Design, synthesis and inhibitory activities of 8-(substituted styrolformamido) phenyl-xanthine derivatives on monoamine oxidase B

Article abstract of DOI:10.1248/cpb.60.385

The design and synthesis of two series of 8-(substituted styrol-formamido)phenyl-xanthine derivatives are described. Their in vitro monoamine oxidase B (MAO-B) inhibition were tested and the effect of substituents on the N-7, phenyl and the substituted positions are discussed. It was observed that compound 9b displayed significant MAO-B inhibition activity and selectivity, fluorine substitution plays a key role in the selectivity of MAO-B inhibition, and the styrol-formamido group at position-3′ may enhance the activity and selectivity of 8-phenyl-xanthine analogues. These results suggest that such compounds may be utilized for the development of new candidate MAO-B inhibitors for treatment of Parkinson's disease.

Full text of DOI:10.1248/cpb.60.385

—
March 2012
Note
Chem. Pharm. Bull. 60(3) 385 390 (2012)
385
Design, Synthesis and Inhibitory Activities of 8-(Substituted styrol-
formamido)phenyl-xanthine Derivatives on Monoamine Oxidase B
,a
Suwen Hu,a,# Siyun Nian,a,# Kuiyou Qin,a Tong Xiao,a Lingna Li,a Xiaolu Qi,a Faqing Ye,*
Guang Liang,a Guoxin Hu,a Jincai He,b Yinfei Yu,c and Bo Songa
b
a School of Pharmacy, Wenzhou Medical College; Wenzhou, Zhejiang 325035, P.R. China: Department of Heurology,
the First Affiliated Hospital of Wenzhou Medical College; Wenzhou, Zhejiang 325000, P.R. China: and c The Optometry
Eye Hospital of Wenzhou Medical College; Wenzhou, Zhejiang 325027, P.R. China.
Received August 28, 2011; accepted December 7, 2011; published online December 16, 2011
The design and synthesis of two series of 8-(substituted styrol-formamido)phenyl-xanthine derivatives
are described. Their in vitro monoamine oxidase B (MAO-B) inhibition were tested and the effect of sub-
stituents on the N-7, phenyl and the substituted positions are discussed. It was observed that compound 9b
displayed significant MAO-B inhibition activity and selectivity, fluorine substitution plays a key role in the
selectivity of MAO-B inhibition, and the styrol-formamido group at position-3ꢀ may enhance the activity and
selectivity of 8-phenyl-xanthine analogues. These results suggest that such compounds may be utilized for the
’
development of new candidate MAO-B inhibitors for treatment of Parkinson s disease.
Key words xanthine derivative; monoamine oxidase B inhibition activity; substituent effect
’
Parkinson s disease (PD) is a neurodegenerative disorder
Recently, xanthine and its derivatives were reported to
characterized pathologically by a marked loss of dopaminergic produce little or no inhibition of MAO-B. In the last decade,
nigrostriatal neurons and clinically by disabling movement when introducing phenyl at position-8, 8-phenyl xanthine ex-
disorders.1) Currently, the therapy of PD is largely focused on hibited MAO-B inhibition activity in vitro with a inhibition
dopamine replacement strategies with the dopamine precur- constant (Ki) value of 86.2μM.2) Petzer et al. found a series
sor levodopa and dopamine agonist drugs. Although these of 8-styryl xanthine analogues exhibiting more powerful in-
strategies are highly effective in controlling the disease at the hibition of MAO-B than 8-phenyl xanthine analogues (CSC
early stages, drug-related complications are associated with Ki=100nM; KW6002 Ki=27nM), indicating the styryl group
long-term treatment. The inadequacies of dopamine replace- which had been considered the main active group of xanthine
—
15)
ment therapy have prompted the research of alternative drug analogues in inhibition of MAO-B1,13
(Fig. 1). And studies
of CSC interaction with the protein pocket of MAO-B found
—
5)
targets.2
Monoamine oxidase (MAO) is an integral protein of the that the caffeine group may embed one hole, and the styryl
outer mitochondrial membrane, and exist in two forms, name- group into the another hole.16)
ly, monoamine oxidase A (MAO-A) and MAO-B. MAO-A
Taking advantage of above information, we speculated
preferentially deaminates aromatic monoamines such as the that condensation products of styrol-formamido group with
neurotransmitters serotonin (5-HT), noradrenaline (NA), an- 8-phenyl xanthine might exhibit potent MAO-B inhibition
dadrenaline (A); MAO-B is one of the most important key activity (Fig. 2). Therefore, two series of 8-(substituted styrol-
enzymes in metabolic pathways in vivo and plays mainly three formamido)phenyl-xanthine derivatives, totally 14 compounds,
roles: firstly, MAO-B decomposes dopamine into 3,4-dihy- were prepared and their in vitro inhibition against MAO-B
droxyphenyl acetic acid and homovanillic acid, meanwhile were determined. These compounds were also designed to
producing the neurotoxic H2O2. Secondly, MAO-B inacti- examine the role of different substitutes in the N-7, phenyl
vates p-phenylethylamine which has the ability to stimulate and substituted positions of phenyl. It is hoped that continued
secrection and reuptake function of dopamine. Thirdly, in research will lead to the development of new lead compounds
the precence of MAO-B, catalyzed oxidation of 1-methyl- from 8-phenyl xanthine as effective MAO-B inhibitors for PD.
4-phenyl-1,2,3,6-terahydropyridine (MPTP) to intermediate
1-methyl-4-phenyl-2,3-dihydropyridin-1-ium (MPDP+) and
Results and Discussion
Chemistry The structure and general synthesis of
then to 1-methyl-4-phenylpyridin-1-ium (MPP+)with neurotox-
icity occurs. Together these suggest that inhibiting the action analogues designed are shown in Chart 1. The 8-(sub-
of MAO-B can not only reduce the degradation of dopamine stituted styrol-formamido)phenyl-xanthine analogues were
and reuptake, improving the concentration of dopamine in the prepared in high yield according to the procedure previ-
brain, but also lower levels of H2O2, and MPP+ to enhance ously reported for the preparation of (E)-8-styrylcaffeinyl
—
12)
—
19)
analogues.1,17
neuroprotective properties.6
The key starting materials for the procedure,
Fig. 1. Chemical Structures of Well-Known MAO-B Inhibitors
*To whom correspondence should be addressed. e-mail: yfq664340@163.com
# These authors contributed equally to this work.
© 2012 The Pharmaceutical Society of Japan

386
Vol. 60, No. 3
1,3-dimethyl-5,6-diaminouracil 4, were allowed to react with to MAO activity indirectly determined by the activity of com-
—
the appropriate carboxylic acid 8a g in the presence of N- pounds on MAO. The relationship between the Ki and IC50 is
(3-dimethylaminopropyl)-N-ethylcarbodiimidehydrochloride described by Cheng–Prusoff equation Ki=IC50/(1+[S]/Km).
(EDAC). The resulting amidyl intermediates underwent
As shown in Tables 1 and 2, all of the 8-(substituted
ring closure when heated under reflux in aqueous sodium styrol-formamido)phenyl-xanthine analogues were found to
hydroxide to yield the corresponding 1,3-dimethyl-8-susti- exhibit MAO-B inhibition activity, especially 9b, 9f, and 10f,
—
—
tuted-7H-xanthinyl analogues 9a g. 9a g were selectively which showed a better selectivity with the inhibition rate of
7N-methylated with an excess of iodomethane and potas- 30.9, 9.6, 17.6 to 0% of MAO-A inhibition, respectively. This
sium carbonate to yield 1,3,7-trimethyl-8-sustituted xanthinyl indicates that fluorine substitution plays a key role in the
—
analogues 10a g. Following crystallization from a suitable selectivity of MAO-B inhibition (except for 10b). In the two
solvent, the structures and purity of all compounds were veri- series of compounds, the inhibition rate of MAO-B were: 9a
1
fied by mass, IR, and H-NMR spectrometry. The trans–trans (20.2%)>9e (10.6%), 9b (30.9%)>9f (9.6%), 9c (33.5%)>9g
geometry about the conjugated ethenyl π-bonds of the com- (21.2%), 10a(25.2%)>10e (3.4%), 10b (32.6%)>10f (17.6%)
pounds were confirmed by proton–proton coupling constants and 10c (18.5%)>10g (8.3%), indicating the styrol-formamido
15.6Hz for the olefinic proton signals.
group at position-3′ may enhance the selectivity more than
Biological Activity On the base of MAO, kynuramine at position at 4′. Distinguishing between the two series of
dihydrobromide was catalytically oxidized to fluorescence compounds, 7N-methylated had no obvious influence on the
4-hydroxyquinoline, and the fluorescence intensity detected selectivity. Focussing only on the MAO-B inhibition activity,
indirectly to obtain the MAO activity. Our compounds had none of the compounds had significant differences, except
various degrees of MAO inhibition activity in vitro, according for 10e. 9b, 9c, 10b yield better activities, with Ki values of
3.42, 3.58, and 3.68, respectivity. Coincidentally, the Ki values
were 9a (5.94)<9e (11.32), 9b (3.42)<9f (12.50), 9c (3.58)<9g
(5.66), 10a (4.76)<10e (34.30), 10b (3.68)<10f (6.82) and 10c
(6.48)<10g (14.46), indicating the styrol-formamido group at
position-3′ may enhance the inhibition more than at position at
4′. 7N-Methylated had no obvious influence on the inhibition.
Fig. 2. Design of New MAO-B Inhibitors Based on the Pharmacophore
Reagents and conditions: (i) NCCH2COOH, acetic anhydride; (ii) NaNO2, CH3COOH/H2O; (iii) Na2S2O4, NH4OH; (iv) SOCl2; (v) triethylamine, CH2Cl2; (vi)
EDC·HCl, 1,4-dioxane/H2O; (vii) NaOH, reflux; (viii) CH3I, K2CO3, DMF.
Chart 1. General Route for the Synthesis of 8-(Substituted styrol-formamido)phenyl-xanthine Analogues

March 2012
387
Table 1. Inhibition Rate of All Tested Compounds on MAO
Experimental
Chemistry Melting points were determined on a XRC-
1 melting apparatus. 1H-NMR spectra were recorded on a
Bruker AC 600 spectrometer. The chemical shifts are pre-
sented in terms of ppm with tetramethylsilane (TMS) as the
internal reference. IR were measured on a Bruker Vector22
spectrometer. Electron-spray ionization mass spectra in posi-
tive mode (ESI-MS) were recorded on a Esquire HCT spec-
trometer. The purity of all novel compounds was checked by
Inhibition rate (%)a), b)
Compounds
MAO-A
MAO-B
9a
9b
3.8 1.22
20.2 1.01
30.9 0.97
33.5 1.48
7.1 0.88
0
0.66
9c
47.2 1.62
16.5 1.51
27.8 0.94
9d
9e
10.6 1.08
9.6 0.74
1
TLC and H-NMR. All reactions were monitored by TLC on
9f
0 0.44
pre-coated Silica Gel F254 plates (purchased from Qingdao
Marine Chemical Factory, China) with detection by UV. All
reagents used were of analytical grade. m-Aminobenzoic acid,
p-aminobenzoic acid, cinnamic acid, and substituted cin-
namic acid were purchased from Wuhan Yuancheng Co., Ltd.
Other reagents were purchased from Wenzhou Jiutai Chemical
Reagent Co., Ltd., China.
General Procedure of 5,6-Diamino-1,3-dimethylpyrim-
idine-2,4(1H,3H)-dione 420) To a stirred solution of 1,3-di-
methylurea (40mmol) 1 in acetic anhydride (30mL) was
added cyanoacetic acid (44mmol), and the resulting mixture
9g
8.0 1.23
2.8 0.41
12.9 1.05
14.3 1.14
29.0 1.26
27.5 0.81
21.2 0.85
25.2 1.22
32.6 0.76
18.5 0.85
29.8 1.56
3.4 0.67
17.6 1.45
8.3 1.04
10a
10b
10c
10d
10e
10f
10g
0
0.32
19.8 1.15
a) Data represent mean values of at least three separate experiments. b)
Percentage inhibition at an inhibitor concentration of 1mM.
Table 2. The Ki and IC50 Values for the Inhibition of MAO-B by All
Tested Compounds
°
was stirred overnight at 70 C. The reaction mixture was con-
centrated, and the resulting oily residue was diluted with H2O
(40mL) and treated with 5N NaOH (15mL). The precipitate
thus formed was collected by filtration, washed with cold
water, and purified by recrystalization from MeOH/H2O to
give 6-amino-1,3-dimethylpyrimidine-2,4(1H,3H)-dione as
a light-yellow solid 2 (5.70g). Compound 2 (25.8mmol) was
Compounds
Exp. IC50 (μM)a), b)
Ki value (μM)a), c)
9a
9b
29.70 1.22
17.10 2.24
17.91 1.08
84.50 1.12
56.60 2.46
62.50 2.08
28.3 2.34
23.8 1.68
5.94
3.42
3.58
16.9
11.32
12.5
5.66
4.76
3.68
6.48
4.02
34.3
6.82
14.46
9c
9d
°
stirred in 50% aquesous AcOH solution (160mL) at 75 C for
9e
30min until the reaction mixture became homogeneous. Once
the reaction mixture was homogeneous, the temperature was
9f
9g
°
reduced to 50 C, and sodium nitrite (51.6mmol) was added
10a
10b
10c
10d
10e
10f
10g
in small portions. After the completion of the addition, the
resulting mixture was cooled to room temperature and stirred
for 1h. The resulting precipitate was collected by filtration,
washed with water, and dried to obtain 6-amino-1,3-dimethyl-
5-nitrosopyrimidine 2,4(1H,3H)-dione 3 (4.64g). A suspen-
sion of compound 3 (6.0mmol) was stirred in 14.5% NH4OH
18.40 1.14
32.43 2.32
20.13 3.06
176.4 4.08
34.09 2.18
72.29 3.24
°
(40mL) at 70 C for 30min until the reaction mixture became
a) Data represent mean values of at least three separate experiments. b) The IC50
values were experimentally determined by fitting the rate data to the one site com-
petition model incorporated into the Prism software package. c) The Ki values were
calculated from the experimental IC50 values according to the equation by Cheng
and Prusoff: Ki=IC50/(1+[S]/Km).
°
homogeneous. The temperature was reduced to 50 C, and
Na2S2O4 (18.0mmol) was added in small portions. During
the addition, the red solution changed to yellow-green and to
light yellow, and the solution was stirred at room temperature
for another 30min. The volume of the reaction mixture was
reduced to half and cooled in an ice bath for 1h, and the
Conclusion
C-8 substituted xanthine analogues have been reported for precipitate was collected by filtration, followed by washing
the therapy of PD through inhibiting MAO-B, so the rational with a small amount of water. Compound 4, 5,6-diamino-1,3-
design and synthsis of the 8-(substituted styrol-formamido)- dimethylpyrimidine-2,4(1H,3H)-dione, was collected as
a
phenyl-xanthine analogues presented here may also be useful white solid (0.69g).
for finding more advanced compounds for anti-PD. In this
General Procedure of 3-(Substituted styrol-formamido)-
paper, two series of 8-(Substituted styrol-formamido)phenyl- benzoic Acid and 4-(Substituted styrol-formamido)benzoic
—
xanthine analogues, totalling 14 compounds, were designed, Acid Cinnamic acid 5a d (24mmol) and fresh thionyl chlo-
synthsized, and evaluated for the inhibition of MAO-B. It ride (26mmol) were heated for 30min, then rotary evaporated
was observed that 9b showed remarkable MAO-B inhibition to remove excessive thionyl chloride to obtain a faint yellow
—
activity and excellent selectivity. We can conclude that in the needle crystal cinnamonyl chloride 6a d. Aminobenzoic acid
structure of 8-phenyl xanthine, styrol-formamido group at po- 7a,b (0.02mmol) were dissolved in dichloromethane (60mL)
sition 3′ may enhance the selectivity and activity on MAO-B and added appropriate triethylamine to be dissolved, stirred
inhibition, and fluorine substituted compounds have clear for 30min at room temperature, then 10mL of dichlorometh-
—
d
selectivity.
ane containing cinnamonyl chloride slowly dropped 6a
(0.02mmol) into the reaction mixture. After the completion
of the dropping, a white precipitate was formed and stirred

388
Vol. 60, No. 3
for 2h, collected by filtration, and crystallized from acetate to m/z: 436.4 [M+H]+.
obtain white crystal substituted styrol-formamidobenzoic acid
N-(4-(1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-
—
°
8-yl)phenyl)cinnamamide (9e): Yield 48.1%, mp >300 C.
(8a g).
General Procedure of 1,3-Dimethyl-(E)-8-substituted 1H-NMR (DMSO-d6, 600MHz) δ: 3.27 (3H, s, –CH3), 3.51
—
1,17 19)
—
styrol-formamido-7H-xanthinyl Analogues (9a g)
(3H, s, –CH3), 6.91 (1H, d, J=15.6Hz, –CH=C), 7.45 (3H,
To a solution of 1,3-dimethyl-5,6-diaminouracil 4 (3.50mmol) m, –ArH), 7.61 (1H, d, J=15.6Hz, –CH=C), 7.65 (2H, d,
and EDAC (5.11mmol) in dioxane–H2O (1:1, 40mL), the ap- J=7.2Hz, –ArH), 7.84 (2H, d, J=8.4Hz, –ArH), 8.12 (2H,
—
propriate substituted styrol-formamidobenzoic acid 8a
g
d, J=8.4Hz, –ArH), 10.46 (1H, s, –NH–C=O), 13.72 (1H, s,
(3.81mmol) was added. The pH of the suspension was adjust- –NH–C). IR (KBr) cm−1: 3157.26, 3291.24, 1625.77, 1645.29,
ed to 5 with 2M aqueous hydrochloric acid and stirring was 1706.03, 2900.70, 2987.95. ESI-MS m/z: 402 [M+H]+.
continued for an additional 8h. The reaction mixture was neu-
(E)-N-(4-(1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-
°
tralized with 1M aqueous sodium hydroxide, cooled to 0 C, purin-8-yl)phenyl)-3-(4-fluorophenyl)acrylamide (9f): Yield
1
°
and the resulting precipitate was collected by filtration. The 43.7%, mp >300 C. H-NMR (DMSO-d6, 600MHz) δ: 3.27
crude product was dissolved in 40mL aqueous sodium hy- (3H, s, –CH3), 3.51 (3H, s, –CH3) 6.79 (1H, d, J=15.6Hz,
—
°
droxide (1M)–dioxane (1:1) and heated for 8h at 90 105 C. –CH=C), 7.30 (2H, m, –ArH), 7.62 (1H, d, J=15.6Hz,
°
The reaction mixture was cooled to 0 C, acidified to a pH of –CH=C), 7.72 (2H, m, –ArH), 7.83 (2H, d, J=9Hz, –ArH),
4 with 4M aqueous hydrochloric acid, and the precipitate was 8.11 (2H, d, J=9Hz, –ArH), 10.45 (1H, s, –NH–C=O), 13.72
collected by filatration. This was follwed by crystallization (1H, s, –NH–C). IR (KBr) cm−1: 3157.26, 3291.24, 1645.29,
from dimethyl sulfoxide (DMSO) to obtain pure 1,3-dimethyl- 1689.47, 1706.03, 2900.70, 2987.95. ESI-MS m/z: 420 [M+H]+.
(E)-8-substituted styrol-formamido-7H-xanthinyl analogues
(E)-N-(4-(1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-
—
(9a g).
purin-8-yl)phenyl)-3-(3-(trifluoromethyl)phenyl)acrylamide
°
(E)-N-(3-(1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H- (9g): Yield 45.5%, mp >300 C. 1H-NMR (DMSO-d6,
purin-8-yl)phenyl)cinnamamide (9a): Yield 46.0%, mp 600MHz) δ: 3.28 (3H, s, –CH3), 3.52 (3H, s, –CH3), 6.99
>300 C. 1H-NMR (DMSO-d6, 600MHz) δ: 3.28 (3H, s, (1H, d, J=15.6Hz, –CH=C), 7.72 (1H, m, –ArH), 7.73 (1H, d,
°
–CH3), 3.52 (3H, s, –CH3), 6.91 (1H, d, J=15.6Hz, –CH=C), J=15.6Hz, –CH=C), 7.79 (1H, d, J=7.8Hz, –ArH), 7.80 (1H,
7.48 (3H, m, –ArH), 7.61 (1H, d, J=15.6Hz, –CH=C), 7.61 d, J=7.8Hz, –ArH), 7.85 (2H, d, J=8.4Hz, –ArH), 8.02 (1H,
(1H, m, -ArH), 7.71 (2H, d, J=9Hz, -ArH), 7.86 (2H, d, s, –ArH), 8.14 (2H, d, J=8.4Hz, –ArH), 10.52 (1H, s, –NH–
J=9Hz, -ArH), 8.44 (1H, s, –ArH), 10.45 (1H, s, –NH–C=O), C=O), 13.76 (1H, s, –NH–C). IR (KBr) cm−1: 3183.19, 3347.31,
13.93 (1H, s, –NH–C). IR (KBr) cm−1: 3152.94, 3364.71, 1651.08, 1685.82, 1698.23, 2900.67, 2988.12. ESI-MS m/z: 470
1648.98, 1683.39, 1696.52, 2902.28, 2984.39. ESI-MS m/z: 402 [M+H]+.
[M+H]+.
General Procedure of 1,3,7-Trimethyl-(E)-8-substituted
—
17 19)
—
(E)-N-(3-(1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H- styrol-formamido-7H-xanthinyl Analogues (10a g)
purin-8-yl)phenyl)-3-(4-fluorophenyl)acrylamide (9b): Yield Iodomethane (0.40mmol) was added to a stirred suspen-
41.0%, mp >300 C. H-NMR (DMSO-d6, 600MHz) δ: 3.28 sion of (0.20mmol) 1,3-dimethyl-(E)-8-substituted styrol-
1
°
—
(3H, s, –CH3), 3.52 (3H, s, –CH3), 6.80 (1H, d, J=15.6Hz, formamido-7H-xanthinyl analogues 9a
g and potassium
–CH=C), 7.30 (2H, d, J=8.4Hz, –ArH), 7.47 (1H, m, –ArH), carbonate (0.50mmol) in N,N-dimethylformamide (10mL).
°
7.62 (1H, d, J=15.6Hz, –CH=C), 7.71 (2H, d, J=8.4Hz, Stirring was continued at 60 C for 1h, and the insoluble ma-
–ArH), 7.86 (2H, m, –ArH), 8.43 (1H, s, –ArH),10.42 (1H, terials were removed by filtration, and sufficient water was
s, –NH-C=O), 13.91 (1H, s, –NH-C). IR (KBr) cm−1: 3159.09, added to the filtrate to precipitate the product collected by
3368.35, 1649.87, 1683.15, 1693.41, 2900.72, 2988.10. ESI-MS filtration. This was followed by crystallization from DMSO to
m/z: 420 [M+H]+.
obtain pure 1,3,7-trimethyl-(E)-8-substituted styrol-formami-
—
(E)-N-(3-(1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H- do-7H-xanthinyl analogues 10a g.
purin-8-yl)phenyl)-3-(3-trifluoromethyl)phenyl)acrylamide
(E)-N-(3-(1,3,7-Trimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-
(9c): Yield 43.2%, mp >300 C. 1H-NMR (DMSO-d6, purin-8-yl)phenyl)cinnamamide (10a): Yield 48.6%, mp
°
600MHz) δ: 3.27 (3H, s, –CH3), 3.52 (3H, s, –CH3), 6.98 >300 C. 1H-NMR (DMSO-d6, 600MHz) δ: 3.26 (3H, s,
°
(1H, d, J=15.6Hz, –CH=C), 7.48 (1H, m, –ArH), 7.70 (1H, –CH3), 3.48 (3H, s, –CH3), 4.03 (3H, s, –CH3), 6.84 (1H,
d, J=15.6Hz, –CH=C), 7.72 (1H, m, –ArH), 7.77 (1H, d, d, J=15.6Hz, –CH=C), 7.45 (3H, m, –ArH), 7.52 (2H, d,
J=7.8Hz, –ArH), 7.87 (2H, m, –ArH), 7.94 (1H, d, J=7.8Hz, J=9Hz, –ArH), 7.61 (1H, m, –ArH), 7.63 (1H, d, J=15.6Hz,
–ArH), 7.99 (1H, s, –ArH), 8.43 (1H, s, –ArH), 10.47 (1H, s, –CH=C), 7.88 (2H, d, J=9Hz, –ArH), 8.18 (1H, s, –ArH),
–NH–C=O), 13.92 (1H, s, –NH–C). IR (KBr) cm−1: 3162.65, 10.47 (1H, s, –NH–C=O). IR (KBr) cm−1: 3340.88, 1630.65,
3305.87, 1649.85, 1683.12, 1693.71, 2900.73, 2988.02. ESI-MS 1650.21, 1697.77, 2900.70, 2970.84, 2988.27. ESI-MS m/z: 416
m/z: 470 [M+H]+.
[M+H]+.
(E)-3-(4-Fluorophenyl)-N-(3-(1,3,7-trimethyl-2,6-dioxo-
(E)-3-(3-Chlorophenyl)-N-(3-(1,3-dimethyl-2,6-dioxo-
2,3,6,7-tetrahydro-1H-purin-8-yl)phenyl)acrylamide
(9d): 2,3,6,7-tetrahydro-1H-purin-8-yl)phenyl)acrylamide
(10b):
Yield 45.5%, mp >300 C. H-NMR (DMSO-d6, 600MHz) δ: Yield 40.5%, mp >300 C. 1H-NMR (DMSO-d6, 600MHz)
3.28 (3H, s, –CH3), 3.52 (3H, s, –CH3), 6.91 (1H, d, J=15.6Hz, δ: 3.26 (3H, s, –CH3), 3.48 (3H, s, –CH3), 4.03 (3H, s, –CH3),
–CH=C), 7.48 (3H, m, –ArH), 7.61 (1H, d, J=15.6Hz, 6.78 (1H, d, J=15.6Hz, –CH=C), 7.31 (2H, d, J=8.4Hz,
–CH=C), 7.61 (1H, m, –ArH), 7.71 (1H, s, –ArH), 7.86 –ArH), 7.53 (2H, m, –ArH), 7.63 (1H, d, J=15.6Hz, –CH=C),
(2H, d, J=8.4Hz, –ArH), 8.44 (1H, s, –ArH), 10.45 (1H, s, 7.72 (2H, d, J=8.4Hz, –ArH), 7.88 (1H, m, –ArH), 8.17 (1H,
–NH–C=O), 13.93 (1H, s, –NH–C). IR (KBr) cm−1: 3209.12, s, –ArH), 10.46 (1H, s, –NH–C=O). IR (KBr) cm−1: 3334.45,
3327.49, 1654.88, 1697.36, 1700.12, 2900.72, 2987.79. ESI-MS 1644.66, 1685.44, 1700.84, 2897.96, 2971.43, 2988.73. ESI-MS
1
°
°

March 2012
389
m/z: 434 [M+H]+.
tube B was added 3.1μL clorgyline (0.0031g+10mL dd H2O,
(E)-3-(3-(Trifluoromethyl)phenyl)-N-(3-(1,3,7-trimethyl-2,6- 1mM) to inhibit MAO-A. Drug solutions in DMSO, added
dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)phenyl)acrylamide to the reaction mixture at six different final concentrations
(10c): Yield 44.6%, mp >300 C. 1H-NMR (DMSO-d6, ranging from 0.5 to 10mM. The mixture was preincubated at
°
°
600MHz) δ: 3.27 (3H, s, –CH3), 3.49 (3H, s, –CH3), 4.04 37 C for 15min. Then kynuramine dihydrobromide (2.19mM,
(3H, s, –CH3), 6.97 (1H, d, J=15.6Hz, –CH=C), 7.54 (2H, m, 30μL) was added to the reaction mixture (final concentration
°
–ArH), 7.71 (2H, m, –ArH), 7.72 (1H, d, J=15.6Hz, –CH=C), 22μM) as substrate. Samples were then incubated at 37 C for
7.78 (1H, d, J=7.8Hz, –ArH), 7.95 (1H, d, J=7.8Hz, –ArH), 30min once more. After incubation, the reaction was termi-
8.02 (1H, s, –ArH), 8.19 (1H, s, –ArH), 10.52 (1H, s, –NH– nated by adding perchloric acid (5M, 0.2mL). After cooling
C=O). IR (KBr) cm−1: 3325.81, 1650.12, 1681.99, 1698.04, and centrifugation at 1500g for 10min, an aliquot of the
2900.76, 2971.86, 2987.78. ESI-MS m/z: 484 [M+H]+.
supernatant (0.5mL) was added to NaOH (1M, 2.5mL). The
(E)-3-(3-Chlorophenyl)-N-(3-(1,3,7-trimethyl-2,6-dioxo- fluorescence intensity was detected with 315nm excitation
2,3,6,7-tetrahydro-1H-purin-8-yl)phenyl)acrylamide
(10d): and 380nm emission wavelengths using a fluorescence spec-
Yield 44.2%, mp >300 C. 1H-NMR (DMSO-d6, 600MHz) trometer. The concentration of 4-hydroxyquinoline was esti-
δ: 3.26 (3H, s, –CH3), 3.48 (3H, s, –CH3), 4.03 (3H, s, –CH3), mated from a corresponding standard fluorescence curve of
6.89 (1H, d, J=15.6Hz, –CH=C), 7.53 (3H, m, –ArH), 7.55 4-hydroxyquinoline. MAO activity (U/mg prot) was calculated
(1H, m, –ArH), 7.61(1H, d, J=15.6Hz, –CH=C), 7.62 (1H, d, as=fluorescence÷0.01÷3h÷sample protein (mg); Inhibition
J=8.4Hz, –ArH), 7.71 (1H, s, –ArH), 7.87 (1H, d, J=8.4Hz, rate of MAO=(MAO inhibition activity/tool drug inhibition
–ArH), 8.18 (1H, s, –ArH), 10.49 (1H, s, –NH–C=O). IR activity)×100%, to MAO-A the tool drug was clorgyline, and
(KBr) cm−1: 3327.72, 1651.64, 1681.32, 1694.79, 2900.71, deprenyl for MAO-B, with inhibition activity of 100%.
2971.80, 2984.12. ESI-MS m/z: 450.6 [M+H]+.
°
N-(4-(1,3,7-Trimethyl-2,6-dioxo-2,3,6,7-tetrahydro-
Acknowledgements This work was supported by Zhejiang
1H-purin-8-yl)phenyl)cinnamamide (10e): Yield 42.3%, Provincial Natural Science Foundation of China (Y2080697),
mp>300 C. 1H-NMR (DMSO-d6, 600MHz) δ: 3.26 (3H, and partly supported by Zhejiang Extremely Key Subject of
°
s, –CH3), 3.47 (3H, s, –CH3), 4.02 (3H, s, –CH3), 6.86 (1H, Pharmacology and Biochemical Pharmaceutics. The authors
d, J=15.6Hz, –CH=C), 7.44 (1H, m, –ArH), 7.47 (2H, d, are very thankful to the Analysis and Testing Center of
J=7.2Hz, –ArH), 7.64 (1H, d, J=15.6Hz, –CH=C), 7.65 (2H, Pharmacy School, Institute of Biological Pharmaceuticals and
d, J=7.2Hz, –ArH), 7.82 (2H, d, J=8.4Hz, –ArH), 7.90 (2H, Natural Products of Wenzhou Medical College and Zhejiang
d, J=8.4Hz, –ArH), 10.50 (1H, s, –NH–C=O). IR (KBr) Provincial Key Laboratory of Biotechnology Pharmaceutical
cm−1: 3381.99, 1630.44, 1646.59, 1695.01, 2900.72, 2971.43, Engineering for experimental assistance.
2987.84. ESI-MS m/z: 416 [M+H]+.
(E)-3-(4-Fluorophenyl)-N-(4-(1,3,7-trimethyl-2,6-dioxo-
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