Design and Synthesis of Amide Derivatives as S-Adenosyl-L-Homocysteine Hydrolase Inhibitors

Abstract

In this study, a series of amide derivatives were synthesized and evaluated for their S-adenosyl-L-homocysteine hydrolase (SAHase) inhibitory activities. The results demonstrated that most of compounds displayed potent SAHase inhibitory activities. Interestingly, compounds 11 and 14 exhibited more potent inhibitory effects than the aristeromycin, one of the most potent SAHase inhibitors known so far. Compounds 12, 13, 15 and 17 exhibited a moderate effect (IC₅₀<10.0 µM). The structure–activity relationship found that compounds with substituted indazole-5-yl group at Ar position and ethylamino group at the side chain showed better SAHase inhibitory activities.

S-Adenosyl-L-homocysteine hydrolase (SAHase) can catalyze the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine (ADO) and L-homocysteine (HCY).¹⁾ Transmethylation reactions are involved in various biological phenomena related to the development of pathological processes.²⁾ S-Adenosyl-L-methionine (AdoMet) seems to be the most versatile methyl donor in mammalian systems. The formation of SAH from AdoMet, and the inhibition of cellular SAHase results in an intracellular accumulation of SAH, which leads to feedback inhibition of AdoMet dependent methylations.³⁾

In recent years, SAHase has become an attractive target for drug design, SAHase inhibitors have been shown to exhibit antiviral,^4,5) antiparasitic,⁶⁾ anti-cancer,⁷⁾ and immunosuppressive effect,^8,9) SAHase inhibitors have also been shown to exhibit plasma homocysteine-lowering effect.^10,11) Numerous SAHase inhibitors have been extensively reported in literature, and all the existing SAHase inhibitors can be divided into three types according to the mechanisms of enzyme inhibition. Most of SAHase inhibitors were type I or type II inhibitor which were irreversible inhibitors,¹⁰⁾ including aristeromycin, neplanocin A and 3-deazaadenosine (3-DZA) (Fig. 1). Type III inhibitors can reversibly bind to the open form of the enzyme, maintaining a similar potency with much reduced toxicity, such as DZ2002^8,9) (Fig. 1). However, most of the existing SAHase inhibitors are adenosine analogues, and most of the work previously reported on inhibitors of this enzyme has focused on systematically altering the structure of adenosine and evaluating the derivatives.¹²⁾ There have been very few studies on new structure as SAHase inhibitors.

Fig. 1. Chemical Structures of Some of the SAHase Inhibitors

Recent studies found the N-(carbamoylmethyl)glycinamide derivatives exhibited SAHase inhibitory activities.¹³⁾ In terms of the structure of these compounds, we chose the different substituted phenylazanediyl diacetic acid derivatives as the core scaffold, which firstly reacted with N-methylisoindolin-2-amine hydrochloride to obtain monoacid derivatives, and then acylation reaction with ethylenediamine derivatives to configure the side chain of this moiety. Accordingly, a novel class of amide derivatives (11–25) was synthesized and their SAHase inhibitory activity were estimated. These compounds were also designed to examine the role of different substitutes at aryl (Ar) position of phenylazanediyl diacetic acid and different substitutes in the side chain. It is hoped that continued research will lead to the development of new lead compounds from N-(carbamoylmethyl)glycinamide derivatives as effective SAHase inhibitors.

Results and Discussion

Chemistry

The general method for the synthesis of the amide derivatives 11–25 was described in Chart 1. Bromination of the compound 1 with N-bromosuccinimide (NBS) gave intermediate 2 in 80% yield.^14,15) Treatment of 2 with N-tert-butoxycarbonyl-N-methylhydrazine to afford compound 3 in 75% yield.¹³⁾ Finally, the deprotection of tert-butoxycarbonyl (Boc) group on amino group by hydrolysis with concentrated hydrochloric acid was carried out at room temperature then transformed to compound 4 as hydrochloride salt in 70% yield.¹⁶⁾ The corresponding aromatic amines 5a–e were firstly alkylated with ethyl bromoacetate under the basic condition to give compounds 6a–e,¹⁷⁾ which were hydrolyzed with potassium hydroxide in ethanol afforded the desired substituted phenylazanediyl diacetic acids 7a–e. The intermediates 8a–e were obtained by dehydration of the substituted phenylazanediyl diacetic acids 7a–e in the presence of acetic anhydride at 90°C.¹⁸⁾ Treatment of 8a–e with 4 under the basic condition to give monacid derivatives 9a–e. The amide derivatives 11–25 were prepared by condensation of monacid derivatives 9a–e and various ethylenediamine derivatives 10 with 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride (EDCI) as condensation agent and 1-hydroxy-benzotriazole (HOBt) as catalyst in N,N-dimethylformamide (DMF) in good yield.^18,19) The chemical structures of all the synthesized compounds were characterized by ¹H-NMR, MS and elemental analysis (C, H, N).

Chart 1. Synthetic Protocol of the Amide Derivatives 11–25

Reagents and conditions: (a) NBS, benzoyl peroxide, CHCl₃, reflux, 5 h, 80%; (b) NH₂–N(CH₃)Boc, Et₃N, DMF, rt, 75%; (c) conc. HCl, 70%; (d) BrCH₂CO₂Et, Na₂HPO₄, NaI, CH₃CN, reflux, 12 h; (e) KOH, EtOH then 2 N HCl, 35–45% in 2 steps; (f) Ac₂O, 90°C, 3 h; (g) Et₃N, THF, rt; (h) EDCI, HOBt, DMF, Et₃N, rt, 34–47% in 3 steps.

Biological Activity

All the newly synthesized compounds, along with the reference compounds aristeromycin and 3-DZA, were screened for their SAHase inhibitory activities using the method which descried in literature.^20,21) The results, expressed as inhibition ration and IC₅₀ values, were summarized in Table 1.

Table 1. The SAHase Inhibitory Activity of 11–25

Compound	Inhibition ration (%)		IC50 (µM)a)
	0.5 µM	2.5 µM
11	62.31±1.48	85.72±1.69	0.48
12	24.24±1.78	54.24±1.12	2.03
13	9.53±1.70	30.19±3.02	7.61
14	69.58±0.72	86.56±1.10	0.44
15	17.24±1.30	38.24±1.46	4.13
16	15.97±2.35	27.15±1.24	25.34
17	49.72±0.20	79.46±2.83	0.73
18	10.92±0.91	21.46±3.38	12.12
19	6.41±0.23	20.16±0.84	14.08
20	4.23±0.14	8.23±1.02	78.82
21	0±0.32	2.63±0.41	>500
22	0±0.96	4.56±0.77	>500
23	8.23±0.92	19.23±1.35	15.60
24	4.56±0.45	6.97±0.87	101.9
25	0±0.43	4.89±0.45	214
Aristeromycin	83.93±0.90	98.35±0.16	0.49b)
3-DZA	2.16±0.47	35.37±3.2	5.52c)

a) Values were determined from logarithmic concentration–inhibition curves and are given as means of three experiments. b) Values in the literature is 0.2 µM, the SAHase from Mycobacterium tuberculosis.²²⁾ c) Values in the literature is 20 µM, the SAHase from Mycobacterium tuberculosis.²²⁾

As shown in Table 1, most of the compounds displayed potent SAHase inhibitory activities. Five compounds (11, 12, 14, 15, 17) displayed more potent SAHase inhibitory activities than the reference inhibitor 3-DZA (IC₅₀=5.52 µM). The most promising compound 11 (IC₅₀=0.48 µM) and 14 (IC₅₀=0.44 µM) exhibited more potent SAHase inhibition effects than the reference inhibitor aristeromycin (IC₅₀=0.49 µM), one of the most potent SAHase inhibitors known so far. Meanwhile, compounds 13, 18 and 19 also exhibited SAHase inhibitory activities in some extent.

All of the amide derivatives 11–25. In most case, compounds with substituted indazole-5-yl group (11–15) displayed potent SAHase inhibitory activities (except compound 16). Particularly, compound 14 was found to be the most active compounds of this series. When the 2-methyl-2H-indazole-5-yl group of compound 14 was replaced with 4-(1H-pyrrol-1-yl) phenyl group as in compound 17, a slight decrease in SAHase inhibition potency was observed. However, when the 2-methyl-2H-indazole-5-yl group of compound 14 was replaced with 4-methylphenyl group as in compound 23, a significant decrease in SAHase inhibition potency was observed. Unfortunately replacement of 2-methyl-2H-indazole-5-yl group of compound 14 with 4-fluorophenyl to afford corresponding compounds 20–22 resulted in a loss of activity, and 4-methylphenyl derivatives 24 and 25 were also devoid of SAHase inhibitory activities. These facts imply that the introduction of substituted indazole-5-yl group on the Ar region obviously affected the SAHase inhibitory activity, and in the present investigation, 4-(1H-pyrrol-1-yl) phenyl group facilitated their inhibitory activities, while 4-fluorophenyl group and 4-methylphenyl group were unfavorable.

Substituted ethylenediamine structure at the side chain also affects the SAHase inhibitory activity. Compounds 11, 14, 17, 20 and 23 bearing ethylamino group at side chain exhibited highest influence on SAHase inhibitory activity, followed by the corresponding compounds with piperidinyl group at side chain (12, 15, 18, 21, 24). However, the corresponding compounds with diethylamino group at side chain (13, 16, 19, 22, 25) displayed lower SAHase inhibition potency. These results indicate that the ethylamino group is considered to be the best substituent at side chain showing potent SAHase inhibitory activity.

Conclusion

In summary, a novel class of amide derivatives were designed, synthesized and evaluated as SAHase inhibitors. The results demonstrated that most of target compounds displayed potent SAHase inhibitory activities. Particularly, compounds 11 and 14 exhibited more potent inhibitory effects than the aristeromycin and 3-DZA. The compounds with 1-methyl-1H-indazole-5-yl group or 2-methyl-2H-indazole-5-yl group at Ar position and ethylamino group at side chain showed better SAHase inhibitory activities. These results showed above are very encouraging, and further investigation of this kind of compounds may be of interest.

Experimental

Chemistry

Melting points were determined on WRS-21 melting point apparatus and were uncorrected. ¹H-NMR spectra were recorded on INOVA 400 (400 MHz) spectrometer with tetramethylsilane (TMS) as an internal standard. Chemical shifts (δ) are in ppm relative to TMS, and coupling constants (J) are expressed in hertz (Hz). Electron-spray ionization mass spectra (ESI-MS) in positive mode were recorded on a HP5989A mass spectrometer. Column chromatography was performed on 200–300 mesh silica gel. Analytical thin layer chromatography (TLC) was performed on precoated silica gel 60 F254 plates and visualization on TLC was achieved by UV light (254, 354 nm). Unless otherwise stated, all commercial reagents and solvents were used without additional purification.

1,2-Bis(bromomethyl)benzene (2)

A mixture of compound 1 (40 g, 0.38 mol), NBS (140.8 g, 0.79 mol), benzoyl peroxide (0.91 g, 3.8 mmol) in CHCl₃ (400 mL) was heated for 5 h under reflux. The reaction mixture was cooled to room temperature and CH₂Cl₂ (400 mL) was added to the mixture. The organic layer was washed with water (2×200 mL) and dried over Na₂SO₄, filtered, and the solvent were removed in vacuo. The residue was recrystallized from n-hexane–ethanol (30 : 1, 620 mL) to give compound 2 (79.0 g, 80%) as white solid, mp 92–93°C. ¹H-NMR (400 MHz, CDCl₃) δ: 4.66 (4H, s), 7.29–7.38 (4H, m).

tert-Butyl Isoindolin-2-yl(methyl) Carbamate (3)

A mixture of compound 2 (59.7 g, 0.22 mol), tert-butyl methylcarbamate (34.7 g, 0.23 mol) and triethylamine (44.5 g, 0.44 mol) in DMF (300 mL) was heated for 2 h at 65°C. The reaction mixture was cooled to room temperature and water (400 mL) was added to the mixture, the aqueous phase was extracted with ethyl acetate (3×300 mL). The combined organic layer was washed with brine and dried over Na₂SO₄, filtered, and the solvent were removed in vacuo to get compound 3 (35.5 g, 65.0%) as white solid, mp 62–64°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 1.35 (9H, s), 2.99 (3H, s), 4.33 (4H, s), 7.20–7.22 (4H, m). ESI-MS m/z: 249.16 [M+H]⁺.

N-Methylisoindolin-2-amine Hydrochloride (4)

Compound 3 (27.5 g, 0.11 mol) was dissolved in concentrated hydrochloric acid (83 mL), and stirred at room temperature for 12 h. The reaction mixture was evaporated under reduced pressure and co-evaporated with ethanol (1000 mL). The residue was recrystallized from ethanol (100 mL) to give compound 4 (14.2 g, 70%) as white solid, mp 160–162°C. ¹H-NMR (400 MHz, DMSO-d₆+D₂O) δ: 2.88 (3H, s), 4.59 (4H, s), 7.41–7.42 (4H, m). ESI-MS m/z: 149.11 [M+H]⁺.

General Procedure for the Synthesis of Substituted Phenylazanediyl Diacetic Acids (7a–e)

A mixture of aromatic amines 5a–e (0.1 mol), ethyl 2-bromoacetate (0.21 mol), Na₂HPO₄ (0.25 mol) and NaI (0.05 mol) in CH₃CN (250 mL) was heated for 12 h under reflux. The reaction mixture was cooled to room temperature and concentrated in vacuo, and the water was added to the residue, the aqueous phase was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over Na₂SO₄, filtered, and the solvent were removed in vacuo to obtain 6a–e. The solution of KOH (0.25 mol) in ethanol (500 mL) was added to the solution of 6a–e in ethanol (100 mL), and the reaction mixture was heated at 60°C for 1 h. The reaction mixture was cooled to room temperature, filtered, and the filtrate was dissolved in water. The pH of the suspension was adjusted to 2 with 2 N HCl. The crystalline product was precipitated, filtrated and dried under vacuum to give 7a–e.

2,2′-((1-Methyl-1H-indazol-5-yl)azanediyl)diacetic Acid (7a): Yield 40% (2 steps), white solid, mp 172–174°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.95 (3H, s), 4.14 (4H, s), 6.87–6.90 (1H, m), 7.47 (1H, d, J=9.2 Hz), 7.81 (1H, s). ESI-MS m/z: 264.12 [M+H]⁺.

2,2′-((2-Methyl-2H-indazol-5-yl)azanediyl)diacetic Acid (7b): Yield 35% (2 steps), white solid, mp 156–158°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.91 (3H, s), 4.12 (4H, s), 6.70 (1H, s), 6.85–6.88 (1H, m), 7.42–7.45 (1H, d, J=9.2 Hz), 7.79 (1H, s). ESI-MS m/z: 264.16 [M+H]⁺.

2,2′-((4-(1H-Pyrrol-1-yl)phenyl)azanediyl)diacetic Acid (7c): Yield 30% (2 steps), brown solid, mp 150–152°C. ¹H-NMR (400 MHz DMSO-d₆+D₂O) δ: 4.13 (4H, s), 6.17–6.20 (2H, m), 6.59–6.66 (2H, m), 7.10–7.26 (2H, m), 7.33 (2H, d, J=8.8 Hz). ESI-MS m/z: 275.12 [M+H]⁺.

2,2′-((4-Fluorophenyl)azanediyl)diacetic Acid (7d): Yield 42% (2 steps), white solid, mp 192–194°C. ¹H-NMR (400 MHz, CDCl₃) δ: 4.12 (4H, s), 6.55–6.57 (2H, m), 6.96–7.01 (2H, m). ESI-MS m/z: 228.06 [M+H]⁺.

2,2′-(p-Tolylazanediyl)diacetic Acid (7e): Yield 45% (2 steps), white solid, mp 202–204°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 2.16 (3H, s), 3.99 (4H, s), 6.37 (2H, d, J=8.8 Hz), 6.97 (2H, d, J=8.8 Hz). ESI-MS m/z: 224.10 [M+H]⁺.

General Procedure for the Synthesis of Amide Derivatives (11–25)

A mixture of substituted phenylazanediyl diacetic acids 7a–e (3.8 mmol) and acetic anhydride (20 mL) was heated to 90°C for 3 h, and the reaction mixture was concentrated in vacuo to get intermediates 8a–e. A solution of triethylamine (9.5 mmol) in tetrahydrofuran (THF) (20 mL) was added to the corresponding intermediates 8a–e, the mixture was stirred at room temperature for 10 min. Compound 4 (3.8 mmol) was added in three portions and the resulting mixture was stirred at room temperature for 12 h. The solution was concentrated and water was added to the residue. The mixture was extracted with ethyl acetate–THF (10 : 1) and the combined organic layer was dried over Na₂SO₄, filtered, then evaporated to remove solvent to obtain corresponding monacid derivatives 9a–e. To a stirred solution of 9a–e in DMF (20 mL) was added triethylamine (9.5 mmol), EDCI (7.6 mmol) and HOBt (7.6 mmol). After the addition was completed, the reaction mixture was stirred for 10 min at room temperature. A solution of corresponding ethylenediamine derivatives 10 (4.56 mmol) was added to the reaction mixture, and the resulting mixture was stirred at room temperature for 12 h. The reaction mixture was poured into water. The pH of the suspension was adjusted to 10 with 5% aqueous NaOH, and the aqueous phase was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over Na₂SO₄, filtered, evaporated, and purified by flash column chromatography (CHCl₃–CH₃OH=8 : 1) to afford amide derivatives 11–25.

2-((2-((2-(Ethylamino)ethyl)amino)-2-oxoethyl)(1-methyl-1H-indazol-5-yl)amino)-N-(isoindolin-2-yl)-N-methylacetamide (11): Yield 39% (3 steps), white solid, mp 178–179°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 0.91 (3H, t, J=7.2 Hz), 2.51–2.61 (4H, m), 2.97 (3H, s), 3.19–3.22 (2H, m), 3.91 (3H, s), 3.98 (2H, s), 4.27–4.38 (4H, m), 4.66 (2H, s), 6.60–6.87 (2H, m), 7.24–7.46 (5H, m), 8.93 (1H, br s). ESI-MS m/z: 464.27 [M+H]⁺. Anal. Calcd for C₂₅H₃₃N₇O₂: C, 64.77; H, 7.18; N, 21.15. Found: C, 64.87; H, 7.24; N, 21.03.

N-(Isoindolin-2-yl)-N-methyl-2-((1-methyl-1H-indazol-5-yl)(2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)amino)acetamide (12): Yield 47% (3 steps), white solid, mp 124–125°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 1.28–1.39 (6H, m), 2.42–2.48 (6H, m), 2.98 (3H, s), 3.22–3.26 (2H, m), 3.95–4.01 (5H, m), 4.37–4.40 (4H, m), 4.65 (2H, s), 6.58 (1H, s), 6.71–6.74 (1H, m), 7.24–7.37 (4H, m), 7.46–7.48 (1H, m), 7.82 (1H, s), 8.89 (1H, br s). ESI-MS m/z: 504.54 [M+H]⁺. Anal. Calcd for C₂₈H₃₇N₇O₂: C, 66.77; H, 7.40; N, 19.47. Found: C, 66.65; H, 7.45; N, 19.56.

2-((2-((2-(Diethylamino)ethyl)amino)-2-oxoethyl)(1-methyl-1H-indazol-5-yl)amino)-N-(isoindolin-2-yl)-N-methylacetamide (13): Yield 39.0% (3 steps), white solid, mp 91–93°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 0.89 (6H, t, J=6.8 Hz), 2.49–2.61 (6H, m), 2.96 (3H, s), 3.23 (2H, t, J=6.4 Hz), 3.94–3.98 (5H, m), 4.36 (4H, s), 4.64 (2H, s), 6.61 (1H, s), 6.74–6.76 (1H, m), 7.25–7.33 (4H, m), 7.43 (1H, d, J=9.2 Hz), 7.79 (1H, s), 8.85 (1H, br s). ESI-MS m/z: 492.32 [M+H]⁺. Anal. Calcd for C₂₇H₃₇N₇O₂: C, 65.96; H, 7.59; N, 19.94 . Found: C, 66.04; H, 7.50; N, 19.87.

2-((2-((2-(Ethylamino)ethyl)amino)-2-oxoethyl)(2-methyl-2H-indazol-5-yl)amino)-N-(isoindolin-2-yl)-N-methylacetamide (14): Yield 34% (3 steps), white solid, mp 131–132°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 0.97 (3H, t, J=7.2 Hz), 2.68–2.78 (4H, m), 2.90 (3H, s), 3.27–3.29 (2H, m), 3.94 (3H, s), 4.00 (2H, s), 4.29 (4H, s), 4.58 (2H, s), 6.61(1H, s), 6.74–6.77 (1H, m), 7.22–7.43 (5H, m), 7.78 (1H, s), 8.98 (1H, br s). ESI-MS m/z: 464.30 [M+H]⁺. Anal. Calcd for C₂₅H₃₃N₇O₂: C, 64.77; H, 7.18; N, 21.15; Found: C, 64.69; H, 7.24; N, 21.09.

N-(Isoindolin-2-yl)-N-methyl-2-((2-methyl-2H-indazol-5-yl)(2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)amino)acetamide (15): Yield 40% (3 steps), white solid, mp 155–157°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 1.25–1.37 (6H, m), 2.41–2.51 (6H, m), 2.98 (3H, s), 3.25 (2H, t, J=6.4 Hz), 3.95–3.98 (5H, m), 4.33 (4H, s), 4.61 (2H, s), 6.61 (1H, s), 6.73–6.76 (1H, m), 7.24–7.45 (5H, m), 7.81 (1H, s), 8.85 (1H, br s). ESI-MS m/z: 504.31 [M+H]⁺. Anal. Calcd for C₂₈H₃₇N₇O₂: C, 66.77; H, 7.40; N, 19.47. Found: C, 66.79; H, 7.34; N, 19.55.

2-((2-((2-(Diethylamino)ethyl)amino)-2-oxoethyl)(2-methyl-2H-indazol-5-yl)amino)-N-(isoindolin-2-yl)-N-methylacetamide (16): Yield 45% (3 steps), white solid, mp 98–99°C. ¹H-NMR (400 MHz, DMSO-d₆+D₂O) δ: 0.90 (6H, t, J=7.2 Hz), 2.50–2.62 (6H, m), 2.93 (3H, s), 3.23 (2H, t, J=6.4 Hz), 3.95–3.99 (5H, m), 4.37 (4H, s), 4.65 (2H, s), 6.60 (1H, s), 6.73 (1H, d, J=8.8 Hz), 7.23–7.31 (4H, m), 7.44 (1H, d, J=8.8 Hz), 7.80 (1H, s), 8.85 (1H, br s). ESI-MS m/z: 492.35 [M+H]⁺. Anal. Calcd for C₂₇H₃₇N₇O₂: C, 65.96; H, 7.59; N, 19.94. Found: C, 65.90; H, 7.64; N, 19.98.

2-((4-(1H-Pyrrol-1-yl)phenyl)(2-((2-(ethylamino)ethyl)amino)-2-oxoethyl)amino)-N-(isoindolin-2-yl)-N-methylacetamide (17): Yield 31% (3 steps), white solid, mp 133–135°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 1.09 (3H, t, J=7.2 Hz), 2.79–2.87 (4H, m), 2.95 (3H, s), 3.20 (2H, t, J=6.4 Hz), 3.99 (2H, s), 4.34 (4H, s), 4.63 (2H, s), 6.18–6.19 (2H, m), 6.50 (2H, d, J=9.2 Hz), 7.11–7.13 (2H, m), 7.25–7.39 (6H, m), 8.90 (1H, br s). ESI-MS m/z: 475.35 [M+H]⁺. Anal. Calcd for C₂₇H₃₄N₆O₂: C, 68.33; H, 7.22; N, 17.71. Found: C, 68.27; H, 7.26; N, 17.77.

2-((4-(1H-Pyrrol-1-yl)phenyl)(2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)amino)-N-(isoindolin-2-yl)-N-methylacetamide (18): Yield 39% (3 steps), white solid, mp 124–126°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 1.40–1.45 (6H, m), 2.49–2.51 (6H, m), 2.98 (3H, s), 3.98 (2H, s), 4.36 (4H, s), 4.65 (2H, s), 6.15–6.18 (2H, m), 6.48 (2H, d, J=9.2 Hz), 7.12–7.13 (2H, m), 7.27–7.35 (6H, m), 8.90 (1H, br s). ESI-MS m/z: 515.33 [M+H]⁺. Anal. Calcd for C₃₀H₃₈N₆O₂: C, 70.01; H, 7.44; N, 16.33. Found: C, 70.08; H, 7.39; N, 16.38.

2-((4-(1H-Pyrrol-1-yl)phenyl)(2-((2-(diethylamino)ethyl)amino)-2-oxoethyl)amino)-N-(isoindolin-2-yl)-N-methylacetamide (19): Yield 44% (3 steps), white solid, mp 70–72°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 1.04 (6H, t, J=7.2 Hz), 2.89–2.94 (9H, m), 3.34–3.37 (2H, m), 3.99 (2H, s), 4.32 (4H, s), 4.61 (2H, s), 6.18–6.19 (2H, m), 6.48–6.51 (2H, m), 7.12–7.14 (2H, m), 7.28–7.32 (6H, m), 8.98 (1H, br s). ESI-MS m/z: 503.32 [M+H]⁺. Anal. Calcd for C₂₉H₃₈N₆O₂: C, 69.29; H, 7.62; N, 16.72. Found: C, 69.34; H, 7.66; N, 16.63.

2-((2-((2-(Diethylamino)ethyl)amino)-2-oxoethyl)(4-fluorophenyl)amino)-N-(isoindolin-2-yl)-N-methylacetamide (20): Yield 51% (3 steps), white solid, mp 74–76°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 1.07 (3H, t, J=7.2 Hz), 2.70–2.77 (4H, m), 2.95 (3H, s), 3.27–3.30 2H, m), 3.94 (2H, s), 4.34 (4H, s), 4.61 (2H, s), 6.40–6.43 (2H, m), 6.97–6.99 (2H, m), 7.25–7.30 (4H, m), 8.90 (1H, br s). ESI-MS m/z: 428.20 [M+H]⁺. Anal. Calcd for C₂₃H₃₀FN₅O₂: C, 64.62; H, 7.07; N, 16.38. Found: C, 64.56; H, 7.01; N, 16.44.

2-((4-Fluorophenyl)(2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)amino)-N-(isoindolin-2-yl)-N-methylacetamide (21): Yield 45% (3 steps), white solid, mp 145–146°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 1.33–1.46 (3H, m), 2.42–2.50 (6H, m), 2.95 (3H, s), 3.20–3.28 (2H, m), 3.90 (2H, s), 4.33 (4H, s), 4.57 (2H, s), 6.38–6.42 (2H, m), 6.97–7.01 (2H, m), 7.25–7.31 (4H, m), 8.78 (1H, br s). ESI-MS m/z: 468.32 [M+H]⁺. Anal. Calcd for C₂₆H₃₄FN₅O₂: C, 66.79; H, 7.33; N, 14.98. Found: C, 66.72; H, 7.28; N, 14.91.

2-((2-((2-(Diethylamino)ethyl)amino)-2-oxoethyl)(4-fluorophenyl)amino)-N-(isoindolin-2-yl)-N-methylacetamide (22): Yield 43% (3 steps), white solid, mp 85–87°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 0.97–1.05 (6H, m), 2.69–2.74 (6H, m), 2.92 (3H, s), 3.25–3.30 (2H, m), 3.91 (2H, s), 4.33 (4H, s), 4.58 (2H, s), 6.39–6.44 (2H, m), 6.96–7.02 (2H, m), 7.24–7.31 (4H, m), 8.77 (1H, br s). ESI-MS m/z: 456.28 [M+H]⁺. Anal. Calcd for C₂₅H₃₄FN₅O₂: C, 65.91; H, 7.52; N, 15.37. Found: C, 65.95; H, 7.48; N, 15.44.

2-((2-((2-(Ethylamino)ethyl)amino)-2-oxoethyl)(p-tolyl)amino)-N-(isoindolin-2-yl)-N-methylacetamide (23): Yield 54% (3 steps), white solid, mp 162–163°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 1.11 (3H, t, J=7.2 Hz), 2.17 (3H, m), 2.73–2.81 (4H, m), 2.99 (3H, s), 3.32–3.35 (2H, m), 3.93 (2H, s), 4.30–4.38 (4H, m), 4.61 (2H, s), 6.32 (2H, d, J=8.4 Hz), 6.96 (2H, d, J=8.4 Hz), 7.26–7.33 (4H, m), 9.06 (1H, s). ESI-MS m/z: 424.20 [M+H]⁺. Anal. Calcd for C₂₄H₃₃N₅O₂: C, 68.06; H, 7.85; N, 16.53. Found: C, 68.13; H, 7.79; N, 16.48.

N-(Isoindolin-2-yl)-N-methyl-2-((2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)(p-tolyl)amino)acetamide (24): Yield 48% (3 steps), white solid, mp 117–119°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 1.23–1.42 (6H, m), 2.16 (3H, s), 2.26–2.32 (6H, m), 2.96 (3H, s), 3.16–3.26 (2H, m), 3.88 (2H, s), 4.28–4.37 (4H, m), 4.56 (2H, s), 6.31 (2H, d, J=8.4 Hz), 6.96 (2H, d, J=8.4 Hz), 7.25–7.33 (4H, m), 8.84 (1H, br s). ESI-MS m/z: 464.19 [M+H]⁺. Anal. Calcd for C₂₇H₃₇N₅O₂: C, 69.95; H, 8.04; N, 15.11. Found: C, 70.01; H, 8.09; N, 15.03.

2-((2-((2-(Diethylamino)ethyl)amino)-2-oxoethyl)(p-tolyl)amino)-N-(isoindolin-2-yl)-N-methylacetamide (25): Yield 46% (3 steps), white solid, mp 137–138°C. ¹H-NMR (400 MHz, DMSO-d₆) δ: 0.96 (6H, t, J=7.2 Hz), 2.16 (3H, s), 2.54–2.61 (6H, m), 2.95 (3H, s), 3.23 (2H, m), 3.89 (2H, s), 4.29–4.37 (4H, m), 4.57 (2H, s), 6.31 (2H, d, J=8.4 Hz), 6.95 (2H, d, J=8.4 Hz), 7.25–7.33 (4H, m), 8.87 (1H, br s). ESI-MS m/z: 452.26 [M+H]⁺. Anal. Calcd for C₂₆H₃₇N₅O₂: C, 69.15; H, 8.26; N, 15.51. Found: C, 69.09; H, 8.29; N, 15.57.

SAHase Inhibition Assay

The activity was measured following the procedure described previously with a slight modification.^19,20) This method involves the hydrolytic conversion of SAH into ADO and HCY. SAHase from human placenta, SAH was obtained from Sigma, adenosine deaminase (ADA) and ThioGlo 1 were obtained from Calbiochem. SAHase and all compounds were cultured in phosphate buffer (pH 8.0) before initiated the reaction. ThioGlo 1 solution was freshly prepared in phosphate buffer (pH 8.0) before initiated the reaction. The reaction mixture (50 µL) contained ethylene-diaminetetraacetic acid (50 mM), ADA (0.03 U), SAH (1.68 µM), SAHase (0.5 mU) and various concentrations of inhibitors. The reaction took place at 37°C for 10 min, and the reaction mixture was quenched through the addition of 50 µL pure ice-cold isopropanol. A solution of 100 µL of 20 µM ThioGlo1 in dimethyl sulfoxide was added to reaction mixture. The plate was maintained in the dark at room temperature for 10 min, and the fluorescence was read using the SpectraMax M5 spectrophotometer with 380 nm excitation and 510 nm emission filters. One unit of inhibitory activity was defined as the amount of the sample needed to inhibit one unit of enzyme activity. IC₅₀ value was determined by lineal regression of the dose–response curves and was defines as the amount of the sample needed to inhibit the 50% of control enzyme activity.

Acknowledgment

The work was supported by ‘New Drug Innovation 2009X09313-006’ from the Ministry of Science and Technology of China.

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