Structure-Activity Relationships of 2,N⁶,5′-Substituted Adenosine Derivatives with Potent Activity at the A_2B Adenosine Receptor

Abstract

2, N⁶, and/or 5′ substituted adenosine derivatives were synthesized via alkylation of 2-oxypurine nucleosides leading to 2-aralkylether derivatives. 2-(3-(Indolyl)ethyloxy)adenosine 17 was found to be a potent agonist of the human A_2BAR in both binding and cAMP assays. Simplification, altered connectivity and mimicking of the indole ring of 17 failed to maintain A_2BAR potency. Introduction of N⁶-ethyl or N⁶-guanidino substitution, shown to favor A_2BAR potency, failed to enhance potency in the 2-(3-(indolyl)ethyloxy)adenosine series. Indole 5″- or 6″-halo substitution was favored at the A_2BAR, but a 5′-N-ethylcarboxyamide did not further enhance potency. 2-(3″-(6″-Bromoindolyl)ethyloxy)adenosine 28 displayed an A_2BAR EC₅₀ value (nM) of 128, i.e. more potent than the parent 17 (299) and similar to 5′-N-ethylcarboxamidoadenosine (140). 28 was a full agonist at A_2B and A_2AARs and a low efficacy partial agonist at A₁ and A₃ARs. Thus, we have identified and optimized 2-(2-arylethyl)oxo moieties in AR agonists that enhance A_2BAR potency and selectivity.

INTRODUCTION

There are four subtypes of adenosine receptors (ARs): A₁, A_2A, A_2B, and A₃.¹ Three of these subtypes already possess selective and potent agonists and antagonists.² Only the A_2BAR remains without a selective agonist. It should be noted, however, that highly potent and selective antagonists have been reported for this subtype.^3–8 A_2BAR antagonists are directed toward clinical use for treating asthma and/or diabetes. Conversely, a selective A_2BAR agonist would provide a useful pharmacological probe for exploring the role of receptor activation. Activation of the A_2BAR is known to induce angiogenesis,⁹ reduce vascular permeabilization,¹⁰ increase production of the anti-inflammatory cytokine IL-10,¹¹ increase chloride secretion in epithelial cells,^12–14 and increase release of inflammatory mediators from human and canine mast cells.^15,16 A_2BAR agonists have been proposed for the treatment of septic shock¹⁷ and cystic fibrosis,¹⁸ and cardiac,¹⁹ pulmonary,²⁰ and kidney¹⁹ diseases associated with remodeling and hyperplasia. Thus, such an agonist may be useful in preventing restenosis. The A_2BAR mediates vasodilation in the corpus cavernosum of rabbit and agonists, therefore, may be useful in treating erectile dysfunction.²¹ Recently Yang et al., describe a proinflammatory phenotype resulting from deletion of the gene encoding the A_2BAR in the mouse, suggesting that activation of the A_2BAR can have anti-inflammatory effects.²² Activation of the A_2BAR also promotes postconditioning salvage of ischemic myocardium.²³

Modulation of A_2BAR potency has been achieved through structural changes at several sites on the adenosine molecule. R-PIA (R-N⁶-(phenylisopropyl)adenosine) 1 was one of the earliest potent agonists to be extensively investigated at ARs and was one of the nucleosides used initially to distinguish the low affinity A_2B and high affinity A_2AARs.²⁴ It was found to activate the A_2BAR with a micromolar potency and later was noted to bind to the A₃AR with nearly the same affinity as at the A_2AAR.

For several decades NECA (5′-N-ethylcarboxamidoadenosine) 2 was considered to be the most potent known agonist at the A_2BAR, with an EC₅₀ of 140 nM.^25–27 A survey of structurally diverse adenosine derivatives as agonists of the human A_2BAR failed to identify a lead that surpassed the potency of 2.²⁸ Cristalli and coworkers have explored the SAR (structure-activity relationship) of 2-substituted 5′-uronamide adenosine derivatives such as (S)-PHP-NECA 4 (EC₅₀ = 220 nM) as potent but relatively nonselective agonists of the A_2BAR.^29–31

Recent reports provided new insights into the SAR of agonists of the A_2BAR. A three-fold enhancement in potency at the A_2BAR was achieved by combining 2 with the N⁶-guanidino modification in 3.³² This structural change produced a three-fold gain in potency at the A₃AR, a 300-fold loss of potency at the A_2AAR, and no change at the A₁AR. Also, while 2-ether derivatives of adenosine were characterized as potent A_2AAR agonists, specific 2-(2-arylethyloxy) ethers were also noted to be particularly potent at the A_2BAR.²⁶ In the present study we characterized the SAR of potent A_2BAR agonists based on compound 17. In particular, 5″ or 6″-functionalization on the indole moiety of 17 led to the achievement of new AR agonists with enhanced potency at the A_2BAR reduced potency at other AR subtypes. Moreover, we have used molecular modeling of the human A_2BAR³¹ to propose a mode of docking of the potent 2-substituted derivatives.

RESULTS

Chemical Synthesis

The structures of the target adenosine 2-ether derivatives 5 – 40 appear in Chart 1 and Table 1. Compounds 5 – 7 and 9 – 16 were studied previously at the human A_2BAR.²⁶ Routes to the synthetic intermediates for the 2-ether component are shown in Schemes 1 and 2. The novel 2-alkoxyadenosines 8 and 17 – 36, N⁶-guanidino-2-(3-indolyl)ethyloxy)adenosine derivatives 37 and 38, N⁶-ethyl-2-(3-indolyl)ethyloxy)adenosine 39 and 5′-N-ethylcarboxamido-2-(3-indolyl)ethyloxyadenosines 40 were prepared via alkylation of 2-oxypurine nucleosides using arylalkyl/alkyl iodides as shown in Schemes 3 – 5.

Chart 1.

Chemical structures of selected adenosine derivatives 1 – 4 previously used as pharmacological reference compounds for characterization of the A_2BAR and novel adenosine derivatives 8, 17 – 40. The remaining adenosine derivatives in the series, 5 – 7 and 9 – 16 (structures in Table 1) were reported previously.²⁶

Table 1.

Potency of various adenosine derivatives in activation of the human A_2BAR expressed in CHO cells, with values expressed either the EC₅₀ (nM) or the percent stimulation at 1 μM (in parentheses). For comparison, binding affinities of the adenosine derivatives at human A₁, A_2A, and A₃ARs expressed in CHO cells (expressed as K_i value or percent displacement at 10 μM) and maximal agonist effects at 10 μM at the A₃AR. Values for compounds 5 – 7 and 9 – 16 are from ref. 25.

5 – 40, R2= H, R3 = CH2OH, unless noted
Compound	Name/Substitution	EC50 at A2BAR (nM) (or% activation)	Ki at A1AR (nM) or % inhib.a	Ki at A2AAR (nM) or % inhib.a	Ki at A3AR (nM) or % inhib.a	Efficacy, A3AR %
Reference Agonists
1	R-PIA	1680±500	2.0±0.3	884±188	8.7±0.9	102±6
2	NECA	140±19	6.8±2.4	2.2±0.6	16.0±5.4	100
3b	6-guanidino-NECA	54.5±13.3	7.0±1.0	628±39	5.1±1.3	100
4c	(S)-PHP-NECA	220	2.1	2.0	0.75
2-Ethers
5d	R1 = H	(−1%)	2640±540	360±139	568±205	99±4
6d		(36%)	(36%)	579±250	578±182	52±3
7d		3490±1490	221±57	9.3±2.9	54.2±14.3	71±3
8		(29%)	960±95	500±50	66 ± 3	42±8
9d		(3%)	2060 ± 630	519 ± 41	352 ± 66	37±8
10d		(13%)	1560 ± 250	413 ± 37	312 ± 47	18±8
11d		(12%)	331±22	58.1±24.9	77.8±13.5	45±5
12d		(64%)	312 ± 24	69.3 ± 4.7	119 ± 8	50±7
13d		(11%)	467±100	56.8±16.3	112±16	74±5
14d		1440±70	141±51	16.1±7.0	130±8	45±9
15d		1780±260	174±20	10.9±4.8	93.3±16.8	80±5
16d		(9%)	280±72	13.3±4.1	101±34	62±15
17b		299±45	148±19	45.0±11.6	232±54	17±3
18		(43%)	(39±6%)	2670±630	1340±230	0±3
19		2580	218±55	95±18	104±40	75±1
20		(49%)	197±47	373±71	513±84	37±8
21		(32%)	47±2%	2570±670	622±19	30±1
22		767	150±50	370±80	490±60	−1±1
23		2180	130±40	390±110	296±8	−4±1
24b		365±73	358±1	502±32	234±24	1±2
25		1870	350±60	900±200	110±20	−5±2
26		(30%)	(38±2%)	(54±8%)	8730±340	−1±5
27		216±59	145±6	29.3±13.7	92.3±7.9	2±5
28b		128±32	253±3	150±20	90±15	20±1
29		(16%)	443±82	39.7±14.4	260±19	−5±1
30		(43%)	210±19	252±109	142±17	−8±5
31		(48%)	579±88	(64±2%)	599±3	13±4
32		(24%)	20±2%	(26±4%)	(66±5%)	3±2
33		1250	1820±330	1400±300	360±50	−3±3
34		896	310 ± 90	450±8	120 ± 20	24±3
35		(0%)	(18±4%)	3870±497	2070 ± 700	3±2
36		(0%)	(41±5%)	(46±2%)	1920 ± 470	13±4
37	R2 = guanidino	(40%)	73.6 ± 8.0	277 ± 74	90 ± 10	58±8
38	R2 = guanidino	(42%)	52±2%	344 ± 72	457 ± 40	24±7
39	R2 = Et	3270	640±110	40±4%	30±10	54±12
40	R3 = CONHEt	989	190±20	250±30	110±20	102±2

^aAll experiments were performed using adherent CHO cells stably transfected with cDNA encoding a human AR. Percent activation of the human A_2B or A₃AR was determined at 10 μM. Binding at A₁, A_2A and A₃ARs was carried out as described in Experimental Procedures. The A₃ receptor activation results were from three separate experiments. The K_i and EC₅₀ values from the present study are expressed as mean±s.e.m., N = 3–5.

^bCompounds 3, MRS3218; 17, MRS3534; 24, MRS3854; 28, MRS3997.

^cData from references 29,31.

^dData from reference 26.

Scheme 1.

Reagents and conditions: (a) TsCl, NaH, THF, 0°C - room temperature; (b) NaI, DMF, 60°C; (c) (i) TsOH-H₂O, MeOH, 60°C (ii) LAH, THF, 4°C - room temperature; (d) EtOH, reflux; (e) oxalyl chloride, Et₂O; (f) LAH, THF, reflux; (g) I₂, PPh₃, imidazole, benzene.

Scheme 2.

Reagents and conditions: (a) i. TsCl, pyridine, CH₂Cl₂; ii. 3-butyn-1-ol, Pd(PPh₃)₂Cl₂, CuI, Et₃N, DMF, 70°C; (b) I₂, PPh₃, imidazole, Et₂O-MeCN, room temperature; (c) LAH, THF, 0°C - room temperature

Scheme 3.

Reagents and conditions: (a) R₁CH₂CH₂-I (47 – 49, 59 – 61, 74 – 77, 85, 86, 1-iodo-3-phenylpropane, 89, 94, 95 and 99, respectively), Cs₂CO₃, DMF, room temperature; (b) saturated NH₃ in EtOH, 120°C; (c) deprotection of tosyl group of 18, 119 – 121, 26, 32, 122 – 126, 21 and 127; KOH, MeOH 70 – 90°C.

Scheme 5.

Reagents and conditions: (a) 2,2-dimethoxypropane, p-TsOH-H₂O, DMF; (b) KMnO₄, KOH, H₂O; (c) PyBop, DIPEA, EtNH₂ HCl, DMF; (d) t-butyl nitrite, 2-PrOH/H₂O (1:1); (e) iodide 47, Cs₂CO₃, DMF, room temperature; (f) saturated NH₃, EtOH, 120°C; (g) 80% AcOH, 80°C; (h) KOH, MeOH, 70°C.

The various arylalkyl iodides used in this study, when not commercially available, were synthesized by the routes shown in Schemes 1 and 2. Tryptophol (3-(2-hydroxyethyl)indole) 41, 5-methoxytryptophol 42 and 5-hydroxy-tryptophol 43 were converted to the corresponding tosylates 44 – 46, respectively, followed by iodination with NaI to give the iodides 47 – 49. Indole-3-acetic acid analogues 50 – 52 having a functional group at the 2 and/or 5 position were transformed to the corresponding esters, which were reduced with lithium aluminum hydride to give the alcohols 53 – 55. Tosylation of the alcohols 53 – 55 followed by iodination with sodium iodide gave the corresponding iodides 59 – 61. Furthermore, other 5 or 6-substituted tryptophols 67 – 70 were prepared by refluxing the corresponding phenylhydrazine hydrochloride salts 63 – 66 and 2-ethoxytetrahydrofuran 62 to effect a Fischer indole ring cyclization.³⁴ Compounds 67 – 70 were converted to the corresponding iodides 74 – 77, respectively, by using the conventional method mentioned above. Compounds 78 and 79 were transformed to the ethyl glyoxylate derivatives 80 and 81, which were reduced with lithium aluminum hydride to give the corresponding tryptophol derivatives 82 and 83, respectively. Compounds 82 and 83 were converted to the corresponding iodides 85 and 86 by using conventional methods.

A tosylated 2-tryptophol 88 was prepared by the palladium-mediated heteroannulation of 2-iodoaniline with 3-butyn-1-ol (Scheme 2). The amino group of 2-iodoaniline 87 was activated by the strong electron-withdrawing sulfonyl group, and the indole cyclization occurred in one pot to give the tosylated 2-tryptophol 88³⁵. Compound 88 was converted to the corresponding iodide 89 with iodine, triphenylphosphine, and imidazole.

Reduction of benzoimidazol-1-yl-acetic acid 90 and benzotriazol-1-yl-acetic acid 91 with lithium aluminum hydride followed by iodination gave the corresponding iodides 94 and 95, respectively.

3-Pyrrolylacetic acid methyl ester 97 was synthesized from pyrrole by the known method³⁶ involving Friedel-Crafts acylation followed by a Willgerodt-Kindler reaction. Reduction of 97 with lithium aluminum hydride produced the alcohol 98, which was converted to the corresponding iodide 99 by using iodine, triphenylphosphine, and imidazole.

The synthesis of the 5′-CH₂OH analogues (Scheme 3) started from 9-(β-D-ribofuranosyl)-2-amino-6-chloropurine 100, which was converted to 9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-6-chloro-2-hydroxypurine 101 as reported.³³ Reaction of the hydroxyl group at the 2 position of 101 with various iodides 47 – 49, 59 – 61, 74 – 77, 85, 86, 1-iodo-3-phenylpropane, 89, 94 and 95, respectively, was carried out in the presence of cesium carbonate to give compounds 102 – 118. Simultaneous removal of the acetyl group and amination at the 6 position of 102 – 118 by using saturated ammonia in ethanol solution afforded compounds 18, 119 – 121, 26, 32, 125 – 126, 30, 8, 21, 35, 36, 127, respectively. Deprotection of the N-tosyl group of the indole or pyrrole ring of 18, 119–121, 26, 32, 122 – 126, 21 and 127 was conducted using potassium hydroxide in methanol to give compounds 17, 33, 34, 22, 24, 31, 23, 25, 27 – 29, 20 and 19, respectively.

Synthesis of N⁶-guanidino derivatives 37 and 38 began with compound 102 (Scheme 4). The guanidinolysis of 102 in the presence of 1,4-diazabicyclo[2.2.2]octane (DABCO) afforded 37 and the tosylate 38. Treatment of 102 with ethyl amine and N,N-diisopropylethylamine in DMF at 140 °C followed by removal of the tosyl group with potassium hydroxide gave the N⁶-ethyl derivative 39.

Scheme 4.

Reagents and conditions: (a) guanidine solution,³² DABCO, EtOH, 110°C; (b) i. EtNH₂ HCl, DIPEA, DMF, 140°C; ii. KOH, MeOH, 80°C.

Synthesis of the 5′-N-ethylcarboxamido derivative 40 began with 100 (Scheme 5). Protection of the 1,2-diol of 100 afforded the acetonide 128. Oxidation of 128 with potassium permanganate gave the corresponding carboxylic acid derivative 129, which was converted to the 5′-N-ethylcarboxamide derivative 130 using benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP). Diazotization of 130 with t-butyl nitrite followed by hydrolysis gave the 2-hydroxy derivative 131 in 47% yield. Coupling of 131 with the iodide 47 in the presence of cesium carbonate in DMF gave the 2-O-alkylated derivative 132, which was followed by displacement of the 6-chloro moiety with ammonia to give 133. Removal of the 2′,3′-O-isopropylidene group of 133 with 80% acetic acid aqueous solution afforded compound 134. Treatment of 134 with potassium hydroxide in methanol resulted in the desired 5′-N-ethyluronamide derivative 40.

Biological Evaluation

The AR binding affinities of novel compounds 8 and 17 – 40 were investigated in comparison to known (1 – 4, 5 – 7, and 9 – 16) nucleosides (Table 1). The SAR proceeded from known agonists with high potency at the A_2BAR, i.e. 1 – 4. A previous difficulty has been targeting the A_2BAR potency distinctly from the usually more potent activity at the A_2AAR. Compound 4 was reported as a potent A_2BAR agonist; nevertheless, it remained two orders of magnitude selective for the A_2AAR in comparison to the A_2BAR.³¹ Among 2 substituted derivatives, 2-ethers were more potent than the corresponding amines or thioethers.²⁶ For example, a 2-phenylethyl ether 7 was only two-fold less potent than R-PIA 1 at the A_2BAR. Nevertheless, the affinity in binding to the A_2AAR was nearly 400-fold greater than the A_2BAR functional potency. Therefore, great improvement was necessary in order to approach A_2BAR-selectivity.

Elongation of the spacer alkyl chain beyond ethyl weakened the affinity against all ARs as shown in 7 – 9. Compound 10 is a variation of 8 in which a methyl group is branched in the alkyl chain, and its affinity was reduced in comparison to 8.

The effect of fluoro substitution of the phenyl ring was also probed. The 2-F 11, 4-F 12, and 3-F 13 analogues were invariant in affinity at A₁, A_2A, and A₃ ARs, with K_i values at these subtypes ranging from 60 to 500 nM. These three analogues were also nearly inactive at the A_2BAR.

Three other aromatic moieties in 2-(2-arylethyloxy) ethers 14 – 16 were reported in a previous study.²⁶ 2-Naphthyl and 2-thienyl moieties were tolerated at the A_2BAR, while a 3-thienyl group resulted in inactivity at that subtype. Those modifications produced relatively minor changes at A₁, A_2A, and A₃ARs in comparison to the phenyl analogue 7.

Interestingly, the 3-indolyl analogue 17 (a tryptophol ether) was 12-fold more potent than 7 at the A_2BAR and 4 – 5 fold less potent than 7 in binding to the A_2A and A₃ARs. Also, 17 was only 2-fold less potent than 2 at the A_2BAR. This considerable enhancement was exploited in subsequent SAR exploration. The corresponding N-tosyl derivative 18, as for similar N-tosyl derivatives, was considerably less potent at all ARs.

The 3-pyrrolyl derivative 19 was the most simple analogue in this study, whose pyrrole ring moiety is a critical component of an indole ring. Compound 19 was tolerated at the A_2BAR, with a potency close to that of the 2-thienyl derivative 15, leading to 1.4-fold and 8-fold decreased potency at the A₁ and A_2AARs, respectively, and 2-fold similar potency at A₃AR.

On the other hand the corresponding 2-indolyl derivative 20 decreased markedly A_2BAR potency compared to 17. Thus, the 3 position was clearly the favored connection point and was utilized in subsequent synthesis. Its N-tosyl derivative 21 was less potent at all ARs, similar to results with 18.

It is known from SAR studies of A_2B agonists that substitution of the 4′-hydroxymethyl group of an adenosine analogue with a 5′-N-ethylcarboxamido group often yields compounds endowed with higher affinity than the parent compound.^31,37 Based on these findings we have prepared the 5′-N-ethyluronamido analogue 40 of 17. Unexpectedly, 40 was 3-fold less potent than the parent 5′-CH₂OH compound 17 at the A_2BAR and showed a 2-fold increased potency at only the A₃AR.

Cristalli and colleagues have established that the N⁶-ethyl analogue of (S)-PHP-adenosine was more potent than the N⁶-methyl and N⁶-isopropyl derivatives at the A_2B subtype.³¹ These findings were applied to 3-indolyl analogue 17, leading to the N⁶-ethyl analogue 39. Compound 39 was somewhat tolerated at A_2BAR and 7-fold more potent than the parent compound 17 at the A₃AR.

Another N⁶-functionalization for 17 was also investigated at ARs. Recently the N⁶-guanidino derivative 3 was reported to display a 3-fold potency enhancement over the parent 5′-N-ethyluronamide 2 at the A_2BAR.³² However, introduction of a N⁶-guanidino group to 17 led to derivative 37 and its tosyl derivative 38 with decreased A_2B potency compared to the parent compounds. Compound 37 showed a 2-fold increased potency at A₁ and A₃ ARs. Thus, the effect of N⁶-guanidinylation to enhance A_2BAR selectivity is not always compatible with other beneficial structures.

Benzoimidazole and benzotriazole analogues 35 and 36 are simple congeners of 17, in which indole ring was substituted with an imidazole or triazole ring, respectively. Compounds 35 and 36 showed disappointingly low potency at all AR subtypes.

The effect of substitution of the indole ring moiety was tested. A 2″-methyl-5″-methoxy indolyl derivative 31 showed reduced potency compare to 17, especially at the A_2BAR. Its N-tosyl derivative 32 lost potency at all ARs. A 5″-methoxy derivative 33 was tolerated at A_2BAR, but the affinities against A₁, A_2A, and A₃ARs were significantly reduced. 5″-Hydroxy analogue 34 was more potent than the 5″-methoxy analogue 33 at all ARs. Bulkiness of the substituent at the 5″ position might be related to the reduced affinity.

The effect on the 5″-halo-substitution of the indole moiety was also investigated. The corresponding 5″-halo analogues 22 – 25 were well tolerated by the A_2BAR. Of these analogues, the 5″-bromo analogue 24 was equipotent to 17 at the A_2BAR, but was 11-fold less potent than 17 in binding to the A_2AAR. Compound 24 was roughly equipotent at all four ARs, however its selectivity was improved compared to the parent compound 17. This series of 5″-halo derivatives showed a tendency toward increased K_i values at A₁ and A_2AARs depending on the bulkiness of halogen atom.

The importance of bromo-substitution of 26 for A_2BAR potency prompted us to design the positional isomers of bromo-substituted indole, leading to the 4″, 6″, or 7″-bromo derivatives 28 – 30. Of these bromo analogues, surprisingly, compound 28 surpassed the A_2BAR potency of the 5″-bromo analogue 24, the parent compound 17, and even 2. Also, 28 displayed improved selectivity compared to 17 and 2. On the other hand the 5″-chloro analogue 27 showed a decreased potency at A_2BAR compared to 28.

Activation curves were determined for 28 in comparison to 2 at the A₁, A_2A, A_2BARs and A₃AR (Figure 1). Compound 28 was a partial agonist at A₁ and A₃ARs and a full agonist at A_2A and A_2BARs.

Figure 1.

Functional effects of compound 28 on adenylate cyclase in CHO cells stably expressing the human ARs. Compound 28 was a full agonist at the A_2A and A_2BARs. In the curves shown the EC₅₀ values for 2 were 21.9 (A_2A) and 110 (A_2B) nM, and for 28 EC₅₀ values of were 39.7 (A_2A) and 109 (A_2B) nM were obtained. The relative maximal efficacy of 28 at the A₁ and A₃ARs was 31.8% and 20.2±1.0% of the full agonist 2, respectively.

Molecular Modeling

A recently published rhodopsin-based molecular model of the human A_2BAR³⁸ was adapted to study the binding mode of compound 28 after docking and energy optimization using Monte Carlo Multiple Minimum (MCMM) calculations.³⁹ The position of the adenosine moiety of 28 in the A_2BAR obtained after MCMM calculations was found to be similar to its initial positions. Furthermore, it was observed that the 2-(6-bromoindol-3-yl)-ethyloxy substituent fits the binding site well (Figure 2). In the resulting model, the oxygen atom of this moiety was found in proximity to the side chain amino group of Asn254 (6.55) and seemed to be involved in H-bonding with this residue. The indole ring occupied a pocket formed by several residues located in TM3 and EL2. In particular, the NH-group of the indole ring was found near the OH-group of Ser165 (EL2). Although, a H-bond between the NH-group of the indole ring and Ser165 was not observed in the model, a formation of this bond seems to be possible due to the rotation of the side chain of Ser165.

Figure 2.

Docking model of compound 28 in the binding site of the human A_2BAR, showing residues in proximity (A) and the Van der Waals surface of the receptor (B).

DISCUSSION

The goal was to prepare novel A_2BAR agonists having high potency and selectivity. Although the most potent agonist 28 is not truly selective for the A_2BAR, it is effectively a mixed A_2AAR/A_2BAR agonist with minimal ability to activate A₁ and A₃ARs.

Initially we found a novel lead compound 17, in which adenosine is substituted with a 3-indolylethyloxy functional group at the 2 position as an A_2BAR agonist having favorable pharmacological properties. The A_2BAR potency (299 nM) of compound was similar to 2 (140 nM). These promising findings encouraged us to optimize the A_2BAR activity and selectivity of 17 by derivatization at the indole 2 position and by modification at the ribose 5′ position or the purine 6 position. Generally, substitution of the 2 position of adenosine is not well tolerated by the A_2BAR, however, (S)-PHP-Ado 13 and (S)-PHP-NECA 4 were known to show higher A_2BAR potencies compared to 2.³¹ Distinct from the 2-ethynyl substituent of 4, exploration of the SAR of a 2-(3-indolylethyloxy) substituent could provide novel insights to molecular recognition at the A_2BAR. We can speculate that the lack of an additive effect on A_2B potency of combining the 3-(indolyl)ethyloxy- and 5′-N-ethyluronamido fragments (i.e., 40 in comparison to 2 and 17) may be due to an unfavorable change in the conformation or position of the ribose ring inside the ligand binding site.

First, we focused on the simplification, altered connectivity and mimicking of the indole ring of 17 as shown in the case of compounds 19, 8, 20, 35 and 36. Unfortunately, these approaches failed to maintain the A_2BAR potency. Next, we tried to transform the 4′-hydroxymethyl moiety to an ethylcarboxamide, which was expected to favorably increase A_2BAR potency. However, the 5′-N-ethyluronamide analogue 40 showed only a 2-fold increased potency at the A₃AR compared to 17. In this respect 40 has quite different pharmacological characteristics from (S)-PHPNECA 4. Also, the 6′ modification of 17, as shown in the case of compound 37 and 38, did not improve potency at the A_2BAR. Finally, we focused on functionalization of the indole moiety. Even minor modifications were examined because of the lack of a prior pharmacological precedent for the indole ring moiety at this position.

Eventually, through probing a relatively restrictive SAR, we achieved the new A_2B agonist 28 that gained an advantage over the parent compounds 17 and 2 for the A_2BAR in both potency and selectivity. In addition, compound 28 produced quite a different selectivity from (S)-PHPNECA 4 and 6-guanidino-NECA 3. Compound 4 showed a high potency/affinity at each AR (Table 1).³¹ Compound 3 displayed a 14 selectivity at A₁ and A₃ARs (A₁: 7.0 nM, A_2A: 628 nM, A_2B: 54.5 nM, A₃: 5.1 nM).³² Compound 28 showed the improved selectivity compared to compounds 2 – 4, providing a novel type of potent A_2BAR agonist. Molecular modeling results with 28 docked in the human A_2BAR demonstrated that in addition to all interactions proposed for adenosine, the 2-(6-bromoindol-3-yl)-ethyloxy fragment can provide additional favorable interactions of the ligand with a distal region of the putative agonist binding site of the receptor.

EXPERIMENTAL PROCEDURES

Chemical Synthesis

Materials and instrumentation

2-Amino-6-chloropurine-9-riboside, tryptophol, 1-iodo-3-phenylpropane, 5-bromoindole-3-acetic acid, 5-methoxyindole-3-acetic acid and other reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO), and 5-fluoroindole-3-acetic acid was purchased from Wako Chemicals USA, Inc. (Richmond, VA). Compound 69 was prepared as reported.³⁴

¹H NMR spectra were obtained with a Varian Gemini 300 spectrometer using CDCl₃, CD₃OD as solvents. Chemical shifts are expressed in δ values (ppm) with tetramethylsilane (δ0.00) for CDCl₃ and (δ3.30) for CD₃OD.

Purity of the nucleosides submitted for biological testing was checked using a Hewlett–Packard 1100 HPLC equipped with a Luna 5μ RP-C18(2) analytical column (250 X 4.6 mm; Agilent Technologies, Santa Clara, CA). System A: linear gradient solvent system: CH₃CN/H₂O from 20/80 to 40/60 in 20 min; the flow rate was 1 mL/min., System B: linear gradient solvent system: CH₃CN/H₂O from 20/80 to 60/40 in 20 min; the flow rate was 1 mL/min., System C: linear gradient solvent system: CH₃CN/5mM TBAP from 20/80 to 60/40 in 20 min., the flow rate was 1 mL/min., System D: linear gradient solvent system: CH₃CN/5mM TBAP from 5/95 to 80/20 in 20 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 >98% purity in the HPLC systems.

TLC analysis was carried out on aluminum sheets precoated with silica gel F₂₅₄ (0.2 mm) 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 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 using polyalanine. 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.

General tosylation procedure for the synthesis of 3-iodoethylindole derivative (44)~(46), (56)~(58), (71)~(73) and (84)

To a solution of the alcohol in THF (tetrahydrofuran) was added sodium hydride (60 %, 3 eq) at 0 °C, and the reaction mixture was stirred at 0 °C for 1 h. To the suspension was added tosyl chloride (3 eq) at 0 °C, the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate and washed with water, dried over MgSO₄ and filtered. The filtrate was evaporated to give a crude oil, which was subjected to column chromatography on silica gel. Elution with a mixture of toluene and acetone (40:1) gave the tosylated derivative.

General iodination procedure for the synthesis of compounds 47 – 49, 59 – 61, 74 – 77, and 85

A solution of the tosylate and sodium iodide (3.5 eq) in N, N-dimethylformamide was stirred overnight at 60 °C. The reaction mixture was diluted with ethyl acetate and washed with water, dried over MgSO₄ and filtered. The filtrate was evaporated to give a crude oil, which was subjected to column chromatography on silica gel. Elution with a mixture of hexanes and ethyl acetate (4:1) gave the iodide.

3-(p-Toluenesulfonyloxyethyl)-1-(p-toluenesulfonyl)indole (44)

The yield was 62%: ¹H NMR (CDCl₃) δ 7.93 (1H, d with small coupling, J = 8.0 Hz), 7.74 (2H, d, J = 8.2 Hz), 7.56 (2H, d, J = 8.5 Hz), 7.12 – 7.34 (7H, m), 4.24 (2H, t, J = 6.6 Hz), 3.01 (2H, t, J = 6.6 Hz), 2.38 (3H, s), 2.32 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₄H₂₃NO₅S₂Na (M+Na)⁺ 492.0915, found 492.0914.

5-Methoxy-3-(p-toluenesulfonyloxyethyl)-1-(p-toluenesulfonyl)indole (45)

The yield was 56 %. ¹H NMR (CDCl₃) δ 7.82 (1H, d, J = 9.1 Hz), 7.71 (2H, d with small coupling, J = 8.5 Hz), 7.53 (2H, d with small coupling, J = 8.2 Hz), 7.27 (1H, s), 7.21 (2H, dd, J = 0.6 and 8.8 Hz), 7.15 (2H, dd, J = 0.6 and 8.5 Hz), 6.89 (1H, dd, J = 2.5 and 9.1 Hz), 6.71 (1H, d, J = 2.2 Hz), 4.23 (2H, t, J = 6.6 Hz), 3.77 (3H, s), 2.97 (2H, dt, J = 0.8 and 6.6 Hz), 2.39 (3H, s), 2.32 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₅H₂₆NO₆S₂ 500.1202 (M+H)⁺, found 500.1207.

1-(p-Toluenesulfonyl)-3-(p-toluenesulfonyloxyethyl)-5-(p-toluenesulfonyloxy) indole (46)

The yield was 57%: ¹H NMR (CDCl₃) δ 7.80 (1H, d, J = 9.1 Hz), 7.71 (2H, d with small coupling, J = 8.5 Hz), 7.82 (2H, d with small coupling, J = 8.5 Hz), 7.58 (2H, d, with small coupling, J = 8.2 Hz), 7.36 (1H, s), 7.31 (2H, dd, J = 0.6 and 8.5 Hz), 7.25 (2H, d, J = 8.4 Hz), 7.19 (2H, d, J = 8.2 Hz), 6.97 (1H, d, J = 2.2 Hz), 6.84 (1H, dd, J = 2.3 and 8.9 Hz), 4.18 (2H, t, J = 6.5 Hz), 2.90 (2H, t, J = 6.5 Hz), 2.46 (3H, s), 2.40 (3H, s), 2.35 (3H, s); HRMS (ESI-MS m/z) calcd for C₃₁H₃₀NO₈S₃ (M+H)⁺ 640.1134 found 640.1099.

3-Iodoethyl-1-(p-toluenesufonyl)indole (47)

The yield was 70%: ¹H NMR (CDCl₃) δ 7.98 (1H, d with small coupling, J = 8.2 Hz), 7.76 (2H, dt, J = 1.9 and 8.5 Hz), 7.45 (2H, m), 7.32 (1H, ddd, J = 1.3, 7.1, and 8.4 Hz), 7.23 – 7.28 (2H, m), 7.20 (2H, d with small coupling, J = 8.0 Hz), 3.41 (2H, t with small coupling, J = 7.1 Hz), 3.24 (2H, t with small coupling, J = 7.3 Hz), 2.33 (3H, s); HRMS (ESI-MS m/z) calcd for C₁₇H₁₇NO₂SI (M+H)⁺ 426.0025, found 426.0016.

3-Iodoethyl-5-methoxy-1-(p-toluenesulfonyl)indole (48)

The yield was 74 %. ¹H NMR (CDCl₃) δ 7.74 (1H, d with small coupling, J = 9.1 Hz), 7.73 (2H, d with small coupling, J = 8.2 Hz), 7.40 (1H, s), 7.20 (2H, dd, J = 0.7 and 8.7 Hz), 6.92 (1H, dd, J = 2.5 and 9.2 Hz), 6.85 (1H, d, J = 2.5 Hz), 3.82 (3H, s), 3.40 (2H, dt, J = 0.7 and 7.6 Hz), 3.20 (2H, t, J = 7.3 Hz), 2.33 (3H, s); HRMS (ESI-MS m/z) calcd for C₁₈H₁₉NO₃SI (M+H)⁺ 456.0130, found 456.0135.

3-Iodoethyl-1-(p-toluenesulfonyl)-5-(p-toluenesulfonyloxy) indole (49)

The yield was 83%. ¹H NMR (CDCl₃) δ 7.85 (1H, d, J = 9.1 Hz), 7.73 (2H, d with small coupling, J = 8.5 Hz), 7.68 (2H, d with small coupling, J = 8.2 Hz), 7.47 (1H, s), 7.30 (2H, d, J = 8.3 Hz), 7.23 (2H, d, J = 8.3 Hz), 7.02 (1H, d, J = 2.5 Hz), 6.91 (1H, dd, J = 2.3 and 8.9 Hz), 3.26 (2H, t with small coupling, J =7.4 Hz), 3.12 (2H, t with small coupling, J = 7.1 Hz), 2.46 (3H, s), 2.36 (3H, s); APCI-MS (m/z) 596.0 (M+H)⁺.

General procedure for the synthesis of 3-hydroxyethylindole derivatives 53 – 55

Esterification and reduction: To a solution of a 2- and/or 5-substituted-indole-3-acetic acid in methanol was added p-toluenesulfonic acid monohydrate (3 eq), and the reaction mixture was stirred at 60 °C overnight. After neutralization with 1N aqueous NaOH the solvent was evaporated leaving an oily residue, which was dissolved in ethyl acetate. The solution was washed with water, dried over MgSO₄ and filtered. The filtrate was evaporated leaving an oily residue, which was subjected to column flush chromatography on silica gel. Elution with a mixture of toluene and acetone (5:1) gave the corresponding ester.

To a solution of the ester in THF was added lithium aluminum hydride (2.8 eq) at 0 °C, and the reaction mixture was stirred at 0 °C for 1 h and at room temperature for 1 h. After addition of ethyl acetate the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was diluted with ethyl acetate and washed with water, dried over MgSO₄ and filtered. The filtrate was evaporated leaving an oily residue, which was subjected to column chromatography on silica gel. Elution with a mixture of toluene and acetone (3:1) gave the pure alcohol.

5-Fluoro-tryptophol (53)

Compound 53 was identical to the known compound reported by Mewshaw et al.⁴⁰

5-Bromo-tryptophol (54)

Compound 54 was identical to the commercially available compound.

3-Hydroxyethyl-5-methoxy-2-methylindole (55)

The yield was 81%: ¹H NMR (CDCl₃) δ 7.27 (1H, br s), 7.16 (1H, d, J = 8.8 Hz), 6.97 (1H, d, J = 2.2 Hz), 6.78 (1H, dd, J = 2.5 and 8.5 Hz), 3.78 – 3.88 (2H, m overlapped with OCH₃), 3.85 (3H, s), 2.94 (2H, t, J = 6.5 Hz), 2.39 (3H, s); HRMS (ESI-MS m/z) calcd for C₁₂H₁₆NO₂ 206.1181, found 206.1190.

5-Fluolo-1-(p-toluenesulfonyl)-3-(p-toluenesulfonyloxyethyl)indole (56)

The yield was 58 %. ¹H NMR (CDCl₃) δ 7.87 (1H, dd, J = 4.1 and 9.1 Hz), 7.72 (2H, d with small couplings, J = 8.5 Hz), 7.55 (2H, d with small coupling, J = 8.2 Hz), 7.36 (1H, S), 7.24 (2H, d with small coupling, J = 8.0 Hz), 7.16 (2H, dd, J = 0.7 and 8.7 Hz), 7.00 (1H, dt, J = 2.4 and 9.0 Hz), 6.89 (1H, dd, J = 2.2 and 8.5 Hz), 4.22 (3H, t, J = 6.5 Hz), 2.95 (3H, t, J = 6.5 Hz), 2.39 (3H, s), 2.34 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₄H₂₃NO₅S₂F (M+H)⁺ 488.1002, found 488.0995.

5-Bromo-3-(p-toluenesulfonyloxyethyl)-1-(p-toluenesulfonyl)indole (57)

The yield was 63%: ¹H NMR (CDCl₃) δ 7.80 (1H, d with small coupling, J = 9.6 Hz), 7.73 (2H, d with small coupling, J = 8.2 Hz), 7.52 (2H, d with small coupling, J = 8.5 Hz), 7.32–7.39 (3H, m), 7.25 (2H, d, J = 8.0 Hz), 7.13 (2H, d, J = 8.0 Hz), 4.23 (2H, t, J = 6.3 Hz), 2.95 (2H, dt, J = 0.8 and 6.5 Hz), 2.39 (3H, s), 2.34 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₄H₂₂NO₅S₂BrLi (M+Li)⁺ 554.0283, found 554.0292.

5-Methoxy-2-methyl-3-(p-toluenesulfonyloxyethyl)-1-(p-toluenesulfonyl)indole (58)

The yield was 30%: ¹H NMR (CDCl₃) δ 8.02 (1H, d, J = 9.1 Hz), 7.58 (2H, d with small coupling, J = 8.2Hz), 7.48 (2H, d with small coupling, J = 8.2 Hz), 7.18 (2H, dd, J = 0.7 and 8.7 Hz), 7.13 (2H, dd, J = 0.6 and 8.5 Hz), 6.84 (1H, dd, J = 2.8 and 9.1 Hz), 6.64 (1H, d, J = 2.5 Hz), 4.11 (2H, t, J = 6.7 Hz), 3.79 (3H, s), 2.90 (2H, t, J = 6.7 Hz), 2.43 (3H, s), 2.39 (3H, s), 2.33 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₆H₂₇NO₆S₂Na (M+Na)⁺ 536.1178, found 536.1186.

3-Iodoethyl-5-fluoro-1-(p-toluenesulfonyl)indole (59)

Yield 70 %. ¹H NMR (CDCl₃) δ 7.92 (1H, ddd, J = 0.6, 4.3 and 9.1 Hz), 7.74 (2H, dt, J = 1.9 and 8.5 Hz), 7.48 (1H, s), 7.22 (2H, d, J = 8.0 Hz), 7.09 (1H, dd, J = 2.5 and 8.5 Hz), 7.04 (1H, dt, J = 2.5 and 9.1 Hz), 3.39 (2H, dt, J = 0.7 and 6.8 Hz), 3.19 (2H, t, J = 7.3 Hz), 2.35 (3H, s); HRMS (ESI-MS m/z) calcd for C₁₇H₁₅NO₂FS 316.0808, found 316.0810.

5-Bromo-3-Iodoethyl-1-(p-toluenesufonyl)indole (60)

The yield was 65 %. ¹H NMR (CDCl₃) δ 7.85 (1H, d, J = 8.8 Hz), 7.74 (2H, d with small coupling, J = 8.2 Hz), 7.57 (1H, d, J = 1.9 Hz), 7.45 (1H, s), 7.41 (1H, dd, J = 1.9 and 8.8 Hz), 7.22 (2H, d, J = 8.2 Hz), 3.88 (2H, t, J = 7.0 Hz), 3.19 (2H, t, J = 7.3 Hz), 2.35 (3H, s); HRMS (ESI-MS m/z) calcd for C₁₇H₁₅NO₂SBrI (M+H)⁺ 502.9025, found 502.9036.

3-Iodoethyl-5-methoxy-2-methyl-1-(p-toluenesufonyl)indole (61)

The yield was 85%: ¹H NMR (CDCl₃) δ 8.07 (1H, d, J = 9.1 Hz), 7.58 (2H, dt, J = 1.8 and 8.5 Hz), 7.17 (2H, d, J = 8.2 Hz), 6.87 (1H, dd, J = 2.5 and 9.1 Hz), 6.79 (1H, d, J = 2.5 Hz), 3.84 (3H, s), 3.26 (2H, m), 3.14 (2H, m), 2.52 (3H, s), 2.33 (3H, s); HRMS (ESIMS m/z) calcd for C₁₉H₂₁NO₃IS (M+H)⁺ 470.0287, found 470.0294.

General synthetic procedure for 3-hydroxyethylindole derivatives (67) – (70) via Fischer indole ring preparation

A solution of substituted phenylhydrazine hydrochloride and ethoxytetrahydrofuran (1.5 eq) in 95 % ethanol was refluxed overnight. The reaction mixture was filtered through celite. The filtrate was evaporated to give a crude solid. The solid was dissolved in ethyl acetate, the solution was washed with water, dried over MgSO₄, and filtered. The filtrate was evaporated to give a crude oil, which was subjected to column chromatography on silica gel. Elution with a mixture of toluene and acetone (2:1) gave the alcohol.

6-Chloro-tryptophol (67)⁴¹, 6-Bromo-tryptophol (68)⁴² and 5-Chloro-tryptophol (69)³⁴ are identical to the known compounds.

5-Iodo-tryptophol (70)

The yield was 32 %. ¹H NMR (CDCl₃) δ 8.07 (1H, br s), 7.95 (1H, m), 7.45 (1H, dd, J = 1.7 and 8.5 Hz), 7.16 (1H, d, J = 8.5 Hz), 7.06 (1H, d, J = 2.2 Hz), 3.89 (2H, br s), 2.98 (2H, dt, J = 0.8 and 6.3 Hz), 1.45 (1H, br s); APCI-MS (m/z) 288.0 (M+H)⁺.

6-Chloro-3-(p-toluenesulfonyloxyethyl)-1-(p-toluenesulfonyl)indole (71)

Yield 32%. ¹H NMR (CDCl₃) δ 7.94 (1H, d, J = 1.7 Hz), 7.74 (2H, d with small coupling, J = 8.5 Hz), 7.51 (2H, d with small coupling, J = 8.2 Hz), 7.29 (2H, d, J = 8.5 Hz), 7.25 (1H, s), 7.19 (1H, d, J = 8.2 Hz), 7.10–7.16 (3H, m), 4.27 (2H, t, J = 6.3 Hz), 2.97 (2H, t, J = 6.3 Hz), 2.40 (3H, s), 2.35 (3H, s); APCI-MS (m/z) 504.1 (M+H)⁺.

6-Bromo-3-(p-toluenesulfonyloxyethyl)-1-(p-toluenesulfonyl)indole (72)

The yield was 30 %. ¹H NMR (CDCl₃) δ 8.10 (1H, d, J = 1.4Hz), 7.74 (2H, d with small coupling, J = 8.2 Hz), 7.51 (2H, d with small coupling, J = 8.2 Hz), 7.23–7.30 (5H, m), 7.13 (2H, dd, J = 1.7 and 8.2 Hz), 4.23 (2H, t, J = 6.3 Hz), 2.97 (2H, t, J = 6.3 Hz), 2.40 (3H, s), 2.35 (3H, s); APCI-MS (m/z) found 548.0 (M+H)⁺.

5-Chloro-3-(p-toluenesulfonyloxyethyl)-1-(p-toluenesulfonyl)indole (73)

The yield was 30 %. ¹H NMR (CDCl₃) δ 7.85 (1H, dd, J = 0.6 and 8.8 Hz), 7.73 (2H, dt, J = 2.1 and 8.7 Hz), 7.53 (2H, dt, J = 1.8 and 8.5 Hz), 7.36 (1H, s), 7.21 – 7.26 (3H, m), 7.19 (1H, dd, J = 0.6 and 1.9 Hz), 7.14 (2H, dd, J = 0.5 and 8.5 Hz), 4.23 (2H, t, J = 6.5 Hz), 2.95 (2H, dt, J = 0.8 and 6.5 Hz), 2.39 (3H, s), 2.34 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₄H₂₂NO₅NaS₂Cl 526.0526, found 526.0535.

6-Chloro-3-iodoethyl-1-(p-toluenesulfonyl)indole (74)

The yield was 67 %. ¹H NMR (CDCl₃) d 8.00 (1H, d, J = 1.7 Hz), 7.76 (2H, d with small coupling, J = 8.5 Hz), 7.43 (1H, s), 7.36 (1H, d, J = 8.5 Hz), 7.25 ~ 7.28 (2H overlapped with CHCl₃), 7.22 (1H, dd, J = 1.8 and 8.4 Hz), 3.39 (2H, t with small coupling, J = 7.3 Hz), 3.21 (2H, t, J with small coupling, J = 7.3 Hz), APCI-MS (m/z) 460.0 (M+H)⁺.

6-Bromo-3-iodoethyl-1-(p-toluenesulfonyl)indole (75)

The yield was 67 %. ¹H NMR (CDCl₃) δ 8.16 (1H, d, J = 1.7 Hz), 7.76 (2H, dd, J = 1.9 and 8.5 Hz), 7.42 (1H, br s), 7.36 (1H, dd, J = 1.7 and 8.5 Hz), 7.30 (1H, d, J = 8.2 Hz), 7.25 (2H, d, J = 8.5 Hz), 3.38 (2H, t with small coupling, J = 7.4 Hz), 3.21 (2H, t with small coupling, J = 7.3 Hz), 2.36 (3H, s); HRMS (ESI-MS m/z) calcd for C₁₇H₁₅BrINO₂S (M)⁺ 502.9052, found 502.9066.

5-Chloro-3-iodoethyl-1-(p-toluenesulfonyl)indole (76)

The yield was 60 %. ¹H NMR (CDCl₃) δ 7.90 (1H, dd, J = 0.6 and 8.8 Hz), 7.74 (2H, d with small coupling, J = 8.2 Hz), 7.47 (1H, s), 7.41 (1H, dd, J = 0.6 and 1.9 Hz), 7.24–7.29 (1H overlaped with CHCl₃), 7.22 (2H, d, J = 8.3 Hz), 3.39 (2H, dt, J = 0.8 and 7.3 Hz), 3.20 (2H, t, J = 7.3 Hz), 2.35 (3H, s); HRMS (ESI-MS m/z) calcd for C₁₇H₁₆NO₂IClS (M+H)⁺ 459.9635, found 459.9625.

5-Iodo-3-iodoethyl-1-(p-toluenesulfonyl)indole (77)

The yield was 68 %. ¹H NMR (CDCl₃) δ 7.77 (1H, d, J = 1.6 Hz), 7.74 (1H, d, J = 8.8 Hz), 7.73 (2H, d with small coupling, J = 8.5 Hz), 7.58 (1H, dd, J = 1.7 and 8.5 Hz), 7.41 (1H, s), 7.22 (2H, d, J = 8.2 Hz), 3.83 (2H, t, J = 7.2 Hz), 3.19 (2H, t, J = 7.3 Hz), 2.35 (3H, s); APCI-MS (m/z) 551.9 (M+H)⁺.

Ethyl 4-bromo-3-indolylglyoxylate (80)

To a solution of 78 (2.94 g, 15.0 mmol) in diethyl ether (60 mL) was added oxalyl chloride (3.01 ml, 34.5 mmol) at 0 °C, and the reaction mixture was stirred at room temperature for 10 h. After evaporation, ethanol (30 mL) was added to the solids, and the solution was stirred at room temperarure overnight. The solvent was evaporated to give a solid. This residue was dissolved in ethyl acetate, washed with water, dried over MgSO₄, and filtered. The filtrate was evaporated to give a crude solid, which was subjected to column chromatography on silica gel. Elution with a mixture of hexanes and ethyl acetate (1:1) gave 80 (2.3g, 52 %).

¹H NMR (CDCl₃) δ 9.55 (1H, br s), 8.24 (1H, d, J = 3.3 Hz), 7.50 (1H, dd, J = 0.8 and 7.7 Hz), 7.42 (1H, dd, J = 0.8 and 8.3 Hz), 7.13 (1H, t, J = 8.0 Hz), 4.41 (2H, q, J = 7.1 Hz), 1.40 (3H, t, J = 7.1 Hz); APCI-MS (m/z) 296.0 (M+H)⁺.

Ethyl 7-Bromo-3-indolylglyoxylate (81)

Compound 81 was obtained from 79 by the similar procedure for the preparation of 80 (yield 70 %).

¹H NMR (CDCl₃) δ 8.96 (1H, br s), 8.55 (1H, d, J = 3.3 Hz), 8.39 (1H, dd, J = 0.6 and 8.0 Hz), 7.48 (1H, dd, J = 0.8 and 8.0 Hz), 7.23 (1H, t, J = 7.8 Hz), 4.43 (2H, q, J = 7.1 Hz), 1.44 (3H, t, J = 7.1 Hz), APCI-MS (m/z) 296.0 (M+H)⁺.

4-Bromo-tryptophol (82)

To a solution of 80 (20 mg, 0.0675 mmol) in THF (1.4 mL) was added lithium aluminum hydride (17.4 mg, 0.459 mmol), and the reaction mixture was refluxed for 2 h. The mixture was diluted with ethyl acetate and washed with water, dried over MgSO₄ and filtered. The filtrate was evaporated to give a crude oil, which was subjected to preparative TLC developed with a mixture of hexanes and ethyl acetate (1:1) to give 82 (10 mg, 63 % yield). ¹H NMR (CDCl₃) δ 8.12 (1H, br s), 7.32 (1H, dd, J = 0.8 and 7.7 Hz), 7.28 (1H, dd, J = 0.8 and 7.7 Hz), 7.14 (1H, d, J = 2.5 Hz), 7.02 (1H, t, J = 7.8 Hz), 3.97 (2H, q, J = 6.1 Hz), 3.29 (2H, dt, J = 0.6 and 6.5 Hz), 1.46 (1H, t, J = 5.6 Hz); HRMS (ESI-MS m/z) calcd for C₁₀H₁₁BrNO (M+H)⁺ 240.0024, found 240.0028.

7-Bromo-tryptophol (83)

Compound 83 was obtained from 81 by the similar procedure for the preparation of 82 (yield 52 %).

¹H NMR (CDCl₃) δ 8.82 (1H, br s), 7.57 (1H, d, J = 8.0 Hz), 7.36 (1H, d, J = 8.0 Hz), 7.16 (1H, d, J = 2.2 Hz), 7.02 (1H, t, J = 7.8 Hz), 3.91 (2H, q, J = 6.2 Hz), 3.02 (2H, dt, J = 0.5 and 6.3 Hz), 1.46 (1H, t, J = 6.0 Hz); HRMS (ESI-MS m/z) calcd for C₁₀H₁₁NOBr (M+H)⁺ 240.0024, found 240.0031.

4-Bromo-3-(p-toluenesulfonyloxyethyl)-1-(p-toluenesulfonyl)indole (84)

The yield was 41 %. ¹H NMR (CDCl₃) δ 7.91 (1H, dd, J = 0.8 and 8.2 Hz), 7.75 (2H, d with small coupling, J = 8.5 Hz), 7.55 (2H, d with small coupling, J = 8.2 Hz), 7.41 (1H, s), 7.28 (1H, dd, J = 1.0 and 7.8 Hz), 7.25 (2H, d, J = 8.2 Hz), 7.11 (2H, d, J = 8.0 Hz), 7.09 (1H, t, J = 8.0 Hz), 4.32 (2H, t, J = 6.5 Hz), 3.25 (2H, t, J = 6.3 Hz), 2.34 (6H, s); APCI-MS (m/z) 548.0 (M+H)⁺.

4-Bromo-3-iodoethyl-1-(p-toluenesulfonyl)-indole (85)

The yield was 67 %. ¹H NMR (CDCl₃) δ 7.96 (1H, dd, J = 0.8 and 8.2 Hz), 7.76 (2H, d with small coupling, J = 8.5 Hz), 7.52 (1H, s), 7.38 (1H, dd, J = 0.8 and 8.0 Hz), 7.23 (2H, dd, J = 0.6 and 8.5 Hz), 7.13 (1H, t, J = 8.1 Hz), 3.45 (4H, s), 2.35 (2H, s); HRMS (ESI-MS m/z) calcd for C₁₇H₁₆NO₂IBrS (M+H)⁺ 509.9130, found 509.9114.

7-Bromo-3-iodoethylindole (86)

The yield was 88 %. ¹H NMR (CDCl₃) δ 8.21 (1H, br s), 7.52 (1H, dd, J = 0.8 and 8.0 Hz), 7.36 (1H, d, J = 7.7 Hz), 7.16 (1H, d, J = 2.2 Hz), 7.02 (1H, t, J = 7.7 Hz), 3.39–3.46 (2H, m), 3.29–3.37 (2H, m); HRMS (ESI-MS m/z) calcd for C₁₀H₁₀NBrI (M+H)⁺ 349.9041, found 349.9036.

2-Hydroxyethyl-1-(p-toluenesulfonyl)-indole (88)

To a solution of N-tosyl-2-iodoanilide (1.172g. 3.14 mmol) in DMF were added 3-butyn-1-ol (1.42 ml, 18.8 mmol), copper iodide (119 mg, 0.628 mmol), triethyl amine (13.56 mL, 97.3 mmol) and Pd(PPh₃)₂Cl₂ (220.3 mg, 0.314 mmol), and the reaction mixture was stirred at 70 °C overnight. The reaction mixture was diluted with ethyl acetate. The solution was washed with water, dried over MgSO₄ and filtered. The filtrate was evaporated to give an oil, which was subjected to column chromatography on silica gel. Elution with a mixture of hexanes and ethyl acetate (1:1) gave 88 (820 mg, 83%). ¹H NMR (CDCl₃) δ 8.16 (1H, d, J = 8.2 Hz), 7.61 (2H, d with small coupling, J = 8.2 Hz), 7.42 (1H, dd, J = 1.7 and 7.4 Hz), 7.28 (1H, dt, J = 2.2 and 7.6 Hz), 7.23 (1H overlapped with Ph), 7.18 (2H, d, J = 8.5 Hz), 6.50 (1H, s), 4.01 (2H, t, J = 6.1 Hz), 3.29 (2H, t, J = 6.2 Hz), 2.33 (3H, s); APCI-MS (m/z) 316.0 (M+H)⁺.

2-Iodoethyl-1-(p-toluenesulfonyl)-indole (89)

To a solution of 88 (638 mg, 2.02 mmol) in a mixture of diethylether (24 mL) and acetnitrile (8 mL) were added triphenylphosphine (1.589 g, 6.06 mmol), imidazole (439 mg, 6.46 mmol) and iodine (1.639 g, 6.46 mmol), the reaction mixture was stirred at 0 °C for 1 h. The reaction mixture was diluted with ethyl acetate. The solution was washed with water, dried over MgSO₄ and filtered. The filtrate was evaporated to give an oil, which was subjected to column chromatography on silica gel. Elution with a mixture of hexanes and ethyl acetate (4:1) gave 89 (847 mg, 98 %).

¹H NMR (CDCl₃) δ 8.14 (1H, d with small coupling, J = 8.2 Hz), 7.60 (2H, dt, J = 2.0 and 8.6 Hz), 7.45 (1H, dd, J = 1.0 and 6.7 Hz), 7.30 (1H, dt, J = 1.5 and 8.0 Hz), 7.23 (1H, dd, J = 1.2 and 7.6 Hz), 7.18 (2H, d with small coupling, J = 8.0 Hz), 6.50 (1H, s), 3.46 – 3.60 (4H, m), 2.33 (3H, s); HRMS (ESI-MS m/z) calcd for C₁₇H₁₇NO₂IS (M+H)⁺ 426,0025 found 426.0035.

Benzoimidazol-1-yl-ethanol (92)

To a solution of benzoimidazol-1-yl-acetic acid (893 mg, 5.06 mmol) in THF (20 mL) was added lithium aluminum hydride (672 mg, 17.7 mmol) at 0 °C, and the reaction mixture was stirred at room temperature for 5 h. After dilution with ethyl acetate the solution was washed with water, dried over MgSO₄ and filtered. The filtrate was evaporated to give a crude oil, which was subjected to column chromatography on silica gel. Elution was with a mixture of chloroform and methanol (8:1) to give 92 (620 mg, 76 %).

¹H NMR (CDCl₃) δ 7.63 (1H, s), 7.38 (1H, dt, J = 1.0 and 8.2 Hz), 7.31 (1H, dt, J = 1.0 and 8.0 Hz), 7.17 (1H, ddd, J = 1.0, 7.1, and 8.1 Hz), 7.06 (1H, ddd, J = 1.1, 7.1, and 8.1 Hz), 4.22 (2H, t, J = 4.9 Hz), 3.99 (2H, t, J = 4.8 Hz); HRMS (ESI-MS m/z) calcd for C₉H₁₁N₂O (M+H)⁺ 163.0871, found 163.0880.

Benzotriazol-1-yl-ethanol (93)

Procedure used for the preparation of 93 from 91 was similar to those used for the preparation of 92 from 90; amorphous solid, the yield was 59 %.

¹H NMR (CDCl₃) δ (1H, d with small coupling, J = 8.5 Hz), 7.61 (1H, d with small coupling, J = 8.2 Hz), 7.50 (1H, ddd, J = 1.1, 7.0 and 8.1 Hz), 7.36 (1H, ddd, J = 1.2, 6.9 and 8.1 Hz), 4.75 (2H, t, J = 5.1 Hz), 4.25 (2H, dd, J = 5.9 and 10.3 Hz), 2.55 (1H, t, J = 6.0 Hz); HRMS (ESI-MS m/z) calcd for C₈H₁₀N₃O 164.0824 found 164.0812.

Benzoimidazol-1-yl-ethyliodide (94)

To a solution of 92 (22 mg, 0.134 mmol) in a mixture of acetonitrile (0.3 mL) and diethylether (0.9 mL) were added triphenylphosphine (105 mg, 0.402 mmol), imidazole (29 mg, 0.428 mmol), and iodine (108 mg, 0.428 mmol) at 0 °C, the reaction mixture was stirred for 2 h. The mixture was diluted with ethyl acetate, washed with water, dried over MgSO₄ and filtered. The filtrate was evaporated to give a crude oil, which was subjected to preparative TLC developed with a mixture of toluene and acetone (2:1) to give 94 (32.3 mg, 88 %).

¹H NMR (CDCl₃) δ 7.97 (1H, s), 7.80–7.87 (1H, m), 7.28–7.42 (3H, m), 4.58 (2H, t, J = 7.0 Hz), 3.51 (2H, t, J = 7.1 Hz); APCI-MS (m/z) 273.0 (M+H)⁺.

Benzotriazol-1-yl-ethyliodide (95)

Procedure used for the preparation of 95 from 93 was similar to those used for the preparation of 94 from 92; amorphous solid, the yield was 88 %. ¹H NMR (CDCl₃) δ 8.09 (1H, dt, J = 1.0 and 8.3 Hz), 7.50 – 7.60 (2H, m), 7.40 (1H, ddd, J = 1.8, 6.3, and 8.1 Hz), 5.02 (2H, t, J = 7.3 Hz), 3.67 (2H, t, J = 7.3 Hz); HRMS (ESI-MS m/z) calcd for C₈H₉N₃I (M+H)⁺ 273.9841, found 273.9833.

3-Hydroxyethyl-1-(p-toluenesulfonyl)pyrrole (98)

To a solution of 97 (1.24 g, 4.21 mmol) in THF (20 mL) was added lithium aluminum hydride (340 mg, 6.32 mmol) at 0 °C. The reaction mixture was stirred for 2 h. After addition of ethyl acetate the mixture was stirred for 30 min. The mixture was diluted with ethyl acetate, washed with water, dried over MgSO₄ and filtered. The filtrate was evaporated to give a crude oil, which was subjected to column chromatography on silica gel. Elution with a mixture of chloroform and methanol (20:1) gave 98 (692 mg, 62 %).

¹H NMR δ 7.74 (2H, d, J = 8.4 Hz), 7.28 (2H, d, J = 8.7 Hz), 7.10 (1H, t, J = 2.7 Hz), 6.99 (1H, m), 6.19 (1H, dd, J = 1.5 and 3.3 Hz), 3.74 (2H, t, J = 6.5 Hz), 2.64 (2H, t, J = 6.5 Hz), 2.40 (3H, s); HRMS (ESI-MS m/z) calcd for C₁₃H₁₆NO₃S (M+H)⁺ 266.0851, found 266.0837.

3-Iodoethyl-1-(p-toluenesulfonyl)pyrrole (99)

The procedure used for preparation of 99 from 98 is similar to those used for the preparation of 89 from 88; the yield was 62 %.

¹H NMR (CDCl₃) δ 7.73 (2H, d, J = 8.2 Hz), 7.29 (2H, d, J = 8.5 Hz), 7.08 (1H, t, J = 2.8 Hz), 6.99 (1H, m), 6.16 (1H, dd, J = 1.6 and 3.3 Hz), 3.24 (2H, t, J = 7.3 Hz), 2.96 (2H, t, J =7.6 Hz), 2.40 (3H, s); HRMS (ESI-MS m/z) calcd for C₁₃H₁₅NO₂SI (M+H)⁺ 375.9868, found 375.9857.

General synthetic procedure for 2-substituted adenosine derivatives

To a solution of 2′, 3′, 5′-triacetyl-6-chloroguanosine in N,N-dimethylformamide were added iodide (1.8 eq) and Cs₂CO₃ (2.7 eq) at room temperature, and the reaction mixture was stirred overnight. After dilution with ethyl acetate the solution was washed with water twice, dried over MgSO₄ and filtered. The filtrate was evaporated to give a crude oil which was purified by column chromatography or preparative TLC on silica gel. Elution or developing with a mixture of toluene and acetone (4:1) gave the 2-substituted 2′, 3′, 5′-triacetyl-6-chloroadenosine derivative.

A solution of 2-substituted 2′, 3′, 5′-triacetyl-6-chloroadenosine derivative in saturated ammonia ethanol solution was stirred in sealed tube overnight at 110 – 120 °C. The solvent was evaporated to give an oil, which was subjected to preparative TLC developed with a mixture of chloroform and methanol (8:1) to give the 2-substituted adenosine derivative.

In case of deprotection of tosyl group of 2-substituent, tosylated adenosine derivative was treated with KOH (20 eq) in methanol overnight at 70 °C in sealed tube. The reaction mixture was concentrated to a small amount of solution, which was subjected to preparative TLC developed with a mixture of chloroform and methanol (5:1) to give the final product.

6-Chloro-2-(3″-(1″-(p-toluenesulfonyl)indolyl)ethyloxy)-3′, 4′, 5′-triacetyladenosine (102)

The yield was 86 %. ¹H NMR (CDCl₃) δ 8.09 (1H, s), 7.98 (1H, d with small coupling, J = 6.6 Hz), 7.76 (2H, d with small coupling, J = 8.2 Hz), 7.61 (1H, d with small coupling, J = 7.1 Hz), 7.53 (1H, s), 7.14 – 7.36 (6H, m), 6.13 (1H, d, J = 4.7 Hz), 5.93 (1H, t, J = 5.1 Hz), 5.65 (1H, t, J = 5.5 Hz), 4.71 (2H, m), 4.39 – 4.49 (2H, m), 4.32 (1H, dd, J = 4.7 and 12.6 Hz), 3.24 (2H, t, J = 7.0 Hz), 2.32 (3H, s), 2.14 (3H, s), 2.09 (3H, s), 2.05 (3H, s); HRMS (ESI-MS m/z) calcd for C₃₃H₃₂N₅O₁₀ClSNa (M+Na)⁺ 748.1456, found 748.1455.

6-Chloro-2-(3″-(5″-methoxy-1″-(p-toluenesulfonyl)indolyl)ethyloxy)- 3′, 4′, 5′-triacetyladenosine (103)

The yield was 72 %. ¹H NMR (CDCl₃) δ 8.09 (1H, s), 7.86 (1H, dd, J = 9.1 Hz), 7.73 (2H, d, J = 8.5 Hz), 7.48 (1H, s), 7.20 (2H, d, J = 8.2 Hz), 7.03 (1H, d, J = 2.5 Hz), 6.92 (1H, dd, J = 2.3 and 8.9 Hz), 6.12 (1H, d, J = 4.7 Hz), 5.93 (1H, t, J = 5.1 Hz), 5.66 (1H, t, J = 5.6 Hz), 4.69 (2H, ddd, J = 3.8, 7.4, and 14.3 Hz), 4.40 – 4.49 (2H, m), 4.31 (1H, dd, J = 4.4 and 12.6 Hz), 3.85 (3H, s), 3.19 (2H, t, J = 6.7 Hz), 2.32 (3H, s), 2.14 (3H, s), 2.09 (3H, s), 2.05 (3H, s); HRMS (ESI-MS m/z) calcd for C₃₄H₃₅N₅O₁₁SCl (M+H)⁺ 756.1742, found 756.1735.

6-Chloro-2-(3″-(5″-(p-toluenesulfonyloxy) -1″-(p-toluenesulfonyl)indolyl)ethyloxy)-3′, 4′, 5′-triacetyladenosine (104)

The yield was 51 %. ¹H NMR (CDCl₃) δ 8.09 (1H, s), 7.83 (1H, d, J = 8.5 Hz), 7.72 (2H, d with small coupling, J = 8.5 Hz), 7.68 (2H, d with small coupling, J = 8.2 Hz), 7.57 (1H, s), 7.25–7.31 (3H, m), 7.22 (2H, d, J = 8.0 Hz), 6.83 (1H, dd, J = 2.3 and 8.9 Hz), 6.12 (1H, d, J = 4.4 Hz), 5.94 (1H, dd, J = 4.7 and 5.5 Hz), 5.67 (1H, t, J = 5.4 Hz), 4.64 (2H, m), 4.40–4.50 (2H, m), 4.31 (1H, dd, J = 5.1 and 12.8 Hz), 3.13 (2H, t, J = 6.7 Hz), 2.42 (3H, s), 2.34 (3H, s), 2.15 (3H, s), 2.08 (3H, s), 2.03 (3H, s); HRMS (ESI-MS m/z) calcd for C₄₀H₃₉ClN₅O₁₃S₂ (M+H)⁺ 896.1674 found 896.1638.

6-Chloro-2-(3″-(5″-fluoro-1″-(p-toluenesulfonyl)indolyl)ethyloxy)-3′, 4′, 5′-triacetyladenosine (105)

The yield was 59 %. ¹H NMR (CDCl₃) δ 8.09 (1H, s), 7.91 (1H, dd, J = 4.4 and 9.1 Hz), 7.74 (2H, d with small coupling, J = 8.5 Hz), 7.57 (1H, s), 7.25–7.31 (1H overlapped with CHCl₃), 7.22 (2H, d, J = 8.0 Hz), 7.04 (1H, dt, J = 2.6 and 9.0 Hz), 6.11 (1H, d, J = 4.7 Hz), 5.94 (1H, t, J = 4.9 Hz), 5.67 (1H, t, J = 5.5 Hz), 4.69 (2H, m), 4.40–4.50 (2H, m), 4.31 (1H, dd, J = 4.9 and 12.9 Hz), 3.18 (2H, t, J = 6.9 Hz), 2.33 (3H, s), 2.15 (3H, s), 2.09 (3H, s), 2.04 (3H, s); HRMS (ESI-MS m/z) calcd for C₃₂H₂₃N5O₁₀SFCl (M+H)⁺ 744.1542, found 744.1522.

2-(3″-(5″-Bromo-1″-(p-toluenesulfonyl)indolyl)ethyloxy)-6-chloro-3′, 4′, 5′-triacetyladenosine (106)

The yield was 47 %. ¹H NMR (CDCl₃) δ 8.09 (1H, s), 7.84 (1H, d, J = 8.8 Hz), 7.75 (1H, s), 7.73 (2H, d with small coupling, J = 6.6 Hz), 7.54 (1H, s), 7.40 (1H, dd, J = 1.8 and 8.9 Hz), 7.22 (2H, d, J = 8.5 Hz), 6.13 (1H, d, J = 4.7 Hz), 5.94 (1H, t, J = 4.9 Hz), 5.67 (1H, t, J = 5.4 Hz), 4.69 (2H, t, J = 6.6 Hz), 4.40–4.50 (2H, m), 4.31 (1H, dd, J = 5.2 Hz), 3.19 (2H, t, J = 6.7 Hz), 2.33 (3H,s), 2.15 (3H, s), 2.09 (3H, s), 2.03 (3H, s); HRMS (ESIMS m/z) calcd for C₃₃H₃₂N₅O₁₀BSClBr (M+H)⁺ 804.0742, found 804.0752.

6-Chloro-2-(3″-(5″-methoxy-2″-methyl-1″-(p-toluenesulfonyl)indolyl)ethyloxy)-3′,4′,5′-triacetyladenosine (107)

The yield was 72 %. ¹H NMR (CDCl₃) δ 8.08 (1H, s), 8.07 (1H, d, J = 9.6 Hz), 7.60 (2H, d with small coupling, J = 8.5 Hz), 7.17 (2H, d, J = 8.0 Hz), 6.98 (1H, d, J = 2.5 Hz), 6.87 (1H, dd, J = 2.8 and 9.1 Hz), 6.12 (1H, d, J = 5.0 Hz), 5.83 (1H, t, J = 5.2 Hz), 4.36 – 4.54 (5H, m), 4.31 (1H, dd, J = 4.1 and 12.4 Hz), 3.86 (3H, s), 3.13 (2H, t, J = 7.8 Hz), 2.61 (3H, s), 2.32 (3H, s), 2.13 (3H,s), 2.06 (3H,s), 2.05 (3H, s); HRMS (ESI-MS m/z) calcd for C₃₅H₃₇N₅O₁₁SCl (M+H)⁺ 770.1899, found 770.1895.

6-Chloro-2-(3″-(6″-chloro-1″-(p-toluenesulfonyl)indolyl)ethyloxy)-3′, 4′, 5′-triacetyladenosine (108)

The yield was 70 %. ¹H NMR (CDCl₃) d 8.09 (1H, s), 7.99 (1H, d, J = 1.9 Hz), 7.76 (2H, d with small coupling, J = 8.5 Hz), 7.53 (1H, d, J = 8.5 Hz), 7.52 (1H, s), 7.20 – 7.28 (3H, m), 6.11 (1H, d, J = 4.7 Hz), 5.95 (1H, t, J = 4.9 Hz), 5.66 (1H, t, J = 5.5 Hz), 4.68 (2H, m), 4.38–4.50 (2H, m), 4.31 (1H, dd, J = 4.5 and 12.2 Hz), 3.20 (2H, t, J = 6.9 Hz), 2.35 (3H, s), 2.14 (3H, s), 2.09 (3H, s), 2.06 (3H, s); APCI-MS (m/z) 760.1 (M+H)⁺.

2-(3″-(6″-Bromo-1″-(p-toluenesulfonyl)indolyl)ethyloxy)-6-chloro-3′, 4′, 5′-triacetyladenosine (109)

The yield was 45 %. ¹H NMR (CDCl₃) δ 8.15 (1H, d, J = 1.6 Hz), 8.09 (1H, s), 7.76 (2H, d with small coupling, J = 8.2 Hz), 7.50 (1H, s), 7.49 (1H, d, J = 9.1 Hz), 7.38 (1H, dd, J = 1.7 and 8.5 Hz), 7.25 (2H, d, J = 8.2 Hz), 6.11 (1H, d, J = 4.7 Hz), 5.95 (1H, t, J = 4.9 Hz), 5.66 (1H, t, J = 5.5 Hz), 4.68 (2H, m), 4.18–4.50 (2H, m), 4.31 (1H, dd, J = 4.5 and 12.5 Hz), 3.20 (2H, t, J = 7.0 Hz), 2.35 (3H, s), 2.14 (3H, s), 2.09 (3H, s), 2.06 (3H, s); HRMS (ESI-MS m/z) calcd for C₃₃H₃₂N₅O₁₀SCl Br(M+H)⁺ 804.0742, found 804.0760.

6-Chloro-2-(3″-(5″-chloro-1″-(p-toluenesulfonyl)indolyl)ethyloxy)- 3′, 4′, 5′-triacetyladenosine (110)

The yield was 46 %. ¹H NMR (CDCl3) δ 8.09 (1H, s), 7.89 (1H, d, J = 9.1 Hz), 7.73 (2H, d with small coupling, J = 8.2 Hz), 7.59 (1H, d, J = 2.2 Hz), 7.24–7.30 (1H, m), 7.22 (2H, d, J = 8.0 Hz), 6.12 (1H, d, J = 4.4 Hz), 5.94 (1H, t, J = 5.1 Hz), 5.67 (1H, t, J = 5.4 Hz), 4.69 (2H, m), 4.40–4.49 (2H, m), 4.31 (1H, dd, J = 4.9 and 13.2 Hz), 3.18 (2H, t, J = 6.7 Hz), 2.33 (3H, s), 2.15 (3H, s), 2.09 (3H, s), 2.04 (3H, s); HRMS (ESI-MS m/z) calcd for C₃₃H₃₁N₅O₁₀SCl₂Na (M+Na)⁺ 782.1066, found 782.1071.

6-Chloro-2-(3″-(5″-iodo-1″-(p-toluenesulfonyl)indolyl)ethyloxy)- 3′, 4′, 5′-triacetyladenosine (111)

The yield was 45 %. ¹H NMR (CDCl₃) δ 8.09 (1H, s), 7.93 (1H, d, J = 1.7 Hz), 7.73 (3H, m), 7.57 (1H, dd, J = 1.8 and 8.7 Hz), 7.50 (1H, s), 7.22 (2H, d, J = 8.0 Hz), 6.13 (1H, d, J = 4.7 Hz), 5.94 (1H, t, J = 5.1 Hz), 5.67 (1H, t, J = 5.4 Hz), 4.68 (2H, t, J = 7.0 Hz), 4.40–4.48 (2H, m), 4.31 (1H, dd, J = 5.1 and 13.3 Hz), 3.18 (2H, t, J = 6.9 Hz), 2.33 (3H, s), 2.15 (3H, s), 2.09 (3H, s), 2.04 (3H, s); HRMS (ESI-MS m/z) calcd for C₃₃H₃₂N₅O₁₀SClI (M+H)⁺ 852.0603, found 852.0566.

6-Chloro-2-(3″-(4″-bromo-1″-(p-toluenesulfonyl)indolyl)ethyloxy)- 3′, 4′, 5′-triacetyladenosine (112)

The yield was 40 %. ¹H NMR (CDCl3) δ 8.09 (1H, s), 7.95 (1H, dd, J = 0.8 and 8.2 Hz), 7.75 (2H, d with small coupling, J = 8.5 Hz), 7.59 (1H, s), 7.39 (1H, dd, J = 0.8 and 8.0 Hz), 7.23 (2H, d, J = 8.5 Hz), 7.12 (1H, t, J = 8.1 Hz), 6.13 (1H, d, J = 5.0 Hz), 5.92 (1H, t, J = 5.1 Hz), 5.64 (1H, t, J = 5.4 Hz), 4.75 (2H, m), 4.38–4.50 (2H, m), 4.33 (1H, dd, J = 4.7 and 12.9 Hz), 3.53 (2H, t, J = 7.0 Hz), 2.34 (3H, s), 2.13 (3H, s), 2.09 (3H, s), 2.05 (3H, s); APCI-MS (m/z) 806.1 (M+H)⁺.

6-Chloro-2-(3″-(7″-bromoindolyl)ethyloxy)- 3′, 4′, 5′-triacetyladenosine (113)

The yield was 10 %. ¹H NMR (CDCl₃) δ 8.06 (1H, s), 7.66 (1H, d, J = 8.0 Hz), 7.35 (1H, d, J = 7.7 Hz), 7.27 (1H overlapped with CHCl3), 7.03 (1H, t, J = 7.7 Hz), 6.11 (1H, d, J = 5.0 Hz), 5.91 (1H, t, J = 5.2 Hz), 5.64 (1H, t, J = 5.2 Hz), 4.70 (2H, m), 4.37–4.46 (2H, m), 4.32 (1H, dd, J = 5.4 and 13.1 Hz), 3.30 (2H, t, J = 7.1 Hz), 2.13 (3H, s), 2.07 (3H, s), 2.07 (3H, s); APCI-MS (m/z) 672.1 (M+Na)⁺.

6-Chloro-2-phenypropoxy-3′, 4′, 5′-triacetyladenosine (114)

The yield was 63 %. ¹H NMR (CDCl₃) δ 8.08 (1H, s), 7.16–7.34 (5H, m), 6.14 (1H, d, J = 4.9 Hz), 5.91 (1H, t, J = 5.4 Hz), 5.63 (1H, t, J = 5.2 Hz), 4.38 – 4.52 (5H, m), 4.31 (1H, dd, J = 3.8 and 11.8 Hz), 2.85 (2H, dd, J = 7.4 and 8.0 Hz), 2.12 – 2.24 (2H, m), 2.15 (3H, s), 2.10 (3H, s), 2.09 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₅H₂₇N₄O₈ClLi (M+Li)⁺ 553.1677, found 553.1661.

6-Chloro-2-(2″-(1″-(p-toluenesulfonyl)indolyl)ethyloxy)- 3′, 4′, 5′-triacetyladenosine (115)

The yield was 40 %. ¹H NMR (CDCl₃) δ 8.16 (1H, d, J = 8.2 Hz), 8.09 (1H, s), 7.63 (2H, d, J = 8.5 Hz), 7.42 (2H, d, J = 7.1 Hz), 7.15 – 7.32 (5H, m), 6.59 (1H, s), 6.16 (1H, d, J = 5.0 Hz), 5.87 (1H, t, J = 5.2 Hz), 5.61 (1H, t, J = 5.2 Hz), 4.83 (2H, m), 4.40 – 4.48 (2H, m), 4.34 (1H, dd, J = 4.9 and 13.2 Hz), 3.58 (2H, t, J = 6.6 Hz), 2.33 (3H, s), 2.12 (3H, s), 2.08 (3H,s), 2.07 (3H,s); HRMS (ESI-MS m/z) calcd for C₃₃H₃₃N₅O₁₀ClS (M+H)⁺ 726.1637, found 726.1640.

6-Chloro-2-(3″-(benzoimidazol-1″-yl)ethyloxy)- 3′, 4′, 5′-triacetyladenosine (116)

The yield was 51 %. ¹H NMR (CD₃OD) δ 8.09 (2H, d, J = 8.0 Hz), 7.79 (1H, dd, J = 1.4 and 7.1 Hz), 7.55 (1H, dd, J = 1.1 and 7.1 Hz), 7.34 (1H, dt, J = 1.4 and 7.4 Hz), 7.28 (1H, dt, J = 1.4 and 7.5 Hz), 6.05 (1H, d, J = 4.7 Hz), 5.92 (1H, t, J = 4.9 Hz), 5.67 (1H, t, J = 5.4 Hz), 4.82 (2H, m), 4.66 (2H, t, J = 5.4 Hz), 4.39 – 4.48 (2H, m), 4.27 (1H, dd, J = 5.2 and 13.2 Hz), 2.16 (3H, s), 2.09 (3H, s), 2.02 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₅H₂₆N₆O₈Cl (M+H)⁺ 573.1501, found 573.1503.

6-Chloro-2-(3″-(benzotriazol-1″-yl)ethoxy)- 3′, 4′, 5′-triacetyladenosine (117)

The yield was 53 %. ¹H NMR (CDCl₃) δ 8.08 (1H, s), 8.03 (1H, dt, J = 0.8 and 8.5 Hz), 7.72 (1H, dt, J = 0.8 and 8.5 Hz), 7.52 (1H, ddd, J = 1.0, 7.1 and 8.1 Hz), 7.36 (1H, ddd, J = 1.1, 7.1 and 8.2 Hz), 6.07 (1H, d, J = 4.4 Hz), 5.89 (1H, dd, J = 4.7 and 5.5 Hz), 5.62 (1H, t, J = 5.4 Hz), 5.11 (2H, m), 5.00 (2H, m), 4.40–4.48 (2H, m), 4.26–4.34 (1H, m), 2.15, 2.10 and 2.05 (each 3H, s); HRMS (ESI-MS m/z) calcd for C₂₄H₂₅N₇O₈Cl (M+H)⁺ 574.1453, found 574.1456.

6-Chloro-2-(3″-(1″-p-toluenesulfonyl)pyrrolyl)ethyloxy)- 3′, 4′, 5′-triacetyladenosine (118)

The yield was 61 %. ¹H NMR (CDCl3) δ 8.08 (1H, s), 7.73 (1H, d, J = 8.5 Hz), 7.27 (2H, d overlapped with CHCl₃), 7.08 (2H, m), 6.28 (1H, dd, J = 1.9 and 3.0 Hz), 6.13 (1H, d, J = 5.0 Hz), 5.91 (1H, t, J = 5.1 Hz), 5.63 (1H, t, J = 5.4 Hz), 4.57 (1H, dd, J = 2.8 and 7.4 Hz), 4.53 (1H, dd, J = 3.0 and 7.7 Hz), 4.38 – 4.45 (2H, m), 4.32 (1H, dd, J = 4.3 and 12.2 Hz), 2.95 (2H, t, J = 7.0 Hz), 2.40(3H, s), 2.15(3H, s), 2.09 (3H, s), 2.07 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₉H₃₁N₅O₁₀SCl (M+H)+ 676.1480, found 676.1450.

2-(3″-(5″-Methoxy-1″-(p-toluenesulfonyl)indolyl)ethyloxy)adenosine (119)

The yield was 56 %. ¹H NMR (CD₃OD) δ 8.14 (1H, s), 7.81 (1H, d, J = 9.3 Hz), 7.67 (2H, dt, J =1.9 and 8.5 Hz), 7.52 (1H, s), 7.15 (2H, dd, J = 0.6 and 8.5 Hz), 7.02 (1H, d, J =2.5 Hz), 6.89 (1H, dd, J = 2.8 and 8.8 Hz), 5.89 (1H, d, J = 6.0 Hz), 4.72 (1H, t, J = 5.6 Hz), 4.55 (2H, dt, J = 1.1 and 6.3 Hz), 4.33 (1H, dd, J = 3.3 and 5.0 Hz), 4.12 (1H, q, J = 3.0 Hz), 3.88 (1H, dd, J = 2.7 and 12.4 Hz), 3.78 (3H, s), 3.74 (1H, dd, J =3.2 and 12.5 Hz), 3.11 (2H, t, J = 6.3 Hz), 2.25 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₈H₃₁N₆O₈S (M+H)⁺ 611.1924, found 611.1899.

2-(3″-(5″-(p-toluenesulfonyloxy) -1″-(p-toluenesulfonyl)indolyl)ethyloxy)adenosine (120)

The yield was 63 %. ¹H NMR (CD₃OD) δ 8.15 (1H, s), 7.86 (1H, d, J = 8.8 Hz), 7.71 (2H, d with small coupling, J = 8.5 Hz), 7.64 (1H, s), 7.60 (2H, d with small coupling, J = 8.2 Hz), 7.28 (2H, d, J = 8.5 Hz), 7.21 (2H, d, J = 8.0 Hz), 7.08 (1H, d, J = 2.5 Hz), 6.93 (1H, dd, J = 2.2 and 9.1 Hz), 5.90 (1H, d, J = 6.1 Hz), 4.71 (1H, t, J = 5.6 Hz), 4.42 (2H, t, J = 6.6 Hz), 4.33 (2H, t, J = 6.6 Hz), 4.33 (1H, dd, J = 3.2 and 5.1 Hz), 4.14 (1H, q, J = 3.0 Hz), 3.90 (1H, dd, J = 2.8 and 12.6 Hz), 3.74 (1H, dd, J = 3.2 and 12.5 Hz), 3.00 (2H, t, J = 6.6 Hz), 2.34 (3H, s), 2.29 (3H, s); HRMS (ESI-MS m/z) calcd for C₃₄H₃₅N₆O₁₀S₂ (M+H)⁺ 751.1856, found 751.1819.

2-(3″-(5″-Fluoro-1″-(p-toluenesulfonyl)indolyl)ethyloxy)adenosine (121)

The yield was 55 %. ¹H NMR (CD₃OD) δ 8.13 (1H, s), 7.91 (1H, dd, J = 4.4 and 9.1 Hz), 7.70 (2H, d with small coupling, J = 8.5 Hz), 7.64 (1H, s), 7.29 (1H, dd, J = 2.5 and 8.8 Hz), 7.16 (2H, d, J = 8.0 Hz), 7.05 (1H, dt, J = 2.5 and 9.1 Hz), 5.89 (1H, d, J = 6.0 Hz), 4.73 (1H, t, J = 5.5 Hz), 4.54 (2H, t, J = 6.2 Hz), 4.33 (1H, dd, J = 3.3 and 5.2 Hz), 4.12 (1H, q, J = 3.0 Hz), 3.89 (1H, dd, J = 2.7 and 12.4 Hz), 3.74 (1H, dd, J = 3.2 and 12.5 Hz), 3.10 (2H, t, J = 6.3 Hz), 2.26 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₇H₂₈N₆O₇SF (M+H)⁺ 599.1724, found 599.1714.

2-(3″-(6″-Chloro-1″-(p-toluenesulfonyl)indolyl)ethyloxy)adenosine (122)

The yield was 51 %. ¹H NMR (CD₃OD) δ 8.13 (1H, s), 7.92 (1H, d, J = 1.7 Hz), 7.72 (2H, d with small coupling, J = 8.2 Hz), 7.61 (1H, s), 7.58 (1H, d, J = 8.5 Hz), 7.25 (1H, dd, J = 1.9 and 8.5 Hz), 7.21 (2H, dd, J = 0.7 and 8.7 Hz), 5.89 (1H, d, J = 6.1 Hz), 4.72 (1H, t, J = 5.5 Hz), 4.55 (2H, m), 4.33 (1H, dd, J = 3.3 and 5.2 Hz), 4.12 (1H, q, J = 2.9 Hz), 3.88 (1H, dd, J = 2.8 and 12.4 Hz), 3.74 (1H, dd, J = 3.3 and 12.4 Hz), 3.12 (2H, t, J = 6.5 Hz), 2.28 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₇H₂₈N₆O₇SCl (M+H)⁺ 615.1429, found 615.1413.

2-(3″-(6″-Bromo-1″-(p-toluenesulfonyl)indolyl)ethyloxy)adenosine (123)

The yield was 50 %. ¹H NMR (CD₃OD) δ 8.13 (1H, s), 8.08 (1H, d, J = 1.7 Hz), 7.71 (2H, dt, J = 2.1 and 8.5 Hz), 7.60 (1H, s), 7.53 (1H, d, J = 8.2 Hz), 7.38 (1H, dd, J = 1.7 and 8.2 Hz), 7.21 (2H, d with small coupling, J = 8.2 Hz), 5.89 (1H, d, J = 6.1 Hz), 4.72 (1H, t, J = 5.5 Hz), 4.54 (2H, m), 4.33 (1H, dd, J = 3.3 and 5.2 Hz), 4.12 (1H, q, J = 3.0 Hz), 3.88 (1H, dd, J = 2.7 and 12.4 Hz), 3.74 (1H, dd, J = 3.3 and 12.4 Hz), 3.12 (2H, t, J = 6.5 Hz), 2.28 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₇H₂₈N₆O₇BrS (M+H)⁺ 659.0924, found 659.0910.

2-(3″-(5″-Chloro-1″-(p-toluenesulfonyl)indolyl)ethyloxy)adenosine (124)

The yield was 62 %. ¹H NMR (CD₃OD) δ 8.13 (1H, s), 7.90 (1H, d, J = 8.8), 7.71 (2H, d with small coupling, J = 8.8 Hz), 7.64 (1H, s), 7.57 (1H, d, J = 1.9 Hz), 7.27 (1H, dd, J = 1.9 and 8.8 Hz), 7.18 (2H, d, J = 8.2 Hz), 5.89 (1H, d, J = 5.8 Hz), 4.73 (1H, t, J = 5.6 Hz), 4.55 (2H, t, J = 6.5 Hz), 4.33 (1H, dd, J = 3.3 and 5.2 Hz), 4.12 (1H, q, J = 3.1 Hz), 3.89 (1H, dd, J = 2.7 and 12.6 Hz), 3.74 (1H, dd, J = 3.2 and 12.5 Hz), 3.11 (2H, t, J = 6.3 Hz), HRMS (ESI-MS m/z) calcd for C₂₇H₂₈N₆O₇SCl (M+H)⁺ 615.1429, found 615.1401.

2-(3″-(5″-Iodo-1″-(p-toluenesulfonyl)indolyl)ethyloxy)adenosine (125)

The yield was 71 %. ¹H NMR (CD₃OD) δ 8.14 (1H, s), 7.89 (1H, d, J = 1.7 Hz), 7.73 (1H, d, J = 9.1 Hz), 7.71 (2H, d with small coupling, J = 8.5 Hz), 7.58 (1H, s), 7.57 (1H, dd, J = 1.8 and 8.7 Hz), 7.18 (2H, d, J = 8.2 Hz), 5.89 (1H, d, J = 5.8 Hz), 4.73 (1H, t, J = 5.5 Hz), 4.54 (2H, t, J = 6.3 Hz), 4.34 (1H, dd, J = 3.3 and 5.2 Hz), 4.12 (1H, q, J = 3.0 Hz), 3.89 (1H, dd, J = 2.9 and 12.5 Hz), 3.75 (1H, dd, J = 3.3 and 12.4 Hz), 3.10 (2H, t, J = 6.3 Hz), 3.27 (3H, s); APCI-MS (m/z) 707.0 (M+H)+.

2-(3″-(4″-Bromo-1″-(p-toluenesulfonyl)indolyl)ethyloxy)adenosine (126)

The yield was 43 %. ¹H NMR (CD₃OD) δ 8.13 (1H, s), 7.95 (1H, dd, J = 0.7 and 8.4 Hz), 7.73 (2H, d with small coupling, J = 8.5 Hz), 7.69 (1H, s), 7.39 (1H, dd, J = 0.8 and 7.7 Hz), 7.19 (2H, dd, J = 0.6 and 8.5 Hz), 7.15 (1H, t, J = 8.1 Hz), 5.89 (1H, d, J = 6.1 Hz), 4.74 (1H, t, J = 5.5 Hz), 4.61 (2H, m), 4.33 (1H, dd, J = 3.3 and 5.2 Hz), 4.12 (1H, q, J = 3.1 Hz), 3.89 (1H, dd, J = 2.8 and 12.4 Hz), 3.75 (1H, dd, J = 3.3 and 12.4 Hz), 3.44 (2H, m), 2.27 (3H, s); APCI-MS (m/z) 659.1 (M+H)⁺.

2-(3″-(1″-(p-toluenesulfonyl)pyrrolyl)ethyloxy)-adenosine (127)

The yield was 69 %. ¹H NMR (CD₃OD) δ 8.12 (1H, s), 7.72 (2H, d with small coupling, J = 8.5 H), 7.29 (2H, d, J = 8.5Hz), 7.08–7.14 (2H, m), 6.30 (1H, dd, J = 1.7 and 3.2 Hz), 5.88 (1H, d, J = 6.0 Hz), 4.71 (1H, t, J = 5.5 Hz), 4.41 (2H, t, J = 6.7 Hz), 4.32 (1H, dd, J = 3.4 and 5.1 Hz), 4.11 (1H, q, J = 3.2 Hz), 3.86 (1H, dd, J = 2.6 and 12.5 Hz), 3.73 (1H, dd, J = 3.3 and 12.7 Hz), 2.85 (2H, t, J = 6.5 Hz), 2.36 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₃H₂₇N6O7S (M+H)⁺ 531.1662, found 531.1667.

(2R, 3S, 4S, 5R)-2-(2′-Amino-6′-chloropurin-9′-yl)-5-hydroxymethyl-3, 4-O-isopropylidene-tetrahydrofuran (128)

To a solution of 2-amino-6-chloropurine-9-riboside (100 mg, 0.331 mmol) in N,N-dimethylformamide (2 mL) were added 2,2-dimethoxypropane (0.242 ml, 1.97 mmol) and p-toluenesulfonic acid monohydrate (188 mg, 0.993 mmol), and the reaction mixture was stirred overnight at room temperature. The reaction was diluted with ethyl acetate, washed with water, dried over MgSO₄ and filtered. The filtrate was evaporated to give an oil, which was subjected to preparative TLC developed with a mixture of toluene and acetone (1:1) to give 128 (56 mg, 50 %):

¹H NMR (CDCl₃) δ 7.81 (1H, s), 5.79 (1H, d, J = 4.9 Hz), 5.68 (1H, dd, J = 1.4 and 11.3 Hz), 5.14 – 5.24 (3H, m), 5.08 (1H, dd, J = 1.4 and 6.0 Hz), 4.51 (1H, d, J = 1.7 Hz), 3.97 (1H, d with small coupling, J = 12.6 Hz), 3.78 (1H, ddd, J = 1.9, 11.3 and 13.2 Hz), 1.64 (3H, s), 1.38 (3H, s); HRMS (ESI-MS m/z) calcd for C₁₃H₁₇N₅O₄Cl (M+H)⁺ 342.0969, found 342.0979.

(2R, 3S, 4S, 5R)- 2-(2′-Amino-6′-chloropurin-9′-yl)-5-carboxy-3, 4-O-isopropylidene-tetrahydrofuran (129)

To a solution of 128 (16.9 mg, 0.0494 mmol) in water (4.5 mL) were added potassium permanganate (70.3 mg, 0.445 mmol) and potassium hydroxide (25 mg, 0.444 mmol), and the reaction mixture was stirred for 1 h. After addition of isopropanol the reaction mixture was filtered. The filtrate was neutralized with 0.1N hydrochloric acid aqueous solution and evaporated to give a crude solid, which was subjected to preparative TLC developed with a mixture of chloroform, methanol and saturated aqueous ammonia (2: 1: 0.3) to give 129 (9 mg, 51 %).

¹H NMR (CD₃OD) δ 8.29 (1H, s), 6.18 (1H, d, J = 1.2 Hz), 5.52 (1H, dd, J = 1.7 and 6.2 Hz), 5.37 (1H, d, J = 6.0 Hz), 4.59 (1H, d, J = 1.7 Hz), 1.55 (3H, s), 1.39 (3H, S); HRMS (ESI-MS m/z) calcd for C₁₃H₁₃N₅O₅Cl (M-H)⁻ 354.0605, found 354.0622.

(2R, 3S, 4S, 5R)- 2-(2′-Amino-6′-chloropurin-9′-yl)-5-ethoxycarboxyamide-3, 4-isopropylidene-tetrahydrofuran (130)

To a solution of 129 (11.9 mg, 0.0334 mmol) in N, N-dimethylformamide (0.8 mL) were added ethylamine hydrochloride (8.1 mg, 0.100 mmol), N, N-diisopropylethylamine (0.035 ml, 0.200 mmol), and (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (22.5 mg, 0.0434 mmol), and the reaction mixture was stirred overnight. The mixture was diluted with ethyl acetate, washed with water, dried over MgSO₄, and filtered. The filtrate was evaporated to give a crude oil, which was subjected to preparative TLC developed with a mixture of chloroform and methanol (10:1) to give 130 (10 mg, 78 %)

¹H NMR (CD₃OD) δ 8.16(1H, s), 6.27 (1H, s), 5.73 (1H, dd, J = 1.9 and 6.3 Hz), 5.43 (1H, d, J = 6.3 Hz), 4.62 (1H, d, J = 1.7 Hz), 2.91 (1H, dt, J = 6.0 and 13.3 Hz), 2.80 (1H, dt, J = 6.0 and 13.3 Hz), 1.55 (3H, s), 1.40 (3H,s), 0.61 (3H, t, J = 7.3 Hz); HRMS (ESIMS m/z) calcd for C₁₅H₂₀N₆O₄Cl (M+H)⁺383.1235, found 383.1229.

(2R, 3S, 4S, 5R)-5-Ethoxycarboxyamide-2-(2′-hydroxy-6′-chloropurin-9′-yl)-3, 4-O-isopropylidene-tetrahydrofuran (131)

To a solution of 130 (10 mg, 0.026 mmol) in a mixture of 2-propanol (0.4 mL) and water (0.4 mL) was added t-butylnitrite (13.3 μl, 0.115 mmol) at 4 °C, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate, washed with water, dried over MgSO₄ and filtered. The filtrate was evaporated to give a crude oil, which was subjected to preparative TLC developed with a mixture of chloroform and methanol (10:1) to give 131 (5 mg, 50 %).

¹H NMR (CDCl₃) δ 7.99 (1H, s), 6.37 (1H, br t, J = 6.1Hz), 6.11 (1H, d, J = 1.6 Hz), 5.72 (1H, dd, J = 1.7 and 6.1 Hz), 5.36 (1H, dd, J = 1.7 and 6.0 Hz), 4.75 (1H, s), 3.05 (2H, m), 1.61 (3H, s), 1.41 (3H, s), 0.77 (3H, t, J = 7.3 Hz); APCI-MS (m/z) 384.1 (M+H)⁺.

(2R, 3S, 4S, 5R)-5-Ethylcarboxyamide-2–2′-(3″-(1″-(p-toluenesufonyl) indolyl)ethyloxy)-6′-chloropurin-9′-yl)-3, 4-isopropylidene-tetrahydrofuran (132)

To a solution of 131 (19.4 mg, 0.0505 mmol) in N, N-dimethylformamide (0.8 mL) was added iodide 47 (43 mg, 0.101 mmol) and cesium carbonate (49.3 mg, 0.151 mmol), and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate, washed with water, dried over MgSO₄ and filtered. The filtrate was evaporated to give a crude oil, which was subjected to preparative TLC developed with a mixture of toluene and acetone (4:1) to give 132 (24 mg, 70 %).

¹H NMR (CDCl₃) δ 7.99 (1H, s), 7.98 (1H, d, J = 8.2 Hz), 7.77 (2H, d with small coupling, J = 8.5 Hz), 7.61 (1H, d with small coupling, J = 7.7 Hz), 7.56 (1H, s), 7.24- 7.35 (2H, m), 7.21 (2H, d, J = 8.5 Hz), 6.27 (1H, t, J = 6.0 Hz), 6.14 (1H, d, J = 2.2 Hz), 5.52 (1H, dd, J = 1.9 and 6.1 Hz), 5.39 (1H, dd, J = 2.1 and 6.2 Hz), 4.60–4.80 (3H, m), 3.27 (2H, t, J = 6.7 Hz), 2.97 (2H, m), 2.32 (3H, s), 1.61 (3H, s), 1.35 (3H, s), 0.69 (3H, t, J = 7.3Hz); HRMS (ESI-MS m/z) calcd for C₃₂H₃₃N₆O₇SCl Na (M+Na)⁺ 703.1718, found 703.1732.

(2R, 3S, 4S, 5R)-5-Ethylcarboxyamide-2-(6′-amino -2′-(3″-(1″-(p-toluenesufonyl) indolyl)ethyloxy)-purin-9′-yl)-3, 4-O-isopropylidene-tetrahydrofuran (133)

A solution of 132 in saturated ammonia ethanol solution was stirred at 120 °C overnight. The solvent was evaporated to give an oil, which was subjected to preparative TLC developed with a mixture of chloroform and methanol (10:1) to give 133 (17 mg, 88 % yield).

¹H NMR (CDCl₃) δ 7.96 (1H, d, J = 7.7 Hz), 7.77 (2H, d, J = 8.2 Hz), 7.68 (1H, s), 7.54–7.60 (2H, m), 7.30 (1H, dt, J = 1.6 and 7.7 Hz), 7.24 (1H, overlapped with CHCl₃), 7.19 (2H, d, J = 8.5 Hz), 6.44 (1H, t, J = 5.9 Hz), 6.07 (1H, d, J = 1.9 Hz), 5.59 (1H, br s), 5.51 (1H, dd, J = 1.9 and 6.0 Hz), 5.43 (1H, dd, J = 1.8 and 6.2 Hz), 4.70 (1H, d, J = 1.9 Hz), 4.61 (2H, m), 3.18 (2H, t, J = 6.7 Hz), 2.96 (2H, m), 2.31 (3H, s), 1.64 (3H, s), 1.31 (3H, s), 0.69 (3H, t, J = 7.3 Hz); HRMS (ESI-MS m/z) calcd for C₃₂H₃₆N₇O₇S (M+H)⁺ 662.2397, found 662.2374.

(2R, 3S, 4S, 5R)-5-Ethylcarboxyamide-2-(6′-amino -2′-(3″-(1″-(p-toluenesufonyl) indolyl)ethyloxy)-purin-9′-yl)-tetrahydrofuran (134)

A solution of 133 (13.4 mg, 0.0202 mmol) in 80 % acetic acid aqueous solution was stirred at 80 °C for 63 h and evaporated to give an oil, which was subjected to preparative TLC developed with a mixture of chloroform and methanol (8:1) to give 134 (9 mg, recovery yield 85 %).

¹H NMR (CD₃OD) δ 8.09 (1H, s), 7.94 (1H, d, J = 7.7 Hz), 7.69 (2H, d with small coupling, J = 8.5 Hz), 7.56 (1H, d with small coupling, J = 7.7 Hz), 7.52 (1H, s), 7.30 (1H, dt, J = 1.3 and 7.7 Hz), 7.16–7.26 (3H, m), 5.93 (1H, d, J = 7.4 Hz), 4.54–4.76 (2H, m), 4.42 (1H, d, J = 1.9 Hz), 4.31 (1H, dd, J = 1.8 and 4.8 Hz), 3.00–3.18 (4H, m), 2.28 (3H, s), 0.83 (3H, t, J = 7.1 Hz); HRMS (ESI-MS m/z) calcd for C₂₉H₃₂N₇O₇S (M+H)⁺ 622.2084, found 622.2095.

2-Phenylpropoxyadenosine (8)

The yield was 66 %. ¹H NMR (CD₃OD) δ 8.11 (1H, s), 7.10 – 7.28 (5H, m), 5.88 (1H, d, J = 6.1 Hz), 4.72 (1H, t, J = 5.5 Hz), 4.30 – 4.34 (1H overlaped with CH₂), 4.29 (2H, t, J = 6.6 Hz), 4.10 (1H, q, J = 3.2 Hz), 3.85 (1H, dd, J = 2.9 and 12.5 Hz), 3.72 (1H, dd, J = 3.3 and 12.4 Hz), 2.78 (2H, t, J = 7.7 Hz), 2.06 (2H, dt, J = 6.4 and 15.3 Hz); HRMS (ESI-MS m/z) calcd for C₁₉H₂₄N₅O₅(M+H)⁺ 402.1777, found 402.1771; HPLC (system A) 14.1 min (99%), (system C) 10.7 min (99%).

2-(3″-Indolylethyloxy)adenosine (17)

The yield was 72 %. ¹H NMR (CD₃OD) δ 8.12 (1H, s), 7.60 (1H, d with small coupling, J = 7.7 Hz), 7.32 (1H, d with small coupling, J = 7.7 Hz), 7.15 (1H, s), 7.07 (1H, dt, J = 1.2 and 7.5 Hz), 7.01 (1H, dt, J = 1.2 and 7.3 Hz), 5.90 (1H, d, J = 5.5 Hz), 4.71 (1H, t, J = 5.5 Hz), 4.58 (2H, m), 4.31 (1H, dd, J = 3.6 and 5.2 Hz), 4.11 (1H, q, J = 3.2 Hz), 3.85 (1H, dd, J = 2.8 and 12.4 Hz), 3.73 (1H, dd, J = 3.4 and 12.2 Hz), 3.24 (2H, t, J = 7.3 Hz); HRMS (ESI-MS m/z) calcd for C₂₀H₂₃N₆O₅ (M+H)⁺ 427.1730, found 427.1711; HPLC (system A) 11.4 min (99 %), (system C) 9.3 min (99 %).

2-(3″-(1″-(p-Toluenesulfonyl)indolyl)ethyloxy)adenosine (18)

The yield was 60 %. ¹H NMR (CD₃OD) δ 8.14 (1H, s), 7.93 (1H, d with small coupling, J = 7.4 Hz), 7.70 (2H, d with small coupling, J = 8.2 Hz), 7.59 (2H, m), 7.29 (1H, dt, J = 1.7 and 8.1 Hz), 7.23 (1H, dt, J = 1.5 and 8.1 Hz), 7.17 (2H, d, J = 8.0 Hz), 5.90 (1H, d, J = 6.0 Hz), 4.73 (1H, t, J = 5.6 Hz), 4.57 (2H, m), 4.33 (1H, dd, J = 3.2 and 5.1 Hz), 4.12 (1H, q, J = 3.0 Hz), 3.88 (1H, dd, J = 2.8 and 12.4 Hz), 3.74 (1H, dd, J = 3.3 and 12.4 Hz), 3.14 (2H, t, J = 6.3 Hz); HRMS (ESI-MS m/z) calcd for C₂₇H₂₉N₆O₇S (M+H)⁺ 581.1818, found 581.1797; HPLC (system B) 16.5 min (99%), (system C) 16.7 min(99%).

2-(3″-Pyrrolylethyloxy)adenosine (19)

The yield was 57 %. ¹H NMR (CD₃OD) δ 8.11(1H, s), 6.63(2H, d, J = 2.2 Hz), 6.04 (1H, t, J = 2.1 Hz), 5.88 (1H, d, J = 5.8 Hz), 4.72 (1H, t, J = 5.5 Hz), 4.42 (2H, t, J = 7.4 Hz), 4.31 (1H, dd, J = 3.4 and 5.1 Hz), 4.10 (1H, q, J =3.2 Hz). 3.85 (1H, dd, J = 3.0 and 12.4 Hz), 3.73 (1H, dd, J = 3.6 and 12.4 Hz), 2.91 (2H, t, J = 7.3 Hz); HRMS (ESI-MS m/z) calcd for C₁₆H₂₁N₆O₅ (M+H)⁺ 377.1573, found 377.1577; HPLC (system A) 4.5 min (99%), (system C) 4.7 min (99%).

2-(2″-Indolylethyloxy)adenosine (20)

The yield was 32 %. ¹H NMR (CD₃OD) δ 8.08 (1H, s), 7.37 (1H, d with small couplings, J = 7.4 Hz), 7.25 (1H, d with small coupling, J = 8.0 Hz), 6.97 (1H, dt, J = 1.4 and 7.1 Hz), 6.88 (1H, dt, J = 1.1 and 7.2 Hz), 6.21 (1H, d, J = 0.8 Hz), 5.86 (1H, d, J = 5.8 Hz), 4.69 (1H, t, J = 5.5 Hz), 4.57 (2H, t, J = 6.6 Hz), 4.30 (1H, dd, J = 3.3 and 5.2 Hz), 4.08 (1H, q, J = 3.3 Hz), 3.83 (1H, dd, J = 2.9 and 12.2 Hz), 3.72 (1H, dd, J = 3.3 and 12.4 Hz), 3.18 (2H, t, J = 6.7 Hz); HRMS (ESI-MS m/z) calcd for C₂₀H₂₃N₆O₅ (M+H)⁺ 427.1730, found 427.1735; HPLC (system A) 14.6 min (99 %), (system C) 10.9 min (99 %).

2-(2″-(1″-(p-Toluenesulfonyl)indolyl)ethyloxy)adenosine (21)

The yield was 55 %. ¹H NMR (CDCl₃) δ 8.10 (1H, d, J = 8.2 Hz), 7.57–7.64 (3H, m), 7.37 (1H, d, J = 7.7 Hz), 7.11–7.26 (4H, m), 6.52 (1H, s), 5.71 (1H, d, J = 6.9 Hz), 5.66 (2H, br s), 5.03 (1H, t, J = 6.9 Hz), 4.62 (1H, m), 4.62 (1H, m), 4.46 (2H, m), 4.27 (1H, s), 3.89 (1H, d, J = 11.5 Hz), 3.74 (1H, d, J = 11.5 Hz), 3.43 (2H, m), 3.19 (1H, br s), 2.30 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₇H₂₉N₆O₇S (M+H)⁺ 581.1818, found 581.1802; HPLC (system A) 23.0 min (99%), (system C) 16.7 min (99%).

2-(3″-(5″-Fluoro-indolyl)ethyloxy)adenosine (22)

The yield was 80 %. ¹H NMR (CD₃OD) δ 8.12 (1H, s), 7.20 ~ 7.30 (3H, m), 6.83 (1H, dt, J = 2.5 and 9.1 Hz), 5.89 (1H, d, J = 5.8 Hz), 4.72 (1H, t, J = 5.5 Hz), 4.55 (2H, t, J = 7.0 Hz), 4.31 (1H, dd, J = 3.3 and 5.2 Hz), 4.11 (1H, q, J = 3.3 Hz), 3.86 (1H, dd, J = 2.8 and 12.4 Hz), 3.73 (1H, dd, J = 3.6 and 12.4 Hz), 3.16 (2H, t, J = 7.1 Hz); HRMS (ESI-MS m/z) calcd for C₂₀H₂₁N₆O₅FNa (M+Na)⁺ 467.1455, found 467.1479; HPLC (system A) 13.1 min (99%), (system C) 10.2 min (99 %).

2-(3″-(6″-Chloro-indolyl)ethyloxy)adenosine (23)

The yield was 54 %. ¹H NMR (CD₃OD) δ 8.08 (1H, s), 7.52 (1H, d, J = 8.5 Hz), 7.27 (1H, d, J = 1.7 Hz), 7.13 (1H, s), 6.95 (1H, dd, J = 1.9 and 8.5 Hz), 5.86 (1H, d, J = 6.0 Hz), 4.67 (1H, t, J = 5.5 Hz), 4.51 (2H, m), 4.28 (1H, dd, J = 3.4 and 5.1 Hz), 4.07 (1H, q, J = 3.2 Hz), 3.81 (1H, dd, J = 2.8 and 12.4 Hz), 3.69 (1H, dd, J = 3.3 and 12.4 Hz), 3.14 (2H, t, J = 7.0 Hz); HRMS (ESI-MS m/z) calcd for C₂₀H₂₂N₆O₅Cl (M+H)⁺ 461.1340, found 461.1339; HPLC (system A) 15.7 min (99 %), (system C) 11.9 min (99 %).

2-(3″-(5″-Bromo-indolyl)ethyloxy)adenosine (24)

The yield was 70 %. ¹H NMR (CD₃OD) δ 8.12 (1H, s), 7.70 (1H, d, J = 1.9 Hz), 7.24 (1H, d, J = 8.8 Hz), 7.20 (1H, s), 7.15 (1H, dd, J = 1.8 and 8.7 Hz), 5.89 (1H, d, J = 5.8 Hz), 4.71 (1H, t, J = 5.5 Hz), 4.56 (2H, t, J = 7.0 Hz), 4.32 (1H, dd, J = 3.6 and 5.2 Hz), 4.11 (1H, q, J = 3.2 Hz), 3.86 (1H, dd, J = 2.8 and 12.4 Hz), 3.73 (1H, dd, J = 3.6 and 12.4 Hz), 3.17 (2H, t, J = 6.9 Hz); HRMS (ESI-MS m/z) calcd for C₂₀H₂₂N₆O₅Br (M+H)⁺ 505.0835, found 505.0822; HPLC (system A) 15.2 min(98 %), (system C) 12.2 min (98%).

2-(3″-(5″-Iodo-indolyl)ethyloxy)adenosine (25)

The yield was 56 %. ¹H NMR (CD₃OD) δ 8.12 (1H, s) 7.89 (1H, dd, J = 0.6 and 1.7 Hz), 7.32 (1H, dd, J = 1.7 and 8.5 Hz), 7.16 (1H, s), 7.15 (1H, dd, J = 0.6 and 8.5 Hz), 5.89 (1H, d, J = 5.8 Hz), 4.71 (1H, t, J = 5.5 Hz), 4.55 (2H, t, J = 7.0 Hz), 4.32 (1H, dd, J = 3.6 and 5.2 Hz), 4.11 (1H, q, J = 3.2 Hz), 3.86 (1H, dd, J = 2.8 and 12.4 Hz), 3.73 (1H, dd, J = 3.3 and 12.4 Hz), 3.16 (2H, t, J = 6.9 Hz); HRMS (ESI-MS m/z) calcd for C₂₀H₂₂N₆O₅I (M+H)⁺ 553.0696, found 553.0681; HPLC (system A) 19.0 min (98 %), (system C) 13.1 min (98 %).

2-(3″-(5″-Bromo-1″-(p-toluenesulfonyl)indolyl)ethyloxy)adenosine (26)

The yield was 68 %. ¹H NMR (CD₃OD) δ 8.14 (1H, s), 7.86 (1H, dd, J = 0.6 and 8.8 Hz), 7.68 – 7.74 (3H, m), 7.63 (1H, s), 7.40 (1H, dd, J = 1.8 and 8.8 Hz), 7.18 (2H, d with small coupling, J = 8.5 Hz), 5.89 (1H, d, J = 6.0 Hz), 4.73 (1H, t, J = 5.6 Hz), 4.55 (2H, t, J = 6.3 Hz), 4.33 (1H, dd, J = 3.3 and 5.2 Hz), 4.12 (1H, q, J = 3.0 Hz), 3.89 (1H, dd, J = 2.9 and 12.5 Hz), 3.74 (1H, dd, J = 3.3 and 12.4 Hz), 3.11 (2H, t, J = 6.2 Hz), 2.27 (3H, s); HRMS (ESI MS m/z) calcd for C₂₇H₂₈N₆O₇BrS (M+H)⁺ 659.0924, found 659.0921.

2-(3″-(5″-Chloro-indolyl)ethyloxy)adenosine (27)

The yield was 54 %. ¹H NMR (CD₃OD) δ 8.12 (1H, s), 7.55 (1H, d, J = 1.9 Hz), 7.28 (1H, dd, J = 0.5 and 8.5 Hz), 7.22 (1H, s), 7.03 (1H, dd, J = 1.9 and 8.5 Hz), 5.89 (1H, d, J = 6.0 Hz), 4.71 (1H, t, J = 5.5 Hz), 4.56 (2H, t, J = 7.0 Hz), 4.31 (1H, dd, J = 3.6 and 5.2 Hz), 4.11 (1H, q, J = 3.2 Hz), 3.86 (1H, dd, J = 2.9 and 12.5 Hz), 3.73 (1H, dd, J = 3.3 and 12.4 Hz), 3.17 (2H, t, J = 7.0 Hz); HRMS (ESI-MS m/z) calcd for C₂₀H₂₂N₆O₅Cl (M+H)⁺ 461.1340, found 461.1332; HPLC (system A) 16.6 min (99 %), (system C) 11.8 min (99 %).

2-(3″-(6″-Bromo-indolyl)ethyloxy)adenosine (28)

The yield was 71 %. ¹H NMR (CD₃OD) δ 8.12 (1H, s), 7.52 (1H, d, J = 8.5 Hz), 7.48 (1H, d, J = 1.7 Hz), 7.17 (1H, s), 7.12 (1H, dd, J = 1.7 and 8.5 Hz), 5.90 (1H, d, J = 5.8 Hz), 4.71 (1H, t, J = 5.5 Hz), 4.56 (2H, m), 4.31 (1H, dd, J = 3.3 and 5.2 Hz), 4.11 (1H, q, J = 3.2 Hz), 3.85 (1H, dd, J = 3.4 and 12.2 Hz), 3.73 (1H, dd, J =3.4 and 12.2 Hz), 3.21 (2H, t, J = 7.2 Hz); HRMS (ESI-MS m/z) calcd for C₂₀H₂₂N₆O₅Br (M+H)⁺ 505.0835 found 505.0840; HPLC (system A) 18.1 min (98 %), (system C) 12.6 min (98 %).

2-(3″-(4″-Bromo-indolyl)ethyloxy)adenosine (29)

The yield was 44 %. ¹H NMR (CD₃OD) δ 8.11 (1H, s), 7.31 (1H, dd, J = 0.8 and 8.0 Hz), 7.24 (1H, s), 7.16 (1H, dd, J = 0.8 and 7.4 Hz), 6.93 (1H, t, J = 7.8 Hz), 5.89 (1H, d, J = 6.0 Hz), 4.71 (1H, t, J = 5.5 Hz), 4.60 (2H, m), 4.31 (1H, dd, J = 3.6 and 5.0 Hz), 4.10 (1H, q, J = 3.2 Hz), 3.85 (1H, dd, J = 2.9 and 12.5 Hz), 3.73 (1H, dd, J = 3.4 and 12.5 Hz), 3.47 (1H, t, J = 7.1 Hz); HRMS (ESI-MS m/z) calcd for C₂₀H₂₂N₆O₅Br (M+H)⁺ 505.0835, found 505.0843; HPLC (system A) 15.3 min (99 %), (system C) 11.7 min (99 %).

2-(3″-(7″-Bromo-indolyl)ethyloxy)adenosine (30)

The yield was 27 %. ¹H NMR (CD₃OD) δ 8.12 (1H, s), 7.60 (1H, d, J = 8.0 Hz), 7.25 (1H, d, J = 7.4 Hz), 7.24 (1H, s), 6.95 (1H, t, J = 7.8 Hz), 5.90 (1H, d, J = 5.8 Hz), 4.71 (1H, t, J = 5.6 Hz), 4.58 (2H, m), 4.31 (1H, dd, J = 3.6 and 4.9 Hz), 4.11 (1H, q, J = 3.2 Hz), 3.85 (1H, dd, J = 2.9 and 12.2 Hz), 3.73 (1H, dd, J = 3.3 and 12.4 Hz), 3.20 (2H, t, J = 6.9 Hz); HRMS (ESI-MS m/z) calcd for C₂₀H₂₂N₆O₅Br (M+H)+ 505.0835, found 505.0837; HPLC (system A) 15.9 min (99 %), (system C) 12.1 min (99 %).

2-(3″-(5″-Methoxy-2″-methylindolyl)ethyloxy)adenosine (31)

The yield was 29 %. ¹H NMR (CD₃OD) δ 8.13 (1H, s), 7.10 (1H, dd, J = 0.6 and 8.8 Hz), 6.96 (1H, d, J = 2.2 Hz), 6.65 (1H, dd, J = 2.3 and 8.7 Hz), 5.90 (1H, d, J = 5.8 Hz), 4.68 (1H, t, J = 5.5 Hz), 4.47 (2H, m), 4.30 (1H, dd, J = 3.6 and 5.2 Hz), 4.10 (1H, q, J = 3.2 and 6.5 Hz), 3.84 (1H, dd, J = 2.9 and 12.2 Hz), 3.72 (1H, dd, J = 3.3 and 12.4 Hz), 3.13 (2H, t, J = 7.3 Hz), 2.37 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₂H₂₆N₆O₈Na (M+Na)⁺ 493.1812, found 493.1792; HPLC (system A) 11.8 min (98%), (system C) 9.4 min (98 %).

2-(3″-(5″–Methoxy-2″-methyl-1″-(p-toluenesulfonyl)indolyl)ethyloxy)adenosine (32)

The yield was 71 %. ¹H NMR (CD₃OD) δ 8.12 (1H,s), 7.96 (1H, d, J = 9.1 Hz), 7.52 (2H, d, J = 8.5 Hz), 7.11 (2H, d, J = 8.5 Hz), 6.92 (1H, d, J = 2.5 Hz), 6.82 (1H, dd, J = 2.5 and 9.1 Hz), 5.87 (1H, d, J = 6.0 Hz), 4.66 (1H, t, J = 5.6 Hz), 4.41 (2H, m), 4.29 (1H, dd, J = 3.2 and 5.1 Hz), 4.10 (1H, q, J =2.9 Hz), 3.82 (1H, dd, J = 2.8 and 12.4 Hz), 3.77 (3H, s), 3.70 (1H, dd, J = 3.2 and 12.5 Hz), 3.06 (2H, t, J = 6.6 Hz), 2.56 (3H, s), 2.21 (3H, s); HRMS (ESI-MS m/z) calcd for C₂₉H₃₃N₆O₈S (M+H)⁺ 625.2081, found 625.2079; HPLC (system B) 17.3 min (99 %), (system C) 17.6 min (99 %).

2-(3″-(5″-Methoxy-indolyl)ethyloxy)adenosine (33)

The yield was 54 %. ¹H NMR (CD₃OD) δ 8.12 (1H, s), 7.20 (1H, d, J = 8.8 Hz), 7.12 (1H, s), 7.05 (1H, d, J = 2.5 Hz), 6.73 (1H, dd, J = 2.5 and 8.8 Hz), 5.89 (1H, d, J = 5.8 Hz), 4.71 (1H, t, J = 5.5 Hz), 4.56 (2H, t, J =7.1 Hz), 4.31 (1H, dd, J =3.3 and 5.2 Hz), 4.10 (1H, q, J = 3.2 Hz), 3.84 (1H, dd, J = 2.8 and 12.4 Hz), 3.72 (1H, dd, J = 3.3 and 12.4 Hz), 3.18 (2H, t, J = 7.1 Hz), HRMS (ESI-MS m/z) calcd for C₂₁H₂₅N₆O₆ (M+H)⁺ 457.1836, found 457.1815; HPLC (system A) 10.7 min (98 %), (system C) 8.8 min (98%).

2-(3″-(5″-Hydroxyindolyl)ethyloxy)adenosine (34)

The yield was 31 %; ¹H NMR (CD₃OD) δ 8.12 (1H, s), 7.15 (1H, dd, J = 0.6 and 8.8 Hz), 7.09 (1H, s), 7.01 (1H, dd, J = 0.5 and 2.5 Hz), 6.65 (1H, dd, J = 2.3 and 8.4 Hz), 5.90 (1H, d, J = 5.8 Hz), 4.72 (1H, t, J = 5.6 Hz), 4.54 (2H, t, J = 7.4 Hz), 4.31 (1H, dd, J = 3.3 and 5.2 Hz), 4.11 (1H, q, J = 3.2 Hz), 3.86 (1H, dd, J = 2.7 and 12.4 Hz), 3.74 (1H, dd, J = 3.3 and 12.4 Hz), 3.13 (2H, t, J = 7.3 Hz); HRMS (ESI-MS m/z) calcd for C₂₀H₂₂N₆O₆Na (M+Na)⁺ 465.1499, found 465.1471; HPLC (system A) 5.5 min (98%), (system C) 5.3 min (98 %).

2-(3″-(Benzoimidazole-1″-yl)ethyloxy)adenosine (35)

The yield was 57 %. ¹H NMR (CD₃OD) δ 8.22 (1H, s), 8.10 (1H, s), 7.62 (2H, d with small coupling, J = 8.2 Hz), 7.31 (1H, dt, J = 1.2 and 7.6 Hz), 7.24 (1H, dt, J = 1.3 and 7.6 Hz), 5.84 (1H, d, J = 5.8 Hz), 4.63 – 4.74 (5H, m), 4.31 (1H, dd, J = 3.2 and 5.1 Hz), 4.11 (1H, q, J = 3.0 Hz), 3.86 (1H, dd, J = 2.8 and 12.4 Hz), 3.73 (1H, dd, J = 3.0 and 12.4 Hz); HRMS (ESI-MS m/z) calcd for C₁₉H₂₂N₇O₅ (M+H)⁺ 428.1682, found 428.1691; HPLC (system A) 4.9 min (99%), (system D) 8.3 min (99 %).

2-(3″-(Benzotriazole-1″-yl)ethyloxy)adenosine (36)

The yield was 40 %. ¹H NMR (CD₃OD) δ 8.10 (1H, s), 7.93(1H, dt, J =1.0 and 7.4 Hz), 7.80 (1H, dt, J =0.8 and 8.2 Hz), 7.52 (1H, ddd, J = 1.0, 7.1 and 8.1 Hz), 7.38 (1H, ddd, J =1.0, 7.1 and 8.1 Hz), 5.83 (1H, d, J = 5.8 Hz), 5.13 (2H, m), 4.82–4.92 (2H, m, overlapped with HDO), 4.64 (1H, t, J = 5.6 Hz), 4.31 (1H, dd, J = 3.3 and 5.2 Hz), 4.10 (1H, q, J = 3.1 Hz), 3.83 (1H, dd, J = 2.8 and 12.6 Hz), 3.72 (1H, dd, J = 3.3 and 12.4 Hz); HRMS (ESI- MS m/z) calcd for C₁₈H₂₁N₈O₅ (M+H)⁺ 429.1635, found 429.1642; HPLC (system A) 4.7 min (99 %), (system C) 4.8 min (99 %).

6-Guanidino-2-(3″-indolylethyloxy)adenosine (37) and 6-Guanidino-2-(3″-(1″-(p-Toluenesulfonyl)indolyl)ethyloxy)adenosine (38)

To a solution of guanidine hydrochloride (98 mg, 1.02 mmol) in acetonitrile (2.2 mL) and N, N-dimethylformamide (1.1 mL) was added sodium hydride (60 %) (41.2 mg, 1.02 mmol) at room temperature, the reaction mixture was stirred overnight. This guanidine solution was added to a mixture of compound 102 (46 mg, 0.0633 mmol) and 1,4- diazabicyclo[2.2.2]octane (14 mg, 0.0126 mmol), and the resulting mixture was stirred overnight at 110 °C and filtered. The filtrate was evaporated to give a crude oil, which was subjected to preparative TLC developed with a mixture of chloroform, methanol and saturated aqueous ammonia (2:1:0.3) to give 37 (2.3 mg, 8 %) and 38 (9 mg, 23 %) as an amorphous solid:

37

¹H NMR (CD₃OD) δ 8.09 (1H, s), 7.60 (1H, d with small coupling, J = 7.7 Hz), 7.32 (1H, d with small coupling, J = 7.2 Hz), 7.16 (1H, s), 7.08 (1H, dt, J = 1.3 and 7.6 Hz), 7.01 (1H, dt, J = 1.2 and 7.3 Hz), 5.90 (1H, d, J = 6.0 Hz), 4.73 (1H, t, J = 5.5 Hz), 4.54 (2H, t, J = 7.1 Hz), 4.32 (1H, dd, J = 3.3 and 4.9 Hz), 4.12 (1H, q, J = 3.0 Hz), 3.87 (1H, dd, J = 2.8 and 12.6 Hz), 3.73 (1H, dd, J = 3.3 and 12.4 Hz), 3.24 (2H, t, J = 7.4 Hz); HRMS (ESI-MS m/z) calcd for C₂₁H₂₅N₈O₅ (M+H)⁺ 469.1948, found 469.1952; HPLC (system B) 8.9 min (97 %), (system D) 7.5 min (97 %).

38

¹H NMR (CD₃OD) δ 8.25 (1H, s), 7.93 (1H, dd, J = 1.4 and 7.4 Hz), 7.71 (2H, d with small coupling, J = 8.5 Hz), 7.61 (1H, dd, J = 1.4 and 7.1 Hz), 7.59 (1H, s), 7.30 (1H, dt, J = 1.2 and 7.6 Hz), 7.24 (1H, dt, J = 1.2 and 7.6 Hz), 7.18 (2H, d with small coupling, J = 8.5 Hz), 5.96 (1H, d, J = 6.0 Hz), 4.74 (1H, t, J = 5.5 Hz), 4.59 (2H, t, J = 6.5 Hz), 4.35 (1H, dd, J = 3.4 and 5.1 Hz), 4.13 (1H, q, J = 3.2 Hz), 3.89 (1H, dd, J = 2.9 and 12.5 Hz), 3.76 (1H, dd, J = 3.6 and 12.4 Hz), 3.18 (2H, t, J = 6.5 Hz), 3.26 (3H, s); HRMS (ESIMS m/z) calcd for C₂₈H₃₁N₈O₇S (M+H)⁺ 623.2036, found 623.2045; HPLC (system A) 16.3 min (97 %), (system C) 8.7 min (97 %).

6-Ethylamino-2-(3″-indolyl)ethyloxy)adenosine (39)

To a solution of 102 (28.3 mg, 0.0389 mmol) in DMF (1.4 mL) in sealed tube were added ethylamine hydrochloride (63.5 mg, 0.779 mmol) and N, N-diisopropylethylamine (0.271 mL), and the reaction mixture was stirred at 140 °C overnight and evaporated to give an oil. The oil was dissolved in methanol (1 mL), KOH (14.6 mg, 0.261 mmol) was added, and the reaction mixture was stirred for 42 h at 80 °C. The solvent was evaporated to give a crude solid which was subjected to preparative TLC developed with a mixture of chloroform and methanol (5:1) to give 39 (2.2 mg, 28 % yield in two steps).

¹H NMR (CD₃OD) δ 8.05 (1H, s), 7.60 (1H, d with small coupling, J = 7.1 Hz), 7.32 (1H, d with small coupling, J = 7.7 Hz), 7.14 (1H, s), 7.07 (1H, dt, J = 1.3 and 7.6 Hz), 7.00 (1H, dt, J = 1.1 and 7.4 Hz), 5.87 (1H, d, J = 6.0 Hz), 4.71 (1H, t, J = 5.6 Hz), 4.60 (2H, t, J = 7.0 Hz), 4.30 (1H, dd, J = 3.2 and 5.1 Hz), 4.11 (1H, q, J = 3.0 Hz), 3.86 (1H, dd, J = 2.6 and 12.5 Hz), 3.73 (1H, dd, J = 3.2 and 12.5 Hz), 3.50–3.66 (2H, br m), 3.22 (2H, t, J = 7.1 Hz), 1.26 (3H, t, J = 7.3 Hz); HRMS (ESI-MS m/z) calcd for C₂₂H₂₇N₆O₅ (M+H)⁺ 455.2043, found 455.2063; HPLC (system A) 19.5 min (99 %), (system C) 13.5 min (99 %).

(2R, 3S, 4S, 5R)-6-Amino-5-Ethylcarboxyamide-2-(3″-indolyl)ethyloxy)-purin-9-yl)-tetrahydrofuran (40)

Potassium hydroxide (12.6 mg, 0.025 mmol) was added to a solution of 134 (7.0 mg, 0.0112 mmol) in methanol (1.5 mL), and the reaction mixture was stirred at 70 °C overnight. The mixture was evaporated to a small amount of solution which was subjected to preparative TLC developed with a mixture of chloroform and methanol (5:1) to give 40 (1.7 mg, 33% yield).

¹H NMR (CD₃OD) δ 8.06 (1H, s), 7.56 (1H, d with small coupling, J = 8.0 Hz), 7.32 (1H, d with small coupling, J = 8.0 Hz), 7.11 (1H, s), 7.08 (1H, dt, J = 1.2 and 8.1 Hz), 7.00 (1H, dt, J = 1.1 and 8.1 Hz), 5.91 (1H, d, J = 7.4 Hz), 4.74 (1H, dd, J = 4.8 and 7.3 Hz), 4.61 (2H, m), 4.41 (1H, d, J = 1.9 Hz), 4.30 (1H, dd, J = 1.7 and 4.9 Hz), 3.15–3.26 (4H, m), 0.99 (3H, t, J = 7.2 Hz); HRMS (ESI MS m/z) calcd for C₂₂H₂₆N₇O₅(M+H)⁺ 53 468.1995, found 468.2015; HPLC (system A) 15.7 min (98 %), (system C) 11.4 min (98 %).

Pharmacological Methods

[¹²⁵I]N⁶-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide (I-AB-MECA; 2000 Ci/mmol), [³H]CCPA (2-chloro-N⁶-cyclopentyladenosine, 42.6 Ci/mmol), [³H]CGS21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamido-adenosine, 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 ARs²⁶ were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, 2 μmol/mL glutamine and 800 μg/mL geneticin. 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. The protein concentration was measured using the Bradford assay.⁴³

Binding assay

Human A₁ and A_2A Receptors

For binding to human A₁ receptors, [³H]CCPA (1 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) in a total assay volume of 200 μL. Nonspecific binding was determined using 10 μM of CPA. For human A_2A receptor binding, membranes (20 μg/tube) from HEK-293 cells stably expressing human A_2A receptors were incubated with 15 nM [³H]CGS21680 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. Reaction was terminated by filtration with GF/B filters.

Human A³ Receptor

For competitive binding assay, each tube contained 100 μL membrane suspension (20 μg protein), 50 μL of [¹²⁵I]I-AB-MECA (0.5 nM), and 50 μL of increasing concentrations of the nucleoside derivative in Tris·HCl buffer (50 mM, pH 7.4) 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 25 °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.^{44, 45} CHO cells that expressed recombinant human 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 μM) and adenosine deaminase (3 units/mL). After 45 min forskolin (10 μM) was added to the medium, and incubation was continued an additional 15 min. The reaction was terminated upon removal of 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.

Statistical analysis

Binding and functional parameters were calculated using Prism 4.0 software (GraphPAD, San Diego, CA, USA). IC₅₀ values obtained from competition curves were converted to K_i values using the Cheng-Prusoff equation.⁴⁶ Data were expressed as mean ± standard error.

Molecular modeling

A published molecular model of the human A_2BAR in which (S)-PHPNECA was docked³⁸ was utilized to study the binding mode of 28. Toward this goal, the ribose and adenine moieties of 28 were superimposed upon the corresponding moieties of (S)-PHPNECA located inside the binding site. Then (S)-PHPNECA was removed from the receptor. The obtained complex of the A_2BAR with 28 was subjected to Monte Carlo Multiple Minimum (MCMM) calculations using MacroModel software.³⁸ The MCMM calculations were performed for 28 and all residues located within 5Å from the ligand, using a shell of residues located within 2Å. The following parameters were used: MMFFs force field, water was used as an implicit solvent, a maximum of 1000 iterations of the Polak-Ribier Conjugate Gradient (PRCG) minimization method was used with a convergence threshold of 0.05 kJ·mol⁻¹·Å⁻¹, the number of conformational search steps = 100, the energy window for saving structures = 1000 kJ·mol⁻¹.