue was evaporated twice with water, and the residue was purified by flash chromatography (50 to 100% ethyl acetate).

(1′R, 2′R, 3′S, 4′R, 5′S)-4′-[2-Chloro-6-(3-iodobenzylamino)purine]-2′,3′-O-dihydroxybicyclo-[3.1.0]hexane(33b)

Yield 15 mg (51% ¹H NMR (CD₃OD), δ: 8.16 (s, 1H), 7.49 (s, 1H), 7.60 (d, 1H, 8.5 Hz), 7.40 (d, 1H, 8.5 Hz), 7.10 (t, 1H, 8.5 Hz), 4.71 (s, 2H), 3.90 (d, 3.3Hz, 1H), 3.65 (s, 1H), 2.05−1.95 (m, 1H), 1.67−1.63 (m, 1H), 1.36 (s, 1H), 1.31−1.27 (m, 1H), 0.95−0.87 (m, 1H), 0.77−0.75 (m, 1H). HRMS calculated for C₁₈H₁₈ClIN₅O₂⁺ (M+H)⁺: 498.0194; found, 498.0194. HPLC: RT 21.6 min (98%) in solvent system A, 17.0 min (98%) in system B.

(1′R, 2′R, 3′S, 4′R, 5′S)-4′-[2-Chloro-6-(3-chlorobenzylamino)purine]-2′,3′-O-dihydroxybicyclo-[3.1.0]hexane(34b)

Yield 58%. ¹H NMR (CD₃OD), δ: 8.16 (br.s., 1H), 7.41 (s, 1H), 7.29 (m, 3H), 4.79 (s, 1H), 4.75 (br. s, 2H), 4.70 (br. t., 1H, J=5.4 Hz), 3.86 (d, 1H, J=6.6 Hz), 1.97 (m, 1H), 1.65 (m, 1H), 1.30 (m, 1H), 0.75 (m, 1H). HRMS (ESI MS m/z): calculated for C₁₈H₁₈Cl₂N₅O₂⁺ (M+H)⁺, 406.0832; found, 406.0825. HPLC RT 20.3 min (98%) in solvent system A, 15.6 min (98%) in system B.

(1′R, 2′R, 3′S, 4′R, 5′S)-4′-[2-Chloro-6-(3-bromobenzylamino)purine]-2′,3′-O-dihydroxybicyclo-[3.1.0]hexane(35b)

Yield 65%. ¹H NMR (CD₃OD): 8.03 (s, 1H), 7.45 (s, 1H), 7.29 (m, 2H), 7.12 (t, 1H, J=7.8 Hz), 4.68 (s, 1H), 4.63 (br. s, 2H), 4.59 (br. t., 1H, J=5.4 Hz), 3.79 (d, 1H, J=6.6 Hz), 1.86 (m, 1H), 1.55 (m, 1H), 1.20 (m, 1H), 0.64 (m, 1H). HRMS (ESI MS m/z) calculated for C₁₈H₁₈BrClN₅O₂⁺ (M+H)⁺, 450.0327; found 450.0315. HPLC RT 20.74 min (98%) in solvent system A, 16.1 min (99%) in system B.

(1′R, 2′R, 3′S, 4′R, 5′S)-4′-[2-Chloro-6-(1-naphthylamino)purine]-2′,3′-O-dihydroxybicyclo[3.1.0]hexane(36b)

Yield 48%. ¹H NMR (CD₃OD): 8.13 (br.d., 2H, J=7.8 Hz), 7.84 (m, 2H), 7.49 (m, 4H), 5.21 (s, 1H), 4.79 (br. s, 1H), 4.78 (br. s, 2H), 4.67 (br. t., 1H, J=5.1 Hz), 3.88 (d, 1H, J=6.6 Hz), 1.93 (m, 1H), 1.62 (m, 1H), 1.25 (m, 1H), 0.73 (m, 1H). HRMS (ESI MS m/z) calculated for C₂₂H₂₁ClN₅O₂⁺ (M+H)⁺, 422.1378; found 422.1385. HPLC RT 21.5 min (97%) in solvent system A, 17.0 min (98%) in system B.

(1′R, 2′R, 3′S, 4′R, 5′S)-4′-[2-Chloro-6-(2,5-dimethoxybenzylamino)purine]-2′,3′-O-dihydroxybicyclo-[3.1.0]hexane(37b)

Yield 44%. ¹H NMR (CD₃OD): 8.4 (very br. s, 1H), 6.95 (s, 1H, J=2.7 Hz), 6.89 (d, 1H, J=9.3 Hz), 6.78 (dd, 1H, J=2.7, 9.0 Hz), 4.80 (s, 1H), 4.75 (br. m, 3H), 3.87 (d, 1H, J=6.3 Hz), 3.83 (s, 3H), 3.71 (s, 3H), 1.95 (m, 1H), 1.64 (m, 1H), 1.29 (m, 1H), 0.74 (m, 1H). HRMS (ESI MS m/z) calculated for C₂₀H₂₃ClN₅O₄⁺ (M+H)⁺, 432.1433; found 432.1439. HPLC RT 18.7 min (98%) in solvent system A, 16.6 min (98%) in system B.

(1′R, 2′R, 3′S, 4′R, 5′S)-4′-[2-Chloro-6-(2-hydroxy-5-methoxybenzylamino)purine]-2′,3′-O-dihydroxybicyclo-[3.1.0]hexane(38b)

Yield 39%. ¹H NMR (CD₃OD): 8.07 (s, 1H), 6.60−6.82 (m, 3H), 4.69 (s, 1H), 4.59 (br. t., 1H, J=6.0 Hz), 4.56 (br. s, 2H), 3.79 (d, 1H, J=6.6 Hz), 3.61 (s, 3H) 1.86 (m, 1H), 1.55 (m, 1H), 1.20 (m, 1H), 0.65 (m, 1H). HRMS (ESI MS m/z) calculated for C₁₉H₂₁ClN₅O₄⁺ (M+H)⁺, 418.1277; found, 418.1277. HPLC RT 16.0 min (100%) in solvent system A, 11.0 min (98%) in system B.

(1′R, 2′R, 3′S, 4′R, 5′S)-4′-[2-Chloro-6-(trans-2-phenylcyclopropylamino)purine]-2′,3′-O-dihydroxybicyclo-[3.1.0]hexane(39b)

Yield 52%. ¹H NMR (CD₃OD): 8.16 (very br.s., 1H), 7.0−7.48 (m, 5H), 4.79 (s, 1H), 4.68 (br. s, 2H), 3.88 (d, 1H, J=5.7 Hz), 2.17 (m, 1H) 1.97 (m, 1H), 1.65 (m, 1H), 1.29 (m, 2H), 0.74 (m, 1H). HRMS (ESI MS m/z) calculated for C₂₀H₂₁ClN₅O₂⁺ (M+H)⁺, 398.1378; found, 398.1372. HPLC RT 20.3 min (99%) in solvent system A, 15.6 min (98%) in system B.

Molecular modeling

Docking study

The homology model of the hA₃AR (PDB id: 1OEA) derived using the X-ray structure of bovine rhodopsin with a 2.8 Å resolution (PDB id: 1F88) as a template was used for the docking study. The previously reported A₃AR conformation,¹⁷ which was bound to Cl-IB-MECA, 2, was used for the antagonist docking. For the docking study of compound 33b, a pharmacophore match among hydrophilic interaction sites, for the 2′-OH, 3′-OH, and N⁶H groups, was performed to obtain an initial geometry. The refinement of the side chain in the binding site was followed by binding site minimization with fixed backbone atoms, complex minimization, and 200 ps molecular dynamics.

Atomic charges were recalculated by Mullekin charge using the DFT/6311G** basis set for ligands 2 and 33b. Finally, the complex structure was minimized using an Amber 7 force field 99 with a fixed dielectric constant (4.0), until the conjugate gradient reached 0.1 kcal mol⁻¹ Å⁻¹. Values of clog P were calculated using ChemDraw Ultra 11.0.

Receptor binding and functional assays

[¹²⁵I]N⁶-(4-Amino-3-iodobenzyl)adenosine-5′-N-methyluronamide (I-AB-MECA; 2000 Ci/mmol), [³H]cyclic AMP (40 Ci/mmol), and other radioligands were purchased from Perkin–Elmer Life and Analytical Science (Boston, MA). [³H]CCPA (2-chloro-N⁶-cyclopentyladenosine) was a custom synthesis product (Perkin Elmer). Test compounds were prepared as 5 mM stock solutions in DMSO and stored frozen.

Cell culture and membrane preparation

CHO (Chinese hamster ovary) cells expressing the recombinant human A₃AR were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, 2 μmol/mL glutamine and 800 μg/mL geneticin. The CHO cells expressing rat A₃ARs were cultured in DMEM and F12 (1:1). Cells were harvested by trypsinization. After homogenization and suspension, cell membranes were centrifuged at 500 g for 10 min, and the pellet was re-suspended in 50 mM Tris·HCl buffer (pH 8.0) containing 10 mM MgCl₂, 1 mM EDTA and 0.1 mg/mL CHAPS (3[(3-cholamidopropyl)dimethylammonio]-propanesulfonic acid). The suspension was homogenized with an electric homogenizer for 10 sec, and was then re-centrifuged at 20,000 g for 20 min at 4°C. The resultant pellets were resuspended in buffer in the presence of adenosine deaminase (3 Units/mL), and the suspension was stored at −80°C until the binding experiments. The protein concentration was measured using the Bradford assay.¹⁸

Binding assays at the A₁ and A_2A receptors

For binding to human A₁ receptors,¹⁹ [³H]R-PIA (N⁶-[(R)-phenylisopropyl]adenosine, 2 nM) or [³H]CCPA (0.5 nM) was incubated with membranes (40 μg/tube) from CHO cells stably expressing human A₁ receptors at 25°C for 60 min in 50 mM Tris·HCl buffer (pH 7.4; MgCl₂, 10 mM) and increasing concentrations of the test ligand in a total assay volume of 200 μl. Nonspecific binding was determined using 10 μM of CPA (N⁶-cyclopentyladenosine). For human A_2A receptor binding,²⁰ membranes (20 μg/tube) from HEK-293 cells stably expressing human A_2A receptors were incubated with [³H]CGS21680 (2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamido-adenosine, 15 nM) and increasing concentrations of the test ligand at 25°C for 60 min in 200 μl 50 mM Tris·HCl, pH 7.4, containing 10 mM MgCl₂. NECA (10 μM) was used to define nonspecific binding. The reaction was terminated by filtration with GF/B filters.

Binding assay at the human A₃ receptor

For the competitive binding assay, each tube contained 50 μL membrane suspension (20 μg protein), 25 μL of [¹²⁵I]I-AB-MECA (1.0 nM),²¹ and 25 μL of increasing concentrations of the test ligands in Tris·HCl buffer (50 mM, pH 8.0) containing 10 mM MgCl₂, 1 mM EDTA. Nonspecific binding was determined using 10 μM of Cl-IB-MECA in the buffer. The mixtures were incubated at 37°C for 60 min. Binding reactions were terminated by filtration through Whatman GF/B filters under reduced pressure using a MT-24 cell harvester (Brandell, Gaithersburgh, MD, USA). Filters were washed three times with 9 mL ice-cold buffer. Radioactivity was determined in a Beckman 5500B γ-counter. IC₅₀ values were converted to K_i values as described.²⁵

Cyclic AMP accumulation assay

Intracellular cyclic AMP levels were measured with a competitive protein binding method.^22,23 CHO cells that expressed the recombinant human or rat A₃AR or the human A₁ or A_2BAR were harvested by trypsinization. After centrifugation and resuspended in medium, cells were planted in 24-well plates in 1.0 mL medium. After 24 h, the medium was removed and cells were washed three times with 1 mL DMEM, containing 50 mM HEPES, pH 7.4. Cells were then treated with the agonist NECA and/or test compound (e.g. 33b) in the presence of rolipram (10 μM) and adenosine deaminase (3 units/mL). After 45 min forskolin (10 μM) was added to the medium, and incubation was continued for an additional 15 min. The reaction was terminated by removing the supernatant, and cells were lysed upon the addition of 200 μL of 0.1 M ice-cold HCl. The cell lysate was resuspended and stored at −20°C. For determination of cyclic AMP production, protein kinase A (PKA) was incubated with [³H]cyclic AMP (2 nM) in K₂HPO₄/EDTA buffer (K₂HPO₄, 150 mM; EDTA, 10 mM), 20 μL of the cell lysate, and 30 μL 0.1 M HCl or 50 μL of cyclic AMP solution (0−16 pmol/200 μL for standard curve). Bound radioactivity was separated by rapid filtration through Whatman GF/C filters and washed once with cold buffer. Bound radioactivity was measured by liquid scintillation spectrometry.

[³⁵S]GTPγS binding assay

[³⁵S]GTPγS binding was measured by a variation of the method described.²⁴ Each assay tube consisted of 200 μL buffer containing 50 mM Tris HCl (pH 7.4), 1 mM EDTA, 1 mM MgCl₂, 1 μM GDP, 1 mM dithiothreitol, 100 mM NaCl, 3 U/ml ADA, 0.2 nM [³⁵S]GTPγS, 0.004% 3-[(3-cholamidopropyl) dimethylammonio]propanesulfonate (CHAPS), and 0.5% bovine serum albumin. Incubations were started upon addition of the membrane suspension (CHO cells stably expressing either the native human A₁AR or A₃AR, 5 μg protein/tube) to the test tubes, and they were carried out in duplicate for 30 min at 25°C. The reaction was stopped by rapid filtration through Whatman GF/B filters, pre-soaked in 50 mM Tris HCl, 5 mM MgCl₂ (pH 7.4) containing 0.02% CHAPS. The filters were washed twice with 3 mL of the same buffer, and retained radioactivity was measured using liquid scintillation counting. Non-specific binding of [³⁵S]GTPγS was measured in the presence of 10 μM unlabelled GTPγS.

Chart 1.

Adenine 9-riboside derivatives as prototypical ligands for the A₃AR.

Chart 2.

4′-Thio and (N)-methanocarba derivatives of adenosine as later generation ligands for the A₃AR.

Abbreviations

AIBN

2,2′-azo-bis-isobutyronitrile

AR

adenosine receptor

CCPA

2-chloro-N⁶-cyclopentyladenosine

CHO

Chinese hamster ovary

Cl-IB-MECA

2-chloro-N⁶-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine

DCC

dicyclohexylcarbodiimide

DMEM

Dulbecco's modified Eagle's medium

EDTA

ethylenediaminetetraacetic acid

GPCR

G protein-coupled receptor

I-ABMECA

2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamidoadenosine

NECA

5′-N-ethylcarboxamidoadenosine

DIPEA

diisopropylethylamine

DCM

dichloromethane

DMF

N,N-dimethylformamide

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HRMS

high resolution mass spectroscopy

MRS1292

(2R,3R,4S,5S)-2-[N⁶-3-iodobenzyl)adenos-9'-yl]-7-aza-1-oxa-6-oxospiro[4.4]-nonan-4,5-diol

PIA

R-N⁶-(phenylisopropyl)adenosine

TEA

triethylamine

TLC

thin layer chromatography