Design and synthesis of amide derivatives as S-adenosyl-L-homocysteine hydrolase inhibitors

Article abstract of DOI:10.1248/cpb.c13-00623

In this study, a series of amide derivatives were synthesized and evaluated for their S-adenosyl-Lhomocysteine 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.

Full text of DOI:10.1248/cpb.c13-00623

112
Note
Chem. Pharm. Bull. 62(1) 112–117 (2014)
Vol. 62, No. 1
Design and Synthesis of Amide Derivatives as S-Adenosyl-
L-Homocysteine Hydrolase Inhibitors
,a,b
Xiangduan Tan,a Panfeng Wang,b Siyun Nian,a and Guoping Wang*
a Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry; Shanghai 200437,
b
China: and Shanghai Shyndec Pharmaceutical Co., Ltd.; Shanghai 200437, China.
Received August 7, 2013; accepted October 20, 2013; advance publication released online October 25, 2013
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 in-
hibitory 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<10.0µM). 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.
Key words S-adenosyl-L-homocysteine hydrolase; S-adenosylhomocysteine; SAHase inhibitor; homocys-
teine; amide derivative
S-Adenosyl-L-homocysteine hydrolase (SAHase) can cata- and evaluating the derivatives.12) There have been very few
lyze the reversible hydrolysis of S-adenosylhomocysteine studies on new structure as SAHase inhibitors.
(SAH) to adenosine (ADO) and L-homocysteine (HCY).1)
Recent studies found the N-(carbamoylmethyl)glycinamide
Transmethylation reactions are involved in various biological derivatives exhibited SAHase inhibitory activities.13) In terms
phenomena related to the development of pathological process- of the structure of these compounds, we chose the different
es.2) S-Adenosyl-L-methionine (AdoMet) seems to be the most substituted phenylazanediyl diacetic acid derivatives as the
versatile methyl donor in mammalian systems. The formation core scaffold, which firstly reacted with N-methylisoindolin-
of SAH from AdoMet, and the inhibition of cellular SAHase 2-amine hydrochloride to obtain monoacid derivatives, and
results in an intracellular accumulation of SAH, which leads then acylation reaction with ethylenediamine derivatives to
to feedback inhibition of AdoMet dependent methylations.3)
configure the side chain of this moiety. Accordingly, a novel
In recent years, SAHase has become an attractive target for class of amide derivatives (11–25) was synthesized and their
drug design, SAHase inhibitors have been shown to exhibit SAHase inhibitory activity were estimated. These compounds
antiviral,4,5) antiparasitic,6) anti-cancer,7) and immunosuppres- were also designed to examine the role of different substitutes
sive effect,8,9) SAHase inhibitors have also been shown to at aryl (Ar) position of phenylazanediyl diacetic acid and dif-
exhibit plasma homocysteine-lowering effect.10,11) Numerous ferent substitutes in the side chain. It is hoped that continued
SAHase inhibitors have been extensively reported in lit- research will lead to the development of new lead compounds
erature, and all the existing SAHase inhibitors can be divided from N-(carbamoylmethyl)glycinamide derivatives as effective
into three types according to the mechanisms of enzyme SAHase inhibitors.
inhibition. Most of SAHase inhibitors were type I or type II
inhibitor which were irreversible inhibitors,10) including aris-
Results and Discussion
teromycin, neplanocin A and 3-deazaadenosine (3-DZA) (Fig.
Chemistry The general method for the synthesis of the
1). Type III inhibitors can reversibly bind to the open form of amide derivatives 11–25 was described in Chart 1. Bromina-
the enzyme, maintaining a similar potency with much reduced tion of the compound 1 with N-bromosuccinimide (NBS) gave
toxicity, such as DZ20028,9) (Fig. 1). However, most of the ex- intermediate 2 in 80% yield.14,15) Treatment of 2 with N-tert-
isting SAHase inhibitors are adenosine analogues, and most of butoxycarbonyl-N-methylhydrazine to afford compound 3 in
the work previously reported on inhibitors of this enzyme has 75% yield.13) Finally, the deprotection of tert-butoxycarbonyl
focused on systematically altering the structure of adenosine (Boc) group on amino group by hydrolysis with concentrated
Fig. 1. Chemical Structures of Some of the SAHase Inhibitors
The authors declare no conflict of interest.
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: wgpsipich@163.com

January 2014
113
Reagents and conditions: (a) NBS, benzoyl peroxide, CHCl3, reflux, 5h, 80%; (b) NH2–N(CH3)Boc, Et3N, DMF, rt, 75%; (c) conc. HCl, 70%; (d) BrCH2CO2Et, Na2HPO4,
NaI, CH3CN, reflux, 12h; (e) KOH, EtOH then 2N HCl, 35–45% in 2 steps; (f) Ac2O, 90°C, 3h; (g) Et3N, THF, rt; (h) EDCI, HOBt, DMF, Et3N, rt, 34–47% in 3 steps.
Chart 1. Synthetic Protocol of the Amide Derivatives 11–25
hydrochloric acid was carried out at room temperature then were screened for their SAHase inhibitory activities using
transformed to compound 4 as hydrochloride salt in 70% the method which descried in literature.20,21) The results, ex-
yield.16) The corresponding aromatic amines 5a–e were firstly pressed as inhibition ration and IC50 values, were summarized
alkylated with ethyl bromoacetate under the basic condi- in Table 1.
tion to give compounds 6a–e,17) which were hydrolyzed with
As shown in Table 1, most of the compounds displayed
potassium hydroxide in ethanol afforded the desired substi- potent SAHase inhibitory activities. Five compounds (11, 12,
tuted phenylazanediyl diacetic acids 7a–e. The intermedi- 14, 15, 17) displayed more potent SAHase inhibitory activities
ates 8a–e were obtained by dehydration of the substituted than the reference inhibitor 3-DZA (IC50=5.52 µM). The most
phenylazanediyl diacetic acids 7a–e in the presence of acetic promising compound 11 (IC50=0.48µM) and 14 (IC50=0.44µM)
anhydride at 90°C.18) Treatment of 8a–e with 4 under the exhibited more potent SAHase inhibition effects than the
basic condition to give monacid derivatives 9a–e. The amide reference inhibitor aristeromycin (IC50=0.49µM), one of the
derivatives 11–25 were prepared by condensation of mona- most potent SAHase inhibitors known so far. Meanwhile,
cid derivatives 9a–e and various ethylenediamine derivatives compounds 13, 18 and 19 also exhibited SAHase inhibitory
10 with 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide activities in some extent.
hydrochloride (EDCI) as condensation agent and 1-hydroxy-
All of the amide derivatives 11–25. In most case, com-
benzotriazole (HOBt) as catalyst in N,N-dimethylformamide pounds with substituted indazole-5-yl group (11–15) displayed
(DMF) in good yield.18,19) The chemical structures of all the potent SAHase inhibitory activities (except compound 16).
1
synthesized compounds were characterized by H-NMR, MS Particularly, compound 14 was found to be the most active
and elemental analysis (C,H,N).
compounds of this series. When the 2-methyl-2H-indazole-
Biological Activity All the newly synthesized compounds, 5-yl group of compound 14 was replaced with 4-(1H-pyrrol-
along with the reference compounds aristeromycin and 3-DZA, 1-yl) phenyl group as in compound 17, a slight decrease in

114
Vol. 62, No. 1
Table 1. The SAHase Inhibitory Activity of 11–25
very encouraging, and further investigation of this kind of
compounds may be of interest.
Inhibition ration (%)
Compound
IC50 (µM)a)
0.5µM
2.5µM
Experimental
Chemistry Melting points were determined on WRS-21
melting point apparatus and were uncorrected. 1H-NMR
spectra were recorded on INOVA 400 (400MHz) spectrom-
eter 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 ion-
ization 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 sil-
ica gel 60 F254 plates and visualization on TLC was achieved
by UV light (254, 354nm). Unless otherwise stated, all com-
mercial reagents and solvents were used without additional
purification.
1,2-Bis(bromomethyl)benzene (2) A mixture of com-
pound 1 (40g, 0.38mol), NBS (140.8g, 0.79mol), benzoyl
peroxide (0.91g, 3.8mmol) in CHCl3 (400mL) was heated for
5h under reflux. The reaction mixture was cooled to room
temperature and CH2Cl2 (400mL) was added to the mixture.
The organic layer was washed with water (2×200mL) and
dried over Na2SO4, filtered, and the solvent were removed in
11
62.31 1.48
24.24 1.78
9.53 1.70
69.58 0.72
17.24 1.30
15.97 2.35
49.72 0.20
10.92 0.91
6.41 0.23
4.23 0.14
85.72 1.69
54.24 1.12
30.19 3.02
86.56 1.10
38.24 1.46
27.15 1.24
79.46 2.83
21.46 3.38
20.16 0.84
8.23 1.02
2.63 0.41
4.56 0.77
19.23 1.35
6.97 0.87
4.89 0.45
98.35 0.16
35.37 3.2
0.48
2.03
12
13
7.61
14
0.44
15
4.13
16
25.34
0.73
17
18
12.12
14.08
78.82
>500
>500
15.60
101.9
214
19
20
21
0
0
0.32
0.96
22
23
24
8.23 0.92
4.56 0.45
25
0 0.43
Aristeromycin
3-DZA
83.93 0.90
2.16 0.47
0.49b)
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 0.2µM, the
SAHase from Mycobacterium tuberculosis.22) c) Values in the literature is 20µM, the
SAHase from Mycobacterium tuberculosis.22)
SAHase inhibition potency was observed. However, when vacuo. The residue was recrystallized from n-hexane–ethanol
the 2-methyl-2H-indazole-5-yl group of compound 14 was (30:1, 620mL) to give compound 2 (79.0g, 80%) as white
1
replaced with 4-methylphenyl group as in compound 23, a solid, mp 92–93°C. H-NMR (400MHz, CDCl3) δ: 4.66 (4H,
significant decrease in SAHase inhibition potency was ob- s), 7.29–7.38 (4H, m).
served. Unfortunately replacement of 2-methyl-2H-indazole-
tert-Butyl Isoindolin-2-yl(methyl) Carbamate (3) A mix-
5-yl group of compound 14 with 4-fluorophenyl to afford ture of compound 2 (59.7g, 0.22mol), tert-butyl methylcarba-
corresponding compounds 20–22 resulted in a loss of activity, mate (34.7g, 0.23mol) and triethylamine (44.5g, 0.44mol)
and 4-methylphenyl derivatives 24 and 25 were also devoid in DMF (300mL) was heated for 2h at 65°C. The reaction
of SAHase inhibitory activities. These facts imply that the in- mixture was cooled to room temperature and water (400mL)
troduction of substituted indazole-5-yl group on the Ar region was added to the mixture, the aqueous phase was extracted
obviously affected the SAHase inhibitory activity, and in the with ethyl acetate (3×300mL). The combined organic layer
present investigation, 4-(1H-pyrrol-1-yl) phenyl group facilitat- was washed with brine and dried over Na2SO4, filtered, and
ed their inhibitory activities, while 4-fluorophenyl group and the solvent were removed in vacuo to get compound 3 (35.5 g,
4-methylphenyl group were unfavorable.
65.0%) as white solid, mp 62–64°C. 1H-NMR (400MHz,
Substituted ethylenediamine structure at the side chain also DMSO-d6) δ: 1.35 (9H, s), 2.99 (3H, s), 4.33 (4H, s), 7.20–7.22
affects the SAHase inhibitory activity. Compounds 11, 14, 17, (4H, m). ESI-MS m/z: 249.16 [M+H]+.
20 and 23 bearing ethylamino group at side chain exhibited
N-Methylisoindolin-2-amine Hydrochloride (4) Com-
highest influence on SAHase inhibitory activity, followed by pound 3 (27.5g, 0.11mol) was dissolved in concentrated
the corresponding compounds with piperidinyl group at side hydrochloric acid (83mL), and stirred at room temperature
chain (12, 15, 18, 21, 24). However, the corresponding com- for 12h. The reaction mixture was evaporated under reduced
pounds with diethylamino group at side chain (13, 16, 19, 22, pressure and co-evaporated with ethanol (1000mL). The
25) displayed lower SAHase inhibition potency. These results residue was recrystallized from ethanol (100mL) to give com-
1
indicate that the ethylamino group is considered to be the best pound 4 (14.2g, 70%) as white solid, mp 160–162°C. H-NMR
substituent at side chain showing potent SAHase inhibitory (400MHz, DMSO-d6+D2O) δ: 2.88 (3H, s), 4.59 (4H, s),
activity.
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 aro-
Conclusion
In summary, a novel class of amide derivatives were de- matic amines 5a–e (0.1mol), ethyl 2-bromoacetate (0.21mol),
signed, synthesized and evaluated as SAHase inhibitors. The Na2HPO4 (0.25mol) and NaI (0.05mol) in CH3CN (250mL)
results demonstrated that most of target compounds displayed was heated for 12h under reflux. The reaction mixture was
potent SAHase inhibitory activities. Particularly, compounds cooled to room temperature and concentrated in vacuo, and
11 and 14 exhibited more potent inhibitory effects than the the water was added to the residue, the aqueous phase was
aristeromycin and 3-DZA. The compounds with 1-methyl-1H- extracted with ethyl acetate. The combined organic layer was
indazole-5-yl group or 2-methyl-2H-indazole-5-yl group at Ar washed with brine and dried over Na2SO4, filtered, and the
position and ethylamino group at side chain showed better solvent were removed in vacuo to obtain 6a–e. The solution
SAHase inhibitory activities. These results showed above are of KOH (0.25mol) in ethanol (500mL) was added to the so-

January 2014
115
lution of 6a–e in ethanol (100mL), and the reaction mixture m), 2.97 (3H, s), 3.19–3.22 (2H, m), 3.91 (3H, s), 3.98 (2H, s),
was heated at 60°C for 1h. The reaction mixture was cooled 4.27–4.38 (4H, m), 4.66 (2H, s), 6.60–6.87 (2H, m), 7.24–7.46
to room temperature, filtered, and the filtrate was dissolved in (5H, m), 8.93 (1H, brs). ESI-MS m/z: 464.27 [M+H]+. Anal.
water. The pH of the suspension was adjusted to 2 with 2N Calcd for C25H33N7O2: C, 64.77; H, 7.18; N, 21.15. Found: C,
HCl. The crystalline product was precipitated, filtrated and 64.87; H, 7.24; N, 21.03.
dried under vacuum to give 7a–e.
N-(Isoindolin-2-yl)-N-methyl-2-((1-methyl-1H-indazol-5-
2,2′-((1-Methyl-1H-indazol-5-yl)azanediyl)diacetic Acid (7a): yl)(2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)amino)-
1
Yield 40% (2 steps), white solid, mp 172–174°C. H-NMR acetamide (12): Yield 47% (3 steps), white solid, mp
(400MHz, DMSO-d6) δ: 3.95 (3H, s), 4.14 (4H, s), 6.87–6.90 124–125°C. 1H-NMR (400MHz, DMSO-d6) δ: 1.28–1.39
(1H, m), 7.47 (1H, d, J=9.2Hz), 7.81 (1H, s). ESI-MS m/z: (6H, m), 2.42–2.48 (6H, m), 2.98 (3H, s), 3.22–3.26 (2H,
264.12 [M+H]+.
m), 3.95–4.01 (5H, m), 4.37–4.40 (4H, m), 4.65 (2H, s), 6.58
2,2′-((2-Methyl-2H-indazol-5-yl)azanediyl)diacetic Acid (7b): (1H, s), 6.71–6.74 (1H, m), 7.24–7.37 (4H, m), 7.46–7.48 (1H,
1
Yield 35% (2 steps), white solid, mp 156–158°C. H-NMR m), 7.82 (1H, s), 8.89 (1H, brs). ESI-MS m/z: 504.54 [M+
(400MHz, DMSO-d6) δ: 3.91 (3H, s), 4.12 (4H, s), 6.70 (1H, H]+. Anal. Calcd for C28H37N7O2: C, 66.77; H, 7.40; N, 19.47.
s), 6.85–6.88 (1H, m), 7.42–7.45 (1H, d, J=9.2Hz), 7.79 (1H, s). Found: C, 66.65; H, 7.45; N, 19.56.
ESI-MS m/z: 264.16 [M+H]+.
2-((2-((2-(Diethylamino)ethyl)amino)-2-oxoethyl)-
2,2′-((4-(1H-Pyrrol-1-yl)phenyl)azanediyl)diacetic Acid (7c): (1-methyl-1H-indazol-5-yl)amino)-N-(isoindolin-2-yl)-N-
1
Yield 30% (2 steps), brown solid, mp 150–152°C. H-NMR methylacetamide (13): Yield 39.0% (3 steps), white solid,
(400MHz DMSO-d6+D2O) δ: 4.13 (4H, s), 6.17–6.20 (2H, m), mp 91–93°C. 1H-NMR (400MHz, DMSO-d6) δ: 0.89 (6H,
6.59–6.66 (2H, m), 7.10–7.26 (2H, m), 7.33 (2H, d, J=8.8Hz). t, J=6.8Hz), 2.49–2.61 (6H, m), 2.96 (3H, s), 3.23 (2H, t,
ESI-MS m/z: 275.12 [M+H]+.
J=6.4Hz), 3.94–3.98 (5H, m), 4.36 (4H, s), 4.64 (2H, s), 6.61
2,2′-((4-Fluorophenyl)azanediyl)diacetic Acid (7d): Yield (1H, s), 6.74–6.76 (1H, m), 7.25–7.33 (4H, m), 7.43 (1H, d,
42% (2 steps), white solid, mp 192–194°C. 1H-NMR (400MHz, J=9.2Hz), 7.79 (1H, s), 8.85 (1H, brs). ESI-MS m/z: 492.32
CDCl3) δ: 4.12 (4H, s), 6.55–6.57 (2H, m), 6.96–7.01 (2H, m). [M+H]+. Anal. Calcd for C27H37N7O2: C, 65.96; H, 7.59; N,
ESI-MS m/z: 228.06 [M+H]+.
19.94 . Found: C, 66.04; H, 7.50; N, 19.87.
2-((2-((2-(Ethylamino)ethyl)amino)-2-oxoethyl)(2-methyl-2H-
2,2′-(p-Tolylazanediyl)diacetic Acid (7e): Yield 45% (2
steps), white solid, mp 202–204°C. 1H-NMR (400MHz, indazol-5-yl)amino)-N-(isoindolin-2-yl)-N-methylacetamide
DMSO-d6) δ: 2.16 (3H, s), 3.99 (4H, s), 6.37 (2H, d, J=8.8Hz), (14): Yield 34% (3 steps), white solid, mp 131–132°C. 1H-NMR
6.97 (2H, d, J=8.8Hz). ESI-MS m/z: 224.10 [M+H]+.
(400MHz, DMSO-d6) δ: 0.97 (3H, t, J=7.2Hz), 2.68–2.78 (4H,
General Procedure for the Synthesis of Amide Deriva- m), 2.90 (3H, s), 3.27–3.29 (2H, m), 3.94 (3H, s), 4.00 (2H,
tives (11–25) A mixture of substituted phenylazanediyl di- s), 4.29 (4H, s), 4.58 (2H, s), 6.61(1H, s), 6.74–6.77 (1H, m),
acetic acids 7a–e (3.8mmol) and acetic anhydride (20mL) 7.22–7.43 (5H, m), 7.78 (1H, s), 8.98 (1H, brs). ESI-MS m/z:
was heated to 90°C for 3h, and the reaction mixture was 464.30 [M+H]+. Anal. Calcd for C25H33N7O2: C, 64.77; H,
concentrated in vacuo to get intermediates 8a–e. A solution 7.18; N, 21.15; Found: C, 64.69; H, 7.24; N, 21.09.
of triethylamine (9.5mmol) in tetrahydrofuran (THF) (20mL)
N-(Isoindolin-2-yl)-N-methyl-2-((2-methyl-2H-indazol-5-
was added to the corresponding intermediates 8a–e, the mix- yl)(2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethyl)amino)-
ture was stirred at room temperature for 10min. Compound acetamide (15): Yield 40% (3 steps), white solid, mp
1
4 (3.8 mmol) was added in three portions and the resulting 155–157°C. H-NMR (400MHz, DMSO-d6) δ: 1.25–1.37 (6H,
mixture was stirred at room temperature for 12h. The solu- m), 2.41–2.51 (6H, m), 2.98 (3H, s), 3.25 (2H, t, J=6.4Hz),
tion was concentrated and water was added to the residue. The 3.95–3.98 (5H, m), 4.33 (4H, s), 4.61 (2H, s), 6.61 (1H, s),
mixture was extracted with ethyl acetate–THF (10:1) and the 6.73–6.76 (1H, m), 7.24–7.45 (5H, m), 7.81 (1H, s), 8.85
combined organic layer was dried over Na2SO4, filtered, then (1H, brs). ESI-MS m/z: 504.31 [M+H]+. Anal. Calcd for
evaporated to remove solvent to obtain corresponding mona- C28H37N7O2: C, 66.77; H, 7.40; N, 19.47. Found: C, 66.79; H,
cid derivatives 9a–e. To a stirred solution of 9a–e in DMF 7.34; N, 19.55.
(20mL) was added triethylamine (9.5mmol), EDCI (7.6mmol)
2-((2-((2-(Diethylamino)ethyl)amino)-2-oxoethyl)-
and HOBt (7.6mmol). After the addition was completed, the (2-methyl-2H-indazol-5-yl)amino)-N-(isoindolin-2-yl)-N-
reaction mixture was stirred for 10min at room temperature. methylacetamide (16): Yield 45% (3 steps), white solid, mp
1
A solution of corresponding ethylenediamine derivatives 10 98–99°C. H-NMR (400MHz, DMSO-d6+D2O) δ: 0.90 (6H,
(4.56mmol) was added to the reaction mixture, and the result- t, J=7.2Hz), 2.50–2.62 (6H, m), 2.93 (3H, s), 3.23 (2H, t,
ing mixture was stirred at room temperature for 12h. The J=6.4Hz), 3.95–3.99 (5H, m), 4.37 (4H, s), 4.65 (2H, s), 6.60
reaction mixture was poured into water. The pH of the sus- (1H, s), 6.73 (1H, d, J=8.8Hz), 7.23–7.31 (4H, m), 7.44 (1H,
pension was adjusted to 10 with 5% aqueous NaOH, and the d, J=8.8Hz), 7.80 (1H, s), 8.85 (1H, brs). ESI-MS m/z: 492.35
aqueous phase was extracted with ethyl acetate. The combined [M+H]+. Anal. Calcd for C27H37N7O2: C, 65.96; H, 7.59; N,
organic layer was washed with brine and dried over Na2SO4, 19.94. Found: C, 65.90; H, 7.64; N, 19.98.
filtered, evaporated, and purified by flash column chroma-
2-((4-(1H-Pyrrol-1-yl)phenyl)(2-((2-(ethylamino)-
tography (CHCl3–CH3OH=8:1) to afford amide derivatives ethyl)amino)-2-oxoethyl)amino)-N-(isoindolin-2-yl)-N-
11–25.
methylacetamide (17): Yield 31% (3 steps), white solid, mp
2-((2-((2-(Ethylamino)ethyl)amino)-2-oxoethyl)(1-methyl-1H- 133–135°C. 1H-NMR (400MHz, DMSO-d6) δ: 1.09 (3H,
indazol-5-yl)amino)-N-(isoindolin-2-yl)-N-methylacetamide t, J=7.2Hz), 2.79–2.87 (4H, m), 2.95 (3H, s), 3.20 (2H, t,
(11): Yield 39% (3 steps), white solid, mp 178–179°C. 1H-NMR J=6.4Hz), 3.99 (2H, s), 4.34 (4H, s), 4.63 (2H, s), 6.18–6.19
(400MHz, DMSO-d6) δ: 0.91 (3H, t, J=7.2Hz), 2.51–2.61 (4H, (2H, m), 6.50 (2H, d, J=9.2Hz), 7.11–7.13 (2H, m), 7.25–7.39

116
Vol. 62, No. 1
(6H, m), 8.90 (1H, brs). ESI-MS m/z: 475.35 [M+H]+. Anal. m), 2.96 (3H, s), 3.16–3.26 (2H, m), 3.88 (2H, s), 4.28–4.37
Calcd for C27H34N6O2: C, 68.33; H, 7.22; N, 17.71. Found: C, (4H, m), 4.56 (2H, s), 6.31 (2H, d, J=8.4Hz), 6.96 (2H, d,
68.27; H, 7.26; N, 17.77.
J=8.4Hz), 7.25–7.33 (4H, m), 8.84 (1H, brs). ESI-MS m/z:
2-((4-(1H-Pyrrol-1-yl)phenyl)(2-oxo-2-((2-(piperidin-1- 464.19 [M+H]+. Anal. Calcd for C27H37N5O2: C, 69.95; H,
yl)ethyl)amino)ethyl)amino)-N-(isoindolin-2-yl)-N- 8.04; N, 15.11. Found: C, 70.01; H, 8.09; N, 15.03.
methylacetamide (18): Yield 39% (3 steps), white solid, mp
2-((2-((2-(Diethylamino)ethyl)amino)-2-oxoethyl)(p-tolyl)-
1
124–126°C. H-NMR (400MHz, DMSO-d6) δ: 1.40–1.45 (6H, amino)-N-(isoindolin-2-yl)-N-methylacetamide (25): Yield 46%
m), 2.49–2.51 (6H, m), 2.98 (3H, s), 3.98 (2H, s), 4.36 (4H, (3 steps), white solid, mp 137–138°C. 1H-NMR (400MHz,
s), 4.65 (2H, s), 6.15–6.18 (2H, m), 6.48 (2H, d, J=9.2Hz), DMSO-d6) δ: 0.96 (6H, t, J=7.2Hz), 2.16 (3H, s), 2.54–2.61
7.12–7.13 (2H, m), 7.27–7.35 (6H, m), 8.90 (1H, brs). ESI-MS (6H, m), 2.95 (3H, s), 3.23 (2H, m), 3.89 (2H, s), 4.29–4.37
m/z: 515.33 [M+H]+. Anal. Calcd for C30H38N6O2: C, 70.01; H, (4H, m), 4.57 (2H, s), 6.31 (2H, d, J=8.4Hz), 6.95 (2H, d,
7.44; N, 16.33. Found: C, 70.08; H, 7.39; N, 16.38.
J=8.4Hz), 7.25–7.33 (4H, m), 8.87 (1H, brs). ESI-MS m/z:
2-((4-(1H-Pyrrol-1-yl)phenyl)(2-((2-(diethylamino)- 452.26 [M+H]+. Anal. Calcd for C26H37N5O2: C, 69.15; H,
ethyl)amino)-2-oxoethyl)amino)-N-(isoindolin-2-yl)-N- 8.26; N, 15.51. Found: C, 69.09; H, 8.29; N, 15.57.
methylacetamide (19): Yield 44% (3 steps), white solid, mp
SAHase Inhibition Assay The activity was measured
70–72°C. 1H-NMR (400MHz, DMSO-d6) δ: 1.04 (6H, t, following the procedure described previously with a slight
J=7.2Hz), 2.89–2.94 (9H, m), 3.34–3.37 (2H, m), 3.99 (2H, modification.19,20) This method involves the hydrolytic con-
s), 4.32 (4H, s), 4.61 (2H, s), 6.18–6.19 (2H, m), 6.48–6.51 version of SAH into ADO and HCY. SAHase from human
(2H, m), 7.12–7.14 (2H, m), 7.28–7.32 (6H, m), 8.98 (1H, brs). placenta, SAH was obtained from Sigma, adenosine deami-
ESI-MS m/z: 503.32 [M+H]+. Anal. Calcd for C29H38N6O2: C, nase (ADA) and ThioGlo 1 were obtained from Calbiochem.
69.29; H, 7.62; N, 16.72. Found: C, 69.34; H, 7.66; N, 16.63.
SAHase and all compounds were cultured in phosphate buffer
2-((2-((2-(Diethylamino)ethyl)amino)-2-oxoethyl)(4- (pH 8.0) before initiated the reaction. ThioGlo 1 solution was
fluorophenyl)amino)-N-(isoindolin-2-yl)-N-methylacetamide freshly prepared in phosphate buffer (pH 8.0) before initiated
1
(20): Yield 51% (3 steps), white solid, mp 74–76°C. H-NMR the reaction. The reaction mixture (50µL) contained ethylene-
(400MHz, DMSO-d6) δ: 1.07 (3H, t, J=7.2Hz), 2.70–2.77 (4H, diaminetetraacetic acid (50mM), ADA (0.03U), SAH (1.68µM),
m), 2.95 (3H, s), 3.27–3.30 2H, m), 3.94 (2H, s), 4.34 (4H, s), SAHase (0.5mU) and various concentrations of inhibitors.
4.61 (2H, s), 6.40–6.43 (2H, m), 6.97–6.99 (2H, m), 7.25–7.30 The reaction took place at 37°C for 10min, and the reaction
(4H, m), 8.90 (1H, brs). ESI-MS m/z: 428.20 [M+H]+. Anal. mixture was quenched through the addition of 50µL pure ice-
Calcd for C23H30FN5O2: C, 64.62; H, 7.07; N, 16.38. Found: C, cold isopropanol. A solution of 100µL of 20µM ThioGlo1 in
64.56; H, 7.01; N, 16.44.
dimethyl sulfoxide was added to reaction mixture. The plate
2-((4-Fluorophenyl)(2-oxo-2-((2-(piperidin-1-yl)ethyl)- was maintained in the dark at room temperature for 10min,
amino)ethyl)amino)-N-(isoindolin-2-yl)-N-methylacetamide and the fluorescence was read using the SpectraMax M5
(21): Yield 45% (3 steps), white solid, mp 145–146°C. 1H-NMR spectrophotometer with 380nm excitation and 510nm emis-
(400MHz, DMSO-d6) δ: 1.33–1.46 (3H, m), 2.42–2.50 (6H, sion filters. One unit of inhibitory activity was defined as the
m), 2.95 (3H, s), 3.20–3.28 (2H, m), 3.90 (2H, s), 4.33 (4H, s), amount of the sample needed to inhibit one unit of enzyme
4.57 (2H, s), 6.38–6.42 (2H, m), 6.97–7.01 (2H, m), 7.25–7.31 activity. IC50 value was determined by lineal regression of the
(4H, m), 8.78 (1H, brs). ESI-MS m/z: 468.32 [M+H]+. Anal. dose–response curves and was defines as the amount of the
Calcd for C26H34FN5O2: C, 66.79; H, 7.33; N, 14.98. Found: C, sample needed to inhibit the 50% of control enzyme activity.
66.72; H, 7.28; N, 14.91.
2-((2-((2-(Diethylamino)ethyl)amino)-2-oxoethyl)(4-
Acknowledgment The work was supported by ‘New Drug
fluorophenyl)amino)-N-(isoindolin-2-yl)-N-methylacetamide Innovation 2009X09313-006’ from the Ministry of Science
1
(22): Yield 43% (3 steps), white solid, mp 85–87°C. H-NMR and Technology of China.
(400MHz, 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, brs). 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 (400MHz,
DMSO-d6) δ: 1.11 (3H, t, J=7.2Hz), 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=8.4Hz), 6.96
(2H, d, J=8.4Hz), 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.
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