α-Ketoamino acid ester derivatives as promising MAO inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.11.007

α-Ketoamino acid ester 2-[2-(2-acetamidophenyl)-2-oxoacetamido] and 2-[4-(2-(2-acetamidophenyl)-2-oxoacetamido)benzamido] derivatives were synthesized via the ring opening of N-acetylisatin under mild conditions. These compounds were then examined for the

Full text of DOI:10.1016/j.bmcl.2014.11.007

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
a
-Ketoamino acid ester derivatives as promising MAO inhibitors
a
Ayman El-Faham a,b, , Zainab Al Marhoon , Ahmed Abdel-Megeed c,d, Sherine N. Khattab b,
⇑
a,f,g,h,i,j
Adnan A. Bekhit e, , Fernando Albericio
⇑
a Department of Chemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia
b Chemistry Department, Faculty of Science, Alexandria University, PO Box 426, Ibrahimia, 12321 Alexandria, Egypt
c Department of Botany and Microbiology, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia
d Department of Plant Protection, Faculty of Agriculture, Saba Basha, Alexandria University, Alexandria, Egypt
e Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt
f Institute for Research in Biomedicine (IRB), Barcelona Science Park, Baldiri Reixac 10, Barcelona 08028, Spain
g CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, 08028 Barcelona, Spain
h Department of Organic Chemistry, University of Barcelona, Martí i Franqués 1-11, Barcelona 08028, Spain
i School of Chemistry & Physics, University of KwaZulu-Natal, Durban 4001, South Africa
j School of Chemistry, Yachay Tech, Yachay City of Knowledge, 100119 Urcuqui, Ecuador
a r t i c l e i n f o
a b s t r a c t
Article history:
a
-Ketoamino acid ester 2-[2-(2-acetamidophenyl)-2-oxoacetamido] and 2-[4-(2-(2-acetamidophenyl)-2-
Received 19 September 2014
Revised 1 November 2014
Accepted 3 November 2014
Available online xxxx
oxoacetamido)benzamido] derivatives were synthesized via the ring opening of N-acetylisatin under mild
conditions. These compounds were then examined for their capacity to inhibit monoamine oxidase
(MAO). The inhibition profile was found to be competitive for compounds 4d, 6a, 6b and 6f, which showed
MAO-A selectivity. Observation of the docked positions of these compounds revealed interactions with many
residues previously reported to have an effect on the inhibition of the enzyme. Our findings indicate that the
Keywords:
N-Acetylisatin
members of this family of
a-ketoamino acid esters are promising MAO inhibitors.
Ó 2014 Elsevier Ltd. All rights reserved.
a-Ketoamino acid ester
OxymaPure
p-Aminobenzoic acid
Monoamine oxidase inhibitors
Monoamine oxidase A and B (MAO-A and -B) are flavin adenine
dinucleotide (FAD)-containing enzymes, which are found in the
outer mitochondrial membranes of neuronal and glial cells, among
others1 particularly abundant in the liver and brain.2 These FAD-
dependent enzymes are responsible for regulation and metabolism
of major monoamine neurotransmitters, such as serotonin (5-OH
tryptamine), nor-adrenaline, and dopamine. They are also involved
in the biodegradation of exogenic amines, such as benzylamine,
tyramine, MPTT, MPP+, and a Parkinsonian syndrome-producing
neurotoxin.1 They catalyze the oxidative deamination of a range
of endogenous and exogenous monoamines.3 The two mammalian
isoforms of MAO-A and -B are encoded by two different genes4 and
distinguished by distinct substrate specificities and sensitivities to
selective inhibitors.5 Thus, MAO-A is selectively inhibited by
clorgyline and preferentially metabolizes serotonin, whereas
MAO-A inhibitors are used in clinical practice as antidepressants
and anxiolytics, while MAO-B inhibitors are used to slow down
the progression of Parkinson’s disease and of symptoms associated
with Alzheimer’s disease. Earlier MAO inhibitors introduced into
clinical practice for the treatment of depression were abandoned
due to adverse side-effects, such the ‘cheese effect’, which is char-
acterized by hypertensive crises.6
For this reason, the interest of many research groups has been
devoted to MAO as a therapeutic target.7,8 Among the compounds
studied as MAO inhibitors, heterocyclic hydrazines and hydrazides
are prevalent. N0-Propan-2-ylpyridine-4-carbohydrazide, under the
trade names of ipronid, iprozid, marsilid, propilniazida and rivivol,
was the first modern antidepressant to be introduced into the market.
In the course of our ongoing studies aimed at the synthesis of
heterocyclic compounds of potential pharmaceutical relevance,9–11
MAO-B is inhibited by
benzylamine and phenylethylamine as substrates. Selective
L
-deprenyl and preferentially uses
here we focused on a novel family of
derivatives as MAO inhibitors.
a-ketoamino acid ester
The rational design of these compounds was based on hybrid
structure of known inhibitors and previous reported substituted
pyridazine-1-yl acetic acid derivatives I which were established
as selective monoamine oxidase-A inhibitors.11 The aim of the
⇑
Corresponding authors.
E-mail addresses: aymanel_faham@hotmail.com (A. El-Faham), adnbekhit@
hotmail.com (A.A. Bekhit).
http://dx.doi.org/10.1016/j.bmcl.2014.11.007
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: El-Faham, A.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.11.007

2
A. El-Faham et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
O
HN R1COOR
O
NH
COCH3
Ph
O
O
H
N
R2
N
K2CO3
N
N
NH2R1COOR.HCl
O
O
CH3CN, rt
N
OtBu
O
3a-f
COCH3
Pargyline
Ar
I
4a-f
1
reference 11
O
NH2R1COOR = Ethyl 4-aminobezoate; 3a
Leucine methyl ester; 3b
H
N
O
R2
Aspartic dimethyl ester;3c
H
N
O
O
Methyl-3-aminopropionate; 3d
Methyl-4-aminobutyrate; 3e
Methyl-6-aminohexanoate; 3f
NH
COCH3
R1
O
II
Scheme 2. Reaction of N-acetylisatin with amino acid esterꢀHCl.
O
O
H
N
O
N
H
HO
Eugenol analog
Figure 1. Planned modification and newly designed MAO inhibitors.
N
O
COOH
H
N
O
O
Iproniazide
reflux 1h/MeOH
+
O
O
NH
COCH3
COOH
N
COCH3
NH2
5
1
4-aminobenzoic acid
NC
COOC2H5
OH
NH2R
present study was tailoring MAO-A inhibitors considering some
factors responsible for selectivity against A isoform12 which are
(i) the presence of electron-rich aromatic moieties (e.g., pargyline),
(ii) the presence of amide functionality (e.g., Iproniazid13), (iii) the
presence of ethoxycarbonyl methylene group (e.g., Eugenol
analog14), (iv) the presence of amino acid moiety (I),11 Figure 1.
N
DIEA/DMF, rt
OxymaPure / DIC
O
H
N
O
NHR
NH
COCH3
6a-i
O
Moreover
a-ketoamino moiety was included to study the effect
of such molecular variation on MAO inhibitory activity.
a
-Ketoamides are compounds of interest in organic chemistry
Ph
and are present in many pharmaceutical compounds.15–18 In paral-
lel, their application in medicinal chemistry has fueled the devel-
opment of several synthetic methods.19–30 The synthesis of an
,
,
RNH-
=
,
COOCH3
HN
,
HN
COOCH3
HN
COOCH3
6a
6b
6c
COOCH3
COOCH3
a
-ketoamide fragment could be achieved by the ring opening of
HN (CH2)2COOCH3
N-acetylisatin 1 by attacking of an amine at C2-carbonyl group of
N-acetylisatin.31–39 Recently, Cheah et al.39 reported the synthesis
of N-glyoxylamide peptide mimics from the reaction of N-acetyli-
,
HN
COOCH3
6d
,
HN
6e
6f
satin with
a-amino esters. The reaction was carried out in
,
,
HN (CH2)5COOCH3
HN (CH2)3COOCH3
HN
COOCH3
6i
DCM/H2O (2:1) in the presence of saturated NaHCO3, giving yields
ranging from 61% to 98%. They claimed that the lower yield in
some cases is due to the formation of glycoxalic acid derivative 2
(Scheme 1).
6g
6h
Scheme 3. Synthesis 4-[2-(2-acetylaminophenyl)-2-oxo-acetylamino] benzoyl
amino acid ester derivatives 6a–i.
Later, our group,40 reported the synthesis of
a
-ketoamides using
CH3CN and K2CO3 in place of DCM-H2O/NaHCO3. Herein we report
on the synthesis of -keto amino acid ester and (4-[2-(2-acetylam-
a
(2-acetamidophenyl)-2-oxoacetamido)benzoic acid 5 (Scheme 3).
inophenyl)-2-oxo-acetylamino]benzoyl amino acid ester deriva-
tives and their capacity to inhibit monoamine oxidase (MAO).
Compound 5 was then coupled with several L-amino acid esters
using OxymaPure/DIC43–47 in DMF as a solvent to afford the prod-
ucts 6a–i in excellent yield and purity (Scheme 3).48 The structure
of all the compounds synthesized was confirmed by IR and NMR
(1H NMR and 13C NMR) and were in agreement with the reported
data.49
The
a-ketoamino acid ester derivatives 4a–f were prepared by
the reaction of
L
-amino acid ester hydrochloride 3a–f with
N-acetylisatin 1, following the reported method.41 The reaction
was performed in CH3CN and K2CO3 at rt to afford the products
4a–f in 80–92% yield (Scheme 2).41 The structures of all the
synthesized compounds were confirmed by IR and NMR (1H NMR
and 13C NMR) and were in agreement with the reported data.40,42
The 4-aminobenzoic acid derivatives 6a–i were prepared by
the reaction 4-aminobenzoic acid with N-acetylisatin 1 using
conventional heating for 1 h in MeOH as a solvent to afford 4-(2-
The final compounds 4a–4f and 6a–6i were evaluated for
MAO-A inhibitory activity in vitro following the method described
by Undenfriend et al.50 The method involves the determination of
MAO-A activity of rat liver mitochondria51 using clorgyline as irre-
versible time-dependent reference standard. The test compounds
or reference standard were preincubated for 60 min with enzymes
O
O
O
H
OH
NaHCO3
N
CONHR'
+
H2N
CONHR'
O
+
DCM/H2O
O
N
O
R
R
NH
COCH3
NH
COCH3
COCH3
1
2
Scheme 1. Synthesis of N-glyoxylamide peptide.
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A. El-Faham et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
before the addition of the corresponding substrate to ensure fair
comparison. Furthermore, the synthesized compounds were tested
to determine their selectivity for MAO-A and MAO-B in the
presence of the specific substrate serotonin or benzylamine,
respectively. Bovine brain mitochondria were isolated following
Basford.52 The compounds were tested to determine their activity
toward MAO-A and -B, following methods of Matsumoto et al.53
the various interactions between the ligands and enzyme active
sites.
MOE (Molecular Operating Environment)54 docking studies of
the inhibitors were performed using the crystal structure of human
MAO-A (PDB ID: 2BXR). Docking of 6b into the MAO-A active site
(Fig. 2) revealed several molecular interactions were considered
to be responsible for the observed affinity. For example five hydro-
gen bond interactions were observed, 3 hydrogen bond interac-
tions with ARG51, one with ALA68, and one with TYR69. In
addition, 16 hydrophobic interactions were observed with GLY20,
GLY22, SER24, GLY49, GLY50, ARG51, THR52, GLY66, GLY67,
ALA68, TYR69, ALA272, PRO274, TYR407, TYR444, MET445.
Consequently, these observations provide a good explanation
for the potent inhibitory activity of compound 6b. From the above-
mentioned data 6b may provide a starting point for the design of
unique compounds with high affinity and selectivity for MAO-A.
The most active compounds 4d, 6a, 6b, and 6f were further
evaluated for oral acute toxicity in male mice using the reported
methods.55,56 The results indicated that the compounds were
nontoxic and well tolerated by the experimental animals up to
250 mg/kg, although no mortality was recorded at this concentration.
and Basford.52 The MAO-A and -B results, expressed as IC50
,
and also the selectivity index are given in Table 1. Compound 6b
showed MAO-A inhibitory activity (% MAO-A inhibition = 2.8 ꢁ
10ꢂ9 0.11) comparable to the standard clorgyline (% MAO-A
inhibition = 2.9 ꢁ 10ꢂ9 0.12) while 4d, 6a, and 6f showed lower
inhibition activity than this compound. These three compounds
showed greater capacity to inhibit MAO-A than MAO-B. Compound
6b is the most selective compound as MAO-A inhibitor, it showed
remarkable selectivity. Compounds 4d and 6f are comparable to
clorgyline in their selectivity as MAO-A inhibitors. In an attempt
to rationalize the MAO-A inhibitory activity observed for 6b, we
performed molecular modeling and conformational alignment
studies. Molecular docking studies further contribute to unveiling
Table 1
Effect of some
a
-ketoamino acid ester derivatives on the activity of MAO-A and MAO-Ba
a The results were expressed as mean S.E.M. Data were analyzed by one-way variance. The Student’s t test for unpaired observations was used. P value = <0.001 and the
significant number of experiments was 6.
b SI-MAO-B IC50/MAO-A IC50.
Figure 2. 3D view from a molecular modeling study of a minimum-energy structure of the complex of 6b (stick) docked in the active site of MAO-A (PDB ID: 2BXR). Dashed
lines depict hydrogen bond interactions. Viewed using a Molecular Operating Environment (MOE) module.
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4
A. El-Faham et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
33. Obafemi, C. A.; Taiwo, F. O.; Iwalewai, E. O.; Akinpelu, D. A. Int. J. Life Sci. Pharma
Res. 2013, 2, 22.
34. Chung, Y. M.; Kim, J. H.; Gong, J. N. Bull. Korean Chem. Soc. 2002, 23, 1363.
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Eur. J. Med. Chem. 2010, 45, 1374.
Moreover, these compounds were tested for toxicity when admin-
istered through parenteral route.57 All the test compounds were
nontoxic up to 100 mg/kg. We conclude that the synthesis and bio-
chemical evaluation of the newly synthesized
a-ketoamino acid
ester derivatives will contribute to the design of a novel class of
reversible MAO-A inhibitors with an excellent therapeutic window.
37. Boechat, N.; Kover, W. B.; Bastos, M. M.; Pinto, A. C.; Maciel, L. C.; Mayer, L. M.
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Acknowledgments
38. Ghazzali, M.; El-Faham, A.; Abdel-Megeed, A.; Al-Farhan, K. J. Mol. Struct. 2012,
1013, 163.
39. Cheah, W. C.; Wood, K.; Black, D. S. C.; Kumar, N. Tetrahedron 2011, 67, 7603.
40. El-Faham, A.; Al Marhoon, Z.; Abdel-Megeed, A.; Siddiqui, M. J. Chem. 2013, 1.
http://dx.doi.org/10.1155/2013/901745. Article ID 901745.
The authors thank the Deanship of Scientific Research at King
Saud University for funding this work through research group
No. RGP-234 (Saudi Arabia). Additionally, this study was also
partially funded by CICYT (CTQ2012-30930), the Generalitat de
Catalunya (2014 SGR 137) (Spain), and the Institute for Research
in Biomedicine Barcelona (IRB Barcelona) (Spain); the National
Research Foundation and the Inyuvesi Yakwazulu-Natali (South
Africa); and SENESCYT (Ecuador).
41. General Method for the synthesis of 4a–f:
Amino acid ester (12 mmol) and K2CO3 (1.66 g, 12 mmol) were added to the
solution of N-acetylisatin 1 (1.89 g, 10 mmol) in CH3CN (50 mL) with intensive
stirring. This mixture was stirred at room temperature overnight. The reaction
mixture was then filtered and washed with 10 mL of acetonitrile. The solvent
was removed under vacuum to dryness, and the crude product was
recrystallized from dichloromethane–hexane to afford the pure product.
Compound 4d was obtained as an off-white solid, mp 90–92 °C; yield 87%. IR
(cmꢂ1): 3288, 3124, 1741, 1672, 1607. 1H NMR (CDCl3): d 2.18 (s, 3H, COCH3),
2.64 (t, 2H, CH2CH2CO), 3.67 (q, 2H, NHCH2), 3.70 (s, 3H, COOCH3), 7.09 (t, 1H),
7.45 (s, 1H, NH), 7.57 (t, 1H), 8.28 (d, 1H), 8.62 (d, 1H), 10.93 (s, 1H, NH).
13CNMR (CDCl3): d 25.5, 33.5, 35.1, 52.1, 118.6, 120.7, 122.6, 134.4, 136.6,
142.2, 163.1, 169.4, 172.6, 191.9. Anal. Calcd for C14H16N2O5: C, 57.53; H, 5.52;
N, 9.58. Found: C, 57.74; H, 5.30; N, 9.72.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
11.007.
42. The amino acid esters were prepared using the method reported in: Kudelko,
A.; Zielin´ ski, W. Tetrahedron 2009, 65, 1200. All the data were in good
agreement with the reported data.
43. Subirós-Funosas, R.; Prohens, R.; Barbas, R.; El-Faham, A.; Albericio, F. Chem.
Eur. J. 2009, 15, 9394.
44. Subirós-Funosas, R.; Khattab, Sh. N.; Nieto-Rodriguez, L.; El-Faham, A.;
Albericio, F. Aldrichim. Acta 2013, 40, 21.
45. Khattab, Sh. N.; Subirós-Funosas, R.; El-Faham, A.; Albericio, F. ChemistryOpen
2012, 1, 147.
References and notes
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48. Synthesis of 4-(2-(2-acetamidophenyl)-2-oxoacetamido)benzoic acid 546: N-Ace-
tylisatin and 4-aminobenzoic acid were refluxed in methanol as a solvent in
the presence of glacial acetic acid (2–3 drops) for 1 h. After cooling, the solid
product was filtered, washed with cold methanol, and then dried under
vacuum to afford the product in a pure form. The product was obtained as a
pale yellow powder, mp: 238–240 °C; yield 86% IR (cmꢂ1): 3270, 1679, 1601.
1H NMR (DMSO-d6): d 1.99 (s, 3H, COCH3), 7.25–7.30 (m, 2H), 7.62–7.68 (m,
2H), 7.90 (d, 2H), 7.95 (d, 2H), 10.55 (s, 1H, NH), 10.99 (s, 1H, NH), 13.00 (br s,
1H, COOH); 13C NMR (DMSO-d6): d 22.8, 118.7, 121.0, 123.1, 124.3, 125.4,
129.6, 130.3, 132.9, 136.8, 141.2, 161.00, 166.1, 168.2, 188.1. Anal. Calcd for
C17H14N2O5: C, 62.57; H, 4.32; N, 8.59. Found: C, 62.31; H, 4.57; N, 8.73.
12. Vallejos, G.; Rezende, M. C.; Cassels, B. K. J. Comput. Aided Mol. Des. 2002, 16, 95.
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General method for the synthesis of 6a–i: Acid 5 (1 mmol), Oxyma (1 mmol), and
DIC (1 mmol) were mixed in DMF (5 mL) at 0 °C. The reaction mixture was
stirred for 5 min at 0 °C to pre-activate the acid and generate the active ester.
DIEA (1 mmol) was added, followed by (1 mmol) amino acid ester. The reaction
mixture was stirred at 0 °C for 1 h and at room temperature overnight. The
mixture was diluted with ethyl acetate (50 mL) and extracted with 1 N HCl
(2 ꢁ 10 mL), 10% NaHCO3 (2 ꢁ 10 mL), and saturated NaCl (2 ꢁ 10 mL). The
organic phase was dried over anhydrous MgSO4 and filtered, and the solvent
was removed under vacuum. The residue was recrystallized from dichloro-
methane–hexane to afford the pure product. All the spectral data were in a
good agreement with the reported data.46
Compound 6a was obtained as a white powder, mp: 174–176 °C; yield 88%. IR
(cmꢂ1): 3288, 3124, 1741, 1672, 1607. 1H NMR (DMSO-d6): d 1.41 (d, 3H,
CHCH3), 2.00 (s, 3H, COCH3), 3.65 (s, 3H, COOCH3), 4.48 (m, 1H, NHCHCH3), 7.45
(t, 1H), 7.64 (d,1H), 7.68 (d, 2H), 7.89 (m, 4H, Ar), 8.73 (s, 1H, NH), 10.55 (s, 1H,
NH), 10.92 (s, 1H, NH); 13C NMR (DMSO-d6): d 17.3, 24.2, 49.9, 52.1, 120.0,
122.4, 124.4, 126.4, 128.9, 129.0, 131.6, 134.21, 139.4, 142.2, 162.3, 166.2,
169.5, 173.8, 190.0. Anal. Calcd for C21H21N3O6: C, 61.31; H, 5.14; N, 10.21.
Found: C, 61.52; H, 5.37; N, 10.41.
Compound 6b was obtained as a white powder, mp: 178–180 °C; yield 81%. IR
(cmꢂ1): 3293, 1747, 1679, 1634, 1608. 1H NMR (CDCl3): d 1.00 (t, 6H, CH (CH3)2),
1.59 (m, 1H, CHCH(CH3)2), 2.27 (s, 3H, COCH3), 3.78 (s, 3H, COOCH3), 4.78 (m, 1H,
CH), 6.61 (d, 1H), 7.17 (t, 1H), 7.64 (t, 1H), 7.78 (d, 1H), 7.86 (d, 2H), 8.50 (d, 1H)
8.64 (d,1H), 8.99 (s, 1H, NH), 10.79 (s, 1H, NH); 13C NMR (CDCl3): d 18.1, 19.1, 25.0,
31.0, 52.4, 57.6, 119.4, 119.8, 120.9, 122.8, 128.4, 130.6, 134.3, 136.8, 140.0,
142.1,160.6, 166.5, 169.4, 172.8, 190.8. (C@O). Anal. Calcd for C23H25N3O6: C,
62.86; H, 5.73; N, 9.56. Found: C, 62.59; H, 5.97; N, 9.44.
Compound 6f was obtained as a white powder, mp: 154–156 °C, yield 88%. IR
(cmꢂ1): 3295, 1742, 1666, 1635, 1608. 1H NMR (DMSO-d6): d 1.99 (s, 3H, COCH3),
2.60 (t, 2H, CH2), 3.49 (q, 2H, CH2), 3.61 (s, 3H, CH3), 7.29–7.85 (m, 8H, Ar), 8.51 (s,
1H, NH), 10.55 (s, 1H, NH), 10.90 (s, 1H, NH). 13C NMR (DMSO-d6); d 23.9, 34.1,
36.1, 52.0, 119.9, 122.4, 124.4, 125.6, 128.6, 130.4, 131.6, 134.3, 138.2, 141.1,
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