Structure–activity relationships of thiazole and thiadiazole derivatives as potent and selective human adenosine A₃ receptor antagonists

Abstract

4-(4-Methoxyphenyl)-2-aminothiazole and 3-(4-methoxyphenyl)-5-aminothiadiazole derivatives have been synthesized and evaluated as selective antagonists for human adenosine A₃ receptors. A methoxy group in the 4-position of the phenyl ring and N-acetyl or propionyl substitutions of the aminothiazole and aminothiadiazole templates displayed great increases of binding affinity and selectivity for human adenosine A₃ receptors. The most potent A₃ antagonist of the present series, N-[3-(4-methoxy-phenyl)-[1,2,4]thiadiazol-5-yl]-acetamide (39) exhibiting a K_i value of 0.79 nM at human adenosine A₃ receptors, showed antagonistic property in a functional assay of cAMP biosynthesis involved in one of the signal transduction pathways of adenosine A₃ receptors. Molecular modeling study of conformation search and receptor docking experiments to investigate the dramatic differences of binding affinities between two regioisomers of thiadiazole analogues, (39) and (42), suggested possible binding mechanisms in the binding pockets of adenosine receptors.

1. Introduction

Extracellular adenosine regulates a number of physiological functions by activation of specific cell membrane receptors, classified as A₁, A_2A, A_2B, and A₃ receptors, which belong to the super family of G-protein coupled receptors.^1,2 Adenosine A₃ receptors, linked to Gi proteins decreasing cAMP production upon receptor activation, have been determined to show important roles in physiological systems, including protective actions against myocardial and brain ischemia,^3,4 antiproliferative effects through cell cycle arrest and the Wnt signaling pathway,^5-7 and airway inflammation with high receptor expression in lung mast cells in mice,^8,9 implicating a potential drug target for ischemia, cancer and asthma. Furthermore, A₃ receptor antagonists may be useful in the treatment of glaucoma.¹⁰

Since the cloning of adenosine A₃ receptors from several species in the early 1990s, many efforts have been devoted to develop agonists and antagonists of the receptors, resulting in the design of potent and selective antagonists. Unlike typical xanthine core antagonists of other adenosine receptor subtypes, several diverse heterocyclic compound classes of selective antagonists for A₃ receptors have been developed. Triazolonaphthyridine,¹¹ 1,4-dihydropyridines,^12,13 pyridines,^14,15 pyrans,¹⁶ triazoloquinazolines,^17,18 flavonoids,¹⁹ triazolopyrimidines,^20,21 triazolopurines,²² isoquinolines and quiazolines,²³ imidazopurinones,²⁴ pyridopurinediones,²⁵ have been synthesized and identified as adenosine A₃ receptor antagonists. Recently, thiazole and thiadiazole analogues have been described as the possible core skeletons of A₃ receptor antagonists with moderate affinity and selectivity.^26,27 In this study, we report new findings of great improvement of the scaffold derivatives with subnanomolar affinity at human adenosine A₃ receptors and high subtype selectivity through an SAR (structure–activity relationship) study combined with molecular modeling approaches.

2. Results and discussion

2.1. Chemistry

The general syntheses of various regioisomers of 4-(4-methoxyphenyl)-2-aminothiazole and 3-(4-methoxyphenyl)-5-aminothiadiazole derivatives are depicted in Schemes 1–3. 2-Aminothiazole analogues (3a–d) were prepared from the cyclization of 2-thiocyanoacetophenones in the presence of alumina supported ammonium acetate as described by Kodomari et al. (Scheme 1).²⁸

Scheme 1.

Synthesis of 2-aminothiazole skeleton.

Scheme 3.

Synthesis of 5-amino[1,3,4]thiadiazole skeleton.

In order to synthesize the 5-amino[1,2,4]thiadiazole derivative 7, 4-methoxybenzonitrile was converted to the 4-methoxybenzamidine hydrochloride derivaive 5, followed by a cyclization reaction using perchloromethyl mercaptan²⁹ in basic aqueous solution to afford the chlorothiadiazole intermediate 6. Substitution reaction of 6 with ammonia and methylamine gave 7 and 8, respectively (Scheme 2).

Scheme 2.

Synthesis of 5-amino[1,2,4]thiadiazole skeleton.

Another regioisomer, 5-amino[1,3,4]thiadiazole derivative 10 was prepared from the reaction of p-anisaldehyde with thiosemicarbazide to give intermediate 9, followed by cyclization in the presence of ferric chloride in aqueous solution (Scheme 3).³⁰ The synthesized and commercially available aminothiazole and aminothiadiazole compounds were subjected in the standard acylation protocols including coupling reagents with acids or anhydrides, or acid chlorides with pyridine as base to afford various derivatives 11–43 to be tested in adenosine receptor binding assays.

3. Biological activity

The binding affinity of each compound at four different adenosine receptor subtypes was determined as K_i values in radioligand binding assays at rat cortical or recombinant human A₁ receptors versus [³H]-(R)-PIA([³H]-(R)-N⁶-(phenylisopropyl)adenosine) and recombinant human A_2A receptors versus [³H]-CGS 21680 ([³H]-2-[4-(2-carboxyethyl)phenyl]ethylamino-5′-(N-ethylcarbamoyl)adenosine).^31,32 Affinity at recombinant human A₃ receptors expressed in CHO cells³³ was determined using [¹²⁵I]AB-MECA (N⁶-(4-amino-3-[¹²⁵I]iodobenzyl)-5′-(N-methyl carbamoyl)-adenosine.³⁴

The representative thiadiazole analogue reported earlier by the IJzerman’s group²⁷ was N-(3-phenyl-1,2,4-thiadiazole-5-yl)-4-methoxybenzamide (LUF5417) with Ki values of 0.032, 2.3, and 0.082 μM at rat A₁, rat A_2A, and human A₃ receptors, respectively. In the course of SAR studies of the derivatives of thiazole and thiadiazole templates, we found that 4-methoxyphenyl and aliphatic acyl group substitutions displayed large enhancement of the affinity and selectivity for human adenosine A₃ receptors compare to the parent compound, LUF5417. For example, 11, a 5-acetamido analogue of phenylthiazole showed a K_i value of 18.3 nM at human adenosine A₃ receptors with high selectivity versus human A₁ and A_2A receptors and 16 with 4-methoxy substitution of the phenyl group of 11, displayed a 6-fold increase of binding affinity at A₃ receptors with a K_i value of 3 nM, while maintaining high selectivity (Table 1). Structural variations and binding assays following aliphatic and aromatic acyl substitutions at the 5-amino group identified a slightly more potent analogue, 20, which contained one more methylene unit than 16 and displayed a K_i value of 2.4 nM. Although most of the aromatic acyl substituted derivatives 26–35 showed lower binding affinities than 16 or 20, the binding affinities were two-digit nanomolar K_i values except for 29, 31, and 32, which possessed an extra phenyl group, suggesting a hydrophobic binding pocket of limited space in the receptor for the recognition of the acylamino moieties.

Table 1.

Binding affinities of thiazole and thiadiazole derivatives at adenosine receptors

Compd	R	X	Y	Ki (nM) or % inhibition at 10 μM
				Human A1	Human A2A	Human A3
LUF5417 27	4-OCH3C6H4	N	H	32 (rat A1)	2,300 (rat A2A)	82±4
11	CH3	CH	H	59%	74%	18.3±4.2
12	(CH3)3CO	CH	H	11%	31%	5,100±1,130
13	NCCH2	CH	H	50%	68%	204±17
14	CH3	CH	4-Cl	0%	27%	50.9±23.5
15	C6H5CH2	CH	4-Cl	0%	0%	100
16	CH3	CH	4-CH3O	35%	33%	3.0±0.8
17	CH3	CH	3-CH3O	34%	61%	4.1±1.8
18	CH3	CH	2-CH3O	18%	63%	82±21
19	CF3	CH	4-CH3O	0%	16%	530±91
20	CH3CH2	CH	4-CH3O	22%	47%	2.4±0.6
21	CH3CH2CH2	CH	4-CH3O	49%	48%	7.8±1.5
22	(CH3)2CH	CH	4-CH3O	49%	56%	16.3±4.1
23	NCCH2	CH	4-CH3O	33%	40%	24.3±3.9
24	(CH3)3C	CH	4-CH3O	69%	20%	31.9±4.2
25	(CH3)3O	CH	4-CH3O	0%	0%	3,260±340
26	C6H5	CH	4-CH3O	0%	14%	28.7±3.3
27	C6H5CH2	CH	4-CH3O	17%	27%	14.2±3.0
28	C6H5CH2CH2	CH	4-CH3O	0%	28%	29.1±9.3
29	p-CH3OC6H4CH2	CH	4-CH3O	0%	0%	1,160±190
30	p-CH3OC6H4CH2CH2	CH	4-CH3O	0%	12%	28.6±7.3
31	(C6H5)2CH	CH	4-CH3O	0%	18%	526±193
32	(C6H5)2CHCH2	CH	4-CH3O	0%	17%	400
33	2-Furan	CH	4-CH3O	41%	35%	31.5±1.3
34	Thiophene-2-CH2	CH	4-CH3O	10%	7%	32.3±7.1
35	2-Thiophene	CH	4-CH3O	21%	23%	69.3±10.0
36				11%	0%	373±74
37	CH3	N	H	56%	78%	2.3±0.2
38	C6H5CH2	N	H	19%	73%	79±26
39	CH3	N	4-CH3O	24%	28%	0.79±0.18
40	C6H5CH2	N	4-CH3O	7%	18%	23.8±0.3
41	CH3CH2	N	4-CH3O	47%	3530±600	1.13±0.14
42	CH3			0%	0%	4,690±1,380
43	C6H5CH2			0%	0%	>100,000

In an effort to further enhance the A₃ binding affinity, we applied the acetamido functionality to a thiadiazole template. N-(3-Phenyl-[1,2,4]thiadiazol-5-yl)-acetamide, 37, showed an 8-fold increase of binding affinity, with a K_i value of 2.3 nM at A₃ receptors, than the corresponding thiazole derivative, 11. Thus, we could assume that 4-methoxyphenyl substitution would afford a further increase of affinity, which encouraged us to explore synthesizing 3-(4-methoxy-phenyl)-[1,2,4]thiadiazol-5-ylamine, 7. An acetylated derivative of 7, that is, 39, was characterized as a selective and potent antagonist for human A₃ receptors with subnanomolar affinity, K_i value of 0.79 nM. The corresponding propionyl analogue, 41, which was expected to be slightly more potent than 37 based on a comparision in the thiazole series of 16 and 20, showed a K_i value of 1.13 nM at A₃ receptors, again with high subtype selectivity. Interestingly, the affinity of another regioisomer of 39, N-(2-(4-methoxyphenyl)-[1,3,4]thiadiazol-5-yl)-acetamide, 42 was dramatically decreased to a K_i value of 4.7 μM.

In terms of species differences, we attempted to determine the receptor binding affinity at rat A₁ receptors, since the parent compound LUF5417 displayed high affinity at the receptors. However, most of the derivatives displayed very weak binding affinities with K_i values > 50 μM at the rat A₁ and A_2A receptors. Also binding to the rat adenosine A₃ receptors was weak. K_i values were 3.03 μM for compound 33 and > 10 μM for compounds 17, 39 and 40.

4. Molecular modeling

In order to explain the different binding affinities of two regioisomers, 39 and 42, we calculated the thermodynamically stable conformations. From the result of the PM3 method, the lowest HF (Heat of Formation) of 1,2,4- and 1,3,4-thiadiazole compounds, 39 and 42, were −20.09 and 12.17 kcal/mol, respectively. Thus, the most potent one, 39, was more thermodynamically stable than the other, poor binding isomer 42 by >30 kcal. The lowest energy conformers of both derivatives were used for docking to the human adenosine A₃ receptor.⁴¹

We used an automated docking procedure (FlexiDock) with manual adjustment to determine the most energetically favorable binding location and orientation of the antagonist 39 (Fig. 1A). The result of FlexiDock applied to 39 displayed the possibility of a few complex models clustering within small energy differences. The complex model with the lowest energy among several docking models was well correlated with SAR and selectivity data in binding to the human A₃ receptor. The bound conformation of the amide showed trans-geometry with 4 kcal higher energy than the lowest one with a cis-amide bond obtained using the PM3 method. The high affinity antagonist 39, a 1,2,4-thiadiazole derivative, interacted through the H-bonding of the CO group with Q167 in EL2, which was a unique residue in human A₃ receptors in relation to other subtypes of adenosine receptors; The homologous residue was E for A₁, L for A₂ adenosine receptors (Fig. 1). This hydrophilic interaction appears to explain why compound 39 is selective for the human adenosine A₃ receptors. In addition, S181^5.42 formed H-bonding with the N atom of the thiadiazole ring. The amino acid corresponding to S181^5.42 in other adenosine receptor subtypes was N. This additional H-bonding to the thiadiazole ring, absent with the thiazole derivatives, could contribute to the increase of human adenosine A₃ receptor affinity. The thiadiazole and phenyl rings were surrounded by many hydrophobic amino acids; F168^EL2, F182^5.43, I186^5.47, and L246^6.51. The methyl group of the 4-methoxy phenyl ring substituent showed a hydrophobic interaction with I186^5.47 and W243^6.48. S247^6.52, which corresponds to H in other adenosine receptor subtypes, and was very close to the O of the methoxy group. The distance between the oxygen atom of the methoxy group and the hydroxyl group of S was 4.2 Å. The methyl of the acetamide was oriented toward EL2, indicating a hydrophobic interaction of methyl with I253^6.58. In proximity of the terminal methyl group, there was S170 in EL2, suggesting that a hydrophilic group in preference to a hydrophobic group, aromatic ring or aliphatic chain, might increase the binding affinity to the human adenosine A₃ receptor.

Figure 1.

The complex of the human adenosine A₃ receptors with two regioisomers in the putative binding site of the receptor model. (A) a 1,2,4-thiadiazole compound 39 and (B) 1,3,4-thiadiazole analogue 42. All ligands are displayed as ball-and-stick models, and the side chains of human A₃ receptors are shown as line models. The H-bonding between ligand and the receptor is displayed in yellow. The human A₃ receptor is represented by a tube model with a different color for each TM domain (TM3 in yellow, TM4 in green, TM5 in cyan, TM6 in blue, TM7 in purple).

In the case of the 1,3,4 analogue 42, there was no H-bonding in its complex when docked in the binding site of its isomer 39, correlating with its binding data; that is, about 6000-fold decrease of the binding affinity for the human adenosine A₃ receptor (Fig. 1B). The total energy of its complex was 9 kcal higher than that of the complex of the 1,2,4-analogue 39. Thus, the molecular modeling results supported the docking of the 1,2,4-thiadiazole 39 being energetically more favorable than the 1,3,4-derivative 42.

Thus, the result of FlexiDock was consistent with the current binding results. However, to confirm the reliability of the docked complexes, additional mutational experiments and more SAR data are needed, because theoretically there are other possible models of complexes within small energy differences.

The adenosine receptor sequence alignment indicated that most of the amino acids in the putative binding site within 5Å of the A₃-selective antagonist 39 were conserved among adenosine receptors. Highly conserved amino acids were T94^3.36, V178^5.39, F168^EL2, F182^5.43, W243^6.48, and N250^6.55. However, some variable residues among the four subtypes of ARs, H95^3.37, Q167^EL2, S170^EL2, S247^6.52, and I253^6.58, were located near R, X, and Y positions of the thiazole compound. Thus, variation of substituents in proximity to R, X and Y positions would be expected to increase the affinity and selectivity for the human A₃ subtype through additional H-bonding or hydrophobic interactions.

4.1. Functional assay of cAMP biosynthesis

The most potent derivative, 39, was further evaluated in a functional assay, which measured the antagonism of the inhibitory effect on cAMP production via Gi protein mediated signal transduction system upon activation of the adenosine A₃ receptor. Compound 39 displayed functional antagonistic properties with antagonism of inhibition of cAMP production by Cl-IB-MECA, a selective A₃ receptor agonist in a concentration-dependent manner with K_B value of 1.7 nM (Fig. 2).

Figure 2.

Antagonism by compound 39 of the inhibition of cyclic AMP production elicited by 100 nM Cl-IB-MECA in CHO cells stably transfected with the human adenosine A₃ receptors. The experiment was performed in the presence of 10 μM rolipram and 3 units/mL adenosine deaminase. Forskolin (10 μM) was used to stimulate cyclic AMP levels. The level of cAMP corresponding to 100% was 220±30 pmol mL⁻¹. The K_B value for compound 39 was calculated to be 1.7 nM.

5. Conclusion

In summary, we have identified antagonists for the human adenosine A₃ receptor having subnanomolar affinity and high selectivity versus other subtype receptors through structure–activity relationship studies of aminothiazole and aminothiadiazole as the lead templates. An assay of cAMP production also demonstrated functional inhibition by the most potent antagonist 39, and speculation of a binding mode of thiadiazole isomers was suggested by molecular docking experiments with a human adenosine A₃ receptor model built from homology modeling of the X-ray crystal structure of rhodopsin. The potent human adenosine A₃ receptor antagonists developed in this study will be useful to investigate biological significance of adenosine receptors and to develop therapeutics for the treatment of inflammatory disease, such as asthma, and glaucoma.

6. Experimental

6.1. Chemistry

Proton nuclear magnetic resonance spectroscopy was performed on a Bruker AVANCE 600, JEOL JNM-LA 300WB spectrometers and spectra were taken in DMSO-d₆ or CDCl₃. Unless noted, chemical shifts are expressed as ppm downfield from tetramethylsilane as the internal standard, and J values are given in Hz. Mass spectroscopy was carried out on an MALDI-TOF and FAB instrument. All thiazole and thiadiazole derivatives showed one spot on thin layer chromatography (Merck silica gel 60; F₂₅₄, 0.25 mm). Elemental analyses determined on a Flash EA 1112series (CE Instruments) analyzer. Elemental and high-resolution mass analyses were performed in Seoul Branch Analytical Laboratory of Korea Basic Science Institute.

6.1.1. General procedure for the synthesis of 2-amino-4-[methoxy (or 4-chloro)-phenyl]-thiazole analogues (3).

1 mmol of 2-bromo-methoxy(or 4-chloro)acetophenone, 5 mmol of KSCN/SiO₂, and 18 mmol of NH₄OAc/Al₂O₃ were added in 15 mL of dry benzene. After stirring at 90°C for 16 h, the yellow mixture was filtered and dried under reduced pressure. The residue was purified by silica gel column chromatography (CHCl₃/MeOH, 50:1–30:1) to afford the desired compound (3a–d). Yield 60%.

6.1.2. 5-Amino-3-(–4′-methoxyphenyl)-1,2,4-thiadiazole (7).

Was synthesized from 4-methoxybenzonitrile 4. A 100 mL 2-necked round-bottom flask was charged with 30 mL of anhydrous MeOH, 1.0 g (7.5 mmol) of the 4-methoxybenzonitrile 4, and 0.04 g (0.75 mmol) of sodium methoxide. The contents of the flask were protected from moisture and stirred magnetically for 48 h. Then, 0.4 g (7.5 mmol) of NH₄Cl was added and stirring was continued for 24 h. Unreacted NH₄Cl was filtered, and methanol was stripped from the filtrate to afford the crude product, 4-methoxybenzamidine 5, which was washed ether to remove unreacted 4-methoxybenzonitrile and used for the next step without further purification. 2.2 g (14.6 mmol) of 5 was dissolved into 50 mL water and mixed with 1 mL of sodium dodecylsulfate (as emulsion) and 3.2 mL (29.2 mmol) of perchloromethyl mercaptan. To the mixture was added a solution of 1.6 g of NaOH in 50 mL of water (0.04 M) dropwise, maintaining the temperature under 10°C. Stirring was continued for another 15 min. The product color was changed to the orange-yellow, which was washed with cold water and dried under reduced pressure. The crude product was purified by silica gel column chromatography (hexanes–EtOAc, 30:1) to give 5-chloro-3-(4′-methoxyphenyl)-1,2,4-thiadiazole 6 (Yield 10%). ¹H NMR (600 MHz, DMSO-d₆) δ 3.84 (3H, s, CH₃O), 7.10 (2H, d, J = 7.2 Hz, phenyl-3H), 8.11 (2H, d, J = 7.2 Hz, phenyl-2H); MS [MALDI-TOF] m/z = 226.7 (M). Compound 6 was converted to 5-amino-3-(4′-methoxyphenyl)-1,2,4-thiadiazole 7 (Yield 34%) and 5-methylamino-3-(4′-methoxyphenyl)-1,2,4-thiadiazole 8 (Yield 74%) in ethanolic ammonia and methylamine at room temperature, respectively. 7: ¹H NMR (300 MHz, DMSO-d₆) δ 3.80 (3H, s, CH₃O), 7.01 (2H, d, J = 9.0 Hz, phenyl-3H), 7.97 (2H, s, NH₂), 8.00 (2H, d, J = 9.0 Hz, phenyl-2H); MS [MALDI-TOF] m/z = 207.2 (M); 8: ¹H NMR (300 MHz, DMSO-d₆) δ 2.97 (3H, d, J = 4.8, CH₃), 3.84 (3H, s, CH₃O), 7.02 (2H, d, J = 9.0 Hz, phenyl-3H), 8.04 (2h, d, J = 9.0 Hz, phenyl-2H), 8.42 (1H, s, NH); MS [MALDI-TOF] m/z = 221.2 (M).

6.1.3. 5-Amino-2-(4′-methoxyphenyl)-1,3,4-thiadiazole (10).

10 g (0.11 mol) of thiosemicarbazide and 13.3 g (0.11 mol) of p-anisaldehyde were dissolved in 30 mL ethanol. The mixture was stirred at room temperature for 7 h and filtered under reduced pressure. The residue was washed with ethanol to remove the side-products, and dried under vacuum oven to afford 19.83 g of 9 (86%). ¹H NMR (600 MHz, DMSO-d₆) δ 3.85 (3H, s, CH₃O), 6.92 (2H, d, J = 8.4 Hz, phenyl-3H), 7.02 (1H, s, NCH), 7.60 (2H, d, J = 8.4 Hz, phenyl-2H), 7.85 (2H, s, NH₂), 10.07 (1H, s, SCNH); MS [MALDI-TOF] m/z = 209.3 (M). A mixture of 1.67 g (8 mmol) of 9, 360 mL of water and 6.0 g (22 mmol) of ferric chloride hexahydrate was stirred and heated at 80–90 °C for one-half hour, then filtered while hot. The filtrate was concentrated under reduced pressure and the residue was treated with 10% aqueous ammonia to an alkaline solution. The insoluble material was filtered, dried and purified by silica gel column chromatography (CHCl₃–MeOH, 20:1). Yield 5.6%. 10: ¹H NMR (300 MHz, DMSO-d₆) δ 3.80 (3H, s, CH₃O), 7.00 (2H, d, J = 9.0 Hz, phenyl-3H), 7.29 (2H, s, NH₂), 7.66 (2H, d, J = 9.0 Hz, phenyl-2H); MS [MALDI-TOF] m/z = 207.3 (M).

6.1.4. General procedure for the preparation of N-acyl substituted thiazole and thiadiazole derivatives. Method A.

A mixture of appropriate aminothiazoles or aminothiadiazoles (1 mmol), pyridine (2 mmol) and acyl chloride (3 mmol) in 5 mL of anhydrous dichloromethane was stirred at room temperature for 1 h. The solvent was removed under reduced pressure, and the residue was partitioned between saturated ammonium chloride solution (30 mL) and CHCl₃ (3×20 mL). The organic layer was dried on Na₂SO₄ and evaporated under vacuum, and the residue was purified by silica gel column chromatography (hexanes–EtOAc, 3:1) to afford the product as solid (11, 14–18, 20–22, 24, 26–29, 31, 33–43).

6.1.5. Method B.

The appropriate aminothiazole (0.242 mmol) was dissolved in absolute EtOH (3 mL), and ethyl cyanoacetate (0.726 mmol) and sodium ethoxide (0.726 mmol) were added. The mixture was refluxed for 3 h, then, after cooling, the solvent was removed under reduced pressure and the crude product was purified by column chromatography on silica gel (CHCl₃/MeOH, 50:1) to afford the product as solid (13 and 23).

6.1.6. Method C.

To the appropriate aminothiazole (0.2 mmol) in 3 mL of absolute dichloromethane was added dimethylaminopyridine (0.3 mmol) and corresponding anhydride (0.4 mmol). The mixture was stirred at room temperature for 3 h and the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel (hexanes–EtOAc, 6:1) to afford the final products (12, 19 and 25).

6.1.7. Method D.

A mixture of 40 mg of 2-amino-4-(4α-methoxyphenyl)-thiazole 3a (0.19 mmol), 74.4 mg of EDAC (0.388 mmol), 71.1 mg of dimethylaminopyridine (0.582 mmol) and appropriate acid (0.388 mmol) in 3 mL of anhydrous dichloromethane was stirred at room temperature for 2 h. The solvent was removed under reduced pressure, and the residue was partitioned between saturated ammonium chloride (30 mL) and CHCl₃ (3×20 mL). The organic layer was dried on Na₂SO₄ and evaporated under vacuum. The residue was purified by column chromatography on silica gel (hexanes–EtOAc, 6:1) to afford the final products as solids (30 and 32).

6.1.8. N-(4-Phenyl-thiazol-2-yl)-acetamide (11).

Yield 20%; ocher yellow powder; ¹H NMR (600 MHz, CDCl₃) δ 1.94 (3H, s, CH3), 7.14 (1H, d, J = 7.0 Hz, phenyl-4H), 7.34 (1H, s, thiazole-3H), 7.41 (2H, dd, J = 6.5, 7.0 Hz, phenyl-3H), 7.81 (2H, d, J = 6.5 Hz, phenyl-2H), 10.67 (1H, s, NH); HRMS calcd for C₁₁H₁₀N₂OS (MH⁺) 219.0514, found 219.0511.

6.1.9. (4-Phenyl-thiazol-2-yl)-carbamic acid tert-butyl ester (12).

Yield 19.1% orange-yellow powder; ¹H NMR (600 MHz, CDCl₃) δ 1.61 (9H, s, (CH₃)₃CO), 7.08 (1H, d, J = 7.0 Hz phenyl-4H), 7.37 (1H, s, thiazole-3H), 7.39-7.41 (2H, dd, J = 6.5, 7.0 Hz, phenyl-3H), 7.89 (2H, d, J = 6.5 Hz, phenyl-2H), 11.45 (1H, s, NH). HRMS calcd for C₁₄H₁₆N₂O₂S (MH⁺) 277.0932, found 277.0933.

6.1.10. 2-Cyano-N-(4-phenyl-thiazol-2-yl)-acetamide (13).

Yield 52% ochre yellow powder; ¹H NMR (600 MHz, CDCl₃) δ 3.1 (2H, s, CH₂), 7.26 (1H, s, thiazole-3H), 7.41–7.44 (1H, d, J = 7.6 Hz, phenyl-4H), 7.47–7.50 (2H, dd, J = 7.1, 7.6 Hz, phenyl-3H) 7.79 (2H, d, J = 7.1 Hz, phenyl-2H), 12.61 (1H, s, NH). HRMS calcd for C₁₂H₉N₃OS (MH⁺) 244.0466, found 244.0461.

6.1.11. N-[4-(4-Chloro-phenyl)-thiazol-2-yl]-acetamide (14).

Yield 86.1% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 2.16 (3H, s, CH₃), 7.50 (2H, d, J = 8.4 Hz, phenyl-3H), 7.67 (1H, s, thiazole-3H), 7.90 (2H, d, J = 8.4 Hz, phenyl-2H), 12,31 (1H, s, NH); MS [MALDI-TOF] m/z = 252.2 (M). Anal. calcd for C₁₁H₉ClN₂OS: C, 52.28; H, 3.59; N, 11.08. Found: C, 51.96; H, 3.44; N, 11.27.

6.1.12. N-[4-(4-Chloro-phenyl)thiazol-2-yl]-2-phenyl-acetamide (15).

Yield 98.6% light green powder; ¹H NMR (300 MHz, DMSO-d₆) δ 3.76 (2H, d, J = 7.5 Hz, CH₂C₆H₅), 7.14–7.48 (5H, m, phenyl-4H + phenyl-3H + phenyl 2H), 7.50 (2H, d, J = 8.7 Hz, phenyl-3H), 7.68 (1H, s, thiazole-3H), 7.90 (2H, d, J = 8.7 Hz, phenyl-2H), 12.52 (1H, s, NH). HRMS calcd for C₁₇H₁₃ClN₂OS (MH⁺) 329.0437, found 329.0433.

6.1.13. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-acetamide (16).

Yield 20% light green powder; ¹H NMR (300 MHz, DMSO-d₆) δ 3.77 (3H, s, CH₃), 6.97 (2H, d, J = 8.7 Hz, phenyl-3H), 7.41 (1H, s, thiazole-3H), 7.80 (2H, d, J = 8.7 Hz, phenyl-2H), 12.19 (1H, s, NH). HRMS cald for C₁₂H₁₂N₂O₂S (MH⁺) 249.0619, found 249.0627.

6.1.14. N-[4-(3-Methoxy-phenyl)-thiazol-2-yl]-acetamide (17).

Yield 62.1% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 2.16 (3H, s, CH₃), 3.79 (3H, s, CH₃O), 6.90 (1H, d, J = 8.1 Hz, phenyl-6′·H), 7.33 (1H, t, J = 8.1 Hz, phenyl-5′·H), 7.43-7.48 (2H, m, phenyl-2′H + phenyl-4′H), 7.63 (1H, s, thiazole-3H), 12.26 (1H, s, NH). HRMS calcd for C₁₂H₁₂N₂O₂S (MH⁺) 249.0619, found 249.0618.

6.1.15. N-[4-(2-Methoxy-phenyl)-thiazol-2-yl]-acetamide (18).

Yield 99.3% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 2.16 (3H, s, COCH₃), 3.91 (3H, s, CH₃O), 7.03 (1H, t, J = 7.5 Hz, phenyl-5′ H), 7.13 (1H, d, J = 7.5 Hz, phenyl-6′ H), 7.28 (1H, dd, J = 7.5, 7.8 Hz, phenyl-4′ H ), 7.62 (1H, s, thiazole-3H), 8.05 (1H, d, J = 7.8 Hz, phenyl-3′ H), 12.18 (1H, s, NH); MS [MALDI-TOF] m/z = 248.2 (M). Anal. calcd For C₁₂H₁₂N₂O₂S: C, 58.05; H, 4.87; N, 11.28. Found: C,57.93; H, 4.49; N, 11.08.

6.1.16. 2,2,2-Trifluoro-N-[4-(4-methoxy-phenyl)-thiazol-2-yl]-acetamide (19).

Yield 20.7% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 3.79 (3H, s, CH₃O), 7.03 (2H, d, J = 9.0 Hz, phenyl-3H), 7.57 (1H, s, thiazole-3H), 7.82 (2H, d, J = 9.0 Hz, phenyl-2H), 14.12 (1H, broad s, NH); MS [MALDI-TOF] m/z = 302.1 (M). Anal. calcd for C₁₂H₉F₃N₂O₂S: C, 47.68; H, 3.00; N, 9.27. Found: C, 47.38; H, 2.80; N, 9.08.

6.1.17. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-propionamide (20).

Yield 74.7% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 1.1 (3H, t, J = 7.5 Hz, CH₃), 2.46 (2H, q, J = 7.5 Hz, CH₂), 3.78 (3H, s, CH₃O), 6.97 (2H, d, J = 9.0 Hz, phenyl-3H), 7.42 (1H, s, thiazole-3H), 7.83 (2H, d, J = 9.0 Hz, phenyl-2H), 12.24 (1H, s, NH); MS [MALDI-TOF] m/z = 262.4 (M). Anal. calcd for C₁₃H₁₄N₂O₂S: C, 59.52; H, 5.38; N, 10.68. Found: C, 59.43; H, 5.52; N, 10.39.

6.1.18. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-butyramide (21).

Yield 82.2% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 0.90 (3H, t. J = 7.3 Hz, CH₃), 1.59–1.66 (2H, m, CH₂CH₃), 2.41 (2H, t, J = 7.3 Hz, COCH₂), 3.78 (1H, s, CH₃O), 6.99 (2H, d, J = 4.8 Hz, phenyl-3H), 7.43 (1H, s, thiazole-3H), 7.82 (2H, d, J = 4.8 Hz, phenyl-2H), 12.36 (1H, s, NH); MS [MALDI-TOF] m/z = 276.1 (M). Anal. calcd for C₁₄H₁₆N₂O₂S: C, 60.85; H, 5.84; N, 10.14. Found: C, 60.53; H, 5.91; N, 9.92.

6.1.19. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-isobutyramide (22).

Yield 71.6% white crystals; ¹H NMR (300 MHz, DMSO-d₆) δ 1.10 (6H, d, J = 7.2 Hz, CH(CH₃)₂), 2.75 (1H, m, CH(CH₃)₂), 3.78 (1H, s, CH₃O), 6.99 (2H, d, J = 8.7 Hz, phenyl-3H), 7.43 (1H, s, thiazole-3H), 7.82 (2H, d, J = 8.7 Hz, phenyl-2H), 12.18 (1H, s, NH); MS [MALDI-TOF] m/z = 276.7 (M). Anal. calcd for C₁₄H₁₆N₂O₂S: C, 60.85; H, 5.84; N, 10.14. Found: C, 60.71; H, 5.89; N, 10.01.

6.1.20. 2-Cyano-N-[4-(4-methoxy-phenyl)-thiazol-2-yl]-acetamide (23).

Yield 91.2% ocher yellow powder; ¹H NMR (300 MHz, DMSO-d₆) δ 3.79 (3H, s, CH₃O), 4.05 (2H, d, J = 3.6 Hz, CH₂), 6.98 (2H, d, J = 8.4 Hz, phenyl-3H), 7.52 (1H, s, thiazole-3H), 7.82 (2H, d, J = 8.4 Hz, phenyl-2H), 12.56 (1H, s, NH). HRMS calcd for C₁₃H₁₁N₃O₂S (MH⁺) 274.0572, found 274.0583.

6.1.21. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-2,2-dimethyl-propionamide (24).

Yield 95% white crystals; ¹H NMR (300 MHz, DMSO-d₆) δ 1.25 (9H, s, C(CH₃)₃), 3.78 (3H, s, CH₃O), 6.99 (2H, d, J = 12.0 Hz, phenyl-3H), 7.43 (1H, s, thiazole-3H), 7.85 (2H, d, J = 12.0 Hz, phenyl-2H), 11.79 (1H, s, NH); MS [MALDI-TOF] m/z = 289.6 (M). Anal. calcd for C₁₅H₁₈N₂O₂S: C, 62.04; H, 6.25; N, 9.65. Found: C, 62.33; H, 6.34; N, 9.43.

6.1.22. [4-(4-Methoxy-phenyl)-thiazol-2-yl]-carbamic acid tert-butyl ester (25).

Yield 12% orange-yellow crystals; ¹H NMR (300 MHz, CDCl₃) δ 1.60 (9H, s, OC(CH₃)₃), 3.84 (3H, s, CH₃O), 6.93 (2H, d. J = 8.4 Hz, phenyl-3H), 7.26 (1H, s, thiazole-3H), 7.80 (2H, d, J = 8.4 Hz, phenyl-2H), 11.44 (1H, s, NH). HRMS calcd for C₁₅H₁₈N₂O₃S (MH⁺) 307.1038, found 307.1032.

6.1.23. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-benzamide (26).

Yield 56.1% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 3.79 (3H, s, CH₃O), 6.99 (2H, d, J = 8.7 Hz, phenyl-3H), 7.51–7.64 (3H, m, COC₆H₅-H3 + COC₆H₅-H4), 7.53 (1H, s, thiazole-3H), 7.87 (2H, d, J = 8.7 Hz, phenyl-2H), 8.14 (2H, d, J = 8.1 Hz, COC₆H₅-H2), 12.83 (1H, s, NH). HRMS calcd for C₁₇H₁₄N₂O₂S (MH⁺) 311.0776, found 311.0773.

6.1.24. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-2-phenyl-acetamide (27).

Yield 41.8% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 3.79 (2H, s, CH₂), 3.79 (3H, s, CH₃O), 6.97 (2H, d, J = 8.7 Hz, phenyl-3H), 7.24-7.35 (5H, m, C₆H₅-2H + C₆H₅-3H + C₆H₅-4H), 7.44 (1H, s, thiazole-3H), 7.83 (2H, d, J = 8.7 Hz, phenyl-2H), 12.47 (1H, s, NH). HRMS calcd for C₁₈H₁₆N₂O₂S (MH⁺) 325.0932, found 325.0931.

6.1.25. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-3-phenyl-propionamide (28).

Yield 80.6% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 2.76 (2H, t, J = 8.1 Hz, CH₂C₆H₅), 2.90 (2H, t, J = 8.1 Hz, COCH₂), 3.78 (3H, s, CH₃O), 6.99 (2H, d, J = 8.7 Hz, phenyl-3H), 7.21-7.29 (5H, m, C₆H₅-2H + C₆H₅-3H + C₆H₅-4H), 7.43 (1H, s, thiazole-3H), 7.80 (2H, d, J = 8.7 Hz, phenyl-2H), 12.25 (1H, s, NH). HRMS calcd for C₁₉H₁₈N₂O₂S (MH⁺) 339.1089, found 339.1081.

6.1.26. 2-(4-Methoxy-phenyl)-N-[4-(4-methoxy-phenyl)-thiazol-2-yl]-acetamide (29).

Yield 61.1% lemon-yellow powder; ¹H NMR (300 MHz, DMSO-d₆) δ 3.69 (2H, s, COCH₂), 3.73 (3H, s, CH₃OC₆H₄CH₂), 3.78 (3H, s, CH₃O), 6.88 (2H, d, J = 8.4 Hz, CH₂C₆H₄OCH₃-3H), 6.97 (2H, d, J = 8.7 Hz, thiazole-C₆H₄-3H), 7.25 (2H, d, J = 8.4 Hz, CH₂C₆H₄OCH₃-2H), 7.44 (1H, s, thiazole-3H), 7.83 (2H, d, J = 8.7 Hz, thiazole-C₆H₄-2H), 12.32 (1H, s, NH); MS [MALDI-TOF] m/z = 354.7 (M). Anal. calcd for C₁₉H₁₈N₂O₃S: C, 64.39; H, 5.12; N, 7.90. Found: C, 64.13; H, 5.01; N, 7.88.

6.1.27. 3-(4-Methoxy-phenyl)-N-[4-(4-methoxy-phenyl)-thiazol-2-yl]-propionamide (30).

Yield 43.4% gray-brown powder; ¹H NMR (300 MHz, DMSO-d₆) δ 2.50 (2H, t, J = 7.5 Hz, CH₂C₆H₄OCH₃), 2.71 (2H, t, J = 7.5 Hz, COCH₂), 3.70 (3H, s, CH₃OC₆H₄CH₂), 3.78 (3H, s, CH₃O), 6.86 (2H, d, J = 8.4 Hz, CH₂C₆H₄OCH₃-3H), 6.99 (2H, d, J = 8.7 Hz, thiazole-C₆H₄-3H), 7.16 (2H, d, J = 8.7 Hz, thiazole-C₆H₄-2H), 7.43 (1H, s, thiazole-3H), 7.82 (2H, d, J = 8.4 Hz, CH₂C₆H₄OCH₃-2H), 12.36 (1H, s, NH); MS [MALDI-TOF] m/z = 367.8(M). Anal. calcd for C₂₀H₂₀N₂O₃S: C, 65.20; H, 5.47; N, 7.60. Found: C, 64.87; H, 5.40; N, 7.66.

6.1.28. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-2,2-diphenylacetamide (31).

Yield 39.5% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 3.78 (3H, s, CH₃O), 5.37 (1H s, CH(C₆H₅)₂), 6.99 (2H, d, J = 8.7 Hz, CH₃OC₆H₄-3H), 7.22–7.40 (10H, m, CH(C₆H₅)₂), 7.48 (1H, s, thiazole-3H), 7.82 (2H, d, J = 8.7 Hz, CH₃OC₆H₄-2H), 12.67 (1H, s, NH). HRMS calcd for C₂₄H₂₀N₂O₂S (MH⁺) 401.1245, found 401.1243.

6.1.29. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-3,3-diphenyl-propionamide (32).

Yield 79.3% light-brown crystals; ¹H NMR (300 MHz, DMSO-d₆) δ 3.26 (2H, d, J = 8.1 Hz, CH₂), 3.78 (3H, s, CH₃O), 4.61 (1H, s, CH(C₆H₅)₂), 6.99 (2H, d, J = 8.7 Hz, CH₃OC₆H₄-3H), 7.14–7.2 (5H, m, phenyl-2H + phenyl-3H + phenyl-4H), 7.25–7.33 (5H, m, phenyl-2H + phenyl-3H + phenyl-4H), 7.39 (1H, s, thiazole-3H), 7.78 (2H, d, J = 8.7 Hz, CH₃OC₆H₄-2H), 12.67 (1H, s, NH); MS [MALDI-TOF] m/z = 413.8 (MS). Anal. calcd for C₂₅H₂₂N₂O₂S: C, 72.44; H, 5.35; N, 6.76. Found: C, 72.26; H, 5.62; N, 6.58.

6.1.30. Furan-2-carboxylic acid [4-(4-methoxy-phenyl)-thiazol-2-yl]-amide (33).

Yield 97.8% light-brown powder; ¹H NMR (300 MHz, DMSO-d₆) δ 3.79 (3H, s, CH₃O), 6.74 (1H, dd, J = 1.5, 2.1 Hz, furan-3H), 7.0 (2H, d, J = 9.0 Hz, phenyl-3H), 7.51 (1H, s, thiazole-3H), 7.71 (1H, d, J = 1.5 Hz, furan-2H), 7.88 (2H, d, J = 9.0 Hz, phenyl-2H), 8.02 (1H, d, J = 2.1 Hz, furan-4H), 12.61 (1H, s, NH). HRMS calcd for C₁₅H₁₂N₂O₃S (MH⁺) 301.0569, found 301.0571.

6.1.31. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-2-thiophen-2-yl-acetamide (34).

Yield 11.7% light-gray powder; ¹H NMR (300 MHz, DMSO-d₆) δ 3.79 (3H, s, CH₃O), 4.03 (2H, s, CH₂), 6.98–7.12 (2H, m, thiophen-4H + thiophen-2H), 7.15 (2H, d, J = 8.7 Hz, phenyl-3H), 7.42 (1H, t, J = 1.5 Hz, thiophen-3H), 7.47 (1H, s, thiazole-3H), 7.98 (2H, d, J = 8.7 Hz, phenyl-2H), 12.51 (1H, s, NH). HRMS calcd for C₁₆H₁₄N₂O₂S₂ (MH⁺) 331.0497, found 331.0492.

6.1.32. Thiophene-2-carboxylic acid [4-(4-methoxyphenyl)-thiazol-2-yl]-amide (35).

Yield 18% light-gray powder; ¹H NMR (300 MHz, DMSO-d₆) δ 3.79 (3H, s, CH₃O), 6.99 (2H, d. J = 8.7 Hz, phenyl-3H), 7.26 (1H, dd, J = 2.7, 3.9 Hz, thiophenyl-3H), 7.52 (1H, s, thiazole-3H), 7.87 (2H, d, J = 8.7 Hz, phenyl-2H), 7.98 (1H, d, J = 3.9 Hz, thiophenyl-2H), 8.31 (1H, d, J = 2.7 Hz, thiophenyl-4H), 12.87 (1H, s, NH). HRMS calcd for C₁₅H₁₂N₂O₂S₂ (MH⁺) 317.0340, found 317.0347.

6.1.33. [3-(4-Methoxy-phenyl)-[1,2,4]-thiadiazol-5-yl]-methyl-amine (36).

Yield 74% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 2.95 (3H, d, J = 4.5 Hz, CH₃), 3.80 (3H, s, CH₃O), 7.01 (2H, d, J = 9.0 Hz, phenyl-3H), 8.03 (2H, d, J = 9.0 Hz, phenyl-2H), 8.43 (1H, broad s, NH). HRMS calcd for C₁₀H₁₁N₃OS (MH⁺) 222.0623, found 222.0623.

6.1.34. N-(3-Phenyl-[1,2,4]thiadiazol-5-yl)-acetamide (37).

Yield 67.8% white crystals; ¹H NMR (300 MHz, DMSO-d₆) δ 2.27 (3H, s, CH₃), 7.51 (1H, d, J = 5.1 Hz, phenyl-4H), 7.54 (2H, d, J = 4.5 Hz, phenyl-2H), 8.16 (2H, dd, J = 4.5, 5.1 Hz, phenyl-3H), 13.10 (1H, broad s, NH); MS [MALDI-TOF] m/z = 219.0 (M). Anal. calcd for C₁₀H₉N₃OS: C, 54.78; H, 4.14; N, 19.16. Found: C, 54.48; H, 4.01; N, 19.02.

6.1.35. 2-Phenyl-N-(3-phenyl-[1,2,4]thiadiazol-5-yl)-acetamide (38).

Yield 41.2% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 3.91 (2H, s, CH₂), 7.21–7.36 (5H, m, phenyl-2H + phenyl-3H + phenyl-4H), 7.51 (1H, d, J = 2.1 Hz, phenyl-4H), 7.54 (2H, d, J = 4.5 Hz, phenyl-2H), 8.17 (2H, dd, J = 2.1, 4.5 Hz, phenyl-3H), 12.93 (1H, s, NH). HRMS calcd for C₁₆H₁₃N₃OS (MH⁺) 296.0799, found 296.0791.

6.1.36. N-[3-(4-Methoxy-phenyl)-[1,2,4]thiadiazol-5-yl]-acetamide (39).

Yield 74.2% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 2.26 (3H, s, CH₃), 3.82 (3H, s, CH₃O), 7.04 (2H, d, J = 8.7 Hz, phenyl-3H), 8.07 (2H, d, J = 8,7 Hz, phenyl-2H), 13.05 (1H, s, NH). HRMS calcd for C₁₁H₁₁N₃O₂S (MH⁺) 250.0572, found 250.0571.

6.1.37. N-[3-(4-Methoxy-phenyl)-]1,2,4]thiadiazol-5-yl]-2-phenyl-acetamide (40).

Yield 81.0% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 3.82 (3H, s, CH₃O), 3.90 (2H, s, CH₂), 7.05 (2H, d, J = 9.0 Hz, phenyl-3H), 7.25–7.36 (5H, m, C₆H₅-2H + C₆H₅-3H + C₆H₅-4H), 8.08 (2H, d, J = 9.0 Hz, phenyl-2H), 13.25 (1H, s, NH). HRMS calcd for C₁₇H₁₅N₃O₂S (MH⁺) 326.0885, found 326.0889.

6.1.38. N-[3-(4-Methoxy-phenyl)-[1,2,4]thiadiazol-5-yl]-propionamide (41).

Yield 52.0% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 1.10 (3H, t, J = 7.5 Hz, CH₃), 2.53 (2H, q, J = 7.5 Hz, CH₂), 3.82 (3H, s, CH₃O), 7.04 (2H, d, J = 9.0 Hz, phenyl-3H), 8.08 (2H, d, J = 9.0 Hz, phenyl-2H), 12.97 (1H, s, NH). HRMS calcd for C₁₂H₁₃N₃O₂S (MH⁺) 264.0728, found 264.0731.

6.1.39. N-[5-(4-Methoxy-phenyl)-[1,3,4]thiadiazol-2-yl]-acetamide (42).

Yield 79.0% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 2.20 (3H, s, CH₃), 3.83 (3H, s, CH₃O), 7.08 (2H, d, J = 9.0 Hz, phenyl-3H), 7.88 (2H, d, J = 9.0 Hz, phenyl-2H), 12.51 (1H, broad s, NH); MS [MALDI-TOF] m/z = 248.9 (M). Anal. calcd for C₁₁H₁₁N₃O₂S: C, 53.00; H, 4.45; N, 16.86. Found: C, 52.97; H, 4.33; N, 16.98.

6.1.40. N-[5-(4-Methoxy-phenyl)-[1,3,4]thiadiazol-2-yl]-2-phenyl-acetamide (43).

Yield 62.3% white powder; ¹H NMR (300 MHz, DMSO-d₆) δ 3.81 (3H, s, CH₃O), 3.90 (2H, s, CH₂), 7.05 (2H, d, J = 9.0 Hz, phenyl-3H), 7.26–7.35 (5H, m, C₆H₅-2H + C₆H₅-3H + C₆H₅-4H), 7.84 (2H, d, J = 9.0 Hz, phenyl-2H), 12.87 (1H, broad s, NH). HRMS calcd for C₁₈H₁₆N₂O₂S (MH⁺) 325.0932, found 325.0933.

6.2. Pharmacology

[¹²⁵I]N⁶-(4-Amino-3-iodobenzyl)adenosine-5′-N-methyluronamide (I-AB-MECA; 2000 Ci/mmol), [³H]-(R)-PIA([³H]-(R)-N⁶-(phenylisopropyl)adenosine), [³H]-CGS 21680 ([³H]-2-[4-(2-carboxyethyl)phenyl]ethylamino]-5′-(N-ethylcarbamoyl)adenosine), and [³H]cyclic AMP (40 Ci/mmol) were from Amersham Biosciences (Piscataway, NJ). All other reagents including Cl-IB-MECA (N⁶-(3-iodobenzyl)-2-chloro-adenosine-5′-N-methyluronamide) were obtained from Sigma (St. Louis, MO, USA).

CHO (Chinese Hamster Ovary) cells expressing the recombinant human A₁, A₃ adenosine receptors and HEK293 cells expressing the recombinant human adenosine A_2A receptors were cultured in Dulbecco Modified Eagle’s Medium (DMEM) and F12 (1:1) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, 2 μmol/mL glutamine and 800 μg/mL geneticin. 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. The suspension was homogenized with an electric homogenizer for 10 s, and was then re-centrifuged at 20,000g for 20 min at 4 °C. The resultant pellets were resuspended in buffer containing 3 U/mL adenosine deaminase, and the suspension was stored at −80 °C. Striatal and forebrain tissues from Wistar rats were homogenized in ice-cold 50 mM TrisHCl buffer, pH 7.4, using an electric homogenizer. The homogenate was centrifuged at 20,000g for 10 min at 4 °C, and the pellet was washed in fresh buffer. The final pellet was stored at −80 °C until the binding experiments. The protein concentration was measured using the Bradford assay.³⁵

For binding to human A₁ receptors, [³H]R-PIA (2 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 N⁶-cyclopentyladenosine. 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 μM Tris–HCl, pH 7.4, containing 10 mM MgCl₂. 5′-N-ethyluronamidoadenosine (10 μM) was used to define nonspecific binding. Reaction was terminated upon filtration over GF/B filters.

For binding to recmbinant A₃ receptors, each tube contained 50 μL membrane suspension (20 μg protein), 25 μL of [¹²⁵I]I-AB-MECA (0.5 nM), and 25 μL of increasing concentrations of the test ligands in Tris–HCl buffer (50 mM, pH 8.0) containing 10 mM MgCl₂, 1 mM EDTA. Nonspecific binding was determined using 10 μM 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.

Intracellular cyclic AMP levels were measured with a competitive protein binding method.^36,37 CHO cells expressing human or rat adenosine A₃ receptors were harvested following trypsinization. After centrifugation and resuspended in medium, cells were planted in 24-well plates in 0.5 mL medium. After 24 h, the medium was removed and cells were washed three times with 1 mL DMEM, containing 50 mM HEPES, pH 7.4. Cells were then treated with agonists and/or test compounds in the presence of rolipram (10 μM) and adenosine deaminase (3 U/mL). After 45 min forskolin (10 μM) was added to the medium, and incubation was continued an additional 15 min. The reaction was terminated by removing the supernatant, and cells were lysed upon the addition of 200 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.

6.3. Molecular modeling

All calculations were performed on a Silicon Graphics (Mountain View, CA) Octane workstation (300 MHz MIPS R12000 (IP30) processor). All ligand structures were constructed with the use of the Sketch Molecule of SYBYL 6.9.³⁸ A conformational search of antagonists 39 and 42 was performed by grid search, rotating three rotatable bonds in 60° increments and the amide bond at 0° or 180°. A random search was also performed, using the following options for all rotatable bonds; 3000 iterations, 3-kcal energy cutoffs, and no chirality checking. In all cases, MMFF force field³⁹ and charge were applied with the use of distance-dependent dielectric constants and the conjugate gradient method until the gradient reached 0.05 kcal·mol⁻¹·Å⁻¹. Representative conformers selected from clusters of low-energy conformers obtained in the conformational search were reoptimized by semiempirical molecular orbital calculations with the PM3 method in the MOPAC 6.0 package.⁴⁰

A human adenosine A₃ receptor model, which was previously built using homology modeling⁴¹ from the X-ray structure of bovine rhodopsin with 2.8 Å resolution⁴² was used for docking studies. For a brief explanation of this method, multiple-sequence alignment data of selected GPCRs were used for the construction of human adenosine A₃ receptor TM domains..⁴³ For the model of the second extracellular loop, EL2, two beta-sheet domains in rhodopsin were first aligned, including the disulfide bond between Cys83 and Cys166, and then other components were added or deleted. To construct the N-terminal region and the intra- and extracellular loops, each alignment was manually adjusted to preserve the overall shape of the loop. N-acetyl and N-methyl amide groups blocked the N-terminal and C-terminal regions, respectively, to prevent electrostatic interactions. All helices with backbone constraints and loops were separately minimized. After combining, all structures were reminimized initially with backbone constraints in the secondary structure and then without any constraints. The Amber all-atom force field⁴⁴ with a fixed dielectric constant of 4.0 was used for all calculations, terminating when the conjugate gradient reached 0.05 kcal·mol⁻¹·Å⁻¹.

For the conformational refinement of the adenosine A₃ receptor, the optimized structures were then used as the starting point for subsequent 50-ps MD, during which the protein backbone atoms in the secondary structures were constrained as in the previous step. The options of MD at 300 K with a 0.2-ps coupling constant were a time step of 1fs and a nonbonded update every 25 fs. The lengths of bonds with hydrogen atoms were constrained according to the SHAKE algorithm.⁴⁵ The average structure from the last 10-ps trajectory of MD was reminimized with backbone constraints in the secondary structure and then without all constraints as described above.

For a starting point for FlexiDock,⁴⁶ the superimposition between 39 and MRS1220 (9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-phenylacetamide),¹⁸ both A₃ selective antagonists, was performed. All heteroatoms except H and S atoms of 39 were fitted into the bound conformation of MRS1220 as a template. Flexible docking was facilitated through the FlexiDock utility in the Biopolymer module of SYBYL 6.9. During flexible docking, the ligand and the side chains of hydrophilic amino acids in the putative binding site were defined as rotatable bonds. After the hydrogen atoms were added to the receptor, atomic charges were recalculated by using Kollman All-atom for the protein and Gasteiger-Hückel for the ligand. H-bonding sites were marked for all residues in the active site and ligands with H-bond donor or acceptor. Ligands were variously pre-positioned in the putative binding cavity guided by several superimposition results. Default FlexiDock parameters were set at 3000-generations for genetic algorithms. To increase the binding interaction, the torsion angles of the side chains within 5 Å of the ligands were manually adjusted from the results of FlexiDock. Finally, the complex structure was minimized by using an Amber force field with a fixed dielectric constant (4.0), until the conjugate gradient reached 0.1 kcal·mol⁻¹·Å⁻¹.

Several possible docking modes of 39, which were consistent with the SAR study, were selected from the energetically favorable models. The most similar conformation of antagonist 42 compared to the bound conformation of antagonist 39 was chosen and superimposed into the putative binding sites of several possible docking models. The energies of the two regioisomer complexes were compared.