Design of (N)-Methanocarba Adenosine 5′-Uronamides as Species-Independent A₃ Receptor-Selective Agonists

Abstract

2-Chloro-5′ -N-methylcarboxamidoadenosine analogues containing the (N)-methanocarba (bicyclo[3.1.0]hexane) ring system as a ribose substitute display increased selectivity as agonists of the human A₃ adenosine receptor (AR). However, the selectivity in mouse was greatly reduced due to an increased tolerance of this ring system at the mouse A₁AR. Therefore, we varied substituents at the N⁶ and C2 positions in search of compounds that have improved A₃AR selectivity and are species independent. An N⁶-methyl analogue was balanced in affinity at mouse A₁/A₃ARs, with high selectivity in comparison to the A_2AAR. Substitution of the 2-chloro atom with larger and more hydrophobic substituents, such as iodo and alkynyl groups, tended to increase the A₃AR selectivity (up to 430-fold) in mouse and preserve it in human. Extended and chemically functionalized alkynyl chains attached at the C2 position of the purine moiety preserved A₃AR selectivity more effectively than similar chains attached at the 3 position of the N⁶–benzyl group.

Adenosine is a protective mediator that has been described as a general endogenous signal for tissue protection and regeneration and even “the signal of life”.^1,2 Adenosine activates four different receptor subtypes - A₁, A_2A, A_2B, and A₃ - which are widely but differentially distributed throughout the body.³ The A₃ adenosine receptor (AR) is located in some neurons, astrocytes, various immune cell populations (neutrophils, eosinophils, mast cells) and potentially muscle cells and endothelial cells.^4-7 The A₃AR is coupled to inhibition of adenylate cyclase and also activates Akt and calcium mobilization.^8,9 Two potent and selective agonists of the A₃AR, IB-MECA 1a and Cl-IB-MECA 1b, are currently in Phase II clinical trials for the treatment of rheumatoid arthritis, several other autoimmune inflammatory diseases, and cancer.^10-12 Protective mechanisms in the envisioned disease applications of A₃AR agonists appear to be a downregulation of the NF-κB system, common to both arthritis and cancer treatments,¹² and a widespread correction of gene dysregulation induced in an inflammatory bowel model.¹³ Cardioprotection by selective A₃AR agonists has been extensively explored in various species.^14-16

Other selective A₃AR agonists have recently been reported, based on introduction of large substituents at the N⁶ and C2 positions and modification of the ribose ring, particularly at the 4′ and 5′ positions.^16-19 For example, the 4′ -thio analogue 2 is among the most selective known A₃AR agonists,17 and 3′ -amino substitution is possible.^å16 Carbocyclic nucleosides have also been developed as A₃AR agonists.^20-22 Conformationally constrained methanocarba (bicyclo[3.1.0]hexane) nucleoside analogues were used to determine that the biologically active conformation of the ribose ring, i.e. that required in order to bind to and activate the receptor, corresponds to a North (N) conformation. Furthermore, the addition of a 5′ -N-methyl or ethyl uronamide group assures that the efficacy of the nucleoside to activate the A₃AR is maintained in combination with various structural changes at the N⁶ and C2 positions, which modulate the AR selectivity profile and might otherwise reduce the A₃AR efficacy. The conformational flexibility and H-bonding in the region of the 5′ -uronamide are critical factors affecting the efficacy at the A₃AR, as deduced from structure activity relationship (SAR) studies and from rhodopsin-based dynamic molecular modeling and ligand docking.²³

In this study, we report that certain known A₃AR agonists are much less selective at the A₃AR in the mouse than in other species, such as human, and are therefore unsuitable for use alone as definitive pharmacological probes in mouse. Thus, additional A₃AR agonists displaying high affinity and selectivity that are independent of species are desired. We previously explored the SAR surrounding both N⁶ and C2 positions of 5′ -N-methylcarboxamido adenosine analogues that contained an (N)-methanocarba ring system as a ribose substitute, with the objective of designing potent, and selective A₃AR agonists.²² There is considerable evidence that appropriate modification of the N⁶ and C2 positions of nucleoside agonists is tolerated at the human A₃AR, while SAR at the mouse ARs has not been systematically explored.^{3,16-19,23-25}

Table 1 lists the structures of the adenosine derivatives that were assayed for binding affinity at ARs from several species. Compounds 3 – 12, previously synthesized and evaluated at human and rat ARs,^21,22 were evaluated at the mouse ARs. Based on indications that the 2-iodo derivative 12 maintained selectivity at the mouse A₃AR, compounds 13 – 19 and 25 – 27 were synthesized. Synthetic routes to the novel derivatives are shown in Schemes 1 and 2. In the synthetic route to each of the new A₃AR agonists, after substitution of the chlorine at C6 in the purine ring of 20 and 21 with a corresponding primary amine R¹NH₂, the 5′ -ester group was aminolyzed with methylamine solution. Resultant compounds of type 22 or 23 were either hydrolyzed to afford agonists 3-10, 12, 13, 19, or underwent Sonogashira coupling²⁶ with corresponding alkynes followed by hydrolysis to afford agonists 15 – 17. Agonist 14 was prepared from agonist 15 through desilylation with tetrabutylammnium fluoride. An N⁶-methoxy derivative 19, based on a recent report by Volpini et al. showing high potency at the human A₃AR of similar derivatives in the ribose series,18 was prepared by substitution of the 6-Cl of 20 with O-methylhydroxylamine followed by deprotection of the 2′ and 3′ hydroxyl groups.

Table 1.

Affinity of a series of (N)-methanocarba adenosine derivatives at three subtypes of ARs in various species. Compounds 1a and 1b are 9-riboside derivatives.

				Affinity			Selectivity
Compound	R1	R2	Species	A1a Ki, nM	A2Aa Ki, nM (or % inhib.)	A3a Ki, nM	A1/A3
1a			Mb,c	5.9	~1000	0.087	68
			H	49.3±3.7	93.1±4.2	1.74±0.36	28d
			Re	54	56	1.1	49
1b			Mb,c	35	~10,000	0.18	190
			Hd	220	5400	1.4	160
			Re	820	470	0.33	2500
3	CH3	Cl	M	55.3±6.0	20,400±3,200	49.0±3.9	1.1
			H	2100±1700	(6%)d,f	2.2±0.6	950
			R	805g	>10,000g	160±30d	5.0
4h	3-Cl-Bn	Cl	M	15.3±5.8	10,400±1,700	1.49±0.46	10.3
			Hc,d	260	2300	0.29	900
			Rd	ND	ND	1.0
5	3-Br-Bn	Cl	M	8.79±0.12	6,390±870	0.90±0.22	9.8
			Hc,d	270	1300	0.38	710
			Rc	ND	ND	0.76
6h	3-I-Bn	Cl	M	7.32±1.5	5,350±860	0.80±0.14	9.2
			Hc	136	784	1.5	91
			R	83.9g	1660g	1.1	76
7	3-(C≡C- CH2OH)-Bn	Cl	M	111±22	(11%)i	1.94±1.1	57.2
			Hd	2600±300	(56%)f	2.9±0.7	900
			Rd	ND	ND	1.6±0.6
8	2,5-(OCH3)2Bn	Cl	M	29.0±3.3	44,700±5,700	1.72±0.04	17
			Hc,d	1600	~10,000	1.4	1100
			Rd	ND	ND	0.87
9	CH2CH(Ph)2	Cl	M	6.83±1.5	1,810±581	1.67±0.09	4.1
			Hc,d	1300±100	1600±100	0.69±0.02	1900
			Rd	ND	ND	10±4
10	c-Pr-Ph	Cl	M	6.60±1 .3	38,200±5,300	2.79±0.89	2.4
			Hc,d	770±50	4800±200	0.78±0.06	990
11	3-Cl-Bn	SCH3	M	98.9±18.8	(32%)i	1.19±0.09	83
			Hd	610	~10,000	1.5	410
12h	3-Cl-Bn	I	M	210±34.4	(40%)i	1.18±0.11	178
			Hd	2200	>10,000	3.6	610
			Rd	ND	ND	3.9
13h	2,5-(OCH3)2Bn	I	M	293±29	(14%)i	1.51±0.36	194
			H	3070±820	(35%)f	1.30±0.27	2360
14	3-Cl-Bn	C≡CH	M	45.6±7.9	(41%)i	0.85±0.08	53.6
			H	174±23	(48%)f	1.30±0.38	134
15	3-Cl-Bn	C≡C- Si(CH3)3	M	159±22	(20%)i	4.46±0.57	35.6
			H	160±40	(52%)f	0.98±0.14	160
16	3-Cl-Bn	C≡C(CH2)2- CH3	M	1390±430	(42%)i	6.06±1.21	229
			H	1040±83	(80%)f	0.82±0.20	1300
17	3-Cl-Bn	C≡C(CH2)3- COOCH3	M	1340±330	(50%)i	4.65±0.53	288
			H	482±23	(49%)f	1.17±0.27	412
18ah	3-Cl-Bn	C≡ C(CH2)3- COOH	M	10,500±1900	(8%)i	24.4±3.1	431
			H	14,900±3500	(43%)f	2.38±0.56	6260
18bh	3-Cl-Bn	C≡ C(CH2)3- CONH-	M	546±62	(31%)i	8.60±1.02	64
			H	454±44	(81%)f	2.17±0.51	209
		(CH2)2NH2
19	OCH3	Cl	M	1,160±130	(2%)f	877±149	1.3
			H	265±45	(2%)f	149±15	1.8
25	3-(C≡ C(CH2)3- COOH)-Bn	Cl	M	703±71	(5%)i	14.4±2.5	49
			H	320±31	(14%)f	17.1±1.2	19
26	3-(C≡ C(CH2)3- CON(CH2)2- NH2)-Bn	Cl	M	151±18	(39%)i	11.9±2.4	13
			H	271±23	(58%)f	5.21±0.91	52
27	3-(C≡ C(CH2)3- CONH- (CH2)2NH- COCH3)-Bn	Cl	M	45.4±3.4	(68%)i	4.65±0.22	9.8
			H	181±22	(80%)f	2.88±0.54	63

^aCompetition radioligand binding assays using [¹²⁵I]N⁶-(4-amino-3-iodobenzyl)adenosine-5′ -N-methyl-uronamide (A₁ and A₃ARs) and [³H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′ -N-ethylcarboxamidoadenosine (A_2AAR) were conducted with membranes prepared from HEK293 cells expressing recombinant mouse A₁, A_2A, or A₃ARs. At rat and human ARs, the A₁ radioligand was either [³H]R-phenylisopropyladenosine or [³H]2-chloro-N⁶-cyclopentyladenosine. Values are expressed as the mean ± SEM. ND, not determined.

^bData from Ge et al.¹⁵

^cEC₅₀ value in activation of the A_2BAR (human or mouse, as indicated) is ≥10 μM.¹⁵,²²

^dData from Tchilibon et al.²²

^eData from Kim et al.²⁴

^fPercent inhibition at 10 μM.

^gData from Lee et al.²¹

ⁱPercent inhibition at 100 μM.

^h4, MRS3558; 6, MRS1898; 12, MRS3609; 13, MRS5128; 18a, MRS5151; 18b, MRS5166.

Some of the 2-alkynyl derivatives contained chemically functionalized, extended alkynyl chains to serve as functionalized congeners for conjugation to other biologically active moieties or to carriers.^27-29 These included carboxylic acids 18a and 25, amines 18b and 26, and an acetylamino derivative 27. The Sonogoshira reaction sequence using methyl hexynoate in combination with an iodo-derivatized nucleoside, either a 2-iodo (Scheme 1) or N⁶-(3-iodobenzyl) (Scheme 2) derivative, provided a mixture of the product methyl ester and corresponding carboxylic acid, which were separated by silica gel column chromatography. The primary amine congeners 18b and 26 were prepared by treatment of the appropriate methyl ester with excess ethylenediamine.

Scheme 2.

^a Synthesis of novel (N)-methanocarba A₃AR agonists containing functionalized alkynyl chains attached at the N⁶-benzyl 3 position. Note that 24 and 25 were both isolated chromatographically from the same reaction.

^a Reagents & conditions: a) HC≡C(CH₂)₃COOCH₃, PdCl₂(PPh₃)₂, CuI, DMF, Et₃N; b) TFA, H₂O, MeOH, Δ; c) ethylenediamine; d) acetic anhydride.

Binding assays were carried out using standard radioligands in Chinese hamster ovary (CHO) cells expressing the human A₁ or A₃ARs and in HEK293 cells expressing the human A_2AAR.²² Also, the mouse ARs were expressed in HEK293 cells for binding assays.¹⁵ A functional assay in guanine nucleotide binding ([³⁵S]GTPγS)^20,31 in membranes of CHO cells expressing the human A₃AR showed that 18a is a full agonist. The pEC₅₀ value of 18a was 7.85 ± 0.15, in comparison to the pEC₅₀ value of NECA of 6.46 ± 0.13.

The binding affinity at the mouse and rat A₃ARs of the N⁶-methyl derivative 3 was considerably weaker than at the human A₃AR (Table 1). Thus, this compound was balanced in affinity at mouse A₁/A₃ARs, with high selectivity in comparison to the mouse A2AAR. Other agonists having this mixed AR selectivity were explored for their cardioprotective properties.²⁹

N⁶-Benzyl derivatives of adenosine have previously been shown to favor selectivity at the A₃AR.²⁴ This observation led to the design of 1a and 1b. Although these two 9-riboside derivatives maintain selectivity for the mouse A₃AR, the selectivity in mouse of similar N⁶-substituted benzyl (N)-methanocarba derivatives 4 – 6 was greatly reduced due to increased tolerance of this bicyclic ring system at the mouse A₁AR. Therefore, we varied substituents at the N⁶ and C2 positions in an effort to reduce affinity at the mouse A₁AR. Extension of the 3-benzyl group as an alkyne in 7 reduced mouse A₁ and A_2AAR affinity without greatly reducing affinity at the mouse, rat, or human A₃ARs, resulting in a 57- fold selectivity for the mouse A₃AR. A 2,5-dimethoxy substitution in 8 showed only a slight enhancement of mouse A₃AR selectivity in comparison to 6, the (N)-methanocarba equivalent of Cl-IB-MECA. Modified N⁶-phenylethyl analogues 9 and 10, although both very potent at the mouse A₃AR, were essentially nonselective in comparison to the mouse A₁AR.

Modification at the adenine C2 position was generally more beneficial than structural changes of the N⁶ substituent, with respect to mouse A₃AR selectivity. 2-Cl was replaced with small hydrophobic groups,²² which greatly increased the selectivity for the mouse A₃AR. For example, a 2-iodo analogue 12 was 178-fold and >180,000-fold selective in binding to the mouse A₃AR in comparison to mouse A₁ and A_2AAR, respectively, with a K_i value of 1.18 nM. The corresponding 2-iodo-N⁶-(2,5-dimethoxybenzyl) analogue 13 was similarly selective. Thus, the 2-iodo modification resulted in increased selectivity for the mouse A₃AR; i.e., the selectivity ratio of 2-iodo compounds (12 and 13) was increased over that of the corresponding 2-chloro analogues (4 and 8, respectively) by 11 – 17-fold. Other hydrophobic substituents at the C2 position, such as an ethynyl group in 14 and its trimethylsilyl adduct 15, provided moderate mouse A₃AR selectivity. Flexible, extended ethynyl chains in 16 and 17 increased the ratio of A₃AR selectivity. 2-Alkynyl groups were previously reported to enhance affinity of adenosine derivatives at the rat and human A₃ARs.^25,30 Compound 18a, a long chain carboxylic acid congener, and its corresponding methyl ester 17 were highly selective for the mouse A₃AR in comparison to the A₁AR by 431- and 288-fold, respectively. Both compounds had greater A₃AR selectivity than Cl-IB-MECA 1b, although they were less potent. A primary amino congener 18b displayed moderate A₃AR selectivity, with a K_i value of 8.6 nM. Functionalized congeners in which a functionalized ethynyl chain was positioned on the N⁶-benzyl moiety were only moderately (a carboxylic acid 25) or weakly (a primary amine 26 and its acetyl analogue 27) selective for the mouse A₃AR. Thus, the attachment of alkynyl chains at the 3 position of the N⁶-benzyl moiety did not preserve A₃AR selectivity as well as the placement of similar chains at the adenine C2 position.

The use of an alkynyl substitutent at the 3-position of an N⁶-benzyl group, to serve as a covalent linking site for conjugation, was shown previously to maintain A₃AR affinity in the series of 9-ribosides.^28,29 In the present study of the (N)-methanacarba series, a variety of adenosine derivatives bearing a 2-alkynyl group have been shown to bind potently and selectively to the A₃AR.

The (N)-methanacarba derivatives that were most potent (K_i 1 – 6 nM) and selective (180 – 290-fold) in binding to the mouse A₃AR were, in order of decreasing affinity: 12, 13, 17, and 16. Compound 18a was the most selective novel agonist in this study at the mouse A₃AR; however, the affinity was intermediate, with a K_i value of 24 nM. The selectivity for the human A₃AR was >6000-fold. Thus, these C2 position-modified bicyclic nucleosides are good candidates for species-independent A₃AR agonists.

In conclusion, the selectivity (but not affinity) of (N)-methanocarba-containing nucleosides as A₃AR agonists was greatly reduced in the mouse due to increased tolerance of this ring system at the mouse A₁AR. Several analogues having varied substitution at the N⁶ and C2 positions were balanced in affinity at mouse A₁/A₃ARs, with high selectivity in comparison to the A_2AAR. Substitution of the 2-chloro atom with larger and more hydrophobic substituents, such as iodo and alkynyl groups, tended to increase the A₃AR selectivity in mouse and preserve it in human. The carboxylic acid 18a and primary amino 18b derivatives are good candidates for use as functionalized congeners for covalent conjugation with retention of biological activity and receptor selectivity. Thus, we have identified novel (N)-methanocarba nucleosides that are A₃AR- selective across several species and are especially suitable for pharmacological studies in the mouse.

Supplementary Material

01. Supplementary data.

Supplementary data (chemical synthesis, additional pharmacological procedures, and functional assay of 18a) associated with this article can be found, in the online version, at doi:.

Chart 1.

Prototypical selective A₃AR agonists.

Scheme 1.

^a Synthesis of novel (N)-methanocarba A₃AR agonists with structural variation at the 2 and N⁶ positions. Note that 17 and 18a were both isolated chromatographically from the same reaction.

^a Reagents & conditions: a) R¹NH₂; b) MeNH₂, EtOH; c) RC≡CH, PdCl₂(PPh₃)₂, CuI, DMF, Et₃N; d) TFA, MeOH, H₂O, Δ; e) TBAF, THF; f) ethylenediamine, MeOH.

ABBREVIATIONS

AR

adenosine receptor

CGS21680

2-[p-(2-carboxyethyl)phenylethylamino]-5′ -N-ethylcarboxamido-adenosine

CHO

Chinese hamster ovary

Cl-IB-MECA

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

CPA

N⁶-cyclopentyladenosine

DMEM

Dulbecco’s modified Eagle’s medium

I-AB-MECA

N⁶-(4-amino-3-iodobenzyl)-5′ -N-methylcarboxamidoadenosine

NECA

5′ -N-ethylcarboxamidoadenosine