Structure–activity relationships of 2-chloro-N⁶-substituted-4′-thioadenosine-5′-N,N-dialkyluronamides as human A₃ adenosine receptor antagonists

Abstract

On the basis of potent and selective A₃ adenosine receptor (AR) antagonist, 2-chloro-N⁶-(3-iodobenzyl)-4′-thioadeno-sine-5′-N,N-dimethyluronamide, structure–activity relationships were studied for a series of 5′-N,N-dialkyluronamide derivatives, synthesized from d-gulonic γ-lactone. From this study, it was revealed that removal of the hydrogen bond-donating ability of the 5′-uronamide was essential for the pure A₃AR antagonism. 5′-N,N-Dimethyluronamide derivatives exhibited higher binding affinity than larger 5′-N,N-dialkyl or 5′-N,N-cycloalkylamide derivatives, indicating that steric factors are crucial in binding to the human A₃AR. A N⁶-(3-bromobenzyl) derivative 6c (K_i = 9.32 nM) exhibited the highest binding affinity at the human A₃AR with very low binding affinities to other AR subtypes.

Adenosine (1) regulates many physiological functions through four subtypes (A₁, A_2a, A_2b, and A₃) of adenosine receptors (AR).¹ Among these subtypes, activation of the A₃AR inhibits adenylate cyclase to lower the level of cAMP.¹ Also, activation of the A₃AR stimulates phospholipase C (PLC) through the β, γ subunit of the G protein, to increase levels of inositol 1,4,5-tris-phosphate (IP₃). Control of A₃AR is thought to be relevant to experimental treatment modalities for a variety of disorders related to these signal transduction pathways, such as cancer, cerebral or cardiac ischemia, glaucoma, allergic conditions, and inflammation.^1,2

On the basis of the structure of adenosine (1), worldwide efforts have been made to discover potent and selective human A₃AR agonists and antagonists.^2,3 Modification on the N⁶- and/or 4′-hydroxymethyl group of 1 resulted in 2 (Cl-IB-MECA) as a potent and selective human A₃AR full agonist⁴ (Chart 1). Bioisosteric replacement of the furanose oxygen of 2 with a sulfur atom produced another potent and selective human A₃AR full agonist 3 (LJ-529).⁵ However, until recently it has been difficult to discover potent and selective human A₃AR full antagonists with a nucleoside skeleton because of the tendency of derivatives of adenosine (1) to act as full agonists. Thus, most of the potent and selective antagonists for the human A₃AR belong to the category of nonpurine heterocyclic compounds,^6–10 which act competitively with adenosine derivatives at the orthosteric binding site of the A₃AR. However, these nonpurine heterocyclic compounds showed weak binding affinity at the rat A₃AR^11,12 and were unsuitable for efficacy evaluation in small animal models and thus not optimal for further drug development. Therefore, it has been highly desirable to discover potent and selective A₃AR full antagonists of which the affinity and selectivity are independent of species. Since it was reported that antagonists with a nucleoside skeleton¹³ could be species-independent, potent, and selective A₃AR full antagonists, we searched for novel nucleoside analogues, among which appending an additional methyl group to the 5′-uronamide nitrogen of compounds 2 and 3 converted these A₃AR full agonists into the potent and selective A₃AR full antagonists 4 and 5.¹⁴ 4′-Thionucleoside analogue 5 was found to be a more potent and selective antagonist than the corresponding 4′-oxonucleoside analogue 4.¹⁴ Thus, on the basis of these findings, we modified the N⁶- and 4′-hydroxymethyl groups of 4′-thionucleoside analogue 5 in order to search for potent and selective A₃AR full antagonists. Herein, we report the structure–activity relationship (SAR) study of 2-chloro-N⁶-substituted-4′-thioadenosine-5′-N,N-dialkylamides at the human A₃AR.

Chart 1.

Synthesis of the key intermediates 11a–j started from d-gulonic γ-lactone (7), as shown in Scheme 1.

Scheme 1.

Reagents and conditions: (a) 2,6-dichloropurine, TMSOTf, Et₃N, CH₃CN–ClCH₂CH₂Cl, rt to 83 °C; (b) R¹NH₂, Et₃N, EtOH, rt; (c) 80% AcOH, 70 °C; (d) TBSOTf, pyridine, 50 °C; (e) NaOMe, MeOH. *N⁶ (CH₃)₂.

d-Gulonic γ-lactone (7) was converted to the glycosyl donor 8, using our previously published procedure.^5,15 Pummerer-type condensation of 8 with 2,6-dichloropurine in the presence of TMSOTf and Et₃N afforded the desired nucleoside 9^5,15 as a single diastereomer.

Treatment of 2,6-dichloropurine derivative 9 with various alkyl or arylalkyl amines gave N⁶-substituted analogues 10a–j. Because of the difficulty in removing the isopropylidene group at the final step, the isopropylidene intermediates 10a–j were converted to the TBS derivatives by treatment with 80% acetic acid followed by reprotection with TBSOTf. The resulting compounds were treated with sodium methoxide to give the key intermediates 11a–j.

Conversion of 11a–j to the target nucleosides 6a–r is illustrated in Scheme 2. The primary hydroxyl groups of 11a–j were oxidized by treatment with PDC in DMF to the carboxylic acids 12a–j, which were converted to various tertiary amides 6a–r¹⁶ by coupling the acids 12a–j with various dialkyl or cycloalkyl amines in the presence of EDC and HOBt.

Scheme 2.

*N⁶ (CH₃)₂.

Binding assays were carried out using standard radioligands in Chinese hamster ovary (CHO) cells stably expressing a human AR subtype,^14,17 and binding affinities of the final nucleosides 6a–r at the three subtypes of human ARs are shown in Table 1. In general, 5′-N,N-dimethylamide derivatives exhibited higher binding affinity than larger 5′-N,N-dialkyl or 5′-N,N-cycloalkylamide derivatives indicating that steric factors are crucial in binding to human A₃AR. The effects on the binding affinity of various N⁶-substituted analogues also having a 5′-N,N-dimethyl group at the 5′-uronamide position were examined. The N⁶-(3-halobenzyl) series generally showed better binding affinity at the human A₃AR than the N⁶-dialkyl or N⁶-cycloalkyl series. Within the N⁶-(3-halobenzyl) series, the rank order of binding affinity at the human A₃AR was 3-bromobenzyl > 3-iodobenzyl > 3-chlorobenzyl > 3-fluorobenzyl. This result indicated that the human A₃AR prefers bromo as an optimal size of 3-halo substitution, and larger (I) or smaller (F) substitution reduced binding affinity. Among compounds tested, compound 6c (K_i = 9.32 nM) exhibited the highest binding affinity at the human A₃AR with very low binding affinities to other AR subtypes.

Table 1.

Potency of 2-chloro-4′-thioadenosine-5′-dialkylamide derivatives in binding at human A₁, A_2A, and A₃ARs expressed in CHO cells

Compound No. (R1,R2,R3)	Ki (nM ± SEM) or % inhibition at 10 μMa
	hA1ARb	hA2AARb	hA3ARb
2 c	222 ± 22	5360 ± 2470	1.4 ± 0.3
3 c	193 ± 46	223 ± 36	0.38 ± 0.07
4 d	5870 ± 930	>10,000	29.0 ± 4.9
5d (3-iodobenzyl, Me, Me)	6220 ± 640	>10,000	15.5 ± 3.1
6a (3-fluorobenzyl, Me, Me)	(16 ± 4%)	(1%)	121 ± 11
6b (3-chlorobenzyl, Me, Me)	(10 ± 8%)	(−1%)	21.3 ± 2.1
6c (3-bromobenzyl, Me, Me)	(37 ± 2%)	(7%)	9.32 ± 3.20
6d (Me2, Me, Me)e	(8 ± 2%)	(−2%)	136
6e (2-methoxyethyl, Me, Me)	(−7 ± 7%)	(1%)	425
6f (cyclopropyl, Me, Me)	(7 ± 16%)	(−8 ± 4%)	109 ± 16
6g (cyclopropylmethyl, Me, Me)	404 ± 120	(0 ± 6%)	30.7 ± 9.4
6h (cyclobutyl, Me, Me)	345 ± 152	(10%)	115 ± 50
6i (cyclopentyl, Me, Me)	(18 ± 21%)	(4 ± 27%)	837
6j (3-iodobenzyl, Me, Propyl)	(50 ± 1%)	(5%)	727
6k (3-iodobenzyl, Me, CH2CH2OH)	237 ± 6	(13%)	126 ± 17
6l (3-iodobenzyl, Et, phenyl)	(18 ± 5%)	(8%)	398
6m (3-iodobenzyl, piperidine)	(27 ± 5%)	(7%)	565
6n (3-iodobenzyl, 4-methylpiperazine)	(19 ± 5%)	(−7%)	667
6o (3-iodobenzyl, azetidine)	(8 ± 6%)	(12%)	43.4 ± 2.6
6p (3-iodobenzyl, pyrrolidine)	(20 ± 3%)	(14%)	117 ± 31
6q (3-iodobenzyl, 4-hydroxypiperidine)	(16 ± 7%)	(10%)	1530
6r (3-iodobenzyl, thiomorpholine)	(19 ± 9%)	(15%)	867

^aAll experiments were done on CHO cells stably expressing one of three subtypes of human ARs. The binding affinity for A₁, A_2A, and A₃ARs was expressed as K_i values and was determined by using agonist radioligands ([³H]CCPA), ([³H]CGS21680), [¹²⁵I]I-AB-MECA, respectively. Values in parentheses are for weak binding, corresponding to an IC₅₀ ⩾ 10 μM. Data are expressed as means ± standard error.

^bK_i in binding, unless noted.

^cK_i values from Ref. 5.

^dK_i values from Ref. 14.

^eN⁶ (CH₃)₂.

The functional efficacy of compounds 5¹⁴ and 6c showed that high binding affinity at human A₃AR was determined by inhibition of forskolin-stimulated cAMP production in AR-transfected CHO cells and measured at a concentration of 10 μM, in comparison to the maximal effect of a full agonist, N-ethyl-5′-N-ethylcarboxamidoadenosine (NECA), at 10 μM. In these functional assays, A₃AR agonism was absent in these compounds, indicating that these analogues are pure A₃AR antagonists, while the concentration–response curve for Cl-IB-MECA (2) indicated full agonism, as previously reported,⁴ with an EC₅₀ of 1.2 nM at the human A₃AR. To probe species differences, the affinity of N,N-5′-dimethylamide derivative 5 was also measured at the rat A₃AR. Although the affinity decreased with respect to the affinity at the human A₃AR, this compound showed moderate affinity (K_i = 321 ± 74 nM) at the rat A₃AR.¹⁴

Molecular modeling of the A₃AR indicated that the hydrogen bond-donating ability of the 5′-uronamide is responsible for the conformational change needed for receptor activation.^18,19 Because the synthesized 5′-N,N-dialkyl derivatives which removed the hydrogen bond-donating ability of the 5′-uronamide could not lead to the conformational change, resulting in the loss of ability to activate the A₃AR, the 5′-N,N-dialkyl series of compounds act as pure antagonists.

In summary, on the basis of potent and selective human A₃AR antagonism of the dimethyl derivative 5, we carried out structure–activity relationship studies of 2-chloro-N⁶-substituted-4′-thioadenosine-5′-N,N-dial-kylamides. From this study, we identified compound 6c as a highly potent and selective human A₃AR antagonist, in which removal of the hydrogen bond-donating ability of the 5′-uronamide was essential for the pure A₃AR antagonism. 5′-N,N-dimethyluronamide derivatives generally exhibited higher binding affinity than larger 5′-N,N-dialkyl or 5′-N,N-dicycloalkylamide derivatives, indicating that steric factors in the 5′ region are important in binding of nucleosides to the human A₃AR. The newly synthesized A₃AR antagonists studied here could be evaluated in models of a number of disorders related to the A₃AR, such as glaucoma, inflammation, and asthma.