1. Introduction

S-adenosyl-L-homocysteine hydrolase (SAHase) is a highly conserved enzyme that catalyses the reversible hydrolysis of Sadenosylhomocysteine (SAH) to L-homocysteine (HCy) and adenosine (Ado) ^[1]. This reaction is reversible,with the equilibrium largely favors the direction of SAH synthesis,although physiologically the reaction proceeds in the direction of hydrolysis because Ado and HCy are readily removed by further metabolism. SAHase dysfunction is associated with various diseases ^[2-4],including vascular disorders,myopathy,fatty liver,cancer,renal insufficiency and diabetic nephropathy. The inhibition of SAHase results in the cellular accumulation of SAH,which is a potent feedback inhibitor of SAM-dependent biological methylation ^[5]. Because SAHase plays a key role in the regulation of transmethylation in all eukaryotic organisms,a number of SAHase inhibitors have been designed as drugs against various diseases,including viral infections ^[6-8],malaria ^[9],cancer ^[10] and tuberculosis ^[11]. SAHase inhibitors have also been shown to have immunosuppressive ^{[12, 13]} and homocysteine-lowering effects ^{[14, 15]}.

Most of the known SAHase inhibitors are adenosine analogs ^[16],including 3-deazaadenosine (3-DZA),aristeromycin and neplanocin A (Fig. 1). Some of them are potent,irreversible inhibitors of SAHase. However,these irreversible inhibitors showed high toxicity and are not suitable for clinical usage. Therefore,it is urgent to develop novel non-adenosine SAHase inhibitors with high efficacy and low toxicity,particularly with different mode of actions compared to currently existing adenosine analogs. Some non-adenosine reversible SAHase inhibitors have been reported recently ^{[17, 18]}. In our previous work,we found that some N-(carbamoylmethyl)glycinamide derivatives,a kind of non-adenosine analogs,showed inhibitory activity against SAHase. We also demonstrated that the compounds with electron withdrawing group at Ar position and ethylamino group at side chain showed better SAHase inhibitory activity ^[17]. Indazole can be considered as a phenyl ring fused with a pyrazole ring,and the pyrazole ring dramatically reduced the electron density of the phenyl ring in indazole. Indazole derivatives possess significant potency in a wide range of biological targets,so there have been increasing amount of synthetic compounds containing an indazole core reported for biological studies ^[19]. In this communication,we are going to report the synthesis and the structure-activity relationship studies of a new generation of N-(carbamoylmethyl)- glycinamide derivatives,which contain an indazole ring instead of a phenyl ring.

2. Experimental 2.1. Chemistry

All solvents and reagents were commercially available and used without further purification. ¹H NMR spectra were determined on INOVA 400 (400 MHz) spectrometer with tetramethylsilane (TMS) as an internal standard. Chemical shifts are reported in parts per million (ppm,δ scale),and coupling constants (J) are expressed in hertz (Hz). Mass spectra were determined on a HP5989A mass spectrometer. Thin layer chromatography (TLC) was performed on precoated plates (0.25 mm,silica gel 60F₂₄₅),and compounds were visualized with UV light. Melting points were performed in open capillary tubes and were uncorrected. Elemental analyses were obtained with a Carlo Erba EA 1108 instrument.

2.1.1. General procedure for preparation of compounds 3a-d

To a solution of indazole amines 1a-d (0.27 mol) in DMF (200 mL) were added ethyl bromoacetate (0.57 mol),K₂CO₃ (0.68 mol) and KI (0.14 mol). The mixture was heated for 12 h under reflux. The mixture was concentrated and the water was added,the aqueous layer was extracted with ethyl acetate. The extract was dried over MgSO₄,filtered and concentrated obtained ethyl ester 2a-d. The product was used in the following step without further purification. The ethyl ester 2a-d was hydrolyzed with the solution of KOH (0.25 mol) in ethanol (500 mL). After being stirred for 1 h at 60 °C,the mixture was cooled to room temperature,filtered,and the filtrate was dissolved in water. The pH of the aqueous layer was acidified with 1 mol/L aqueous HCl. The crystalline product was precipitated,filtrated and dried under vacuum to give compounds 3a-d.

2.1.2. General procedure for preparation of compounds 8a-y

To a solution of 3a-d (5.00 mmol) in acetic anhydride (25 mL) was heated to 90 °C for 3 h,and concentrated under reduced pressure to obtain intermediates 4a-d. To a solution of 4a-d in THF (20 mL) was added triethylamine (9.5 mmol) and stirred for 10 min at room temperature. Then various isoindolin-2-amine derivatives or indane-2-amine derivatives 5 (5.00 mmol) was added to the reaction mixture and stirred at room temperature for 12 h. The resulting mixture was concentrated and water was added to the residue. The mixture was extracted with ethyl acetate and the combined organic layer was dried over Na₂SO₄,filtered,and then evaporated to remove solvent to obtain compounds 6 which were used in the following step without further purification. To a stirred solution of 6 in DMF (25 mL) was added EDCI (10 mmol), HOBt (10 mmol),triethylamine (12.5 mmol),and the resulting mixture was stirred at room temperature for 1 h. Then,to the reaction mixture was added ethylenediamine derivatives 7 (5 mmol),and the resulting mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with ethyl acetate (100 mL),and the organic layer was washed with H2O (15 mL ×2),and dried over Na₂SO₄. The insoluble material was filtered off,and the solution was concentrated under reduced pressure and purified by silica gel column chromatography (CHCl₃/ CH₃OH = 10:1 to 1:10,v/v) to afford 8a-y.

The characterization data for the synthesized compounds are deposited in [4TD$DIF]Supporting information.

2.2. In vitro inhibitory activity against SAHase

The method of the enzyme inhibitory activity involves the hydrolytic conversion of SAH into Ado and HCy. SAHase was obtained 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) and ThioGlo 1 solution was freshly prepared in phosphate buffer (pH 8.0) before initiating the reaction. The reaction mixture (50 mL) containing ethylenediaminetetraaceticacid (50 mmol/L),ADA (0.03 U),SAH (1.68 mmol/L), SAHase (0.5 mU) and various concentrations of test compounds was incubated at 37 °C for 10 min,and the reaction was quenched by the addition of 50 mL pure ice-cold isopropanol. A solution of 100 mL of 20mmol/L ThioGlo1 in DMSO was added to reaction mixture. The plate was maintained in dark at room temperature for 10 min,and then the fluorescence was read on 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 defined as the amount of the sample needed to inhibit the 50% of control enzyme activity.

3. Results and discussion 3.1. Synthesis

The synthesis of target compounds is outlined in Scheme 1. Indazole amines 1a-d were used as the starting material,and the reaction of compounds 1a-d with ethyl bromoacetate under basic conditions gave compounds 2a-d ^[20],hydrolysis of 2a-d gave bisacetic acids 3a-d. The intermediates 4a-d were obtained by dehydration of the bisacetic acids 3a-d in the presence of acetic anhydride at 90 °C ^[21]. The reaction of 4a-d with various isoindolin-2-amine derivatives or indane-2- amine derivatives (5) yielded monoacid derivatives 6. Treatment of 6 with various ethylenediamine derivatives (7) using 1-ethyl-(3- (3-dimethylamino)propyl)-carbodiimide hydrochloride (EDCI) as a coupling agent and 1-hydroxy-benzotriazole (HOBt) as a catalyst in N,N-dimethylformamide (DMF) gave products in satisfactory isolated yield ([2TD$DIF]30%-50% in 3 steps). Evaporation of the solvent and column chromatography on silica gel (CHCl₃/CH₃OH) afforded target compounds 8a-y ^[22]. All the titled compounds containing an indazole moiety were new compounds and were characterized with ¹H NMR,ESI-MS and elemental analysis (C,H,N).

3.2. Biological evaluation

The 3-DZA and aristeromycin were used as the positive controls and the human placenta SAHase was employed as the enzyme source. The protocol of the inhibitory activity against SAHase was described in previous research ^{[17, 23, 24]}. Results are described in Table 1.

We firstly investigated the effect on SAHase inhibitory activity of different substituted indazolyl groups at the Ar position, including 1-methyl-1H-indazol-5-yl,1-methyl-1H-indazol-6-yl, 1,3-dimethyl-1H-indazol-6-yl and 2-methyl-2H-indazol-5-yl. Most of the target compounds showed good activity against SAHase. Seven compounds (8a,8b,8m,8r,8t,8u,8w) displayed more potent SAHase inhibitory activity than the reference inhibitor 3-DZA (IC₅₀ = 5.39 mmol/L). Meanwhile,compounds 8d and 8v also exhibited SAHase inhibitory activity close to that of 3- DZA. Eleven compounds (8c,8g,8h,8j,8l,8n,8o,8q,8s,8x,8y) exhibited a moderate effect (IC₅₀ < 20.0 mmol/L). Interestingly, four compounds (8b,8m,8r,8w) showed better or similar inhibitory effects compared to the positive control aristeromycin, one of the most potent SAHase inhibitors known so far.

Compounds 8e,8f,8g,8i and 8q,with an indane-2-amino group on the side chain,exhibited weak inhibitory activity against SAHase. A slight increase in SAHase inhibition potency was observed at a concentration of 2.5 mmol/L after replacing the indane-2-amino group in compound 8i with an isoindolin-2- amino group as in compound 8j. These facts imply that the introduction of an indane-2-amino group at the side chain was not crucial to enzymatic activity. Compounds 8rand 8u,with no substituent on the isoindolin ring,showed good activity with an IC₅₀ value of 0.6 mmol/L and 0.84 mmol/L,respectively. However, compounds 8sand 8twith an electron-withdrawing group on the isoindolin ring displayed weak SAHase inhibition potency, suggesting that electron-withdrawing groups on the isoindolin ring can diminish the SAHase inhibition potency. Substituted ethylenediamine structures on the other side chain also affect the SAHase inhibitory activity. Compound 8w,with an ethylamino group on side chain exhibited the most potent activity in the enzymatic assay (IC₅₀ = 0.44 mmol/L),However,the corresponding compounds with a diethylamino group ( 8x) or a piperidinyl group ( 8y) on the side chain were less potent. These results indicate that the ethylamino group on the side chain enhances the SAHase inhibitory activity.

4. Conclusion

In summary,a series of novel amide derivatives containing an indazole moiety were synthesized,and the structure-activity relationship was studied. The enzyme assay suggested that some of these new amide derivatives were stronger SAHase inhibitors than the positive controls 3-DZA and aristeromycin. Among the tested ones, 8w showed the most potent inhibitory effect. These results provide a novel lead for the discovery of more potent nonadenosine analogs as SAHase inhibitors.

Acknowledgments

This research was supported by National Natural Science Foundation of China (No. 81560653) and Guangxi Natural Science Foundation[5TD$DIF] of China (No. 2015GXNSFBA139124).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2016.03.028.