New coumarin derivatives: Design, synthesis and use as inhibitors of hMAO

Article abstract of DOI:10.1016/j.bmc.2014.05.002

A series new 2H-chromene-3-carboxamides (4a-4i) and S-2H-chromene-3- carbothioates (5j-5t) were synthesized and evaluated as monoamine oxidase A and B inhibitors. Among them, compound 5k (IC₅₀ = 0.21 μM, IC _{50 iproniazid} = 7.65 μM) showed the most activity and higher MAO-B selectivity (189.2-fold vs 1-fold) with respect to the MAO-A isoform. The need to clarify at a 3D level some important molecular aspects of discussed SAR, we undertaked a number of docking simulations to better assess. The steric effect was analyzed interms of both posing and scoring by investigating the nature of the binding interactions. The docking results of active compound 5k with hMAO-B complex indicated that conserved residue ILE 199 was important for ligand binding via Sigma-Pi interaction.

Full text of DOI:10.1016/j.bmc.2014.05.002

Accepted Manuscript
New coumarin derivatives: design、synthesis and use as inhibitors of hMAO
Xu He, Yan-Yan Chen, Jing-Bo Shi, Wen-Jiang Tang, Zhi-Xiang Pan, Zhi-
Qiang Dong, Bao-An Song, Jun Li, Xin-Hua Liu
PII:
S0968-0896(14)00342-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.05.002
BMC 11563
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 January 2014
22 April 2014
2 May 2014
Please cite this article as: He, X., Chen, Y-Y., Shi, J-B., Tang, W-J., Pan, Z-X., Dong, Z-Q., Song, B-A., Li, J., Liu,
X-H., New coumarin derivatives: design、synthesis and use as inhibitors of hMAO, Bioorganic & Medicinal
Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.05.002
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New coumarin derivatives: design、synthesis and use as inhibitors of hMAO
Xu Hea, Yan-Yan Chena, Jing-Bo Shia, Wen-Jiang Tang a, Zhi-Xiang Pana, Zhi-Qiang
Donga, Bao-An Songb, Jun Lia,*, Xin-Hua Liu a,b, c*
a School of Pharmacy, Anhui Medical University, Hefei, 230032, P. R. China
b Key Laboratory of Green Pesticide and Agriculture Bioengineering, Ministry of
Education, Guizhou University, Guiyang, 550025, P. R. China
c
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, P. R. China
Abstract: A series new 2H-chromene-3-carboxamides (4a~4i) and S-2H-chromene-3-
carbothioates (5j~5t) were synthesized and evaluated as monoamine oxidase A and B
inhibitors. Among them, compound 5k (IC50 =0.21 µM, IC50 iproniazid = 7.65 µM) showed
the most activity and higher MAO-B selectivity (189.2-fold vs 1-fold) with respect to the
MAO-A isoform. The need to clarify at a 3D level some important molecular aspects of
discussed SAR, we undertaked a number of docking simulations to better assess. The steric
effect was analyzed interms of both posing and scoring by investigating the nature of the
binding interactions. The docking results of active compound 5K with hMAO-B complex
indicated that conserved residue ILE 199 was important for ligand binding via Sigma-Pi
interaction.
Keywords: Coumarin; Design; Synthesis; Inhibitors; hMAO-B
_________________
*Corresponding author.
Jun Li Tel.: +86 0551-5161001; fax: +86 0551-5161001. E-mail: lijun@ahmu.edu.cn.
Xin-Hua Liu Tel.: +86 551 5161115; fax: +86 551 5161115. E-mail: xhliuhx@163.com.
1

1. Introduction
Monoamine oxidases (MAOs) are a protein family of flavin-containing amine
oxidoreductases that play an important role in the regulation and metabolism of several
neurotransmitters, and their inhibitors (MAOIs) could be useful in the treatment of
1,2
psychiatric and neurological diseases.
Nowadays, the therapeutic interest of MAOIs
falls into two major categories. MAO-A inhibitors have been used mostly in the treatment
of mental disorders, in particular depression and anxiety, 3,4 while MAO-B inhibitors could
be used in the treatment of Parkinson’s disease and perhaps, Alzheimer’s disease. 5,6
Despite the considerable progress in understanding the interactions of the two enzyme
forms with their amino acid sequences and substrate specificity, few general rules were
available for the rational design of potent and selective inhibitors of MAO-A and MAO-B.
Recently, the crystal structures of the two isoforms of human MAO were determined by
Binda et al, which elucidates the mechanism underlying the selective interactions between
these proteins and their ligands, probes the catalytic mechanism 2. This result also provides
a better understanding of the pharmacophoric requirements needed for a rational design of
potent and selective enzyme inhibitors. 7-10
Coumarins are a large family of compounds, of natural and synthetic origin, that display a
variety of pharmacological properties. Recently, coumarins and their derivatives were
extensively studied to their antioxidative and enzymatic inhibition properties. Furthermore,
the coumarins derivatives used as selective MAO inhibitors have also widely reported. 11-18
Rendenbach-Muller et al.19 discovered the importance of the C7 position in simple
7-substitued 3,4-dimethylcoumarines for the selectivity between the MAO A and B
isoforms. A sulfonic ester linkage yielded MAO A selective inhibitors (Figure 1A,
Esuprone), Gnerre et al. 20 identified the potent and selective MAOA ligand (Figure 1B), a
phenolic linker with a spaced heteroaromatic group was found to be preferred for MAO B
selectivity (Figure 1C, LU53439).
(Figure 1)
2

Structure-activity relationship of recent research showed that substitution in position 3 of
21,22
the coumarin nucleus modulated MAO-B inhibitory activity.
When introducing an
alkylamide group in that position (Figure 2A), which could provide them with additional
strong selectivity inhibitory activity toward hMAO-B. 23
Based on the X-ray structure MAO-B complex (2BYB. pdb), computer-generated docking
molecular models of 2H-chromene-3-carboxamide derivatives were analyzed, when the
position of the carbonyl group with interchangeable (Figure 2B), which favor the
formation of a stable binding, should help increase activity against MAO-B. Because –NH-
and -S- is a classic isostere, there have been reported that C-S-C has good permeability 24,
so we incorporated it into the design of the title compound (Figure 2C), that is we also
focused on S-2H-chromene-3-carbothioate series. Therefore, on the basis of rational design,
we synthesized a series novel 2H-chromene derivatives used as selective and efficient
MAO inhibitors.
(Figure 2)
2. Results and discussion
2.1 Chemistry
Coumarin title derivatives 4 were synthesized according to the protocol outlined in Scheme
1. Among them, compounds 1 were prepared starting from a condensation of
substituted-salicylaldehyde and the malonate. The reaction was performed in a dry schlenk
tube, with piperidine as catalyst. Using simple sodium hydroxide and hydrochloric acid,
proved to be an efficient alternative method for the synthesis of compounds 2. The key
intermediate compounds 3 were obtained through the conventional thionyl chloride and
compounds 2. The compounds 2 and 4 were prepared according to references 25 and 26.
(Scheme 1)
2.2 Crystal structure analysis
3

The structures of compounds 4a and 5k were determined by X-ray crystallography. Crystal
o
data of 4a: Colorless crystals, yield, 81%; mp 199~200 C; C13H13NO3, M =245,
monoclinic; Space group C2/c; a =21.9525 (19), b =8.8693 (9), c =13.4159 (17) (Å); α =
90, β = 103.775(11), γ = 90(°), V =2537.7 (5) nm3, T =293(2) K, Z =8, Dc =1.128 g/cm3,
F(000)=1040, Reflections collected / unique = 2502 / 968, Data / restraints / parameters =
2502/0/968, Goodness of fit on F2 =0.910, Fine, R1= 0.0716, wR(F2) = 0.2503. Crystal data
o
of 5k: Colorless crystals, yield, 77%; mp 158~159 C; C15H10O4S, M =286, monoclinic;
Space group P121/n1 ; a =8.4897 (9), b =5.3343 (5), c =28.708 (7) (Å); α = 90, β =
91.517(13), γ = 90(°), V =1299.6 (4) nm3, T =293(2) K, Z =4, Dc =1.463 g/cm3,
F(000)=592, Reflections collected / unique = 4889 / 2562, Data / restraints / parameters =
2562/0/181, Goodness of fit on F2 =1.017, Fine, R1= 0.0612, wR(F2) = 0.1127. The
molecular structures of compounds 4a and 5k were shown in Figure 3.
(Figure 3)
2.3 Inhibition of hMAO
The potential effects of the synthesized compounds on hMAO activity were investigated
by measuring their effects on the production of hydrogen peroxide (H2O2) from p-tyramine,
using the Amplex Red MAO assay kit (Molecular Probes, Inc., Eugene, Oregon, USA) and
MAO isoforms in microsomes prepared from insect cells infected with Recombinant
baculovirus containing cDNA inserts for hMAO-A or hMAO-B. The inhibition of hMAO
27
activity was evaluated using the general method described by L. Santana.
The test
compounds did not show any interference with the reagents used for a biochemical assay.
The results of hMAO-A and hMAO-B inhibition studies with title compounds were
reported in Table 1 together with the MAO-B selectivity index.
It is obvious from the data that compounds 5j, 5k and 5o exhibited high activity against
hMAO-B with IC50 values of 6.50 1.40, 0.21 0.08 and 5.68 0.19 µM respectively,
surpassing that of the positive control Iproniazide (7.65 0.48). Among them, compound 5k
4

(IC50 =0.21 µM) showed the most activity and higher hMAO-B selectivity (189.2-fold vs
1-fold) with respect to the hMAO-A isoform. Inspection of the chemical structures, it can
be concluded that among all the synthetic compounds, two classifications can be made, one
series is 2H-chromene-3-carboxamides which include compounds 4a~4i, and the other
series is S-2H-chromene-3-carbothioates which include compounds 5j~5t (see Scheme 1).
From the data presented in Table 1, we found that most compounds showed obvious
activity against hMAO with high selectivity toward hMAO-B. Furthermore,
S-2H-Chromene-3-carbothioate derivatives which include compounds 5j~5t showed
higher inhibitor activity against hMAO-B. This pointed out the direction for us to further
optimize the structures of 2H-chromene derivatives as selective and potential hMAO-B
inhibitors.
(Table 1)
2.4 Molecular docking
In an effort to elucidate the mechanism by which the title compound exhibited strong
inhibitory selectivie activity against hMAO-B and to establish an SAR based on our
experimental studies, molecule docking of the compound 5k into the ligand binding model
site of MAO-B was performed on the binding model based on the MAO-B complex
structure (2BYB. pdb). 9,28 The binding model of compound 5k with MAO-B complex was
depicted in Figure 4. The 2D and 3D pictures of binding were depicted in Figure 4A and
Figure 4B. In order to reveal that the molecule was well filled in the active pocket, the
enzyme surface was shown in Figure 4C.
Several bonding interactions anchoring compound 5k to the active site tightly might
explain its good inhibitory activity. There are two Pi-Pi interactions between title
compound and amino acids around it. One is between the furan-ring of compound 5k and
benzene-ring of TYR: 398 with length of 3.91 Å, the other is between the furan-ring of
compound 5k and benzene-ring of TYR: 435 with length of 4.55 Å. There is one Sigma-Pi
interaction between title compound and amino groups, which is between benzene-ring of
5

compound 5k and hydrogen atom of ILE: 199 with length of 2.66 Å.
(Figure 4)
3. Conclusions
In summary, we designed and synthesized some 2H-chromene derivatives as selective and
efficient MAO-B inhibitors, followed by chemical synthesis and biological evaluated for
them. Among them, compounds 4a and 5k were determined by X-ray. Enzymatic assays
revealed that compound 5k exhibited strong selectivity inhibitory activity toward hMAO-B,
more than 189-fold and 1-fold inhibition levels with respect to the MAO-A isoform. We
undertake a number of docking simulations to better assess, at the MAO-B binding site, the
effect on binding of some examined substituents at position 3 of coumarin. The steric
effect was analyzed along with an analysis of the enzymatic room available at position 3
and the subtle conformational resubstituents experienced by the 3-position. Among them,
the binding mode of high active compound 5k on the hMAO-B indicated that conserved
residue ILE 199 was important for ligand binding via Sigma-Pi interaction. In brief, based
on reasonable molecular design, we found that there was clear SAR against hMAO-B.
These results are helpful in the rational design of more efficient and selective hMAO
inhibitors in the future.
4. Experimental section
4.1 Chemistry
The reactions were monitored by thin layer chromatography (TLC) on Merck pre-coated
silica GF254 plates. Melting points (uncorrected) were determined on a XT4MP apparatus
1
(Taike Corp., Beijing, China). H NMR spectra were collected on PX400 spectrometer at
room temperature with TMS and solvent signals allotted as internal standards. Chemical
shifts are reported in ppm (δ). Elemental analyses were performed on a CHN–O-Rapid
instrument, and were within 0.4% of the theoretical values. Mass spectra were performed
on an Agilent 1260-6221 TOF mass spectrometer.
6

4.2 General procedure for the synthesis of title compounds 4,5
To a solution of 2-oxo-2H-chromene-3-carbonyl chloride 3 (2 mmol) and DMAP
o
(4-dimethylaminopyridine, 2 mmol) in chloroform (30 mL) at 10 C was added dropwise
substituted amines (compounds 4) or substituted ethiol (compounds 5) (3.0 mmol) for 30
min. The reaction mixture was stirred at room temperature for 2~3 h and washed with 20
ml H2O, 10 mL 1% NaHCO3, respectively, then dried on anhydrous MgSO4. The solvent
was removed in vacuo and the crude residue was purified by chromatography on SiO2
(acetone/petroleum, 4: 1, v/v) to give title compounds 4a~4i and 5j~5t as a colorless solid.
Compounds 1~3 were prepared according to references 23 and 29.
(Scheme 1)
4a: N-isopropyl-2-oxo-2H-chromene-3-carboxamide, colorless crystals, yield, 81%; mp
199-200 oC; 1H NMR (400 MHz, CDCl3) δ 8.90 (s, 1H, chromen C4-H), 8.66 (s, 1H, -NH-),
7.67 (dd, J = 17.1, 7.9 Hz, 2H, ArH), 7.45~7.30 (m, 2H, ArH), 4.26 (d, J = 6.7 Hz, 1H),
1.28 (d, J = 6.5 Hz, 6H). TOF-HRMS: m/z [M + H]+ calcd for C13H13NO3: 232.25; found:
232.0966. Anal. calcd for C13H13NO3: C, 67.52; H,5.67; N, 6.06%. Found: C, 67.33; H,
5.45; N, 5.91%.
4b: N-tert-butyl-2-oxo-2H-chromene-3-carboxamide, colorless crystals, yield, 85%; mp
203-204 oC; 1H NMR (400 MHz, CDCl3) δ 8.87 (s, 1H, chromen C4-H), 8.76 (s, 1H, -NH-),
7.64 (m, 2H, ArH), 7.49~7.31 (m, 2H, ArH), 1.47 (s, 9H). TOF-HRMS: m/z [M + H]+ calcd
for C14H15NO3: 246.27; found: 246.1124. Anal. calcd for C14H15NO3: C, 68.56; H, 6.16; N,
5.71%. Found: C, 68.84; H,6.02; N, 5.87%.
4c: N-sec-butyl-6-nitro-2-oxo-2H-chromene-3-carboxamide, colorless crystals, yield, 80%;
o
1
mp 209-210 C; H NMR (400 MHz, CDCl3) δ 8.91 (s, 1H, chromen C4-H), 8.67 (s, 1H,
-NH-), 7.67 (m, 2H, ArH), 7.38 (m, 2H, ArH), 4.11 (dt, J = 13.6, 7.0 Hz, 1H), 1.64~1.56
(m, 2H), 1.25 (d, J = 6.6 Hz, 3H), 0.97 (t, J = 7.4 Hz, 3H). TOF-HRMS: m/z [M + H]+
calcd for C14H14N2O5: 291.27; found: 291.5507. Anal. calcd for C14H14N2O5: C, 57.93; H,
4.86; N, 9.65%. Found: C, 60.11; H, 5.10; N, 9.88%.
4d: 6,8-dibromo-N-isopropyl-2-oxo-2H-chromene-3-carboxamide, colorless crystals, yield,
7

o
84%; mp 237-239 C; 1H NMR (400 MHz, CDCl3) δ 8.78 (s, 1H, chromen C4-H), 8.51 (s,
1H, -NH-), 7.97 (s, 1H, ArH), 7.76 (s, 1H, ArH), 4.25 (dd, J = 13.3, 6.7 Hz, 1H), 1.28 (d, J
= 6.5 Hz, 6H). TOF-HRMS: m/z [M + H]+ calcd for C13H11Br2NO3: 390.04; found:
389.9197. Anal. calcd for C13H11Br2NO3: C, 40.13; H, 2.85; N, 3.60%. Found: C, 40.28; H,
3.06; N, 3.79%.
4e: 6,8-dibromo-N-sec-butyl-2-oxo-2H-chromene-3-carboxamide, colorless crystals, yield,
o
86%; mp 222-224 C; 1H NMR (400 MHz, CDCl3) δ 8.78 (s, 1H, chromen C4-H), 8.51 (s,
1H, -NH-), 7.99 (d, J= 1.9 Hz, 1H, ArH), 7.77 (d, J= 1.7 Hz, 1H, ArH), 4.16~4.01 (m, 1H),
1.65~1.58 (m, 2H), 1.25 (d, J= 6.5 Hz, 3H), 0.97 (t, J= 7.4 Hz, 3H). TOF-HRMS: m/z [M +
Na]+ calcd for C14H13Br2NO3: 426.07; found: 425.9151. Anal. calcd for C14H13Br2NO3: C,
41.72; H, 3.25; N, 3.48%. Found: C, 41.90; H, 3.14; N, 3.61%.
4f: 6,8-dibromo-N,N-dihexyl-2-oxo-2H-chromene-3-carboxamide, colorless crystals, yield,
o
80%; mp 128-129 C; 1H NMR (400 MHz, CDCl3) δ 7.92 (s, 1H, chromen C4-H), 7.64 (s,
1H, ArH), 7.59 (s, 1H, ArH), 3.47 (s, 2H), 3.17 (t, J= 7.4 Hz, 2H), 1.64 (d, J= 6.9 Hz, 2H),
1.55~1.49 (m, 2H), 1.34 (s, 6H), 1.26~1.16 (m, 6H), 0.90 (d, J= 6.6 Hz, 3H), 0.82 (t, J=
6.5 Hz, 3H). TOF-HRMS: m/z [M + H]+ calcd for C22H29Br2NO3: 516.28; found: 516.0565.
Anal. calcd for C22H29Br2NO3: C, 51.28; H, 5.67; N, 2.72%. Found: C, 51.49; H, 6.00; N,
2.88%.
4g: N-isopropyl-6-nitro-2-oxo-2H-chromene-3-carboxamide, colorless crystals, yield, 75%;
o
1
mp 248-249 C; H NMR (400 MHz, CDCl3) δ 8.98 (s, 1H, chromen C4-H), 8.62 (s, 1H,
-NH-), 8.51 (dd, J = 9.2, 2.4 Hz, 2H, ArH), 7.55 (d, J = 9.1 Hz, 1H, ArH), 4.27 (dd, J =
13.3, 6.7 Hz, 1H), 1.29 (d, J = 6.6 Hz, 6H). TOF-HRMS: m/z [M + Na]+ calcd for
C13H12N2O5: 299.24; found: 299.0638. Anal. calcd for C13H12N2O5: C, 56.32; H, 4.38; N,
10.14%. Found: C, 56.60; H, 4.11; N, 10.01%.
4h: N-tert-butyl-6-nitro-2-oxo-2H-chromene-3-carboxamide, colorless crystals, yield, 78%;
mp 216-217 oC; 1H NMR (400 MHz, CDCl3) δ 8.94 (s, 1H, chromen C4-H), 8.66~8.43 (m,
3H), 7.54 (d, J= 9.1 Hz, 1H, ArH), 1.48 (s, 9H). TOF-HRMS: m/z [M + Na]+ calcd for
C14H14N2O5: 313.27; found: 313.0793. Anal. calcd for C14H14N2O5: C, 57.93; H, 4.86; N,
9.65%. Found: C, 58.15; H, 4.55; N, 9.93%.
4i: N,N-dihexyl-6-nitro-2-oxo-2H-chromene-3-carboxamide, colorless crystals, yield, 79%;
8

o
1
mp 129-130 C; H NMR (400 MHz, CDCl3) δ 8.45 (s, 1H, chromen C4-H), 8.42 (s, 1H,
ArH ), 7.82 (s, 1H, ArH), 7.50 (d, J= 8.7 Hz, 1H, ArH), 3.48 (s, 2H), 3.18 (t, J= 7.5 Hz,
2H), 1.70~1.62 (m, 2H), 1.56 (s, 2H), 1.35 (s, 6H), 1.25~1.15 (m, 6H), 0.91 (t, J= 6.0 Hz,
3H), 0.81 (t, J= 6.5 Hz, 3H). TOF-HRMS: m/z [M + H]+ calcd for C22H30N2O5: 403.48;
found: 403.2222. Anal. calcd for C22H30N2O5: C, 65.65; H, 7.51; N, 6.96%. Found: C,
65.82; H, 7.33; N, 7.11%.
5j: S-4-bromophenyl 2-oxo-2H-chromene-3-carbothioate, colorless crystals, yield, 74%;
mp 211-212 oC; 1H NMR (400 MHz, CDCl3) δ 8.56 (s, 1H, chromen C4-H), 7.73~7.67 (m,
2H, ArH), 7.59 (d, J = 8.0 Hz, 2H, ArH), 7.40 (dd, J = 21.7, 7.6 Hz, 4H, ArH).
TOF-HRMS: m/z [M + H]+ calcd for C16H9BrO3S: 362.21; found: 360.95. Anal. calcd for
C16H9BrO3S: C, 53.20; H, 2.51%. Found: C, 53.40; H, 2.69%.
5k: S-furan-2-ylmethyl 2-oxo-2H-chromene-3-carbothioate, colorless crystals, yield, 77%;
o
1
mp 158-159 C; H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H, chromen C4-H), 7.67 (d, J =
7.2 Hz, 2H, ArH), 7.37 (m, 3H, ArH), 6.29 (s, 2H, furan-H), 4.32 (s, 2H, -CH2-).
TOF-HRMS: m/z [M + H]+ calcd for C15H10O4S:287.3; found: 287.03. Anal. calcd for
C15H10O4S: C, 62.93; H, 3.52 %. Found: C, 63.07; H, 3.61 %.
5l: S-pyrimidin-2-yl 2-oxo-2H-chromene-3-carbothioate, colorless crystals, yield, 68%; mp
199-200 oC; 1H NMR (400 MHz, CDCl3) δ 8.86 (d, J = 4.8 Hz, 2H, pyrimidine-H), 8.58 (s,
1H, chromen C4-H), 7.72~7.67 (m, 2H, ArH), 7.43~7.34 (m, 3H, ArH and pyrimidine-H).
TOF-HRMS: m/z [M + H]+ calcd for C14H8N2O3S: 285.29; found: 285.17. Anal. calcd for
C14H8N2O3S: C, 59.15; H, 2.84; N, 9.85%. Found: C, 59.33; H, 3.01; N, 10.08%.
5m: S-butyl 2-oxo-2H-chromene-3-carbothioate, colorless crystals, yield, 73%; mp
103-104 oC; 1H NMR (400 MHz, CDCl3) δ 8.53 (s, 1H, chromen C4-H), 7.69~7.64 (m, 2H,
ArH), 7.36 (m, 2H, ArH), 3.05 (t, J = 7.4 Hz, 2H), 1.70~1.63 (m, 2H), 1.46 (m, 2H), 0.95 (t,
J = 7.3 Hz, 3H). TOF-HRMS: m/z [M + H]+ calcd for C14H14O3S: 263.32; found: 263.07.
Anal. calcd for C14H14O3S: C, 64.10; H, 5.38%. Found: C, 64.25; H, 5.60%.
5n: S-4-bromophenyl 6,7-dibromo-2-oxo-2H-chromene-3-carbothioate, colorless crystals,
o
1
yield, 83%; mp 171-172 C; H NMR (400 MHz, CDCl3) δ 8.41 (s, 1H, chromen C4-H),
8.03 (s, 1H, ArH), 7.77 (s, 1H, ArH), 7.60 (d, J = 8.1 Hz, 2H, ArH), 7.36 (d, J = 8.2 Hz, 2H,
ArH). TOF-HRMS: m/z [M + Na]+ calcd for C16H7Br3O3S: 542.00; found: 541.75. Anal.
9

calcd for C16H7Br3O3S: C, 37.03; H,1.36 %. Found: C, 37.28; H, 1.59 %.
5o: S-furan-2-ylmethyl 6,7-dibromo-2-oxo-2H-chromene-3-carbothioate, colorless crystals,
o
1
yield, 75%; mp 193-195 C; H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H, chromen C4-H),
8.00 (s, 1H, ArH), 7.75 (s, 1H, ArH), 7.35 (s, 1H, furan-H), 6.30 (d, J = 3.4 Hz, 2H,
furan-H), 4.32 (s, 2H, -CH2-). TOF-HRMS: m/z [M + Na]+ calcd for C15H8Br2O4S: 467.09;
found: 465.84. Anal. calcd for C15H8Br2O4S: C, 40.37; H, 1.82 %. Found: C, 40.53; H,
1.96 %.
5p: S-butyl 6,7-dibromo-2-oxo-2H-chromene-3-carbothioate, colorless crystals, yield, 70%;
o
1
mp 146-147 C; H NMR (400 MHz, CDCl3) δ 8.37 (s, 1H, chromen C4-H), 7.99 (s, 1H,
ArH), 7.74 (s, 1H, ArH), 3.06 (t, J = 7.4 Hz, 2H), 1.68~1.64 (m, 2H), 1.48~1.42 (m, 2H),
0.95 (t, J = 7.3 Hz, 3H). TOF-HRMS: m/z [M + Na]+ calcd for C14H12Br2O3S: 443.12;
found: 442.87. Anal. calcd for C14H12Br2O3S: C, 40.02; H, 2.88 %. Found: C, 40.29;
H,3.01 %.
5q: S-4-bromophenyl 6-nitro-2-oxo-2H-chromene-3-carbothioate, colorless crystals, yield,
o
1
79%; mp 188-189 C; H NMR (400 MHz, CDCl3) δ 8.61 (d, J = 7.5 Hz, 2H, chromen
C4-H and , ArH), 8.54 (d, J = 9.1 Hz, 1H, ArH), 7.59 (dd, J = 16.6, 8.5 Hz, 3H, ArH), 7.37
(d, J = 7.9 Hz, 2H, ArH). TOF-HRMS: m/z [M + Na]+ calcd for C16H8BrNO5S: 429.21;
found: 427.92. Anal. calcd for C16H8BrNO5S: C, 47.31; H, 1.99; N, 3.45 %. Found: C,
47.69; H, 2.02 N, 3.18%.
5r: S-furan-2-ylmethyl 6-nitro-2-oxo-2H-chromene-3-carbothioate, colorless crystals, yield,
o
1
70%; mp 183-184 C; H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H,chromen C4-H), 8.60 (s,
1H, chromen C6-H), 8.52 (d, J = 9.1 Hz, 1H, ArH), 7.53 (d, J = 9.2 Hz, 1H, ArH), 7.35 (s,
1H, furan-H), 6.31 (s, 2H, furan-H), 4.35 (s, 2H). TOF-HRMS: m/z [M + Na]+ calcd for
C15H9NO6S: 354.3; found: 354.00. Anal. calcd for C15H9NO6S: C, 54.38; H, 2.74; N,
4.23 %. Found: C, 54.60; H, 3.01; N, 4.17 %.
5s: S-butyl 6-nitro-2-oxo-2H-chromene-3-carbothioate, colorless crystals, yield, 68%; mp
141-142 oC; 1H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H, chromen C4-H), 8.56 (s, 1H, ArH),
8.51 (dd, J = 9.1, 2.5 Hz, 1H, ArH), 7.53 (d, J = 9.1 Hz, 1H, ArH), 3.08 (t, J = 7.4 Hz, 2H),
1.70~1.63 (m, 2H), 1.46 (dd, J = 14.9, 7.5 Hz, 2H), 0.95 (t, J = 7.3 Hz, 3H). TOF-HRMS:
m/z [M + Na]+ calcd for C14H13NO5S: 330.32; found: 330.04. Anal. calcd for C14H13NO5S:
10

C, 54.71; H, 4.26; N, 4.56 %. Found: C, 54.89; H, 4.50; N, 4.73 %.
5t: S-4-bromophenyl 2-oxo-6-(trifluoromethyl)-2H-chromene-3-carbothioate, colorless
o
crystals, yield, 80%; mp 165-166 C; 1H NMR (400 MHz, CDCl3) δ 8.49 (s, 1H, chromen
C4-H), 7.37 (J = 8.1 Hz, 2H, ArH), 7.40~7.53 (m, 2H, ArH), 7.11 (d, J = 8.2 Hz, 2H, ArH),
6.98 (d, J = 8.1 Hz, 1H, ArH). TOF-HRMS: m/z [M + H]+ calcd for C17H8BrF3O3S: 430.21;
found: 430.30. Anal. calcd for C17H8BrF3O3S: C, 47.57; H, 1.88 %. Found: C, 47.29; H,
2.01 %.
4.3 Crystallographic Studies
X-ray single-crystal diffraction data for compounds 4a and 5k were collected on a Bruker
SMART APEX CCD diffractometer at 296(2) K using MoKα radiation (λ = 0.71073 Å)
by the ω scan mode. The program SAINT was used for integration of the diffraction
profiles. The structures were solved by direct methods using the SHELXS program of the
SHELXTL package and refined by full-matrix least-squares methods with SHELXL.30 All
non-hydrogen atoms of compounds 4a, 5k were refined with anisotropic thermal
parameters. All hydrogen atoms were generated theoretically onto the parent atoms and
refined isotropically with fixed thermal factors.
4.4 hMAO activity assay
Enzymatic MAO-A and MAO-B activity of compounds was determined by a fluorimetric
method. 31 Briefly, 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4) containing various
concentrations of the test drugs (new compounds or reference inhibitors). The appropriate
amounts of recombinant hMAO-A or hMAO-B was adjusted to the same reaction velocity
in the presence of both isoforms the same concentration of substrate: 165 pmol of
p-tyramine/min (hMAO-A: 1.1 µg protein; specific activity: 150 nmolofp-tyramine
oxidizedtop hydroxyphenylacetaldehyde/min/mgprotein; hMAO-B: 7.5 µg protein; specific
activity: 22 nmol of p-tyramine transformed/min/mg protein). The mixture was incubated
for 15 min at 37 °C, placed in the dark fluorimeter chamber. After this incubation period,
the reaction was started by adding 200 µM of 10-acetyl-3,7-dihydroxyphenoxazine reagent
11

(Amplex Red assay kit, Molecular Probes, Inc., Eugene, OR), 1 U/mL horseradish
peroxidase, and 1 mM p-tyramine. The production of H2O2 and, consequently, ofresorufin,
o
was quantified at 37 C in a multidetection microplate fluorescence reader based on the
fluorescence generated (excitation, 545 nm, emission, 590nm) over a 15 min period, during
which the fluorescence increased linearly.
4.5 General procedure for molecular docking
Discovery Studio 3.1 (DS 3.1, Accelrys Software Inc., San Diego, California, USA).
Crystal structure of telomerase (PDB entry 2BYB) was used as template. Hydrogen atoms
were added to protein model. The added hydrogen atoms were minimized to have stable
energy conformation and to also relax the conformation from close contacts. The active
site was defined and sphere of 4 Å was generated around the active site pocket, with the
active site pocket of BSAI model using C-DOCKER, a molecular dynamics (MD)
simulated-annealingbased algorithm module from DS 3.1 Random substrate conformations
are generated using high-temperature MD. Candidate poses are then created using random
rigid-body rotations followed by simulated annealing. The structure of protein, substrate
were subjected to energy minimization using CHARMm forcefield as implemented in DS
3.1. A full potential final minimization was then used to refine the substrate poses. Based
on C-DOCKER, energy docked conformation of the substrate was retrieved for
postdocking analysis.
5. Acknowledgments
The authors wish to thank the National Natural Science Foundation of China (No.
21272008), Science and Technological Fund of Anhui Province for Outstanding Youth
(1408085J04), Anhui Provincial Natural Science Foundation, (1308085MH137), The
Governor Fund Of Guizhou Province (20120037) and the Research Fundation of Doctor,
Anhui Medical University (XJ201120, XJ201121).
12

6. Supporting information
Crystallographic data (excluding structure factors) for the structure had been deposited
with the Cambridge Crystallographic Data Center as supplementary publication No. CCDC
976175 (4a) and CCDC 981020 (5k). This data can be obtained free of charge via the URL
http:// www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: (+44) 1223 336033; e-mail: deposit@ccdc.cam.ac.uk)
References and notes
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Eur. J. Med. Chem. 2011,46, 4846-4852.
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14

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15

Figure Captions
Table 1. IC50 values and hMAO-B selectivity index for the title compounds a
Scheme 1. Synthesis of title compounds 4,5
Figure 1. MAO inhibitors in the coumarin class
Figure 2.
Figure 3.
General design for the novel coumarin derivatives
ORTEP drawing of compounds 4a and 5k
Figure 4.
Molecule docking of the compound 5k into the ligand binding model site of
MAO-B was performed on the binding model based on the MAO-B complex structure
(2BYB. pdb). Panel is a view into the active site cavity
(A). The 2D picture of binding was depicted
(B). The 3D picture of binding was depicted
(C). In order to reveal that the molecule was well filled in the active pocket, the enzyme
surface was shown
16

Table 1 IC50 values and hMAO-B selectivity index for the title compounds a
Compound
MAO-A (µM)
MAO-B (µM)
SI MAO-Bc
4a
4b
4c
4d
35.51 1.09
40.36 1.25
10.46 0.82
7.90 0.65
17.44 1.85
10.35 0.26
45.37 2.20
7.41 0.55
b
3.4d
5.1 d
3.4 d
2.2 d
0.9 d
1.4 d
_
b
22.69 0.98
42.31 2.49
10.08 0.71
b
4e
4f
4g
4h
4i
b
b
b
b
_
_
5j
5k
22.69 0.98
39.74 0.85
b
b
6.50 1.40
0.21 0.08
7.68 0.31
16.01 1.14
25.18 1.51
5.68 0.19
b
3.5 d
189.2 d
7.8 d
3.8d
2.4 d
10.6 d
_
5l
5m
5n
5o
5p
5q
5r
5s
5t
b
b
b
b
20.71 1.50
13.09 0.58
b
2.6 d
2.9 d
48.27 1.94
b
b
15.21 1.47
b
3.9 d
_
Reagent blank
b
Iproniazide
6.97 0.53
7.65 0.48
0.9
a Each IC50 value is the mean SEM from three experiments (n =3)
b Inactive at 60 µM (highest concentration tested)
c SI MAO-B =[IC50(MAO-A)]/[IC50(MAO-B)]
d
Values obtained under the assumption that the corresponding IC50 against either MAO-A
or MAO-B is the highest concentration tested (60 µM)
17

O
O
CHO
OH
COOH
O
(A)
(B)
O
R1
R1
R1
O
O
2
1
O
C
R2
COCl
O
(C)
(D)
R1
R1
O
O
O
3
4
(E)
O
C
R3
S
R1
O
O
5
H
N
R1=6-H, R2=
R1=H, R2=
R1=6-NO2,
R2=
(4b)
(4c)
(4a)
N
H
N
H
H
R1=6, 8- 2Br, R2=
R1=6, 8- 2Br, R2=
R1=6, 8- 2Br, R2=
R1=6-NO2, R2=
(4e)
N
N
(4d)
(4f)
(4i)
N
H
H
R1=6-NO2, R2=
R1=H, R3=
R1=6-NO2, R2=
R1=H, R3=
(4h)
N
N
N
H
(4g)
N
O
(5l)
R1=H, R3=
R1=H, R3=
(5j)
Br
(5k)
N
R1=6, 8- 2Br, R3=
R1=6-NO2, R3=
R1=6, 8- 2Br, R3=
R1=6-NO2, R3=
(5m)
O
(5o)
Br
Br
(5n)
(5q)
O
(5r)
R1=6, 8- 2Br, R3=
R1=6-NO2, R3=
(5p)
(5s)
R1=6-CF3, R3=
(5t)
Br
Scheme 1. Synthesis of title compounds 4,5
Reagent and conditions: (A) CH2(COOC2H5)2, piperidine, reflux 2 h; (B) NaOH, reflux 3
o
h, HCl, pH=2; (C) SO2Cl, reflux 1 h; (D) Substituted amines, CHCl3, 25~40 C, 2~3 h; (E)
Substituted ethiols, CHCl3, DMAP, 25 oC, 3~4 h.
18

Figure 1.
O
S
O
O2N
S
O
O
O
O
O
O
S
O
O
O
O
O
N
N
A (Esuprone)
C (LU 53439)
B
Figure 2.
H
N
R
O
O
O
A
O
O
R
S
N
H
R
O
O
O
O
B
C
19

Figure 3.
4a
5k
20

Figure. 4A
Figure. 4B
21

Figure. 4C
22

Graphical abstract
Novel coumarin derivatives: design、synthesis and use as inhibitors of hMAO
Xu Hea, Yan-Yan Chena, Zhi-Xiang Pana, Zhi-Qiang Donga, Bao-An Songb, Jun Lia,*,
Xin-Hua Liua,b, c*
a School of Pharmacy, Anhui Medical University, Hefei, 230032, P. R. China
b Key Laboratory of Green Pesticide and Agriculture Bioengineering, Ministry of
Education, Guizhou University, Guiyang, 550025, P. R. China
c
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, P. R. China
5k
Novel coumarine derivatives as potential MAO-B selectivity inhibitors were designed and
synthesized. Enzymatic assays revealed that compound 5k exhibited strong selectivity
inhibitory activity toward hMAO-B.
23