(N)-Methanocarba 2,N6-Disubstituted Adenine Nucleosides as Highly Potent and Selective A3 Adenosine Receptor Agonists

Susanna Tchilibon; Bhalchandra V Joshi; Soo-Kyung Kim; Heng T Duong; Zhan-Guo Gao; Kenneth A Jacobson

doi:10.1021/jm049580r

. Author manuscript; available in PMC: 2012 Oct 3.

Published in final edited form as: J Med Chem. 2005 Mar 24;48(6):1745–1758. doi: 10.1021/jm049580r

(N)-Methanocarba 2,N⁶-Disubstituted Adenine Nucleosides as Highly Potent and Selective A3 Adenosine Receptor Agonists

Susanna Tchilibon ¹, Bhalchandra V Joshi ¹, Soo-Kyung Kim ¹, Heng T Duong ¹, Zhan-Guo Gao ¹, Kenneth A Jacobson ^1,^*

PMCID: PMC3463111 NIHMSID: NIHMS31393 PMID: 15771421

Abstract

A series of ring-constrained (N)-methanocarba-5′-uronamide 2,N⁶-disubstituted adenine nucleosides have been synthesized via Mitsunobu condensation of the nucleobase precursor with a pseudosugar ring containing a 5′-ester functionality. Following appropriate functionalization of the adenine ring, the ester group was converted to the 5′-N-methylamide. The compounds, mainly 2-chloro substituted derivatives, were tested in both binding and functional assays at human adenosine receptors (ARs), and many were found to be highly potent and selective A₃AR agonists. Selected compounds were compared in binding to the rat A₃AR to assess their viability for testing in rat disease models. The N⁶-(3-chlorobenzyl) and N⁶-(3-bromobenzyl) analogues displayed K_i values at the human A₃AR of 0.29 and 0.38 nM, respectively. Other subnanomolar affinities were observed for the following N⁶ derivatives: 2,5-dichlorobenzyl, 5-iodo-2-methoxybenzyl, trans-2-phenyl-1-cyclopropyl, and 2,2-diphenylethyl. Selectivity for the human A₃AR in comparison to the A₁AR was (fold): the N⁶-(2,2-diphenylethyl) analogue 34 (1900), the N⁶-(2,5-dimethoxybenzyl) analogue 26 (1200), the N⁶-(2,5-dichlorobenzyl) and N⁶-(2-phenyl-1-cyclopropyl) analogues 20 and 33 (1000), and the N⁶-(3-substituted benzyl) analogues 17, 18, 28, and 29 (700–900). Typically, even greater selectivity ratios were obtained in comparison with the A_2A and A_2BARs. The (N)-methanocarba-5′-uronamide analogues were full agonists at the A₃AR, as indicated by the inhibition of forskolin-stimluated adenylate cyclase at a concentration of 10 µM. The N⁶-(2,2-diphenylethyl) derivative was an A₃AR agonist in the (N)-methanocarba-5′-uronamide series, although it was an antagonist in the ribose series. Thus, many of the previously known groups that enhance A₃AR affinity in the 9-riboside series, including those that reducing intrinsic efficacy, may be adapted to the (N)-methanocarba nucleoside series of full agonists.

Introduction

There are four subtypes of adenosine receptors (ARs): A₁, A_2A, A_2B, and A₃.¹ Each of these subtypes is associated with specific experimental therapeutic modalities.² Selective adenosine receptor antagonists and agonists are under development for treating such diverse conditions including inflammation, cardiac ischemia, stroke, asthma, diabetes, cardiac arrhythmias, and other disorders.^3–7 Agonists selective for the A₃AR are potentially useful for the treatment of stroke, neurodegenerative diseases, myocardial infarction, and cancer.^4,5,8–10 In colon carcinoma, a cytostatic effect of an A₃AR agonist appears to be related to its downstream activation of the Wnt pathway.¹⁰ A₃AR antagonists are potentially useful for the treatment of glaucoma.¹¹

Prototypical high affinity ligands for the A₃AR include the N⁶-(3-iodobenzyl) adenosine analogues 1 – 5 (Chart 1).¹² The moderately A₃AR-selective nucleoside IBMECA 2a (N⁶-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine) is a full agonist,¹³ which is currently in phase 2 clinical trials for the treatment of melanoma and colon carcinoma.¹⁰ Also, chronically administered 2a was neuroprotective in a model of ischemic brain damage in gerbils.⁵ The corresponding 2-chloro derivative Cl-IB-MECA 2b was the first highly selective A₃AR agonist¹⁴ and has been shown to be cardioprotective in a variety of in vitro and in vivo models.^4,15,16 In an SAR study of adenosine derivatives,¹³ the N⁶-(3-iodobenzyl) substituent was found to enhance the selectivity for the A₃AR. Also, the 2-chloro modification has been found to enhance potency in a variety of AR agonists. When present as the sole modifications of adenosine, as in the 9-riboside derivatives 1a and 1b, they reduced the intrinsic efficacy at the A₃AR, to the extent that 2-chloro-9-riboside 1b was an antagonist of this subtype. Rigidity introduced in the ribose moiety also tends to reduce A₃AR efficacy, such as the (N)-methanocarba ring system, a bridged carbocyclic ring system present in the partial agonists 3 and 4 and in the full agonist 5.^17,18 Structural features that reduced efficacy included N⁶-benzyl groups, small moieties at the 2-position, and sterically constrained moieties in the ribose region. For each of these efficacy-diminishing factors, the 5′-uronamide group tended to restore the observed loss of A₃AR efficacy, as observed in the full agonism displayed by 2 and 5. There is still a need for the development of more highly selective A₃AR agonists than the widely used A₃AR agonists 2a and 2b, for which some literature reports have indicated limited pharmacological selectivity.^19,25

The present study characterizes the SAR (structure activity relationship) of a broad class of highly potent and selective A₃AR agonists based on compound 5. The principal design element of these agonists is a modified ribose moiety. The analogues contain the (N)-methanocarba (bicyclo[3.1.0]hexane) ring system, which is a rigid ribose substitute lacking the ether oxygen. This ring system maintains the 2′-exo-(N) ring-twist conformation of the ribose-like ring (pseudosugar moiety), which has been demonstrated to be favored in A₃AR binding (more so than at other AR subtypes).²⁰ These agonists also contain a flexible 5′-uronamide group, necessary to assure full activation of the A₃AR.¹² We utilized a new synthetic route for the synthesis of these derivatives^21,22 which allows for a more versatile substitution of the adenine moiety than the previous synthetic route.²³ In addition, we incorporated N⁶-substituents that have been shown previously to favor A₃AR binding affinity—mainly N⁶-benzyl and N⁶-phenylethyl²⁴ groups—and included new variations thereof. These derivatives were characterized biologically at the four human AR subtypes expressed in Chinese hamster ovary (CHO) cells²⁵ in binding and functional assays to assess their selectivity within the same species. The results indicate that we achieved the design of many new, sterically-constrained and selective A₃AR agonists with nanomolar and subnanomolar affinities. Several of these high affinity agonists were docked in the putative binding site of a rhodopsin²⁶-based model of the human A₃AR.^18,24,27

RESULTS

Chemical Synthesis

The novel derivatives 15 and 17 - 36 were synthesized as shown in Schemes 1 – 3. Most analogues contained a 2-chloro substituent, although 2-amino 15, 2-iodo 35, and 2-alkylthio 36 groups were also included.

Scheme 1 — Reagents and conditions: i) 2,6-dichloropurine, DIAD, TPP, THF, r.t.; ii) RNH₂, TEA, MeOH; iii) 40% aq. MeNH₂, MeOH; iv) 10% CF₃COOH in MeOH, H₂O, 70°C; v) 6-chloro-2-iodopurine or 6-chloro-2-methylthiopurine or 6-chloro-2-aminopurine, DIAD, TPP, THF, r.t.; vi) 3-chlorobenzylamine, TEA, MeOH.

Scheme 3 — Reagents and conditions: i) *tert*-butyl nitrite, methyl disulphide, acetonitrile, r.t. ii) aq. NaOH, i-PrOH,THF.

The general synthetic route follows the method of Joshi et al.^21,22 The protected (N)-methanocarba ring system 37 containing a carbonyl group at the 5′-position was prepared in 8 steps from isopropylidene erythronolactone 36 (Scheme 1). Compound 37 and the nucleobase precursor, 2,6-dichloropurine, were condensed using a Mitsunobu coupling.²⁸ Substitution of the 6-chloro of 38 was then carried out by treating with an excess of the appropriate amine, such as a substituted benzylamine, to provide the series of protected nucleoside 5′-esters 39 – 54. Following appropriate substitution at the N⁶ position, the 5′-esters were treated with an excess of methylamine, and the isopropylidene group was removed from the 2′- and 3′-hydroxyl groups upon acid treatment to provide the target compounds 16 - 33.

The various benzylamine derivatives used in this study, when not commercially available, were synthesized by the methods shown in Scheme 2. 2,5-Dichlorobenzylamine 64 and 2-chloro-5-iodobenzylamine 65 were prepared from the corresponding benzyl bromides. Compound 60 was prepared from 2-chloro-5-iodotoluene by bromination with N-bromosuccinimide in CCl₄.²⁹ 60 and 61 were treated with potassium phthalimide in N,N-dimethylformamide at 60°C to obtain compounds 62 and 63 as white solids in 95% yield. Deprotection with hydrazine gave 64 and 65 in 80% yield.³⁰

5-Iodo-2-methoxybenzylamine 68 was prepared starting from 5-iodosalicylaldehyde 66. Accordingly, compound 66 was methylated with methyl iodide in the presence of K₂CO₃ in N,N-dimethylformamide and subsequently, the formyl moiety was transformed to methylamino by reductive amination with NaCNBH₄ in the presence of ammonium acetate.³¹

Compounds 72 and 73 were synthesized through a Sonogashira-type reaction in high yield using CuI and (Ph₃P)PdCl₂ as catalysts. ³² For the preparation of both 5-chloro-2-benzyloxybenzylamine 76 and 5-chloro-2-(aminocarbonylmethyloxy)benzylamine 80, 5-chloro-2-hydroxybenzamide 74 was used as starting material. Compound 74 was treated with benzyl bromide in the presence of K₂CO₃ in N,N-dimethylformamide to obtain 5-chloro-2-benzyloxybenzamide 75 in 98% yield. Finally, the amide 75 was reduced to the corresponding amine 76 with LiAlH₄ in tetrahydrofuran.

Reduction of compound 74 with LiAlH₄ in tetrahydrofuran gave 5-chloro-2-hydroxybenzylamine 77 in 88% yield. This preparation showed an improvement over the reported procedure, based on the hydrogenation of 5-chloro-2-hydroxybenzaldehyde oxime.³³ The amine 77 was protected as a t-butylcarbamate 78, and the hydroxyl group was alkylated with 2-bromoacetamide in the presence of K₂CO₃ in N,N-dimethylformamide. The subsequent deprotection of the amino group with 15% trifluoroacetic acid in dichloromethane gave the final product 80 as a white solid.

Substitution at the 2-position by groups other than chloro was possible and accomplished by condensing the appropriate 2-substituted nucleobases with the (N)-methanocarba sugar moiety 37 to provide the protected nucleoside esters 55 and 56 Scheme 1). Successive treatment with 3-chlorobenzylamine and methylamine followed by subsequent acid hydrolysis afforded the 2-iodo and 2-thiomethyl analogues 35 and 36. Alternately, the 2-amino substituted analogue was obtained by first condensing the 2-amino substituted nucleobase with 37 followed by the direct interaction with excess methylamine and subsequent hydrolysis to afford 15.

The requisite 6-chloro-2-iodopurine was prepared by a literature procedure,³⁵ while 6-chloro-2-methylthiopurine was prepared as delineated in Scheme 3. Accordingly, 6-chloro-2-methylthiopurin-9-yl-methyl 2,2-dimethylpropionate³⁴ 81 was diazotized with tert-butyl nitrite, and the resulting diazo intermediate was trapped with an excess of methyl disulfide to afford 82, which upon hydrolysis of the pivaloylmethyloxy protecting group with aqueous NaOH, gave the requisite 2-methylthiopurine 83.

Biological Activity

The AR binding affinities of both newly-synthesized (15 and 17 – 36) and known (1 – 14 and 16) nucleoside derivatives (Table 1) was investigated. The known adenosine agonist derivatives included 9-ribosides 1, 6 – 12, (N)-methanocarba derivatives 3 - 5, and 9-riboside 5′-N-methyluronamide analogues 2a, 2b, 14, and 16. Binding affinity at the human A₃AR, and in selected cases the rat A₃AR, was studied using the radioiodinated agonist ¹²⁵I-N⁶-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide, ³⁶ and binding at the human A₁AR and A_2AAR utilized the selective agonists [³H]R-N⁶-[phenylisopropyl]adenosine and [³H]2-[p-(carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine, respectively. Functional assays of the human A_2BAR and A₃AR consisted of measuring cyclic AMP production in intact CHO cells expressing the ARs (Table 2).²⁵ The functional effect of A₃AR was inhibition of forskolin-stimulated cAMP production, and the A_2BAR effect was stimulation of basal cAMP production.

Table 1.

Potency of adenosine derivatives at human A₁, A_2A, A_2B and A₃ARs and the rat A₃AR and maximal agonist effects at human A₃ARs expressed in CHO cells.^a



Compound	N⁶-R′	C2-R	K_i (hA₁AR) nM ^a or % displ. at 10 µM	K_i (hA_2AAR) nM ^a or % displ. at 10 µM	% Activation (hA_2BAR)^b at 10 µM	K_i (hA₃AR) nM^a	% Activation (hA₃AR)^b at 10 µM	K_i (rA₃AR)
	Riboside derivatives
6^c	CH₃	H	6000 ± 2000	17%	16	9.3 ± 0.4	96 ± 3	6400 ± 1600^d
7^c	CH₃	NH₂	480 ± 20	15%	11	39 ± 2	98 ± 3	>10,000
1a ^c	3-iodobenzyl	H	7.4 ± 1.7	140 ± 20	58	5.8 ± 0.4	46 ± 8	9.5 ± 1.4
1b ^c	3-iodobenzyl	Cl	17 ± 2	200 ± 30	16	1.8 ± 0.1	0	2.7 ± 1.2
8	3-chlorobenzyl	H	20 ± 3	1400 ± 200	ND	4.4 ± 1.7^d	80 ± 3^d	35 ± 20^d
9 ^c	5-chloro-2- methyloxybenzyl	H	9.2 ± 0.5	400 ± 10	57	1.3 ± 0.2	53 ± 3	ND
10 ^c	trans-2-phenyl-1- cyclopropyl	H	120 ± 30	2500 ± 700	26	0.86 ± 0.09	101 ± 5	400 ± 30
11^d	2-methylbenzyl	H	39 ± 8	760 ± 110	ND	47 ± 11	100 ± 3	38 ± 15
12 ^d	3-pyridylmethyl	H	42 ± 4	2300 ± 120	ND	4.5 ± 1.1	100 ± 6	290 ± 70
13 ^f	2,2-diphenylethyl	H	50 ± 16	510 ± 50	70	3.9 ± 0.7	0	540 ± 200
	5′-N-Methyluronamido *riboside derivatives*
2a	3-iodobenzyl	H	51 ± 5	2900 ± 600^d	0^h	1.8 ± 0.7^d	100	1.1 ± 0.3ⁱ
2b	3-iodobenzyl	Cl	220 ± 20	5400 ± 2500^d	0^h	1.4 ± 0.3^d	100 ± 4	0.33 ± 0.08ⁱ
	(N)-Methanocarba derivatives
3	3-iodobenzyl	H	35 ± 3	860 ± 70	ND	9.2 ± 0.7^d	13 ± 1	ND
4	3-iodobenzyl	Cl	65 ± 17	1600 ± 400	ND	1.9 ± 0.7^d	3 ± 2	ND
	5′-N-Methyluronamido *(N)-methanocarba derivatives*
14	CH₃	Cl	2100 ± 1700	6%	ND	2.2 ± 0.6	104 ± 7	160 ± 30
15	CH₃	NH₂	1600 ± 200	17%	ND	1.6 ± 0.3	117 ± 21	0.87 ± 0.24
16	3-iodobenzyl	H	700 ± 270	6200 ± 100	ND	2.4 ± 0.5^e	100^e	ND
5	3-iodobenzyl	Cl	136 ± 22 ^g	784 ± 97^g	ND	1.5 ± 0.2^e	100^e	1.1 ± 0.1
17	3-bromobenzyl	Cl	270 ± 70	1300 ± 100	38	0.38 ± 0.11	100 ± 11	0.76 ± 0.08
18	3-chlorobenzyl	Cl	260 ± 60	2300 ± 100	38	0.29 ± 0.04	103 ± 7	1.0 ± 0.10
19	3-fluorobenzyl	Cl	640 ± 140	5100 ± 200	28	2.4 ± 0.1	101 ± 5	1.6 ± 0.1
20	2,5-dichloro-, benzyl	Cl	540 ± 70	1300 ± 100	32	0.56 ± 0.06	102 ± 3	ND
21	2-chloro-5- iodobenzyl	Cl	340 ± 20	480 ± 20	58	0.83 ± 0.19	105 ± 6	ND
22	5-chloro-2- methoxybenzyl	Cl	240 ± 50	1200 ± 100	37	1.5 ± 0.0	107 ± 15	ND
23	5-chloro-2- (aminocarbonyl- methyloxy)benzyl	Cl	81 ± 10	800 ± 70	ND	5.3 ± 0.4	106 ± 12	ND
24	5-chloro-2- benzyloxybenzyl	Cl	1200 ± 200	850 ± 160	49	5.2 ± 1.3	110 ± 10	11 ± 3
25	5-iodo-2- methoxybenzyl	Cl	200 ± 20	430 ± 30	46	0.58 ± 0.12	101 ± 10	0.87 ± 0.24
26	2,5-dimethoxy- benzyl	Cl	1600 ± 200	52%	0	1.4 ± 0.2	107 ± 10	ND
27	2-methylbenzyl	Cl	710 ± 300	3800 ± 200	24	3.7 ± 0.6	100 ± 3	ND
28	3-methylbenzyl	Cl	450 ± 80	3700 ± 600	46	0.63 ± 0.18	106 ± 3	ND
29	3-(3-hydroxypropynyl) benzyl	Cl	2600 ± 300	56%	28	2.9 ± 0.7	102 ± 5	1.6 ± 0.6
30	2-chloro-5-(3- hydroxypropynyl) benzyl	Cl	14%	12%	6	217 ± 27	108 ± 5	ND
31a	4-aminobenzyl	Cl	4100 ± 100	28%	22	14 ± 3	98 ± 10	ND
31b	4-amino-3- iodobenzyl	Cl	48 ± 2	1100 ± 100	ND	3.1 ± 0.1	103 ± 7	ND
32	3-pyridylmethyl	Cl	2600 ± 1200	33%	4	11 ± 3	106 ± 7	ND
33	trans-2-phenyl-1- cyclopropyl	Cl	770 ± 50	4800 ± 200	48	0.78 ± 0.06	110 ± 7	ND
34	2,2-diphenylethyl	Cl	1300 ± 100	1600 ± 100	46	0.69 ± 0.02	107 ± 9	10 ± 4
35	3-chlorobenzyl	I	2200 ± 600	43%	ND	3.6 ± 0.8	107 ± 3	3.9 ± 0.4
36	3-chlorobenzyl	SCH₃	610 ± 40	52%	ND	1.5 ± 0.2	100 ± 4	ND

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^a)

All AR experiments were performed using adherent CHO cells stably transfected with cDNA encoding the human or rat ARs. Percent activation of the human A₃AR was determined at 10 µM. Binding at human A₁ and A_2AARs in this study was carried out as described in Methods using [³H]R-PIA or [³H]CGS 21680 as a radioligand. Values from the present study are expressed as mean ± s.e.m., n = 3–5.

^b)

Percent activity at 10 µM, relative to 10 µM Cl-IB-MECA (A₃).

^c)

Data from Ohno et al. 18

^d)

Data from Gao et al. 12, 17

^e)

Data from Lee et al. 23

^f)

Data from Tchilibon et al.24

^g)

Data from Jacobson et al.40

^h)

Data from de Zwart et al.54

ⁱ⁾

Data from Kim et al.14

ND not determined.

Cristalli and coworkers³⁷ and other investigators^18,23,38 have established that N⁶-methylation of adenosine analogues tends to increase the human A₃AR affinity and selectivity. In the (N)-methanocarba series, the N⁶-methyl-2-chloro derivative 14 was 3-fold more potent in A₃AR binding in the present study than reported previously.²³ The replacement of 2-chloro with a 2-amino group to provide 15 increased the human A₃AR affinity slightly. Compound 15 was 24-fold more potent in A₃AR binding than the 9-riboside equivalent 7.¹⁸

The 3-iodobenzyl group has long been known to enhance affinity and/or selectivity at the A₃AR.^13,14 In the (N)-methanocarba series, compound 16, reported previously,^12,23 was confirmed to be selective for the human A₃AR (300-fold in comparison to the human A₁AR). Addition of the 2-chloro group resulting in the previously reported agonist 5 having slightly enhanced A₃AR affinity, as was seen also for the pairs of compounds 1a and 1b and compounds 2a and 2b. Substitution at the 3-position with other halo atoms further increased human A₃AR affinity (unlike the ribose 5′-uronamide case, in which 3-iodo was optimal¹³). The optimal human A₃AR affinity in this series was achieved with the 3-chloro substitution in 18 with a K_i value of 0.29 nM. Compound 18 was selective for the human A₃AR in comparison to the human A₁ and A_2AAR by 890- and 7800-fold, respectively. The A₃AR selectivity of 18 in comparison to the A_2BAR was >10,000-fold. There was a trend toward an increase of human A₃AR affinity and a decrease of affinity at the other AR subtypes with decreasing size of the halo atom, except for the 3-fluoro analogue, which displayed a K_i value at the A₃AR of 2.4 nM. The affinity of the 3-halobenzyl analogues at the rat A₃AR was less variable than at the corresponding human receptor, and compounds 5 and 17 – 19 all displayed K_i values within the range of 0.8 – 1.6 nM. Dihalo substitution of the benzyl ring in the 2,5-dichloro analogue 20 and the 2-chloro-5-iodobenzyl analogue 21 maintained subnanomolar affinity at the human A₃AR. The 5-chloro substitution combined with an ether group at the 2-position (i.e. methyl ether 22, aminocarbonylmethyl ether 23, or benzyl ether 24) also maintained moderately high affinity at the A₃AR. The bulky benzyl ether 24 was 230-fold selective for the human A₃AR in comparison to the A₁AR. The 5-iodo equivalent of the 5-chloro-2-methoxy derivative 22, i.e. compound 25, was 3-fold more potent at the A₃AR upon halogen exchange. However, the affinity enhancement observed upon replacing m-chloro with m-iodo was not general. For example, with the mono-substituted benzyl derivatives compounds 5 and 18, the 3-chloro analogue 18 was 5-fold more potent than the corresponding 3-iodo 5. Replacement of this 5-chloro atom in compound 22 with methoxy, e.g., 26, did not change the A₃AR affinity; however, the A₃AR selectivity improved.

The N⁶-(2-methylbenzyl) group is present in metrifudil 11, a nonselective adenosine agonist that previously had been in clinical trials for its hypotensive effect.³⁹ In the 2-chloro-(N)-methanocarba series, this substitution in 27 provided high affinity at the A₃AR and selectivity (190-fold in comparison to the A₁AR). Placement of the methyl group at the 3-position in 28 enhanced the affinity at the A₃AR by 3-fold to give a K_i value of 0.63 nM. Inclusion of a 3-hydroxypropynyl group at the meta position of the benzyl ring in 29 produced with high affinity binding to both the human and rat A₃AR, but curiously when a 2-chloro substitution of the benzyl ring was included in 30 the affinity was significantly (76-fold) reduced.

The high affinity radioiodinated tracer used in this study for binding to the A₃AR, I-AB-MECA, contains an N⁶-(4-aminobenzyl) group.³² Compound 31a in the present series of 2-chloro-(N)-methanocarba derivatives contained the same substituent group, and displayed a K_i value at the A₃AR of 14 nM. The compound was A₃AR selective by 300-fold in comparison to the A₁AR. Following direct iodination of 31a to form 31b, the A₃AR affinity increased by 4-fold, however the selectivity was greatly reduced.

More diverse aromatic substitution at the 6-position was present in compounds 32– 34. N⁶-(3-pyridylmethyl)adenosine 12 displayed a relatively high affinity at the A₃AR.¹⁷ Unlike the various N⁶-benzyl analogues, the 3-pyridylmethyl substitution in the (N)-methanocarba analogue 32 resulted in a 2.5-fold reduction in affinity in comparison to the simple 9-riboside 12. The trans-2-phenyl-1-cyclopropyl analogue 33 (K_i 0.78 nM) was equipotent to the 9-riboside 10, while the 2,2-diphenylethyl analogue 34 (K_i 0.69 nM) was 6-fold more potent than the corresponding 9-riboside 13. In the case of 34, applying the (N)-methanocarba 5′-uronamide modification converted an A₃AR antagonist 13 into a full agonist.

High selectivity for the A₃AR was achieved for many of the (N)-methanocarba analogues. Selectivity for the A₃AR in comparison to the A₁AR was (fold): the N⁶-(2,2-diphenylethyl) analogue 34 (1900), the N⁶-(2,5-dimethoxybenzyl) analogue 26 (1200), the N⁶-(2,5-dichlorobenzyl) and N⁶-(2-phenyl-1-cyclopropyl) analogues 20 and 33 (1000), and the N⁶-(3-substituted benzyl) analogues 17, 18, 28, and 29 (700–900).

The N⁶-moiety providing the highest affinity at the human A₃AR was introduced into several analogues bearing substituents at the 2-position of the adenine ring. Although the 2-chloro group provided some enhancement of affinity over no substituent, the 2-iodo 35 and 2-methylthio 36 analogues were 12- and 5-fold less potent, respectively, than the corresponding 2-chloro analogue 18. Nevertheless, the selectivity for the human A₃AR in comparison to the human A₁AR remained high (590- and 390-fold, respectively).

Molecular Modeling

To determine an energetically favorable binding region and orientation of compound 18, the previously reported hA₃AR complex with agonist 2b was used as the starting geometry for the ribose binding position.¹² Different positions of the N⁶-substituent were examined with the ribose moiety anchored in the putative binding site, and the resulting energies of complex with different conformers were compared. The optimized docking mode of compound 18 is shown in Figure 1A. Residues that were within 5 Å proximity to the ligand in this putative binding site were the same residues for docked A₃AR agonists containing a ribose moiety.^{12, 62}

The complex of the hA₃AR with (A) 18, (B) 24, and (C) 29 in the putative binding site. The nucleosides are displayed as ball-and-stick models, and the side chains of hA₃AR are shown as stick models. The H-bonding between ligand and hA₃AR is displayed in yellow. The A₃AR is represented by a tube model with a different color for each TM domain (TM3 in yellow, TM4 in green, TM5 in cyan, TM6 in blue, TM7 in purple). Residues that interacted with ligand in the putative binding site were; T94 (3.36), N150 (EL2), Q167 (EL2), F168 (EL2), S181 (5.42), M177 (5.38), V178 (5.39), N250 (6.55), I268 (7.39), S271 (7.42), and H272 (7.43).

As shown in Figure 1, the purine ring of 18 was surrounded by a hydrophobic pocket defined by L91 (3.33) and L246 (6.51). In addition, certain orientations placed atoms within H-bonding distance, such as the exocyclic amine acting as donor and the side chain carbonyl group of N250 (6.55) at 2.7 Å, and between the purine N³ atom and the side chain of Q167 (EL2) at 2.8 Å. The Cl atom at the C-2 position was surrounded by hydrophobic residues, L91 (3.33), the methyl group of T87 (3.29) and the aromatic ring of F168 in EL2, and was also proximal to the hydrophilic residue Q167 (EL2). The 2′-OH group of the ribose moiety was within H-bonding distance from the backbone carbonyl group of I268 (7.39) at 2.8 Å and the side chain of H272 (7.43) at 2.5 Å. The 3′-OH group could form an H-bond with the backbone carbonyl group of S271 (7.42) at 2.7 Å. Thus, there were some difference in putative H-bonding of the hydroxyl groups in the (N)-methanocarba analogues compared with 9-ribosides such as 2b, which displayed a putative H-bond between the 3′-OH group and H272 (7.43).^12,14 Also, putative H-bonding was observed between the 5′-amide nitrogen and T94 (3.36) at 2.7 Å and between the 5′-carbonyl group and 3′-hydroxyl group at 2.3 Å. For binding at the N⁶-position, the benzyl moiety showed an additional hydrophobic interaction with F168 (EL2), and the chloro atom of the benzyl substituent was in proximity to M177 (5.38). This putative hydrophobic interaction was consistent with the high binding affinity of 28, which has a 3-methyl substituent on the benzyl ring. The receptor docking model suggested that 18 displayed more favorable van der Waals and electrostatic interactions than with the complexes of the N⁶-5-chloro-2-benzyloxybenzyl derivative 24 and the N⁶-3-(3-hydroxypropynyl)benzyl analogue 29.^63,64

Discussion

Activation of the A₃AR is increasingly associated with critical biological processes, such as its action on T-cells,⁴¹ dendritic cells,⁴² mast cells,^43–45 natural killer cells,⁴⁶ epithelial cells,⁴⁷ and in the brain.^48,49 This was accomplished by designing agonist nucleosides, with an awareness of the structural determinants of full agonism and of the preference for the (N)-ribose ring conformation in binding to the A₃AR. The goal was to prepare novel, fully efficacious A₃AR agonists having high affinity and selectivity. The (N)-methanocarba substitution of the ribose ring, resulting in rigidification of the pseudosugar moiety in a 2′-exo (N)-envelope conformation,²⁰ preserves or enhances affinity at the A₃AR to a greater degree than at other AR subtypes. However, the (N)-methanocarba substitution alone also appeared to reduce A₃AR efficacy; thus, 3 and 4 were partial agonists. Therefore, we have included in these analogues the 5′-uronamido modification, which restores the loss of intrinsic efficacy observed with the (N)-methanocarba ring system and with various adenine substitutions (see below),¹² without reducing the associated A₃AR selectivity. The N-methyl 5′-uronamide was used, since this corresponds to the optimal 5′-substitution observed for the ribose series.¹³

At the N⁶-position and 2-position of the adenine ring, we have incorporated groups known to be associated with high A₃AR potency, for example substituted N⁶-benzyl groups.¹³ These groups also tend to enhance A₃AR potency in both human and rat, unlike the N⁶-methyl group, which favors human but not rat A₃ARs.¹⁸ The N⁶-(3-iodobenzyl) group itself may lead to a reduction of efficacy, even in the absence of multiple substitutions of the adenine moiety. In the presence of an N⁶-substitution, such as 3-iodobenzyl or cyclopentyl, a 2-chloro group in adenosine analogues, in certain cases, leads to further reduced efficacy at the A₃AR.⁹ The N⁶-substituted 2-chloroadenosine analogues, however, were full agonists when combined with the 5′-uronamido modification in the present (N)-methanocarba series.

The meta-position of the N⁶-benzyl ring in the ribose series is known as a favorable site for substitution. In general, analogues that were substituted at both 5- and 2-positions in the present study were highly potent and selective A₃AR agonists. The comparable compounds with or without a 2-chloro group on the benzyl ring were generally similar in A₃AR affinity (cf. m-iodo analogues 5 and 21 and m-chloro analogues 18 and 20).

A study of novel binary drugs⁵⁰ that activate both A₁ and A₃ARs has concluded that a linear propynyl group favors A₃AR selectivity when placed at the 3-position of the N⁶-benzyl ring in the ribose series. We wished to test if this finding extended to the (N)-methanocarba series. Compound 29 was similar in A₃AR binding affinity (Ki 2.9 nM) to the other N⁶-benzyl substituted analogues. The loss of affinity upon 2-chloro substitution of the same N⁶-benzyl moiety in 30 (Ki 220 nM) may be explained in terms of restricted rotation of this ring due to the propynyl group, which might place the 2-chloro atom in an unfavorable position.

Several analogues were included that contain a substituted N⁶-(2-phenylethyl) moiety, known to produce high A₃AR affinity. However, as confirmed in the present structural series, such modifications favor human but not rat A₃ARs. The adenosine derivative (N⁶-(1S,2R)-(2-phenyl-1-cyclopropyl)adenosine), with a K_i value in binding to the hA₃AR of 0.63 nM (38-fold more potent than the 1R,2S isomer) and high agonist potency,^{17, 24} constitutes a lead for the development of N⁶ derivatives of adenosine with high hA₃AR affinity. We have utilized this modification in its trans, racemic form (as occurs in analogue 10 in Table 1), in the design of the highly hA₃AR selective agonist 33. We have not resolved the diastereomers of 33, but it is likely that the 1S,2R-form is the more potent isomer.²⁴ The N⁶-(2,2-diphenylethyl) analogue 34 was a full agonist and the most selective for the hA₃AR in the present series. This suggests that the loss of A₃AR efficacy seen in related nucleosides²³ can be overcome and that further derivatization of the N⁶-(2-phenylethyl) moiety might provide useful A₃AR agonists.

Other reported A₃AR agonists include the 3′-amino-3′-deoxy derivatives reported by DeNinno and coworkers.^51,52 We have examined whether N₆ groups found to favor A₃AR affinity in that structural series, such as the N⁶-[5-chloro-2-(aminocarbonylmethoyloxy)benzyl] group in 23, also led to high affinity within the present series. Indeed, 23 and the 2-benzyloxy ether 24 were highly potent at the human A₃AR. Thus, sterically bulky substitution at the 2-position of the benzyl group of the (N)-methanocarba nucleosides is tolerated at the A₃AR. Furthermore, we provided a molecular model that assigns a putative binding region for this moiety. The 3′-amino-3′-deoxy modification of the ribose reported previously in A₃AR ligands was not included in the present study. Other adenosine A₃AR agonists are based on 5′-methylthio, 5′-vinyl, and similar groups on the ribose moiety,^38,52 however, these groups were also not included in the present study, in favor of those incorporating the 5′-uronamide group.

In conclusion, many of the previously known substituent groups that enhance A₃AR affinity, while in some cases reducing intrinsic efficacy in the 9-riboside series, may be adapted to the (N)-methanocarba-5′-uronamide nucleoside series of full agonists. The affinity and selectivity of the agonists in the (N)-methanocarba-5′-uronamide series often exceeded those of the corresponding ribosides. According to rhodopsin-based modeling, the binding regions of both the N⁶-arylalkyl and the ribose (or pseudosugar) moieties were shown to be similar, but not identical, in the 9-riboside and (N)-methanocarba-5′-uronamide series. The putative orientation of the N⁶-benzyl group within the binding site depended on whether bulky substituent groups were present. An additional potential advantage of the (N)-methanocarba nucleosides may be avoiding metabolic transformations to which 9-ribosides would be subject,²⁰ which is a topic for future studies. It will now be useful to examine these agonists in other pharmacological and disease models^4,10,15 in order to determine the generality of the selectivity for the A₃AR and to further validate the therapeutic utility of these selective agonists.

Experimental Procedures

Chemical Synthesis

Materials and instrumentation

Compound 11 was purchased from Sigma (St. Louis, MO), and compounds 3 and 4 were prepared as reported.^{13, 14} Compounds 9 and 10 were prepared as reported.^{18, 24}

Reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO). ¹H NMR spectra were obtained with a Varian Gemini 300 spectrometer using CDCl₃, CD₃OD or DMSO-d₆ as solvents. The chemical shifts are expressed as ppm downfield from TMS. All melting points were determined with a Thomas-Hoover apparatus (A. H. Thomas Co.) and are uncorrected.

Purity of compounds was checked using a Hewlett–Packard 1100 HPLC equipped with a Luna 5µ RP-C18(2) analytical column (250 X 4.6 mm; Phenomenex, Torrance, CA). System A: linear gradient solvent system: H₂O/CH₃CN from 95/5 to 20/80 in 20 min; the flow rate was 1 mL/min. System B: linear gradient solvent system: 5 mM TBAP/CH₃CN from 80/20 to 20/80 in 20 min, then isocratic for 2 min; the flow rate was 1 mL/min. Peaks were detected by UV absorption with a diode array detector. All derivatives tested for biological activity showed >96% purity in the HPLC systems.

TLC analysis was carried out on aluminum sheets precoated with silica gel F₂₅₄ (0.2mm) from Aldrich. Low-resolution mass spectrometry was performed with a JEOL SX102 spectrometer with 6-kV Xe atoms following desorption from a glycerol matrix or on LC/MS 1100 Agilent, 1100 MSD, with Waters Atlantis Column C18. High resolution mass measurements were performed on a proteomics optimized Q-TOF-2 (Micromass-Waters) using external calibration using polyalanine. Observed mass accuracies are those expected based on known instruments performance as well as the trends in observed masses of standard compounds measured at intervals during the series of measurements. Reported masses are observed masses uncorrected for this time dependent drift in mass accuracy.

Bromomethyl-2-chloro-5-iodobenzene (60)

A mixture of 2-chloro-5-iodo-toluene (0.5 g, 1.98 mmol), N-bromosuccinimide (0.422 g, 2.35 mmol) and benzoyl peroxide (21.5 mg, 0.089 mmol) in dry CCl₄ (5 mL) was stirred and heated to reflux for 3 h. After cooling, the mixture was filtered, and the red filtrate was washed with a saturated solution of sodium thiosulfate (2 × 10 mL). The organic phase was washed with brine, dried over Na₂SO₄, filtered and concentrated. The residue was purified by flash chromatography on silica gel (petroleum ether), to give 60 as a white solid (361 mg, 55%). ¹H NMR (CDCl₃, 300 MHz) δ 7.76 (d, J= 2.4 Hz, 1H), 7.56 (dd, J= 8.7, 2.1 Hz, 1H), 7.12 (d, J= 8.4 Hz, 1H), 4.49 (s, 2H). MS (m/e) (positive FAB) 332.1 (M + H)⁺, m.p. 94–95 °C.

N-(2-Chloro-5-iodobenzyl) phthalimide (63)

Bromomethyl-2-chloro-5-iodo-benzene (60) (400 mg, 1.2 mmol) and potassium phthalimide (693 mg, 1.5 mmol) were stirred in dry DMF (20 mL) and heated to 80°C for 3 hours. After cooling, the suspension was filtered and concentrated in vacuo, and the residue was partitioned between water (30 mL) and Et₂O (30 mL). The aqueous phase was extracted with ether (20 mL × 3). The combined organic phases were dried over Na₂SO₄, filtered and concentrated to give 63 (455 mg, 95%). ¹H NMR (CDCl₃, 300 MHz) δ 7.92-7.86 ( (m, 2H), 7.78-7.75 (m, 2H), 7.54-7.50 (m, 2H), 7.10 (d, J= 8.4 Hz, 1H), 4.93 (s, 2H). MS (m/e) (API-ES) 397.9 (M)⁺, m.p. 134–136°C.

N-(2,5-Dichlorobenzyl phthalimide) (62)

Compound 62 was prepared by the same procedure as compound 63. ¹H NMR (CDCl₃, 300 MHz) δ 7.93−7.89 (m, 2H), 7.79-7.75 (m, 2H), 7.32 (d, J= 6 Hz, 1H), 7.21-7.16 (m, 2H), 4.96 (s, 2H). MS (m/e) (API-ES) 306.12 (M)⁺, m.p. 145–146°C.

2-Chloro-5-iodo-benzylamine hydrochloride (65)

Compound 63 (350mg, 0.88 mmol) was dissolved in dry EtOH (15 mL) and hydrazine (0.1 mL) was added. The stirred mixture was refluxed for 24 h, cooled and the EtOH evaporated. The residue was dissolved in Et₂O (3 mL) and treated with HCl / Et₂O. The precipitated hydrochloride salt was filtered and triturated with dry Et₂O (3 × 2mL), obtaining 200 mg of product (yield 75%). ¹H NMR (D₂O, 300 MHz) 7.88 (s, 1H), 7.80 (dd, J= 8.4, 1.8 Hz, 1H), 7.31 ( (d, J=7.8 Hz, 1H), 4.27 (s, 2H). MS (m/e) (positive FAB) 268.1 (M−Cl)⁺, m.p. 207–210°C.

2,5-Dichlorobenzylamine hydrochloride (64)

64 was prepared by the same procedure as compound 65. ¹H NMR (D₂O, 300 MHz) δ 7.59−7.47 (m, 3H), 4.33 (s, 2H). MS (m/e) (positive FAB) 177.1. (M−Cl)⁺, m.p. >220°C.

2-Methoxy-5-iodobenzaldehyde (67)

5-Iodo-salicylaldehyde (1.0 g, 4.0 mmol) was dissolved in DMF (10 mL) and to the stirred solution K₂CO₃ (0.828 g, 6.0 mmol) and CH₃I (1.14 g, 8.0 mmol) were added. The mixture was stirred at room temperature overnight. The suspension was concentrated in vacuo, and the residue was partitioned between water (30 mL) and Et₂O (30 mL). The aqueous phase was separated and extracted with ether (20 mL × 2). The combined organic phases were dried over Na₂SO₄, filtered and concentrated to give 67 (1.02 g, 98%). ¹H NMR (CDCl³, 300 MHz) δ 10.34 (s, 1H), 8.09 (s, 1H), 7.82-7.78 (m, 1H), 7.26 (s, 1H), 6.78 (d, J= 8.7 Hz, 1H), 3.91 (s, 3H). MS (m/e) (positive FAB) 262.1 (M+H)⁺, m.p. 140–142°C.

2-Methoxy-5-iodobenzylamine (68)

To a stirred solution of 67 (500 mg, 1.9 mmol) and ammonium acetate (1.5 g, 19.4 mmol) in dry methanol (6 mL) NaCNBH₃ (170 mg, 2.66 mmol) was added in one portion. The resulting mixture was stirred at room temperature for 48 h. Concentrated HCl was added until pH< 2. The methanol was evaporated and the resulting white residue was dissolved in water (10 mL) and washed with Et₂O (2 × 10 mL). The aqueous phase was then basified with aqueous KOH (45%), saturated with NaCl and extracted with CH₂Cl₂ (10 mL × 4). The combined organic phases were dried over Na₂SO₄, filtered and concentrated to give 68 as a yellow oil (200 mg, 40%). ¹H NMR (CDCl₃, 300 MHz) δ 7.55−7.45 (m, 2H), 6.46- 6.58 (m, 1H), 3.82-3.72 (m, 5H), 1.63 (br s, 2H). MS (m/e) (positive FAB) 264.1 (M+H)⁺, m.p. 95–97°C.

3-(3-Hydroxypropynyl)-benzylamine (72)

Cuprous iodide (1.06 mg, 0.0056 mmol) was added to a mixture of (PPh₃)₂PdCl₂ (7.84 mg, 0.011 mmol) and 3-iodobenzylamine (262 mg, 1.12 mmol) in dry diethylamine (7 mL) under a nitrogen atmosphere. Then a solution of propargyl alcohol (41.2 µL, 0.73 mmol) in dry diethylamine (3 mL) was added. The resulting solution was stirred at room temperature for 3 h. The solvent was concentrated in vacuo and the residue was partitioned between water (20 mL) and CHCl₃ (20 mL). The aqueous phase was separated and extracted with CHCl₃ (20 mL × 2). The combined organic phases were dried over Na₂SO₄, filtered and concentrated. The residue was purified by PTLC (chloroform/methanol 9:1) to give 72 (106 mg, 90% yield). ¹H NMR (CDCl₃, 300 MHz) δ 7.39−7.26 (m, 4H), 4.48 (s, 2H), 3.85 (s, 2H), 1.73 (br s, 2H). MS (m/e) (positive FAB) 162.1 (M+H)⁺.

2-Chloro-5-(3-hydroxypropynyl)-benzylamine (73)

Compound 73 was prepared by the same procedure as compound 72, using compound 65 as starting material. ¹H NMR (CDCl₃, 300 MHz) δ 7.48 (s, 1H), 7.28−7.25 (m, 3H), 4.48 (s, 2H), 3.92 (s, 2H), 1.92 (br s, 2H). MS (m/e) (positive FAB) 196.1 (M+H)⁺.

5-Chloro-2-(methoxybenzyl)-benzamide (75)

5-Chloro-2-hydroxy-benzamide (1.5 g, 8.7 mmol) was dissolved in DMF (120 mL) and to the stirred solution K₂CO₃ (1.38 g, 10 mmol) and benzylbromide (1.71 g, 10 mmol) were added. The mixture was stirred at room temperature overnight. The suspension was concentrated in vacuo, and the residue was partitioned between water (30 mL) and Et₂O (30 mL). The aqueous phase was separated and extracted with ether (20 mL × 2). The combined organic phases were dried over Na₂SO₄, filtered and concentrated. The residue was purified by flash chromatography on silica gel (petroleum ether/ ethyl acetate 90/10), to give 75 as a white solid (2.15 g, 95%). ¹H NMR (CDCl₃, 300 MHz) δ 8.20 (d, J= 3 Hz, 1H), 7.67 (br s, 1H), 7.44−7.38 (m, 6H), 7.99 (d, J= 9 Hz, 1H), 5.85 (br s, 1H), 5.17 (s, 2H). MS (m/e) (positive FAB) 262.1 (M+H)⁺. m.p. 110-112°C.

5-Chloro-2-(methoxybenzyl)-benzylamine hydrochloride (76)

To a suspension of LiAlH₄ (280 mg, 7.37 mmol) in dry tetrahydrofuran (20 mL) under nitrogen atmosphere, a solution of 75 (1.0 g, 3.83 mmol) in THF (10 mL) was added and the mixture was refluxed for 3 h. After cooling, the excess of LiAlH₄ was destroyed with a saturated solution of sodium sulfate. The mixture was filtered on MgSO₄ and the filtrate concentrated in vacuo. The residue was partitioned between water (30 mL) and CH₂Cl₂ (30 mL). The aqueous phase was separated and extracted with CH₂Cl₂ (20 mL × 2). The combined organic phases were dried over Na₂SO₄, filtered and concentrated. The residue was dissolved in Et₂O (5 mL) and treated with HCl / Et₂O. The precipitated hydrochloride salt was filtered and triturated with dry Et₂O (3 × 2mL), obtaining 760 mg of product (yield 70%). ¹H NMR (CD₃OD, 300 MHz) δ 7.50−7.33 (m, 7H), 7.15 (d, J= 9 Hz, 1H), 5.22 (s, 2H), 4.13 (s, 2H). MS (m/e) (API-ES) 248 (M-Cl)⁺. m.p 119-121°C.

5-Chloro-2-hydroxy-benzylamine (77)

To a suspension of LiAlH₄ (437 mg, 11.5 mmol) in dry tetrahydrofuran (30 mL) under nitrogen atmosphere, a solution of 5-chloro-2-hydroxybenzamide 74 (1.5 g, 5.7 mmol) in THF (10 mL) was added and the mixture was refluxed for 3 h. After cooling, the excess of LiAlH₄ was destroyed with a saturated solution of sodium sulfate. The mixture was filtered on MgSO₄ and the filtrate concentrated in vacuo. The residue was partitioned between water (30 mL) and CH₂Cl₂ (30 mL). The aqueous phase was separated and extracted with CH₂Cl₂ (20 mL × 2). The combined organic phases were dried over Na₂SO₄, filtered and concentrated. The obtained solid was recrystallized from ethanol obtaining 787 mg of pure product (yield 88%). ¹H NMR (CDCl₃, 300 MHz) δ 7.10 (dd, J= 6.6, 2.7 Hz, 1H), 6.94 (d, J= 2.4 Hz, 1H), 6.76 (d, J= 8.7 Hz, 1H), 4.10 (br s, 2H), 1.61 (br s, 2H). MS (m/e) (API-ES) 158 (M +H)⁺, m.p. 160-162°C.

(5-Chloro-2-hydroxy-benzyl)-carbamic acid tert-butyl ester (78)

Compound 77 (300 mg, 1.9 mmol) was dissolved in a dry solution at 10% of triethylamine in MeOH (12 mL) and a solution of di-t-butylcarbamate (1 M in THF, 3.8 mL, 3.8 mmol) was added. The solution was stirred at 45°C for 1 h and then concentrated in vacuo. The residue was purified by flash chromatography on silica gel (petroleum ether/ ethyl acetate 90/10), to give 78 as a white solid (350 mg, 80%). ¹H NMR (CDCl₃, 300 MHz) δ 9.02 (br s, 1H), 7.10 (dd, J= 8.4, 2.4 Hz, 1H), 6.94 (d, J= 2.4 Hz, 2H), 6.76 (d, J= 8.7 Hz, 1H), 5.23 (br s, 1H), 4.17 (d, J= 6.9 Hz, 2H), 1.44 (s, 9H). MS (m/e) (API-ES) 258 (M +H)⁺, m.p 124–126°C.

(2-Carbamoylmethoxy-5-chloro-benzyl)-carbamic acid tert-butyl ester (79)

5-Chloro-2-hydroxy-benzyl)-carbamic acid tert-butyl ester 78 (210 mg, 0.8 mmol) was dissolved in DMF (6 mL) and to the stirred solution K₂CO₃ (138 mg, 1 mmol) and 2-bromoacetamide (138 mg, 1 mmol) were added. The mixture was stirred at room temperature overnight. The suspension was concentrated in vacuo, and the residue was partitioned between water (20 mL) and CH₂Cl₂ (10 mL). The aqueous phase was separated and extracted with CH₂Cl₂ (20 mL × 2). The combined organic phases were dried over Na₂SO₄, filtered and concentrated. The residue was purified by flash chromatography on silica gel (petroleum ether/ ethyl acetate 80/20), to give 79 as a white solid (226 mg, 90%). ¹H NMR (CDCl₃, 300 MHz) δ 7.88 (br s, 1H), 7.27-7.18 (m, 2H), 6.76 (d, J= 8.7 Hz, 1H), 5.54 (br s, 1H), 4.78 ( br s, 1H), 4.47 (s, 1H), 4.36 (d, J= 6.3 Hz), 1.43 (s, 9H). MS (m/e) (API-ES) 337.0 (MNa)⁺. m.p 142–143°C.

5-Chloro-2-(aminocarbonyl-methyloxy)-benzylamine (80)

Compound 79 (200 mg, 0.63 mmol) was treated with a solution at 15% of TFA in CH₂Cl₂ at room temperature for 45 min. The solution was concentrated in vacuo and water (10 mL) was added. The aqueous phase was washed with Et₂O, basified with aqueous 2N NaOH, and extracted with CHCl₃ (10 mL × 3) obtaining 130 mg of a white solid (80), (yield 96%). ¹H NMR (CDCl₃, 300 MHz) δ 8.37 (br s, 1H), 7.29-7.22 (m, 2H), 6.82 (d, J= 9.3 Hz, 1H), 5.44 (br s, 1H), 4.60 (s, 2H), 3.89 (s, 2H). MS (m/e) (API-ES) 215 (M+H)⁺, m.p. 135–137°C.

General procedure for the synthesis of compounds 39–54 and 16–33

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Bromobenzylamino)-2-chloro-purin-9-yl]-2',3'-Oisopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (39)

3-Bromobenzylamine hydrochloride (122 mg, 0.55 mml) was added to a solution of 38 (50 mg, 0.12 mmol) and triethylamine (1 mL) in methanol (3 mL). The mixture was stirred at room temperature for 3 h. Then it was concentrated in vacuo to dryness and the residue was purified by PTLC (chloroform/methanol 15:1) to give 39 (55 mg, 82%). ¹H NMR (CDCl₃, 300 MHz) δ 7.70 (s, 1H), 7.52 (s, 1H), 7.31-7.18 (m, 2H), 6.18 (br s, 1H), 5.87 (d, J= 7.2 Hz, 1H), 4.85-4.71 (m, 4H), 4.32-4.16 (m, 2H), 2.24-2.19 (m, 1H), 1.75-1.70(m, 1H), 1.55-1.49 (m, 4H), 1.35(t, J = 7.8, 3H), 1.29 (s, 3H). MS (m/e) (positive FAB) 564.1 (M+H)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Fluorobenzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (40)

¹H NMR (CDCl₃, 300 MHz) δ 7.65 (s, 1H), 7.34-7.26 (m, 1H), 7.15-6.57 (m, 3H), 6.22 (br s, 1H), 5.87 (d, J= 8.4 Hz, 1H), 4.85 (br s, 3H), 4.72 (d, J= 7.2 Hz, 1H), 4.34-4.17 (m, 2H), 3.48 (d, J= 5.1 Hz, 1H), 2.24-2.18 (m, 1H), 1.75-1.69 (m, 1H), 1.55 (s, 3H), 1.34 (t, J = 7.8, 3H), 1.29 (s, 3H). MS (m/e) (ASI-ES) 502.1 (M+H)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Chlorobenzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (41)

¹H NMR (CDCl₃, 300 MHz) ¹H NMR (CDCl₃, 300 MHz) δ 1.20-1.42 (m, 3H), 1.44-1.83 (m, 8H), 2.20-2.26 (m, 1H), 4.05-4.38 (m, 2H), 4.82-4.94 (m, 3H), 5.38 (d, J = 4.5 Hz, 1H), 5.86 (d, J = 8 Hz, 1H), 6.15 (br s, 1H), 7.21-7.40 (m, 4H), 7.67 (s, 1H), MS (m/e) (positive-FAB) 518.1 (M+1)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2,5-Dichlorobenzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (42)

¹H NMR (CDCl₃, 300 MHz) δ 7.68 (s, 1H), 7.51 (s, 1H), 7.33 (d, J= 8.7 Hz, 1H), 7.23-7.19 (m, 1H), 6.28 (br s, 1H), 5.57 (d, J= 8.4 Hz, 1H), 4.85 (br s, 3H), 4.7 (d, J= 6 Hz, 1H), 4.32-4.16 (m, 2H), 2.21-2.19 (m, 1H), 1.75-1.70 (m, 1H), 1.55-1.50 (m, 4H), 1.35 (t, J = 7.8 Hz, 3H), 1.28 (s, 3H). MS (m/e) (positive FAB) 554.1 (M+H)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2-Chloro-5-iodo-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (43)

¹H NMR (CDCl₃, 300 MHz) δ 7.85 (s, 1H), 7.67 (s, 1H), 7.57-7.53 (dd, J= 8.1, 1.8 Hz, 1H), 7.12 (d, J= 8.4 Hz, 1H), 6.32 (br s, 1H), 5.58 (d, J= 8.4 Hz, 1H), 4.85 (br s, 3H), 4.73 (d, J= 6 Hz, 1H), 4.33-4.14 (m, 2H), 2.24-2.19 (m, 1H), 1.75-1.70 (m, 1H), 1.55-1.50 (m, 4H), 1.35 (t, J = 7.8 Hz, 3H), 1.29 (s, 3H). MS (m/e) (positive FAB) 644.1 (M)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(5-Chloro-2-mehoxybenzylamino)-2-chloro-purin-9-yl]-2',3'-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (44)

¹H NMR (CDCl₃, 300 MHz) δ 1.11-1.23 (m, 6H), 1.36-1.81 (m, 5H), 1.09-1.23 (m, 1H), 3.86 (s, 3H), 4.08-4.41 (m, 2H), 4.60-4.83 (m, 3 H), 5.30 (s, 1H), 5.85 (d, J = 4.5 Hz, 1H), 6.21-6.29 (m, 1H), 6.81 (d, J = 12 Hz, 1H), 7.21-7.42 (m, 3H), 7.67 (br s, 1H), MS (m/e) (positive-FAB) 548.1 (M+1)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(5-Chloro-2-(aminoycarbonylmethyloxy)-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (45)

¹H NMR (CDCl₃, 300 MHz) δ 7.66 (s, 1H), 7.36 (s, 1H), 7.30-7.27 (m, 1H), 7.07-7.05 (m, 1H), 6.81 (d, J= 8.7 Hz, 1H), 6.12 (br s, 1H), 5.85 (d, J= 8.4 Hz, 1H), 5.65 (br s, 1H), 5.01-4.85 (br s, 3H), 4.73 (d, J= 6 Hz, 1H), 4.50 (s, 1H), 4.34-4.21 (m, 2H), 2.23-2.18 (m, 1H), 1.74-1.69 (m, 1H), 1.55-1.50 (m, 4H), 1.34 (t, J = 7.8 Hz, 3H), 1.29 (s, 3H). MS (m/e) (API-ES) 591.1 (M+H)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(5-Chloro-2-benzyloxy-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (46)

¹H NMR (CDCl₃, 300 MHz) δ 7.62 (s, 1H), 7.39-7.30 (m, 6H), 7.18 (dd, J= 8.7, 2.7 Hz, 1H), 6.86 (d, J= 8.7 Hz, 1H), 6.33 (br s, 1H), 5.86 (d, J= 8.4 Hz, 1H), 5.81 (s, 2H), 4.85 (br s, 3H), 4.70 (d, J= 6 Hz, 1H), 4.32-4.19 (m, 2H), 2.23-2.18 (m, 1H), 1.74-1.69 (m, 1H), 1.55-1.50 (m, 4H), 1.34 (t, J = 7.8 Hz, 3H), 1.28 (s, 3H). MS (m/e) (positive FAB) 624.2 (M+H)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(5-Iodo-2-methoxy-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (47)

¹H NMR (CDCl₃, 300 MHz) δ 7.71-7.64 (m, 2H), 7.57-7.53 (m, 2H), 6.21 (br s, 1H), 5.57 (d, J= 8.4 Hz, 1H), 4.84 (s, 2H), 4.75-4.69 (m, 2H), 4.32-4.20 (m, 2H), 3.85 (s, 3H), 2.23-2.18 (m, 1H), 1.74-1.69 (m, 1H), 1.55-1.50 (m, 4H), 1.34 (t, J = 7.8 Hz, 3H), 1.28 (s, 3H). MS (m/e) (positive FAB) 640.1 (M+H)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2,5-Dimethoxy-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (48)

¹H NMR (CDCl₃, 300 MHz) δ 7.62 (s, 1H), 7.03 (s, 1H), 7.79 (s, 2H), 6.39 (br s, 1H), 5.57 (d, J= 8.4 Hz, 1H), 4.83-4.69 (m, 4H), 4.31-4.19 (m, 2H), 3.83 (s, 3H), 3.76 (s, 3H), 2.21-2.17 (m, 1H), 1.73-1.68 (m, 1H), 1.55-1.49 (m, 4H), 1.35 (t, J = 7.8 Hz, 3H), 1.28 (s, 3H). MS (m/e) (positive FAB) 544.2 (M+H)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2-Methyl-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (49)

¹H NMR (CDCl₃, 300 MHz) δ 7.58 (s, 1H), 7.32 (d, J= 6.9 Hz, 1H), 7.23-7.16 (m, 2H), 6.07 (br s, 1H), 5.57 (d, J= 8.4 Hz, 1H), 4.83-4.69 (m, 4H), 4.32-4.16 (m, 2H), 2.37 (s, 3H), 2.23-2.18 (m, 1H), 1.74-1.69 (m, 1H), 1.55-1.50 (m, 4H), 1.35 (t, J = 7.8 Hz, 3H), 1.28 (s, 3H). MS (m/e) (positive FAB) 498.3 (M)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Methyl-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (50)

¹H NMR (CDCl₃, 300 MHz) δ 7.56 (s, 1H), 7.26-7.09 (m, 4H), 6.27 (br s, 1H), 5.87 (d, J= 8.4 Hz, 1H), 4.83-4.71 (m, 4H), 4.30-4.20 (m, 2H), 2.33 (s, 3H), 2.22-2.17 (m, 1H), 1.74-1.69 (m, 1H), 1.55-1.50 (m, 4H), 1.34 (t, J = 7.8 Hz, 3H), 1.28 (s, 3H). MS (m/e) (positive FAB) 498.3 (M)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-(3-Hydroxypropynyl)-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (51)

¹H NMR (CDCl₃, 300 MHz) δ 7.56 (s, 1H), 7.38 (s, 1H), 7.34-7.26 (m, 2H), 6.73 (br s, 1H), 5.87 (d, J= 8.4 Hz, 1H), 4.82-4.70 (m, 4H), 4.46 (d, J= 6 Hz, 2H), 4.32-4.20 (m, 2H), 2.41 (br s, 1H), 2.23-2.18 (m, 1H), 1.75-1.70 (m, 1H), 1.55-1.50 (m, 4H), 1.35 (t, J = 7.8 Hz, 3H), 1.28 (s, 3H). MS (m/e) (positive FAB) 538.2 (M+1)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2-Chloro-5-(3-hydroxypropynyl)-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (52)

¹H NMR (CDCl₃, 300 MHz) δ 7.63 (s, 1H), 7.48 (s, 1H), 7.32-7.22 (m, 2H), 6.63 (br s, 1H), 5.87 (d, J= 8.4 Hz, 1H), 4.88-4.79 (m, 3H), 4.71 (d, J= 4.8 Hz, 1H), 4.45 (d, J= 6 Hz, 2H), 4.32-4.19 (m, 2H), 2.35 (br s, 1H), 2.23-2.18 (m, 1H), 1.75-1.68 (m, 2H), 1.55 (s, 3H), 1.35 (t, J = 7.8 Hz, 3H), 1.28 (s, 3H). MS (m/e) (positive FAB) 572.1 (M+1)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(4-Amino-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (53)

¹H NMR (CDCl₃, 300 MHz) δ 7.59 (s, 1H), 7.107 (d, J= 8.1 Hz, 2H), 6.65 (d, J= 8.1 Hz, 2H), 6.12 (br s, 1H), 5.87 (d, J= 8.4 Hz, 1H), 4.84 (s, 1H), 4.73-4.58 (m, 3H), 4.33-4.20 (m, 2H), 3.17 (br s, 2H), 2.26-2.18 (m, 1H), 1.75-1.69 (m, 1H), 1.56-1.50 (m, 4H), 1.35 (t, J = 7.8 Hz, 3H), 1.28 (s, 3H). MS (m/e) (positive FAB) 499.2 (M+1)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Pyridylmethylamino)-2-chloro-purin-9-yl]-2',3'-Oisopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (54)

¹H NMR (CDCl₃, 300 MHz) δ 8.66 (br s, 1H), 8.54 (br s, 1H), 7.73 (d, J= 6.6 Hz, 1H), 7.67 (s, 1H), 7.34-7.27 (m, 1H) 6.26 (br s, 1H), 5.88 (d, J= 8.4 Hz, 1H), 4.85 (br s, 3H), 4.72 (d, J= 4.8 Hz, 1H), 4.45 (d, J= 6 Hz, 2H), 4.33-4.20 (m, 2H), 2.24-2.18 (m, 1H), 1.75-1.70 (m, 2H), 1.56 (s, 3H), 1.35 (t, J = 7.8 Hz, 3H), 1.28 (s, 3H). MS (m/e) (positive FAB) 485.2 (M+1)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Bromobenzylamino)-2-chloro-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methyl amide (17)

The ester 39 (45 mg, 0.08 mmol) was dissolved in methanol (3 mL) and treated with an aqueous solution of methylamine (1 mL, 40%). This mixture was stirred at room temperature overnight, then the solvent was evaporated to dryness, and the white residue was purified by PTLC (chloroform/methanol 9:1) to give the uronamide 17 (19.6 mg, 40%). ¹H NMR (CDCl₃, 300 MHz) δ 7.68 (s, 1H), 7.51 (s, 1H), 7.31-7.19 (m, 2H), 6.83 (br s, 1H), 6.30 (br s, 1H), 5.67 (d, J= 6.9 Hz, 1H), 4.79-4.78 (m, 4H), 2.92 (d, J=4.5 Hz, 3H), 2.07-2.02 (m, 1H), 1.70-1.65 (m, 2H), 1.55 (s, 3H), 1.27 (s, 3H). MS (m/e) (positive FAB) 549.1 (M+1)⁺.

This intermediate (18 mg, 0.03 mmol) was treated with a solution of trifluoroacetic acid in MeOH (5 mL, 10%) and H₂O (0.5 mL) and the mixture was heated at 70°C for 3 h. The solution was cooled and the solvent removed to dryness by coevaporation with toluene in vacuo. The white residue was purified by PTLC (chloroform/methanol 9:1) to give the final product 17 (10 mg, 70%). ¹H NMR (CDCl₃, 300 MHz) δ 7.83 (s, 1H), 7.52-7.50 (m, 1H), 7.43-7.39 (m, 1H), 7.31-7.17 (m, 2H), 6.90 (br s, 1H), 6.63 (br s, 1H), 4.95-4.78 (m, 5H), 4.07 (d, J= 6Hz, 1H), 2.90 (d, J= 5.1 Hz, 3H), 2.24-2.19 (m, 1H), 1.72 (br s, 2H), 1.36-1.32 (m, 1H). HRMS (M+1)⁺: calculated 507.0547, found 507.0686. HPLC (System A) 20.4 min (99%) (System B), 12.9 min (99%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Chlorobenzylamino)-2-chloro-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (18)

¹H NMR (CDCl₃, 300 MHz) δ 1.17-1.39 (m, 1H), 1.45-1.61 (m, 1H), 2.01-2.09 (m, 1H), 2.81 (d, J=4.5 Hz, 1H), 3.91-4.08 (m, 2H), 4.78-5.05 (m, 5H), 6.71 (br s, 1H), 7.04 (br s, 1H), 7.11-7.29 (m, 4H), 7.74 (s, 1H), HRMS (M+1)⁺: calcd 463.0974, Found 463.1052. HPLC (System A) 14.8 min (99%) (System B), 12.5 min (99%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Fluorobenzylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (19)

7.84 (s, 1H), 7.34-7.26 (m, 1H), 7.15-6.95 (m, 3H), 6.86 (br s, 1H), 6.56 (br s, 1H), 4.88-4.80 (m, 4H), 4.08 (d, J= 6Hz, 1H), 2.91 (d, J= 5.1 Hz, 3H), 2.27-2.23 (m, 1H), 1.78 (br s, 2H), 1.36-1.32 (m, 1H). HRMS (M+1)⁺: calculated 447.1347, found 447.1378. HPLC (System A) 13.6 min (99%) (System B), 16.8 min (98%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2,5-Dichlorobenzylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methyl amide (20)

¹H NMR (CDCl₃, 300 MHz) δ 7.94 (s, 1H), 7.60 (s, 1H), 7.43 (d, J= 8.4 Hz, 1H), 7.34-7.30 (m, 1H), 7.04 (br s, 1H), 6.61 (br s, 1H), 5.12-4.92 (m, 1H), 431-4.22 (m, 1H), 3.03 (d, J= 5.1 Hz, 3H), 2.31-2.27 (m, 1H), 1.54-1.46 (m, 1H). HRMS (M+1)⁺: calculated 497.0662, found 497.0797. HPLC (System A) 17.2 min (98%) (System B), 13.8 min (97%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2-Chloro-5-iodo-benzylamino)-2-chloro-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (21)

¹H NMR (CDCl₃, 300 MHz) δ 7.83 (m, 2H), 7.55 (d, J= 8.4 Hz, 1H), 7.12 (d, J= 8.4 Hz, 1H), 6.86 (br s, 1H), 6.43 (br s, 1H), 4.96-4.80 (m, 3H), 4.13-4.08 (m, 2H), 2.91 (d, J= 5.1 Hz, 3H), 2.24-2.19 (m, 1H), 1.72 (br s, 2H), 1.36-1.32 (m, 1H). HRMS (M+1)⁺: calculated 589.0018, found 589.0250. HPLC (System A) 16.6 min (98%) (System B), 14.6 min (97%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(5-Chloro-2-methoxy-benzylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (22)

¹H NMR (CDCl₃, 300 MHz) δ 1.12-1.42 (m, 2H), 2.21-2.27 (m, 1H), 2.91 (d, J = 4.8 Hz, 3H), 3.86 (s, 3H), 4.01-4.22 (m, 2H), 4.65-4.83 (m, 3H), 4.95-5.01 (m, 1H), 6.42 (br s, 1H), 6.81 (d, J = 12Hz, 1H), 6.95 (br s, 1H), 7.21-7.43 (m, 3H), 7.81 (s, 1H). HRMS (M+1)⁺: calculated 493.1080, found 493.1158. HPLC (System A) 15.1 min (99%) (System B), 12.9 min (98%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(5-Chloro-2-(aminocarbonylmethyloxy)-benzylamino)-2-chloro-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (23)

¹H NMR (CD₃OD, 300 MHz) δ 8.04 (s, 1H), 7.38 (s, 1H), 7.25 (d, J=8.4 Hz, 1H), 6.94 (d, J= 8.4 Hz, 1H), 5.07 (d, J= 6.3 Hz, 1H) 4.59 (s, 2H), 4.99 (d, J= 6.3 Hz, 1H) 2.99-2.68 (m, 5H), 2.05-2.02 (m, 1H), 1.38-1.34 (m, 1H). HPLC (System A) 12.3 min (97%) (System B), 9.1 min (98%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(5-Chloro-2-benzyloxy-benzylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (24)

¹H NMR (CDCl₃, 300 MHz) δ 7.80 (s, 1H), 7.41-7.31 (m, 6H), 7.18 (dd, J= 8.7, 2.7 Hz, 1H), 6.90-6.84 (m, 2H), 6.57 (br s, 1H), 5.09 (s, 2H), 4.87-4.77 (m, 5H), 4.07 (d, J= 6Hz, 1H), 2.90 (d, J= 5.1 Hz, 3H), 2.54-2.21 (m, 1H), 1.92 (br s, 2H), 1.36-1.32 (m, 1H). HRMS (M+1)⁺: calculated 569.1471, found 569.1625. HPLC (System A) 17.2 min (98%) (System B), 16.7 min (98%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(5-Iodo-2-methoxy-benzylamino)-2-chloro-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (25)

¹H NMR (CDCl₃, 300 MHz) δ 7.79 (s, 1H), 7.67 (s, 1H), 7.54 (dd, J= 8.7, 2.7 Hz, 1H), 6.94 (br s, 1H), 6.65 (d, J= 8.7 Hz, 1H), 6.48 (br s, 1H),4.97-4.57 (m, 5H), 4.07 (m, 1H), 3.85 (s, 3H), 2.91 (d, J= 5.1 Hz, 3H), 2.24-2.19 (m, 1H), 1.94-1.85 (br s, 2H), 1.38-1.33 (m, 1H). HRMS (M+1)⁺: calculated 585.0514 found 585.0639. HPLC (System A) 18.7 min (98%) (System B), 13.9 min (99%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2,5-Dimethoxy-benzylamino)-2-chloro-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (26)

¹H NMR (CDCl₃, 300 MHz) δ 7.77 (s, 1H), 6.99-6.97 (m, 2H), 6.78-6.75 (m, 2H), 6.90 (br s, 1H), 6.63 (br s, 1H), 4.91 (d, J= 6.3 Hz, 1H), 4.77-4.74 (m, 3H), 4.05 (d, J= 6Hz, 1H), 3.82 (s, 3H), 3.75 (s, 3H), 2.90 (d, J= 5.1 Hz, 3H), 2.21-2.15 (m, 3H), 1.36-1.32 (m, 1H). HRMS (M+1)⁺: calculated 489.1653 found 489.1771. HPLC (System A) 17.5 min (98%) (System B), 11.3 min (98%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2-Methyl-benzylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (27)

¹H NMR (CDCl₃, 300 MHz) δ 7.76 (s, 1H), 7.31 (d, J= 6.9 Hz, 1H), 7.22-7.14 (m, 3H), 6.97 (br s, 1H), 6.20 (br s, 1H), 5.0 7 (br s, 1H), 4.79 (br s, 2H), 4.34 (br s, 1H), 4.11 (d, J= 6Hz, 1H), 3.14 (br s, 1H), 2.92 (d, J= 5.1 Hz, 3H), 2.37 (s, 3H), 2.19-2.14 (m, 3H), 1.97 (br s, 1H), 1.38-1.33 (m, 1H). HRMS (M+1)⁺: calculated 443.1598 found 443.1704. HPLC (System A) 14.3 min (99%) (System B), 12.1 min (99%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Methyl-benzylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (28)

¹H NMR (CDCl₃, 300 MHz) δ 7.79 (s, 1H), 7.23-7.09 (m, 4H), 6.93 (br s, 1H), 6.42 (br s, 1H), 4.95 (d, J= 6.3 Hz, 1H), 4.79 4.70 (m, 3H), 4.09 (d, J= 6Hz, 1H), 2.91 (d, J= 5.1 Hz, 3H), 2.33 (s, 3H), 2.22-2.17 (m, 1H), 1.97 (br s, 2H), 1.37-1.31 (m, 1H). HRMS (M+1)⁺: calculated 443.1598 found 443.1633. HPLC (System A) 14.5 min (99%) (System B), 12.0 min (99%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-(3-Hydroxypropynyl)-benzylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (29)

¹H NMR (CDCl₃, 300 MHz) δ 8.02 (s, 1H), 7.70 (s, 1H), 7.61 (br s, 1H), 7.33-7.22 (m, 2H), 5.62 (br s, 1H), 5.20 (br s, 1H), 5.11-5.03 (m, 1H), 4.94 (br s, 1H), 4.80 (s, 1H), 4.67 (d, J= 6Hz, 1H), 4.45-4.31 (m, 1H), 3.96 (br s, 1H), 2.97 (d, J= 5.1 Hz, 3H), 2.27-2.24 (m, 1H), 2.07 (br s, 1H), 1.36-1.32 (m, 1H). HRMS (M+1)⁺: calculated 483.1547 found 483.1596. HPLC (System A) 16.8 min (97%) (System B), 9.2 min (98%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2-Chloro-5-(3-hydroxypropynyl)-benzylamino)-2-chloro-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (30)

¹H NMR (CDCl₃, 300 MHz) δ 7.93 (s, 1H), 7.75 (s, 1H), 7.38-7.26 (m, 2H), 5.37-5.26 (m, 1H), 4.80-4.75 (m, 2H), 4.65-4.59 (m, 1H), 4.50-4.41 (m, 2H), 4.02 (br s, 1H), 3.72-3.70 (m, 1H), 3.10-3.07 (m, 1H), 2.99 (d, J= 5.1 Hz, 3H), 2.29-2.23 (m, 1H), 2.01 (br s, 1H), 1.36-1.32 (m, 1H). HRMS (M+1)⁺: calculated 517.1158 found 517.1278. HPLC (System A) 17.3 min (98%) (System B), 11.1 min (98%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(4-Amino-benzylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (31a)

¹H NMR (CD₃OD, 300 MHz) δ 8.02 (s, 1H), 7.15 (d. J= 8.1 Hz, 2H), 6.70 (d. J= 8.1 Hz, 2H), 5.09 (d, J= 6.6 Hz, 1H), 4.81 (s, 1H), 4.61 (s, 2H), 4.02 (d, J= 6.6 Hz, 1H), 2.88 (s, 3H), 2.07-2.05 (m, 1H), 1.40-1.35 (m, 1H). HRMS (M+1)⁺: calculated 444.1551 found 444.1688. HPLC (System A) 10.8 min (98%) (System B), 7.1 min (98%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Pyridylmethylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (32)

¹H NMR (CD₃OD, 300 MHz) δ 8.57 (s, 1H), 8.37 (s, 1H), 7.99 (s, 1H), 7.85 (d. J= 8.1 Hz, 1H), 7.38-7.34 (m, 1H), 5.05 (d, J= 6.6 Hz, 1H), 4.76 ( br s, 3H), 3.97 (d, J= 6.6 Hz, 1H), 2.26 (s, 3H), 2.04-1.99 (m, 1H), 1.35-1.295 (m, 2H). HRMS (M+1)⁺: calculated 430.1394 found 430.1467. HPLC (System A) 9.9 min (99%) (System B), 2.7 min (97%).

6-Chloro-2-methylthiopurin-9-ylmethyl 2,2-dimethylpropionate (82)

To a stirred solution of 2-amino-6-chloropurin-9-yl-methyl 2,2-dimethylpropionate 81 (0.566 g, 2 mmol) in acetonitrile (2 mL) was added methyl disulfide (0.94 g, 10 mmol), and tert-butyl nitrite (90%, 1.14 g, 10 mmol) and the resulting reaction mixture was stirred at room temperature for 8 h. The reaction mixture was concentrated in vacuo, and the resulting crude product was subjected to silica gel column chromatography (AcOEt/petroleum ether = 1/10), which furnished 82 (0.364 g, 55%): ¹H NMR (CDCl₃, 300 MHz) δ 8.19 (s, 1H), 6.12 (s, 2H), 2.65 (s, 3H), 1.17 (s, 9H). MS (m/e) (positive-FAB) 315 (M+1)⁺.

6-Chloro-2-methylthiopurine (83)

To a solution of 6-chloro-2-methylthiopurin-9-ylmethyl 2,2-dimethylpropionate (82) (0.314 g, 1 mmol) in i-PrOH (10 mL) and THF (25 mL) was added 2N aq NaOH (2 mL) and the resulting reaction mixture was stirred at room temperature for 12 hr. The reaction mixture was concentrated under reduced pressure and the residue obtained was purified by silica gel column chromatography (AcOEt/ petroleum ether = 3/7) which afforded 83 (0.112 g, 56%): ¹H NMR (DMSO-d₆, 300 MHz) δ 8.54 (s, 1H), 2.58 (s, 3H) MS (m/e) (positive-FAB) 200.9 (M+1)⁺.

(1'S,2'R,3'S,4'S,5'S)-4'-[6-Chloro-2-iodo-purin-9-yl]-2',3'-isopropylidenebicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (55)

To a solution of triphenylphosphine (0.262 g, 1 mmol) and 6-chloro-2-iodopurine (0.175 g, 0.63 mmol) in THF (3 mL) was added DIAD (0.202 g, 1 mmol), and the reaction mixture was stirred for 10 min. A solution of the alcohol 37 (0.121 g, 0.5 mmol) in THF was added to the reaction mixture, and the mixture was further stirred for 10 h. The solvent was removed in vacuo, and the residue obtained was purified by silica gel column chromatography (AcOEt/ petroleum ether = 4/6) which afforded 55 (0.136 g, 54%): ¹H NMR (CDCl₃, 300 MHz) δ 1.30-136 (m, 4H), 1.57 (s, 6H), 1.75-180 (m, 1H), 2.12-2.22 (m, 1H), 4.1-4.2 (m, 2H), 4.75 (d, J = 14 Hz, 1H), 4.92 (s, 1H), 5.85 (d, J = 15 Hz, 1H), 7.98 (s, 1H), MS (m/e) (positive-FAB) 505.0 (M+1)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-Chloro-2-methylthio-purin-9-yl]-2',3'-isopropylidenebicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (56)

To a solution of triphenylphosphine (0.262 g, 1 mmol) and 6-chloro-2-methylthiopurine 83 (0.125 g, 0.63 mmol) in THF (3 mL) was added DIAD (0.202 g, 1 mmol) and the reaction mixture was stirred for 10 min. After which time alcohol 37 (0.121 g, 0.5 mmol) in THF was added and the reaction mixture was further stirred for 10 hr. The solvent was removed in vacuo, and the residue obtained was purified by silica gel column chromatography (AcOEt/petroleum ether = 4/6) which afforded 56 (0.112 g, 53%): ¹H NMR (CDCl₃, 300 MHz) δ 1.25-1.55 (m, 4H), 1.56 (s, 6H), 1.75-1.82 (m, 1H), 2.25-2.32 (m, 1H), 2.64 (s, 3H), 4.18-4.29 (m, 2H), 4.82 (d, J= 12 Hz, 1H), 4.92 (s, 1H), 5.85 (d, J = 11Hz, 1H), 7.90 (s, 1H), MS (m/e) (positive-FAB) 425.10 (M+1)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-Chloro-2-amino-purin-9-yl]-2',3'-isopropylidenebicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (57)

To a solution of triphenylphosphine (0.262 g, 1 mmol) and 2-amino-6-chloropurine (0.169 g, 1 mmol) in THF (3 mL) was added DIAD (0.202 g, 1 mmol) and the reaction mixture was stirred for 10 min. After which time alcohol 37 (0.121 g, 0.5 mmol) in THF was added to the reaction mixture was further stirred for 10 h. The solvent was removed in vacuo, and the residue obtained was purified by silica gel column chromatography (AcOEt/ petroleum ether = 5/5) which afforded 57 (0.041 g, 21%): ¹H NMR (CDCl₃, 300 MHz) δ 1.20-1.55 (m, 6H), 1.52 (m, 4H), 1.75-1.85 (m, 1H), 2.12-2.24 (m, 1H), 4.11-4.24 (m, 2H), 4.67 (d, J = 11 Hz, 1H), 4.77 (s, 1H), 5.23 (br s, 2H), 5.88 (d, J = 12 Hz, 1H), 7.72 (s, 1H), MS (m/e) (positive-FAB) 394.2 (M+1)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Chlorobenzylamino)-2-iodo-purin-9-yl]-2',3'-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (58)

¹H NMR (CDCl₃, 300 MHz) δ 1.11-1.22 (m, 4H), 1.23-1.79 (m, 7H), 2.18-2.24 (m, 1H), 4.10-4.21 (m, 2H), 4.66-4.87 (m, 3H), 5.30 (s, 1H), 5.85 (d, J = 4.5 Hz, 1H), 6.05-6.16 (m, 1H), 7.21-7.26 (m, 4H), 7.58 (s, 1H), MS (m/e) (positive-FAB) 610.1 (M+1)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Chlorobenzylamino)-2-iodo-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (35)

¹H NMR (CDCl₃, 300 MHz) δ 1.15-1.22 (m, 1H), 1.65-1.70 (m, 1H), 2.08-2.13 (m, 1H), 2.94 (d, J = 4.5 Hz, 3H), 4.02-4.23 (m, 2H), 4.61-4.82 (m, 3H), 5.02-5.12 (m, 1H), 6.22-6.31 (m, 1H), 6.61-6.72 (m, 1H), 7.11-7.21 (m, 4H), 7.76 (s, 1H), MS (m/e) (positive-FAB) 515.2 (M+1)⁺. HPLC (System A) 15.7 min (98%) (System B), 13.5 min (99%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Chlorobenzylamino)-2-methylthio-purin-9-yl]-2',3'-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (59)

¹H NMR (CDCl₃, 300 MHz) δ 1.15-1.40 (m, 7H), 1.42-1.81 (m, 5H), 2.53 (s, 3H), 4.15-4.38 (m, 2H), 4.78-4.92 (m, 4H), 5.84 (d, J = 8.5 Hz), 6.02 (br s, 1H), 7.19-7.40 (m, 4H), 7.56 (s, 1H), MS (m/e) (positive-FAB) 555.0 (M+1)⁺.

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Chlorobenzylamino)-2-methylthio-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (36)

¹H NMR (CDCl₃, 300 MHz) δ 1.12–1.18 (m, 1H), 1.62-176 (m, 1H), 2.22-2.29 (m, 1H), 2.51 (s, 3H), 2.90 (d, J = 3.5 Hz, 3H), 4.16 (d, J = 4.5 Hz, 1H), 4.65-4.98 (m, 4H), 6.21-6.32 (m, 1H), 6.71-6.81 (m, 1H), 7.11-7.42 (m, 4H), 7.72 (s, 1H), MS (m/e) (positive-FAB) 475.1 (M+1). HPLC (System A) 15.4 min (98%) (System B), 13.1 min (99%).

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-Methylamino-2-amino-purin-9-yl]-2',3'-dihydroxybicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (15)

To a stirred solution of compound 57 (0.039, 0.1 mmol) in MeOH was added aq. CH₃NH₂ solution (0.5 mL, 40%) and the reaction mixture was stirred for 12 h. It was concentrated to dryness, and the residue was dissolved in the mixture containing 10% trifluoroacetic acid /MeOH (4 mL) and H₂O (0.5 mL) was heated at 70°C for 3 h. The solvent was removed, and the residue was dried by coevaporation with dry toluene. The residue was purified using preparative TLC (CHCl₃- MeOH, 80:20) to afford 15 (0.011 g, 36%): ¹H NMR (DMSOd₆, 300 MHz) δ 1.11-1.23 (m, 1H), 1.45-1.58 (m, 1H), 1.62-1.78 (m, 1H), 2.65 (d, J = 3.5 Hz, 3H), 2.91 (br s, 3H), 3.18 (d, J = 4 hz, 1H), 3.84-3.88 (m, 1H), 4.56 (s, 1H), 4.75 (d, J = 10 Hz, 1H), 5.08 (t, J = 4.5 Hz, 1H), 5.24 (d, J = 6.5 Hz), 5.82 (br s, 2H), 7.1 (br s, 1H), 7.55- 7.75 (m, 2H), MS (m/e) (positive-FAB) 334.1 (M+1). HPLC (System A) 6.6 min (98%) (System B), 5.0 min (98%).

Pharmacological Methods

[¹²⁵I]N⁶-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide (I-AB-MECA; 2000 Ci/mmol), [³H]R-PIA (R-N⁶-[phenylisopropyl]adenosine, 34 Ci/mmol), [³H]CGS21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine, 47 Ci/mmol) and [³H]cyclic AMP (40 Ci/mmol) were from Amersham Pharmacia Biotech (Buckinghamshire, UK).

Cell culture and membrane preparation

CHO (Chinese hamster ovary) cells expressing the recombinant human A₃AR²⁵ were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 µmol/ml glutamine and 800 µ g/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, cells 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. 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 3 Units/ml adenosine deaminase, and the suspension was stored at −80°C until the binding experiments. Striatal and forebrain tissues from Wistar rats were homogenized in ice-cold 50 mM Tris·HCl buffer, pH 7.4, using an electric homogenizer. The homogenate was centrifuged at 20,000 g for 10 min at 4°C, and the pellet was washed in fresh buffer. The final pellet was stored at −80°C until the binding experiments. The protein concentration was measured using the Bradford assay.⁵⁵

Binding assay using [¹²⁵I]4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide

Each tube in the competitive binding assay³⁶ contained 100 µl membrane suspension (20 µg protein), 50 µl [¹²⁵I]4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide (1.0 nM), and 50 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 µ M of 5′-N-ethylcarboxamidoadenosine 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.

Cyclic AMP accumulation assay

Intracellular cyclic AMP levels were measured with a competitive protein binding method.^56,57 CHO cells that expressed recombinant human and rat A₃ARs were harvested by trypsinization. After centrifugation and resuspension in medium, cells were plated in 24-well plates in 1.0 ml medium. After 24 hr, 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 agonists and/or test compounds in the presence of rolipram (10 µ M) and adenosine deaminase (3 units/mL). After 45 min forskolin (10 µ M) was added to the medium, and incubation was continued an additional 15 min. The reaction was terminated by removing the supernatant, and cells were lysed upon the addition of 200 µ L 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 µ L of the cell lysate, and 30 µ L 0.1 M HCl or 50 µ L of cyclic AMP solution (0–16 pmol/200 µ L 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.

Statistical analysis

Binding and functional parameters were calculated using Prism 5.0 software (GraphPAD, San Diego, CA, USA). IC₅₀ values obtained from competition curves were converted to K_i values using the Cheng-Prusoff equation.⁵⁸ Data were expressed as mean ± standard error.

Molecular modeling

All calculations were performed on a Silicon Graphics Octane workstation (300 MHz MIPS R12000 (IP30) processor). All ligand structures were modified from the lowest energy conformation of Cl-IB-MECA¹² using the “Sketch Molecule” of SYBYL 6.9.⁵⁹ In all cases, MMFF force field⁶⁰ and charge were applied using distance-dependent dielectric constants and conjugate gradient method until the terminal gradient reached 0.05 kcal/mol/Å.

A human A₃AR model (PDB code: 1o74) constructed by homology to the x-ray structure of bovine rhodopsin with 2.8 Å resolution²⁶ was used for the docking study. Compound 18, 24, and 29 in Table 2 were docked within the human A₃AR model. The atom types of all ligands were manually assigned with Amber all-atom force field,⁶¹ and their charges were calculated before docking. The starting geometry of ligand conformation was chosen from the human A₃AR complex model with Cl-IB-MECA, which was already validated by point-mutation.¹² The ribose binding position of this series was fixed, using an atom-by-atom fitting method for the carbon atoms of the ribose ring. To determine the binding region of N⁶-derivatives, the flexible bond of N⁶-C-C_ar-C_ar angle important for the interaction of benzyl substituent was variously searched in the putative binding cavity, rotating by 60° increments. The same values of C₅-C₆-N₆-C (234°) and C₆-N₆-C-C_ar (282°) angles from the Cl-IB-MECA complex were used, and the χ (O4′-C1′-N9-C8) angle was also fixed in an anti-conformation. Several starting conformations without any steric bump were selected for further optimization. The initial structures of all complexes were optimized using the Amber force field with fixed dielectric constant of 4.0 and terminating gradient of 0.1 kcal • mol⁻¹ • Å⁻¹.

Acknowledgements

We thank Dr. Victor Marquez (NCI, Frederick, MD) and Brian A. Harris (NIDDK) for helpful discussions and Dr. D. Eric Anderson and Dr. Victor Livengood (NIDDK) for mass spectral determinations. We are grateful to the Cystic Fibrosis Foundation for support provided to Susanna Tchilibon. B.V. Joshi thanks Gilead Sciences (Foster City, CA) for financial support.

References

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[R62] 62.Residues that were within 5 Å proximity to the ligand in this putative binding site were: L91 (3.33), T94 (3.36), H95 (3.37), N150 (EL2), Q167 (EL2), F168 (EL2), S181 (5.42), M177 (5.38), V178 (5.39), F182 (5.43), W243 (6.48), L246 (6.51), S247 (6.52), N250 (6.55), I268 (7.39), S271 (7.42), and H272 (7.43)

[R63] 63.Because of intramolecular H-bonding of the benzyloxy and exocyclic amino groups of the 5-chloro-2-benzyloxybenzyl derivative 24, there was no H-bonding at N⁶H with N250 (6.55). In addition, no H-bonding at the 3′-OH and 5′-NH groups was shown in its docking complex. Compared to 18, the binding of the N⁶-benzyl group in 24 was more shifted toward TM4, and the additional benzyl substituent at 2-position of its benzyl ring was directed towards upper region of TM6 and EL2, which accommodated the bulky substituent. The movement of the N⁶-benzyl binding region resulted in weak H-bonding interactions at the hydroxyl and 5′-uronamide groups of methanocarba ring, thus decreasing the binding affinity.

[R64] 64.The docking complex of the N⁶-3-(3-hydroxypropynyl)benzyl analogue 29 displayed additional H-bonding of the terminal hydroxyl group with the side chain of N150 in EL2. Thus, the extended hydroxypropynyl group penetrated through a hydrophobic region in proximity to the N⁶-benzyl ring, and its hydroxyl group entered a hydrophilic region.

PERMALINK

(N)-Methanocarba 2,N6-Disubstituted Adenine Nucleosides as Highly Potent and Selective A3 Adenosine Receptor Agonists

Susanna Tchilibon

Bhalchandra V Joshi

Soo-Kyung Kim

Heng T Duong

Zhan-Guo Gao

Kenneth A Jacobson

Abstract

Introduction

Chart 1.

RESULTS

Chemical Synthesis

Scheme 1.

Scheme 3.

Scheme 2.

Biological Activity

Table 1.

Molecular Modeling

Figure 1.

Discussion

Experimental Procedures

Chemical Synthesis

Materials and instrumentation

Bromomethyl-2-chloro-5-iodobenzene (60)

N-(2-Chloro-5-iodobenzyl) phthalimide (63)

N-(2,5-Dichlorobenzyl phthalimide) (62)

2-Chloro-5-iodo-benzylamine hydrochloride (65)

2,5-Dichlorobenzylamine hydrochloride (64)

2-Methoxy-5-iodobenzaldehyde (67)

2-Methoxy-5-iodobenzylamine (68)

3-(3-Hydroxypropynyl)-benzylamine (72)

2-Chloro-5-(3-hydroxypropynyl)-benzylamine (73)

5-Chloro-2-(methoxybenzyl)-benzamide (75)

5-Chloro-2-(methoxybenzyl)-benzylamine hydrochloride (76)

5-Chloro-2-hydroxy-benzylamine (77)

(5-Chloro-2-hydroxy-benzyl)-carbamic acid tert-butyl ester (78)

(2-Carbamoylmethoxy-5-chloro-benzyl)-carbamic acid tert-butyl ester (79)

5-Chloro-2-(aminocarbonyl-methyloxy)-benzylamine (80)

General procedure for the synthesis of compounds 39–54 and 16–33

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Bromobenzylamino)-2-chloro-purin-9-yl]-2',3'-Oisopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (39)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Fluorobenzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (40)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Chlorobenzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (41)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2,5-Dichlorobenzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (42)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2-Chloro-5-iodo-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (43)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(5-Chloro-2-mehoxybenzylamino)-2-chloro-purin-9-yl]-2',3'-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (44)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(5-Chloro-2-(aminoycarbonylmethyloxy)-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (45)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(5-Chloro-2-benzyloxy-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (46)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(5-Iodo-2-methoxy-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (47)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2,5-Dimethoxy-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (48)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2-Methyl-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (49)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Methyl-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (50)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-(3-Hydroxypropynyl)-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (51)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2-Chloro-5-(3-hydroxypropynyl)-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (52)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(4-Amino-benzylamino)-2-chloro-purin-9-yl]-2',3'-O-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (53)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Pyridylmethylamino)-2-chloro-purin-9-yl]-2',3'-Oisopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (54)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Bromobenzylamino)-2-chloro-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methyl amide (17)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Chlorobenzylamino)-2-chloro-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (18)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Fluorobenzylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (19)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2,5-Dichlorobenzylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methyl amide (20)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2-Chloro-5-iodo-benzylamino)-2-chloro-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (21)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(5-Chloro-2-methoxy-benzylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (22)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(5-Chloro-2-(aminocarbonylmethyloxy)-benzylamino)-2-chloro-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (23)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(5-Chloro-2-benzyloxy-benzylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (24)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(5-Iodo-2-methoxy-benzylamino)-2-chloro-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (25)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2,5-Dimethoxy-benzylamino)-2-chloro-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (26)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2-Methyl-benzylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (27)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Methyl-benzylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (28)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-(3-Hydroxypropynyl)-benzylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (29)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(2-Chloro-5-(3-hydroxypropynyl)-benzylamino)-2-chloro-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (30)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(4-Amino-benzylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (31a)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Pyridylmethylamino)-2-chloro-purin-9-yl]- 2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid methylamide (32)

6-Chloro-2-methylthiopurin-9-ylmethyl 2,2-dimethylpropionate (82)

6-Chloro-2-methylthiopurine (83)

(1'S,2'R,3'S,4'S,5'S)-4'-[6-Chloro-2-iodo-purin-9-yl]-2',3'-isopropylidenebicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (55)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-Chloro-2-methylthio-purin-9-yl]-2',3'-isopropylidenebicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (56)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-Chloro-2-amino-purin-9-yl]-2',3'-isopropylidenebicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (57)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Chlorobenzylamino)-2-iodo-purin-9-yl]-2',3'-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (58)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Chlorobenzylamino)-2-iodo-purin-9-yl]-2',3'-dihydroxy-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (35)

(1'S, 2'R, 3'S, 4'S, 5'S)-4'-[6-(3-Chlorobenzylamino)-2-methylthio-purin-9-yl]-2',3'-isopropylidene-bicyclo[3.1.0]hexane-1'-carboxylic acid ethyl ester (59)

(N)-Methanocarba 2,N⁶-Disubstituted Adenine Nucleosides as Highly Potent and Selective A3 Adenosine Receptor Agonists

Binding assay using [¹²⁵I]4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide