Design and synthesis of 8-substituted benzamido-phenylxanthine derivatives as MAO-B inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.12.094

Monoamine oxidase-B (MAO-B) inhibitor has been used as neuroprotectants to treat the motor deficits of Parkinson's disease (PD). We designed and synthesized a class of 8-substituted benzamido-phenylxanthine derivatives as MAO-B inhibitors. The compounds have various inhibitory effects, with compound 6a having a Ki value of 0.26 lM. Their promising activity in vitro suggests potential use in the treatment of PD.

Full text of DOI:10.1016/j.bmcl.2011.12.094

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1739–1742
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of 8-substituted benzamido-phenylxanthine derivatives
as MAO-B inhibitors
Bo Song a, Tong Xiao a, Xiaolu Qi a, Ling-Na Li a, Kuiyou Qin a, Siyun Nian a, Guo-Xin Hu a, Yinfei Yu b,
Guang Liang a,c, Faqing Ye a,c,
⇑
a School of Pharmacy, Wenzhou Medical College, Wenzhou 325000, PR China
b The Affiliated Eye Hospital, Wenzhou Medical College, Wenzhou 325000, PR China
c Bioorganic & Medicinal Chemistry Research Center, Wenzhou Medical College, Wenzhou 325000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Monoamine oxidase-B (MAO-B) inhibitor has been used as neuroprotectants to treat the motor deficits of
Parkinson‘s disease (PD). We designed and synthesized a class of 8-substituted benzamido-phenylxan-
thine derivatives as MAO-B inhibitors. The compounds have various inhibitory effects, with compound
Received 14 August 2011
Revised 3 December 2011
Accepted 20 December 2011
Available online 28 December 2011
6a having a Ki value of 0.26
l
M. Their promising activity in vitro suggests potential use in the treatment
of PD.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Monoamine oxidase-B inhibitors
Xanthine derivative
Parkinson’s disease
Neuroportective effect
Inhibitory effect
Cl
Parkinson’s disease (PD) is a degenerative disorder of the
central nervous system, mainly caused by the loss of dopaminergic
neurons of the substantia nigria, which is a region of the midbrain.
Early in the course of the disease, the most obvious symptoms are
movement related, including shaking, rigidity, slowness of move-
ment and difficulty with walking and gait. The main families of
drugs useful for treating motor symptoms are levodopa, usually
combined with a dopa decarboxylase inhibitor or catechol-O-
methyltransferase inhibitor, dopamine agonists and MAO-B inhib-
itors (Scheme 1).1
MAO-B is an enzyme located in the mitochondrial outer mem-
brane, and plays an important role in the metabolism of dopamine
and neuroactive and vasoactive amines in the central nervous sys-
tem. Inhibition of this enzyme may enhance the elevation of dopa-
mine levels in adjunctive treatment with levodopa,2,3 and can
delay the onset of relapse following levodopa monotherapy.4,5
Furthermore, MAO-B may decelerate the denaturation of neurons
and protect against the neurotoxic effects of MPTP catalyzed by
MAO-B. MAO-B is therefore an important target for the develop-
ment of new anti-Parkinson’s drugs and MAO-B inhibitors are
promising drugs in the treatment of PD.
O
N
N
N
N
N
CSC(1)
O
N
(2)
OCH3
O
N
OCH3
N
N
N
O
KW-6002(3)
Scheme 1. The structure of MAO-B inhibitor CSC (1) and (E)-2-styryl-1-methyl-
benzimidazole (2) and KW-6002 (3).
inhibitor dissociation constant) of 128 nM and 11 lM, respec-
tively.6 Earlier studies suggested that structural features of impor-
tance for MAO-B inhibition are in the heterocycle containing
nitrogen.7,8 This suggests that choosing caffeine, or xanthine and
theophylline, as the core structure of the compound may be
responsible for potency as an MAO-B inhibitor. There is also a
structure–activity relationship (SAR) that has shown that substitu-
tion at C-8 of the caffeine ring with an electron deficient group pro-
duces a higher affinity MAO-B inhibitor.7 Further, the trans
configuration of the styryl double bond and the substitution of
(E)-8-(3-Chlorostyryl) caffeine (CSC) and KW-6002 (Scheme 1)
have been reported as inhibitors of MAO-B with a Ki value (enzyme
⇑
Corresponding author. Tel.: +086 0577 86689793; fax: +086 0577 86689983.
E-mail addresses: yfq664340@163.com, xt651259@126.com (F. Ye).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.094

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B. Song et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1739–1742
Table 1
The Ki values for the inhibition of 8-(benzamido)-phenyl xanthine derivativesa
O
R2
O
N
R2
O
N
HN
R1
O
R1
N
N
N
N
N
NH
N
O
O
5a-5c,6a-6c
7d-7f,8d-8f
R1
R2
Inhibition ratio (%)
ExpIC50
(l
M)
Ki Value (lM)
b
b
Compound
5a
5b
5c
6a
6b
6c
7d
7e
7f
H
CH3
H
H
H
CH3
CH3
CH3
H
H
H
ND
ND
ND
42.3
32.1
67.4
53.7
58.2
27.9
41.6
35.1
40.5
34.4
30.8
13.9 1.02
17.2 2.21
8.9 1.52
2.77
3.97
0.26
1.63
1.18
4.21
2.87
3.49
2.95
3.56
3.92
OCH3
H
CH3
OCH3
H
CH3
OCH3
H
CH3
OCH3
11.3 1.03
10.7 4.52
18.9 3.54
14.0 2.56
16.0 1.11
14.5 1.38
16.7 2.02
17.8 1.21
8d
8e
8f
CH3
CH3
CH3
a
The enzyme source used was MAO from mouse striata.
Not determined.
b
O
H
m
O
N
R1
NH2
m
HOOC
Cl
R1
HOOC
4a-4f
4a m=4,R1=H; 4b m=4,R1=CH3; 4c m=4,R1=OCH3;
4d m=3,R1=H; 4e m=3,R1=CH3; 4f m=3,R1=OCH3;
Scheme 2. General method for the synthesis of substituted benzamido benzoic acid (4a–f). Reagents and conditions: CH2Cl2, N(CH3CH2)3, rt.
the xanthine ring were important structural feature for potent
MAO-B inhibition.6 The trans double bond, however, is sensitive
to light, being readily isomerized to the corresponding cis isomer,9
which is expected to be inactive versus MAO-B.
In this study, we chose the xanthine ring as the core scaffold
and replaced the styryl group with a substituted phenyl ring. The
main aim was to determine whether a diaryl amide group would
confer potency versus MAO-B. Accordingly, a new small series A
of 8-(benzamido)-phenyl xanthine derivatives (5a–c, 6a–c, 7d–f
and 8d–f) was synthesized, and their enzyme inhibitor dissociation
constant (Ki value) (Table 1) was estimated. Ki value is particularly
useful for expressing the potency of an inhibitor because. Unlike
IC50, it is independent of substrate concentration.
condition, neither the substrate nor the test compounds absorb.
After treatment,14 samples were measured using a fluorimeter. In
order to blind the affinity of the synthesized compounds to the
MAO-A, clorgyline was added during the test as a specific MAO-A
inhibitor.
GraphPad Prism Software was used to calculate the IC50 values
for MAO-B inhibition and were presented as the mean SD. Fitting
the data to the Michaelis-Menten equation using a nonlinear least-
squares fitting routine, and then the Ki values were obtained by
using the Cheng-Prusoff equation. The Ki values of Series A are pre-
sented in Table 1.
As shown in Table 1, compound 6a, which lacks any substituent
in the benzamide region, had a Ki value of 0.26, 42-fold active than
Here we report the synthesis and inhibition studies of the de-
signed compounds. The synthesis of the target compounds is
shown in two schemes. First was the preparation of the corre-
sponding substituted benzamido benzoic acid 4a–f.10 The general
procedure for their synthesis is depicted in Scheme 2.
the positive KW-6002 (Ki = 11
(Ki = 70 nM). Compound 6b and 6c with Ki values of 1.63 and
1.18 M, respectively, also showed good inhibition. The other com-
pounds 7d–f, 8d–f were not so effective. We also observed that,
compared with the xanthine derivatives of m-substituted in the
8-phenyl, the p-substituted derivatives possessed stronger inhibi-
tion, possibly due to steric clashes of the substituent in the binding
pocket of the enzyme.
lM), and was a quarter of CSC
l
According to a previously reported protocol,11 the preparation
of the substituted 8-(benzamido)-phenylxanthines was followed
the conditions described below (Scheme 3).12
In order to evaluate the therapeutic potential of these com-
pounds as MAO-B inhibitors for the treatment of PD, we measured
their IC50 and Ki values against MAO-B. The measurement is based
on the MAO-catalyzed oxidation of Kynuramine to the correspond-
ing fluorescent 4-hydrxyquinoline which can be measured using a
fluorimeter (315 nm excitation and 380 nm emission).13 Under this
Compounds such as 6a are significantly more active than the
modest MAO-B inhibitor 8-(3-chlorophenyl) caffeine, which has
a Ki value of 36 l
M.15 The enhanced potency is perhaps due to
more favorable interations of the diaryl amide group with MAO-
B. Furthermore, compared with the theophylline derivatives and
caffeine derivatives, we found that with methylation on C-7 of

B. Song et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1739–1742
1741
O
O
NH2
NH2
NH
N
N
i
ii
O
NH
O
N
NH2
O
N
3a
2a
1a
4a-4c
iii
iii 4d-4f
O
O
O
N
HN
R1
O
H
N
H
N
R1
N
N
NH
N
N
O
N
O
5a-5c
iv
7d-7f
iv
O
R2
O
N
O
N
R2
R1
O
HN
R1
N
N
N
N
N
N
NH
O
O
6a-6c
8d-8f
Scheme 3. General method for the synthesis of 8-substituted (benzamido)-phenyl xanthine derivatives. Reagents and conditions: (i) NCCH2COOH,(CH3CO)2O, 70 °C; (ii)
NaNO2, CH3COOH/H2O (1:1), 70 °C, 1 h; Na2S2O4, 14.5% NH4OH, 70 °C, 1 h; (iii) EDCI, MeOH, rt, 8 h; (iv) CH3I, K2CO3, DMF, 1 h.
3. Finberg, J. P.; Wang, J.; Bankiewicz, K.; Harvey-White, J.; Kopin, I. J.; Goldstein,
D. S. J. Neural. Transm. Suppl. 1998, 52, 282.
4. Shoulson, I.; Oakes, D.; Fahn, S., et al Ann. Neurol. 2002, 51, 607.
5. Petzer, Jacobus P.; Castagnoli, Neal, Jr.; Schwarzschild, Michael A.; Chen, Jiang-
Fan; Cornelis J. Neurotherapeutics 2009, 6, 145.
the theophylline ring increased the inhibition ratios to a certain
extent, which indicated that the caffeine structure could improve
the compound’s inhibition of MAO-B.
In summary, we have designed and synthesized 12 new xan-
thine derivatives with a 8-(benzamido)-phenyl connected to the
xanthine ring. The compounds we synthesized have different
inhibitory effects. Target compound 6a, in particular, showed 42-
fold higher MAO-B inhibitory activity than the positive KW-6002.
We also found that the caffeine structure may be favorable for
designing a potent MAO-B inhibitor. Considering the size prefer-
ence for substituents, compounds with smaller groups tend to be
more potent MAO-B inhibitors. Small groups may easily fit into
the active site of MAO-B. In comparing these new compounds with
previous styrenes, the diaryl amide can replace the styryl bond and
can enhance MAO-B inhibition. Optimization of this scaffold to-
ward the design of a more potent MAO-B inhibitor needs further
research.
6. den Berg, Deidre’van; Zoellner, Kevin R.; Modupe; Ogunrombi, O. Bioorg. Med.
Chem. 2007, 15, 3693.
7. Petzer, J. P.; Steyn, S.; Castagnoli, K. P.; Chen, J. F.; Schwarzschild, M. A.; Van der
Schyf, C. J.; Castagnoli, N., Jr. Bioorg. Med. Chem. 2003, 11, 1299.
8. Castagnoli, N.; Petzer, J. P.; Steyn, S.; Castagnoli, K.; Chen, J. F.; Schwarzschild,
M. A.; Van der Schyf, C. J. Neurol. 2003, 61, S63.
9. Petzer, Jacobus P.; Steyn, Salome; Castagnoli, KayP; Chen, Jiang-Fan;
Schwarzschild, Michael A.; Van der Schyf, Cornelis J.; Castagnoli, Neal Bioorg.
Med. Chem. 2003, 11, 1302.
10. A solution of aminobenzoic acid in dichloromethane at room temperature was
treated with triethylamine and stirred for 30 min to obtain a pellucid solution.
The substituted benzoyl chloride was then slowly added. After precipitates
were observed, the mixture was stirred at room temperature for another 2 h.
The suspension was concentrated under reduced pressure, and the residue was
purified by hot acetic acid to give compounds 4a–f.
11. Kalla, Rao V.; Elzein, Elfatih; Perry, Thao; Li, Xiaofen; Palle, Venkata;
Varkhedkar, Vaibhav; Gimbel, Arthur; Maa, Tennig; Zeng, Dewan; Zablocki,
Jeff J. Med. Chem. 2006, 49, 3684.
12. 5,6-Diamino-1,3-dimethyl uracil (3a) was prepared by treating the
symmetrically substituted urea (1a) with cyanoacetic acid in the presence of
acetic anhydride to provide the corresponding substituted 6-amino uracil (2a)
in good yields. Nitrosation of the 6-amino uracil derivatives (2a) was achieved
by the slow addition of solid sodium nitrite to a solution of the amino uracil in
50% aqueous acetic acid at 70 °C. The nitroso uracil was reduced with Na2S2O4
in 15% ammonium hydroxide to furnish 5,6-diamido-1,3-dimethyluracil (3a).
The coupling of the 3a with 4a–f followed by base-induced cyclization that
Acknowledgments
This research was financed in part by Zhejiang Science & Tech-
nology Funding (Y2080697 to F.Q.Y.; Y204089 to F.Q.Y.). We are
grateful to Professor Guang Liang for the assistance in this work.
furnished
the
substituted
8-(benzamido)-phenylxanthines,
further
methylation was achieved by adding iodomethane followed the conditions
described in Scheme 3.
Supplementary data
13. Kmil, M. Biochem. Pharmaco1. 1965, 14, 1685.
14. Briefly, mouse cerebral homogenate was used as the MAO-B source,
kynuramine served as an enzyme substrate, and various concentrations of
the test compounds were used.16 The compounds were prepared in 4% DMSO,
since higher DMSO levels can inhibit MAO-B.17 The mouse cerebral
homogenate (0.2 mL) was treated with 2.5 mL sodium phosphate buffer (PBS,
pH 7.4,0.05 M) and 0.4 mL Tritor (20%) was preincubated for 15 min at room
temperature with clorgyline as a specific irreversible MAO-A inhibitor.18 The
test compounds were then added, followed by treatment with kunuramine
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.12.094. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
Reference and notes
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1742
B. Song et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1739–1742
Finally, 0.5 mL of supernatant was measured and taken using
fluorimeter (315 nm excitation and 380 nm emission).
a
cuvette
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