Synthesis of some pyridazinylacetic acid derivatives as a novel class of monoamine oxidase-A inhibitors

Article abstract of DOI:10.1248/cpb.56.1717

A series of new pyridazinylacetic acid derivatives were synthesized and have been investigated for their ability to inhibit the activity of the A and B isoforms of monoamine oxidase (MAO). All compounds were found to be more selective to the MAO-A isoform with compound 5d having the highest SI values. Computational study performed with a docking technique indicated the potential of these compounds in pyridazine-based MAO-A inhibitor drug development.

Full text of DOI:10.1248/cpb.56.1717

December 2008
Notes
Chem. Pharm. Bull. 56(12) 1717—1721 (2008)
1717
Synthesis of Some Pyridazinylacetic Acid Derivatives as a Novel Class of
Monoamine Oxidase-A Inhibitors1)
Sherine Nabil KHATTAB,a Adnan Ahmed BEKHIT,b Ayman EL-FAHAM,a Abdel Moneim EL MASSRY,a
,a
*
and Adel AMER
a Department of Chemistry, Faculty of Science, University of Alexandria; Alexandria 21321, Egypt: and b Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria; Alexandria 21521, Egypt.
Received May 3, 2008; accepted August 23, 2008; published online September 25, 2008
A series of new pyridazinylacetic acid derivatives were synthesized and have been investigated for their abil-
ity to inhibit the activity of the A and B isoforms of monoamine oxidase (MAO). All compounds were found to be
more selective to the MAO-A isoform with compound 5d having the highest SI values. Computational study per-
formed with a docking technique indicated the potential of these compounds in pyridazine-based MAO-A in-
hibitor drug development.
Key words monoamine oxidase inhibitor; pyridazine; acetic acid; amino acid
Monoamic oxidase (MAO) (EC 1.4.3.4) is an integral the benzyl group was retained in the new model compounds
flavin-containing enzyme of the outer mitochondrial mem- in Fig. 1.
brane which is responsible for regulation and metabolism of
The new substituted pyridazine-1-yl acetic acid derivatives
major monoamine neurotransmitters such as serotonin (5-OH (2, 3, 5) were designed as selective monoamine oxidase-A in-
tryptamine), noradrenaline and dopamine. It is also involved hibitors. The synthesis and biological evaluation of these
in the biodegradation of exogenic amines such as benzyl- compounds were described. As a preliminary result of the
amine, tyramine, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri- biological investigation, compound 5d showed the highest
dine (MPTT), 1-methyl-4-phenyl pyridium (MPPꢀ), and a MAO-A selectivity and could be the lead compound for de-
Parkinsonian-producing neurotoxin.2) It is found in two dif- signing more potent and selective MAO-A inhibitors.
ferent isoforms designated as MAO-A and MAO-B which
First, the 4-benzyl-2H-pyridazin-3-one derivatives 1 were
are encoded by two different genes3) and distinguished by prepared. The reaction of the 4-benzyl-2H-pyridazin-3-one
different substrate specificities and sensitivities to the selec- derivatives 1 with bromo ethyl acetate in dimethyl form-
tive inhibitors.4)
amide in the presence of potassium iodide and sodium bi-
The new generation of MAO inhibitors is characterized by carbonate afforded the (5-benzyl-6-oxo-6H-pyridazin-1-yl)-
their relative specificities for the MAO subtypes and in some
cases by the reversibility of their actions. In spite of consid-
erable progress in our understanding of the interactions of
the two enzyme forms with their preferred substrates and in-
hibitors, no general rules are yet available for the rational de-
sign of potent and selective inhibitors of MAO. This is partly
due to the fact that the mechanism of interaction of several
new drugs with MAOs has not been fully characterized.
Therefore, the discovery of several selective MAO inhibitors
has relied on serendipity. Preferential MAO-A inhibitors
have been recognized as therapeutically useful antidepres-
sants5), while MAO-B inhibitors have been found to be bene-
ficial in the treatment of Parkinson's disease and Alzheimer’s
disease.6,7)
Recently, we have demonstrated a series of 3-benzyl-2-
substituted quinoxalines as selective MAO-A inhibitors bear-
ing substituted amino or hydrazino functionalities at position
2 (e.g. 2-morpholinoethylamino, 2-hydroxyethylamino, hy-
drazino, etc).8) The rational design of these compounds was
based on a hybrid structure of known inhibitors. The aim of
the present study was to design MAO-A inhibitors while tak-
ing into consideration various factors responsible for selec-
tivity against the A isoform,9) namely i) the presence of elec-
tron-rich aromatic moieties (e.g. Bazinaprine10)), ii) the pres-
ence of hydrazido functionality (e.g. Iproniazid11)), iii) the
presence of an ethoxycarbonyl methylene group side chain of
an aromatic system (e.g. Eugenol analog12)); exchanging the
Fig. 1. Planned Modification and Newly Designed MAO-A Inhibitor
© 2008 Pharmaceutical Society of Japan
ester group with an amido group was also considered, and iv)
∗ To whom correspondence should be addressed. e-mail: aamer@sci.alex.edu.eg

1718
Vol. 56, No. 12
Table 1. Effect of Some Pyridazinyl Acetic Acid Derivatives 2, 3 and 5 on
the Activity of MAO-A and MAO-B
% MAO-A
inhibition
Selectivity
index (SI)a)
Compound
MAO-A IC50
MAO-B IC50
2a
2b
2c
2d
3a
3b
3c
5a
5b
5c
5d
5e
5f
43.75ꢃ1.22
29.45ꢃ1.98
40.65ꢃ1.94
34.86ꢃ2.47
11.64ꢃ1.56
33.70ꢃ3.44
29.84ꢃ4.12
37.95ꢃ1.87
30.96ꢃ3.12
27.12ꢃ2.54
57.65ꢃ1.74
30.98ꢃ2.84
38.73ꢃ1.82
32.92ꢃ2.88
30.99ꢃ2.68
36.42ꢃ2.88
7.3ꢄ10ꢁ9ꢃ0.04 9.2ꢄ10ꢁ4ꢃ0.55
1.8ꢄ10ꢁ8ꢃ0.09 2.2ꢄ10ꢁ4ꢃ0.06
8.1ꢄ10ꢁ9ꢃ0.02 7.4ꢄ10ꢁ4ꢃ0.21
9.7ꢄ10ꢁ9ꢃ0.02 4.6ꢄ10ꢁ4ꢃ0.18
6.2ꢄ10ꢁ7ꢃ0.04 1.9ꢄ10ꢁ4ꢃ0.08
9.8ꢄ10ꢁ9ꢃ0.06 3.6ꢄ10ꢁ5ꢃ0.27
2.3ꢄ10ꢁ8ꢃ0.05 8.2ꢄ10ꢁ4ꢃ0.22
9.4ꢄ10ꢁ9ꢃ0.16 4.7ꢄ10ꢁ5ꢃ0.07
9.7ꢄ10ꢁ9ꢃ0.08 7.1ꢄ10ꢁ5ꢃ0.36
2.9ꢄ10ꢁ8ꢃ0.14 1.4ꢄ10ꢁ4ꢃ0.18
5.4ꢄ10ꢁ9ꢃ0.02 8.1ꢄ10ꢁ4ꢃ0.43
9.6ꢄ10ꢁ9ꢃ0.12 2.7ꢄ10ꢁ4ꢃ0.28
1.4ꢄ10ꢁ8ꢃ0.16 2.3ꢄ10ꢁ4ꢃ0.14
2.8ꢄ10ꢁ8ꢃ0.01 2.6ꢄ10ꢁ4ꢃ0.52
2.6ꢄ10ꢁ8ꢃ0.25 7.5ꢄ10ꢁ4ꢃ0.28
1.7ꢄ10ꢁ8ꢃ0.13 4.3ꢄ10ꢁ4ꢃ0.33
126027
12222
91358
47422
306
3673
35652
5000
7319
4827
150000
28125
16428
9285
5g
5h
5i
28846
25294
a) SIꢂMAO-B IC50/MAO-A IC50. ∗ The results were expressed as meanꢃS.E.M.
Data were analyzed by one way of variance. Student’s t test for unpaired observations
was used. p valueꢂꢅ0.001 and was significant. Number of experiments was 6.
pressed as IC50 (Table 1). The selectivity index is also given
in Table 1. The results revealed that compound 5d showed
MAO-A inhibition greater than that of pargyline HCl (%
MAO-A inhibitionꢂ39.14ꢃ1.32), while compounds 2a, 2c,
2d, 3b, 5a, 5f and 5i exhibited inhibition activity more or
Chart 1
acetic acid ethyl ester and its derivatives 2. The esters 2a, 2b less comparable to the standard, pargyline HCl. These com-
and 2c were hydrolysed by alcoholic potassium hydroxide to pounds showed higher MAO-A inhibitory activity than
give the corresponding acids 3a, 3b and 3c, while the ester MAO-B inhibitory activity. Thus, compound 5d appears to
2d was hydrolyzed by acid hydrolysis by the use of 5 N HCl. be the most selective MAO-A inhibitor.
The structures of compounds 2a—d and 3a—d were estab-
In an attempt to understand the reason for the observed
MAO-A inhibitory activity, a molecular modelling study of
lished by IR, 1H-NMR, 13C-NMR, and elemental analyses.
The reaction of 2-(5-benzyl-6-oxo-3-phenylpyridazin- compounds 2a, 3a and 5d, as well as conformational align-
1(6H)-yl)acetic acid 3a and its derivatives 3b and 3c with ment studies were performed in order to rationalize the ob-
different L-amino acid t-butyl esters 4a—e in dimethylform- tained biological results. Molecular docking studies can fur-
amide in the presence of triethylamine and HATU as cou- ther assist in understanding in detail the various interactions
pling reagent at 0 °C yielded the derivatives 5 in high yield between the ligands and enzyme active sites in detail.
(Chart 1). The structures of compounds 5 were established
Molecular operating environment (MOE)18) docking stud-
1
by IR, H-NMR, 13C-NMR and elemental analyses. The in- ies of the inhibitors were performed using human MAO-A
frared spectra of these compounds showed absorption bands crystal structure (PDB ID: 2BXR).
of the ester carbonyl groups at the 1731—1732 cmꢁ1 range,
Docking of compound 2a into the MAO-A active site re-
of the amide carbonyl groups are approximately at 1659 vealed that several molecular interactions were considered to
cmꢁ1 and at 1690 cmꢁ1 (the first one is the one conjugated be responsible for the observed affinity: Hydrophobic inter-
with the adjacent double bond), and of the N–H at the actions were observed with each of the following residues:
3308—3313 cmꢁ1 range. In addition, 1H-NMR spectra PHE208, ILE335, LEU337, MET350. Docking of the corre-
showed only one exchangeable signal for the NH group. For sponding acid derivative 3a showed less hydrophobic interac-
further structure elucidation, the DEPT technique was per- tions with the backbone of MAO-A enzyme with the follow-
formed for compound 5c as a prototype.
ing residues: VAL93, LEU97, PHE208. On the other hand,
The newly synthesized compounds 2, 3, 5 were evaluated docking of compound 5d into the MAO-A active site (Fig. 2)
for their MAO-A inhibitory activity in vitro by the method showed that the molecular interactions responsible for the
described by Undenfriend et al.13 by determination of the observed affinity are: i) two hydrogen bond interactions with
MAO-A activity of rat liver mitochondria14) (Table 1).
the following residue: SER209. ii) Hydrophobic interactions
Furthermore, the synthesized compounds 2, 3, 5 were with the following residues: PHE352, Val93, Leu97, ILE180,
tested to determine their activity toward MAO-A and MAO- PHE208, ILE325, ILE335, ILE337, TRP441. These results
B selectivity in the presence of a specific substrate,15) sero- showed that compound 5d bound to the MAO-A active site
tonin or benzylamine respectively. Bovine brain mitochon- with a position and orientation that were superior of those of
dria were isolated according to Basford.15) The compounds 2, 2a and 3a.
3, 5 were tested to determine their activity toward MAO-A
It is worth to mentioning that the test compounds were fur-
and MAO-B according to the methods of Matsumoto et al.16) ther tested for their oral acute toxicity in male mice using a
and Bradford.17) The MAO-A and MAO-B results are ex- literature method.18,19) The results revealed that the test com-

December 2008
1719
Fig. 2. 3D View from a Molecular Modelling Study, of Minimum-Energy Structure of the Complex of 5d (Stick) Docked in the Active Site of MAO-A
(PDB ID: 2BXR)
Dashed lines depict hydrogen bond interactions. The space filling model at the left side represents FAD coenzyme. Viewed using Molecular Operating Environment (MOE)
module.
tained as colorless crystals, 0.36 g (68.6%), mp 178—180 °C (lit. mp 179—
pounds proved to be non-toxic and well tolerated by the ex-
perimental animals up to 250 mg/kg, although no mortality
was recorded at 500 mg/kg. Furthermore, these compounds
were evaluated for their toxicity when administered via the
181 °C).21,22)
4-Benzyl-6-(4-chlorophenyl)pyridazin-3(2H)-one (1b): This compound
was obtained as colorless crystals, 0.47 g (79.2%), mp 218—220 °C. Anal.
Calcd for C17H13ClN2O: C, 68.81; H, 4.41; N, 9.44. Found: C, 68.81; H,
parenteral route.20) The results showed that all of the test 4.12; N, 9.56.
4-Benzyl-6-(thiophen-2-yl)pyridazin-3(2H)-one (1c): This compound was
compounds were non-toxic up to 125 mg/kg.
From the above mentioned data and its non toxic behav-
iour,18—20) 5d may provide a starting point for the design of
unique compounds with high affinity and selectivity for
MAO-A.
obtained as colorless crystals, 0.39 g (72.7%), mp 135—137 °C. Anal. Calcd
for C15H12N2OS: C, 67.14; H, 4.51; N, 10.44. Found: C, 67.08; H, 4.51; N,
10.56.
4-Benzyl-6-(pyridin-3-yl)pyridazin-3(2H)-one (1d): This compound was
obtained as colorless crystals, 0.47 g (89.3%), mp 214—216 °C. Anal. Calcd
for C16H13N3O: C, 72.99; H, 4.98; N, 15.96. Found: C, 72.77; H, 5.02; N,
15.66.
Experimental
General Procedure for the Preparation of Compounds (2a—d) 4-
Benzylpyridazin-3(2H)-one derivatives 1 (2 mmol) were dissolved in dry
DMF (5 ml), NaHCO3 (3.02 g, 36 mmol) and KI (0.432 g, 2.6 mmol) and the
mixture was stirred at room temperature for 15 min. Excess of ethyl bro-
moacetate (1.33 g, 8 mmol) was then added and the reaction mixture was
kept stirring at room temperature for 84 h. DMF was removed under reduced
pressure. The residue was suspended in water (150 ml) and extracted with
methylene chloride (100 ml). The organic layer was dried over anhydrous
sodium sulphate, filtered and the solvent was removed under reduced pres-
sure. The crude product was crystallized from ethanol.
Chemistry Melting points were determined with a Mel-Temp apparatus
and are uncorrected. Magnetic resonance spectra (1H-NMR and 13C-NMR
spectra) were recorded on a JEOL 500 MHz spectrometer with chemical
shift values reported in d units (part per million) relative to an internal stan-
dard (tetramethylsilane). Splitting patterns were designated as follows: s:
singlet; d: doublet; t: triplet; q: quartet, m: multiplet. Infrared (IR) data were
obtained on a Perkin-Elmer 1600 series Fourier transform instrument as KBr
pellets. Mass spectra were recorded on a QP 1000EX GC/MS (Shimadzu
Corp.). Elemental analyses were performed on a Perkin-Elmer 2400 elemen-
tal analyzer, and the values were within ꢃ0.3% of the theoretical values.
The reactions were followed-up and the purity of the compounds was
checked by TLC on silica gel-protected aluminium sheets (Type 60 GF254,
Merck) and the spots were detected by exposure to a UV-lamp at l 254 nm
for several seconds. The compounds were named using Chem. Draw Ultra
version 6 (Cambridgesoft Corporation). The following abbreviations are
used throughout the manuscript: HATU, N-[(dimethylamino)-1H-1,2,3-
triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluo-
rophosphate N-oxide, Et3N, triethylamine, and DMF, N,N-dimethylform-
amide were used through out the manuscript.
Ethyl 2-(5-Benzyl-6-oxo-3-phenylpyridazin-1(6H)-yl)acetate (2a): This
compound was obtained as colorless crystals, 0.64 g (92.0%), mp 92—93 °C
(lit. mp 96 °C)22); IR (KBr) cmꢁ1: 1760 (CꢂO ester), 1651 (CꢂO amide);
1H-NMR (CDCl3) d: 1.29 (t, 3H, CH3, Jꢂ6.9 Hz), 3.99 (s, 2H, CH2), 4.26
(q, 2H, CH2, Jꢂ6.9 Hz), 4.98 (s, 2H, CH2), 7.26—7.42 (2m, 9H, PyrH,
ArH), 7.63—7.65 (m, 2H, ArH). MS [EI]: m/e 348 (Mꢀ, 100%), 302 (22),
274 (95), 246 (38), 203 (25), 104 (16), 91 (36), 77 (31). Anal. Calcd for
C21H20N2O3: C, 72.40; H, 5.79; N, 8.04. Found: C, 72.60; H, 5.95; N, 7.88.
Ethyl 2-(5-Benzyl-3-(4-chlorophenyl)-6-oxopyridazin-1(6H)-yl)acetate
(2b): This compound was obtained as colorless crystals, 0.71 g (92.9%), mp
All molecular modeling studies were carried out on an Intel Pentium
1.6 GHz processor, 512 MB memory with Windows XP operating system
using Molecular Operating Environment (MOE 2005.06; Chemical Comput-
ing Group, Montréal, Canada) as the computational software. All minimiza-
1
127—128 °C; IR (KBr) cmꢁ1: 1761 (CꢂO ester), 1650 (CꢂO amide); H-
NMR (CDCl3) d: 1.29 (t, 3H, CH3, Jꢂ6.9 Hz), 3.98 (s, 2H, CH2), 4.26 (q,
2H, CH2, Jꢂ6.9 Hz), 4.98 (s, 2H, CH2), 7.22 (s, 1H, PyrH), 7.26, 7.35, 7.37,
7.57 (m, d, m, d, 9H, ArH). MS [EI]: m/e 384, 382 (Mꢀ, 33, 93%), 354 (50),
308 (90), 203 (30), 196 (23), 189 (20), 139 (57), 115 (63), 113, (27), 111
(30), 91 (40), 51 (100). Anal. Calcd for C21H19ClN2O3: C, 65.88; H, 5.00; N,
7.32. Found: C, 66.15; H, 5.22; N, 7.04.
tions were performed with MOE until
a RMSD gradient of 0.05
kcal molꢁ1 Åꢁ1 with MMFF94X forcefield and the partial charges were auto-
matically calculated. Docking and molecular modeling calculations were
carried out at the Department of Pharmaceutical and Medicinal Chemistry,
Faculty of Pharmacy, Assiut University, Assiut, Egypt.
Ethyl 2-(5-Benzyl-6-oxo-3-(thiophen-2-yl)pyridazin-1(6H)-yl)acetate
(2c): This compound was obtained as colorless crystals, 0.65 g (91.8%), mp
General Procedure for the Preparation of 4-Benzylpyridazin-3(2H)-
1
113—114 °C; IR (KBr) cmꢁ1: 1761 (CꢂO ester), 1652 (CꢂO amide); H-
one Derivatives (1a—d)
A solution of 2-benzyl-2-hydroxy-4-oxo-4-
NMR (CDCl3) d: 1.29 (t, 3H, CH3, Jꢂ6.9 Hz), 3.96 (s, 2H, CH2), 4.25 (q,
2H, CH2, Jꢂ6.9 Hz), 4.92 (s, 2H, CH2), 7.00 (dd, 1H, ThH-4, Jꢂ5.4,
3.9 Hz), 7.18 (s, 1H, PyrH), 7.19 (d, 1H, ThH-3, Jꢂ3.9 Hz), 7.26—7.37 (m,
6H, ThH-5, ArH). 13C-NMR (CDCl3–HMQC) d: 14.23, 36.17, 54.02, 61.81,
arylbutanoic acid (2 mmol) in ethanol (20 ml) was refluxed with 90% hy-
drazine hydrate (2 ml) for 3 h. The product which was separated out on con-
centration was collected by filtration, dried, and recrystallized from ethanol.
4-Benzyl-6-phenylpyridazin-3(2H)-one (1a): This compound was ob-

1720
Vol. 56, No. 12
125.68, 126.95, 127.11, 127.76, 127.91, 128.96, 129.57, 136.94, 139.24,
tert-Butyl 2-(2-(5-Benzyl-6-oxo-3-phenylpyridazin-1(6H)-yl)acetamido)-
141.11, 143.63, 160.22, 167.50. MS [EI]: m/e 356, 355, 354 (Mꢀ, 46, 36, 3-(4-tert-butoxyphenyl)propanoate (5a): The crude compound was re-
100%), 310 (51), 309 (33), 308 (99), 245 (42),203 (68), 202 (85), 138 (32),
101 (86), 51 (71). Anal. Calcd for C19H18N2O3S: C, 64.39; H, 5.12; N, 7.90. as a white powder, 0.52 g (87.4%), mp 75—76 °C; IR (KBr) cmꢁ1: 3313
Found: C, 64.10; H, 4.86; N, 8.17.
(NH), 1732 (CꢂO ester), 1691 (CꢂO amide), 1659 (CꢂO amide); 1H-NMR
cryrstallized from methylene chloride/hexane and the product was obtained
Ethyl 2-(5-Benzyl-6-oxo-3-(pyridin-3-yl)pyridazin-1(6H)-yl)acetate (2d): (CDCl3) d: 1.26, 1.34 (2s, 18H, 6CH3), 3.06 (s, 2H, CH2), 3.97 (s, 2H, CH2),
This compound was obtained as colorless crystals, 0.59 g (84.5%), mp
4.73 (m, 1H, CH), 4.95 (m, 2H, CH2), 6.72 (br d, 1H, NH, D2O exchange-
able), 6.76 (d, 2H, ArH, Jꢂ8.2 Hz), 6.97 (d, 2H, ArH, Jꢂ8.2 Hz), 7.25—
112—113 °C; IR (KBr) cmꢁ1: 1760 (CꢂO ester), 1651 (CꢂO amide) cmꢁ1
;
1H-NMR (CDCl3) d: 1.28 (t, 3H, CH3, Jꢂ6.9 Hz), 3.99 (s, 2H, CH2), 4.25 7.39 (m, 9H, PyrH, ArH), 7.65 (m, 2H, ArH). Anal. Calcd for C36H41N3O5:
(q, 2H, CH2, Jꢂ6.9 Hz), 4.97 (s, 2H, CH2), 7.24—7.28 (m, 2H, PyrH, ArH),
7.31—7.33 (m, 4H, ArH), 7.72 (dt, 1H, PyH-5, Jꢂ7.7, 2.3 Hz), 8.06 (d, 1H,
C, 72.58; H, 6.94; N, 7.05. Found: C, 72.43; H, 6.75; N, 6.89.
tert-Butyl 2-(2-(5-Benzyl-6-oxo-3-phenylpyridazin-1(6H)-yl)acetamido)-
PyH-4, Jꢂ7.7 Hz), 8.09 (br s, 1H, PyH-2), 8.57 (d, 1H, PyH-6, Jꢂ7.7 Hz). 3-phenylpropanoate (5b): The crude compound was recryrstallized from
MS [EI]: m/e 349 (Mꢀ, 45%), 275 (25), 247 (34), 204 (22), 115 (55), 105 methylene chloride/hexane and the product was obtained as a white powder,
(28), 91 (45), 78 (100). Anal. Calcd for C20H19N3O3: C, 68.75; H, 5.48; N, 0.47 g (89.9%), mp 95—96 °C; IR (KBr) cmꢁ1: 3310 (NH), 1731 (CꢂO
1
12.03. Found: C, 69.03; H, 5.70; N, 11.84.
General Procedure for the Preparation of Compounds (3a—c) Ester
ester), 1690 (CꢂO amide), 1659 (CꢂO amide) ; H-NMR (CDCl3) d: 1.38
(s, 9H, 3CH3), 3.10 (m, 2H, CH2), 3.97 (s, 2H, CH2), 4.87 (m, 1H, CH),
2 (1 mmol) was dissolved in 10% KOH in ethanol (20 ml). The reaction mix- 4.87, 4.97 (2d, 2H, CH2, Jꢂ14.9 Hz), 6.73 (br d, 1H, NH, D2O exchange-
ture was refluxed for 3 h and then neutralized with 1 N HCl solution. The
able), 7.09, 7.14, 7.27, 7.38 (4m, 14H, PyrH, ArH), 7.63 (m, 2H, ArH); 13C-
precipitate formed was filtered off and washed with water to give the acid 3.
NMR (CDCl3) d: 28.03, 36.30, 37.87, 53.82, 56.17, 82.59, 126.28, 126.93,
2-(5-Benzyl-6-oxo-3-phenylpyridazin-1(6H)-yl)acetic Acid (3a): This 127.15, 127.74, 128.33, 128.92, 129.03, 129.61, 129.65, 134.77, 136.07,
compound was obtained as white powder, 0.29 g (90.5%), mp 180—181 °C; 136.96, 143.87, 145.37, 160.58, 166.36, 170.12. MS [EI]: m/e 319
IR (KBr) cmꢁ1: 3087—2565 (br, OH), 1728 (CꢂO acid), 1646 (CꢂO (MꢀꢁC14H20O, 2%), 303 (18), 275 (13), 91 (28), 57 (100). Anal. Calcd for
1
amide); H-NMR (CDCl3) d: 3.98 (s, 2H, CH2), 5.02 (s, 2H, CH2), 7.23— C32H33N3O4: C, 73.40; H, 6.35; N, 8.02. Found: C, 73.20; H, 6.64; N, 8.16.
7.39, 7.62—7.63 (2m, 11H, ArH, PyrH), 13.3 (s, 1H, COOH, D2O ex-
changeable). MS [EI]: m/e 320 (Mꢀ, 35%), 274 (27), 203 (32), 178 (12),
104 (35), 102 (43), 91 (46), 77 (100). Anal. Calcd for C19H16N2O3·H2O: C,
67.44; H, 5.36; N, 8.28. Found: C, 67.18; H, 5.14; N, 8.53.
tert-Butyl 2-(2-(5-Benzyl-6-oxo-3-phenylpyridazin-1(6H)-yl)acetamido)-
4-methylpentanoate (5c): The crude compound was purified by column
chromatography (ethylacetate/hexane, 1 : 2) and the product was obtained as
a pale yellow semi-solid, 0.43 g (87.9%); IR (KBr) cmꢁ1: 3305 (NH), 1732
(CꢂO ester), 1690 (CꢂO amide), 1661 (CꢂO amide); 1H-NMR (CDCl3) d:
2-(5-Benzyl-3-(4-chlorophenyl)-6-oxopyridazin-1(6H)-yl)acetic Acid
(3b): This compound was obtained as white powder, 0.32 g (90.2%), mp 0.89, 0.90 (2d, 6H, 2CH3, Jꢂ6.2 Hz), 1.42 (s, 9H, 3CH3), 1.52 (m, 1H, CH),
194—195 °C; IR (KBr) cmꢁ1: 3030—2581 (br, OH), 1729 (CꢂO acid), 1.63 (m, 2H, CH2), 3.98 (s, 2H, CH2), 4.53 (m, H, CH), 4.95 (br s, 2H, CH2),
1626 (CꢂO amide); 1H-NMR (DMSO-d6) d: 3.86 (s, 2H, CH2), 4.81 (s, 2H, 6.67 (br d, 1H, NH, D2O exchangeable), 7.26—7.38 (m, 9H, PyrH, ArH),
CH2), 7.18 (t, 1H, ArH, Jꢂ7.6 Hz), 7.27 (t, 2H, ArH, Jꢂ7.6 Hz), 7.31 (d, 7.65 (m, 2H, ArH); 13C-NMR (CDCl3/DEPT) d: 22.33, 22.83 (2CH3), 25.01
2H, ArH, Jꢂ7.6 Hz), 7.53 (d, 2H, ArH, Jꢂ8.4 Hz), 7.81 (d, 2H, ArH,
(CH), 28.05 (3CH3), 36.33, 41.98 (2CH2), 51.73 (CH), 56.24 (CH2), 82.08
Jꢂ8.4 Hz), 7.96 (s, 1H, PyrH), 13.2 (s, 1H, COOH, D2O exchangeable). MS (C), 126.25, 127.11, 127.79, 128.90, 128.99, 129.57 (10CH), 134.80,
[EI]: m/e 356, 354 (Mꢀ, 21, 82%), 308 (17), 280 (100), 252 (51), 178 (24), 137.02, 143.79, 145.32 (4C), 160.65, 166.44, 171.87 (3CꢂO). Anal. Calcd
115 (61), 113 (5), 111 (13), 110 (30), 91 (67), 77 (27). Anal. Calcd for for C29H35N3O4: C, 71.14; H, 7.21; N, 8.58. Found: C, 70.90; H, 6.97; N,
C19H15ClN2O3: C, 64.32; H, 4.26; N, 7.90. Found: C, 64.09; H, 3.98; N, 8.74.
7.76.
2-(5-Benzyl-6-oxo-3-(thiophen-2-yl)pyridazin-1(6H)-yl)acetic Acid (3c):
Di-tert-butyl 2-(2-(5-Benzyl-6-oxo-3-phenylpyridazin-1(6H)-yl)ac-
etamido)pentanedioate (5d): The crude compound was purified by column
This compound was obtained as white powder, 0.29 g (89.0%), mp 249— chromatography (ethylacetate/hexane, 1 : 2) and the product was obtained as
250 °C; IR (KBr) cmꢁ1: 3076—2576 (br, OH), 1728 (CꢂO acid), 1626 a pale yellow semi-solid, 0.45 g (80.2%); IR (KBr) cmꢁ1: 3310 (NH), 1735
(CꢂO amide); 1H-NMR (DMSO-d6) d: 3.85 (s, 2H, CH2), 4.73 (s, 2H, (CꢂO, ester), 1731 (CꢂO ester), 1690 (CꢂO amide), 1660 (CꢂO amide);
CH2), 7.12 (dd, 1H, ThH-4, Jꢂ4.6, 3.5 Hz), 7.19 (t, 1H, ArH, Jꢂ6.9 Hz),
7.25—7.31 (m, 4H, ThH-3, H-5, ArH), 7.61 (t, 2H, ArH, Jꢂ6.9 Hz), 7.94 (s,
1H, PyrH), 13.25 (s, 1H, COOH, D2O exchangeable). MS [EI]: m/e 328,
1H-NMR (CDCl3) d: 1.41, 1.43 (2s, 18H, 6CH3), 1.92, 2.11 (2m, 2H, CH2),
2.21—2.35 (m, 2H, CH2), 3.98 (s, 2H, CH2), 4.52 (m, H, CH), 4.90, 4.98
(2d, 2H, CH2, Jꢂ14.9 Hz), 6.92 (br d, 1H, NH, D2O exchangeable), 7.26—
327, 326 (Mꢀ, 10, 28, 100%), 325 (30), 252 (46), 178 (32), 165 (24), 115 7.36 (m, 9H, PyrH, ArH), 7.64 (m, 2H, ArH). MS [EI]: m/e 561 (Mꢀ, 2%),
(37), 91 (65), 77 (31). Anal. Calcd for C17H14N2O3S·H2O: C, 59.29; H, 4.68; 505 (Mꢀꢁ56, 2), 303 (100), 275 (34), 115 (13), 91 (12), 57 (94). Anal.
N, 8.13. Found: C, 59.03; H, 4.39; N, 8.31.
Calcd for C32H39N3O6: C, 68.43; H, 7.00; N, 7.48. Found: C, 68.58; H, 7.20;
N, 7.36.
Di-tert-butyl 2-(2-(5-Benzyl-6-oxo-3-phenylpyridazin-1(6H)-yl)ac-
etamido)succinate (5e): The crude compound was purified by column chro-
2-(5-Benzyl-6-oxo-3-(pyridin-3-yl)pyridazin-1(6H)-yl)acetic Acid (3d):
The ester 2d (1 mmol) was dissolved in 5 N HCl (20 ml). The reaction mix-
ture was refluxed for 6 h. The water was removed in vacuo. This compound
was dried under vacuo, to obtain a hygroscopic white powder, 0.28 g matography (ethylacetate/hexane, 1 : 2) and the product was obtained as a
(87.2%), mp 225—226 °C; 1H-NMR (DMSO-d6) d: 4.07 (s, 2H, CH2), 4.88
(s, 2H, CH2), 7.20—7.46 (m, 4H, PyrH, ArH), 7.63 (d, 2H, ArH, Jꢂ7.6 Hz),
7.86—7.92 (m, 1H, PyH-5), 8.01—8.08 (m, 2H, PyH-4, PyH-2), 8.53—8.62 amide); H-NMR (CDCl3) d: 1.40, 1.43 (2s, 18H, 6CH3), 2.78, 2.87 (2dd,
(m, 1H, PyH-6), 12.9 (br s, 1H, COOH, D2O exchangeable).
pale yellow semi-solid, 0.41 g (75.0%) yield; IR (KBr) cmꢁ1: 3310 (NH),
1737 (CꢂO ester), 1731 (CꢂO ester), 1691 (CꢂO amide), 1659 (CꢂO
1
2H, CH2, Jꢂ17.2, 4 Hz), 3.98 (s, 2H, CH2), 4.71 (m, H, CH), 4.96 (m, 2H,
General Procedure for the Reaction of 2-(5-Benzyl-6-oxo-3-arylpyri- CH2), 7.07 (br d, 1H, NH, D2O exchangeable), 7.26, 7.36 (2m, 9H, PyrH,
dazin-1(6H)-yl)acetic Acid with L-Amino Acid Esters (5a—i) A mix- ArH), 7.64 (m, 2H, ArH); 13C-NMR (CDCl3) d: 27.97, 28.10, 36.26, 37.44,
ture of 2-(5-benzyl-6-oxo-3-arylpyridazin-1(6H)-yl)acetic acid 3 (1 mmol), 49.40, 55.86, 81.63, 82.47, 126.31, 127.09, 127.75, 128.85, 128.99, 129.48,
HATU (0.38 g, 1 mmol) and triethylamine (Et3N) (0.28 ml, 2 mmol) was 129.63, 134.89, 137.07, 143.84, 145.31, 160.55, 166.49, 169.51, 170.28. MS
stirred at 0 °C for 3 min in DMF (2 ml) and then amino acid t-butyl ester 4
(1 mmol) was added. The reaction mixture was stirred at 0 °C for 1 h and left
overnight at room temperature. In the case of L-leucine t-butyl ester, L-glu-
tamic di-t-butyl ester and L-aspartic di-t-butyl ester the reaction mixture was
[EI]: m/e 547 (Mꢀ, 3%), 435 (19), 418 (11), 303 (82), 276 (19), 102 (10), 91
(22), 96 (14), 64 (100), 57 (27). Anal. Calcd for C31H37N3O6: C, 67.99; H,
6.81; N, 7.67. Found: C, 68.10; H, 7.08; N, 7.42.
tert-Butyl 2-(2-(5-Benzyl-3-(4-chlorophenyl)-6-oxopyridazin-1(6H)-
diluted with ethyl acetate (80 ml), and the mixture was washed with 5% yl)acetamido)-3-(4-tert-butoxyphenyl)propanoate (5f): The crude compound
aqueous citric acid solution (2ꢄ10 ml), saturated sodium bicarbonate solu-
tion (2ꢄ10 ml) and saturated sodium chloride solution (2ꢄ10 ml). The or-
was recryrstallized from methylene chloride/hexane and the product was ob-
tained as a white powder, 0.54 g (85.7%), mp 58—59 °C; IR (KBr) cmꢁ1
:
ganic layer was dried over anhydrous sodium sulphate, filtered and the sol- 3311 (NH), 1731 (CꢂO, ester), 1690 (CꢂO, amide), 1659 (CꢂO, amide);
vent was removed in vacuo. The crude product was purified with a silica gel
column using ethyl acetate/hexane (1 : 2) as the eluant. In the case of L-(O-t-
Bu)-tyrosine-t-butyl ester and L-phenylalanine-t-butyl ester, the reaction
1H-NMR (CDCl3) d: 1.28, 1.36 (2s, 18H, 6CH3), 3.05 (m, 2H, CH2), 3.95
(m, 2H, CH2), 4.72 (dd, 1H, CH, Jꢂ13.0, 6.1 Hz), 4.86, 4.96 (2d, 2H, CH2,
Jꢂ14.6 Hz), 6.69 (br d, 1H, NH, D2O exchangeable), 6.77 (d, 2H, ArH,
mixture was poured into ice water, filtered, washed with 5% aqueous citric Jꢂ8.4 Hz), 6.98 (d, 2H, ArH, Jꢂ8.4 Hz), 7.19 (s, 1H, PyrH), 7.28, 7.36 (2m,
acid solution, saturated sodium bicarbonate solution, and water, and then 7H, ArH), 7.58 (d, 2H, ArH, Jꢂ8.4 Hz). MS [EI]: m/e 573 (Mꢀꢁ56, 4%),
dried and recrystallized.
337 (9), 107 (18), 57 (100). Anal. Calcd for C36H40ClN3O5: C, 68.61; H,

December 2008
1721
6.40; N, 6.67. Found: C, 68.36; H, 6.22; N, 6.79.
mals up to 250 mg/kg, although no mortality was recorded at 500 mg/kg.
tert-Butyl 2-(2-(5-Benzyl-3-(4-chlorophenyl)-6-oxopyridazin-1(6H)- Moreover, these compounds were tested for their parenteral toxicity.21) The
yl)acetamido)-3-phenyl-propanoate (5g): The crude compound was re-
results revealed that all the test compounds were non-toxic up to 125 mg/kg.
cyrstallized from methylene chloride/hexane and the product was obtained We could conclude that the synthesis and biochemical evaluation of the
as a white powder, 0.53 g (95.0%), mp 54—55 °C; IR (KBr) cmꢁ1: 3312 newly synthesized pyridazines led to the design of a novel class of MAO-A
(NH), 1730 (CꢂO ester), 1691 (CꢂO amide), 1660 (CꢂO amide); 1H-NMR inhibitors with good safety margins.
(CDCl3) d: 1.39 (s, 9H, 3CH3), 3.10 (m, 2H, CH2), 3.96 (s, 2H, CH2), 4.75
(dd, 1H, CH, Jꢂ12.6, 6.9 Hz), 4.85, 4.94 (2d, 2H, CH2, Jꢂ15.3 Hz), 6.68
(br d, 1H, NH, D2O exchangeable), 7.08 (s, 1H, PyrH), 7.09—7.38 (d, 3m,
12H, ArH), 7.57 (d, 2H, ArH, Jꢂ8.4 Hz). MS [EI]: m/e 557 (Mꢀ, 8%), 501
Modeling Studies Computer-assisted simulated docking experiments
were carried out under MMFF94X in human MAO-A crystal structure (PDB
ID: 2BXR). Docking simulation study of the synthesized compounds (2a,
3a, 5d).
(Mꢀꢁ56, 5), 337 (39), 309 (28), 297 (12), 115 (18), 57 (100). Anal. Calcd 1) Enzyme structures were checked for missing atoms, bonds and contacts.
for C32H32ClN3O4·H2O: C, 66.72; H, 5.95; N, 7.29. Found: C, 66.44; H, 2) Hydrogen atoms were added to the enzyme structure. Water molecules
5.69; N, 7.52.
and bound ligands were manually deleted.
3) The ligand molecules were constructed using the builder module and
were energy minimized.
tert-Butyl 2-(2-(5-Benzyl-6-oxo-3-(thiophen-2-yl)pyridazin-1(6H)-yl)ac-
etamido)-3-(4-tert-butoxyphenyl)propanoate (5h): The crude compound was
recryrstallized from methylene chloride/hexane and the product was ob- 4) The active site was generated using the MOE-Alpha Site Finder.
tained as a white powder, 0.56 g (93.2%), mp 49—50 °C; IR (KBr) cmꢁ1
:
5) Dummy atoms were created from the obtained alpha spheres.
6) Ligands were docked within the MAO-A active site using the MOE-
Dock with simulated annealing used as the search protocol and
MMFF94X molecular mechanics forcefield for 8000 iterations.
3313 (NH), 1731 (CꢂO ester), 1691 (CꢂO amide), 1660 (CꢂO amide); 1H-
NMR (CDCl3) d: 1.27, 1.35 (2s, 18H, 6CH3), 3.05 (m, 2H, CH2), 3.95 (m,
2H, CH2), 4.72 (dd, 1H, CH, Jꢂ13.0, 6.9 Hz), 4.82, 4.92 (2d, 2H, CH2,
Jꢂ15.3 Hz), 6.67 (br d, 1H, NH, D2O exchangeable), 6.79 (d, 2H, ArH, 7) The lowest energy conformation was selected and subjected to an en-
Jꢂ7.7 Hz), 7.00 (br d, 3H, ArH), 7.15 (s, 1H, PyrH), 7.20 (d, 1H, ThH-3,
Jꢂ3.9 Hz), 7.28, 7.35 (2m, 6H, ThH-4, H-5, ArH). MS [EI]: m/e 546
ergy minimization using MMFF94X forcefield.
(Mꢀꢁ55, 3%), 545 (Mꢀꢁ56, 2), 326 (14), 309 (26), 281 (14), 107 (19), 91 References and Notes
(11), 57 (100). Anal. Calcd for C34H39N3O5S: C, 67.86; H, 6.53; N, 6.98.
Found: C, 68.04; H, 6.76; N, 6.81.
1) Part 19 of the series: Synthetic Reactions and Structural Studies of
Heterocycles Containing Nitrogen, for part 18 see ref. 8.
2) Kalgutkar A. S., Dalvie D. K., Castagnoli N., Taylor T. J., Chem. Res.
Toxicol., 14, 1139—1162 (2001).
3) Bach A. W. C., Ian N. C., Johnson D. L., Abell C. W., Bembenek M.
E., Kwan S. W., Seeburg P. H., Shih J. C., Proc. Nat.l Acad. Sci.
U.S.A., 85, 4934—4938 (1988).
4) Fowler C. J., Tipton K. F., J. Pharm. Pharmacol., 36, 111—115
(1984).
5) Pacher P., Kohegyi E., Kecskemeti V., Furst S., Curr. Med. Chem., 8,
89—100 (2001).
tert-Butyl 2-(2-(5-Benzyl-6-oxo-3-(thiophen-2-yl)pyridazin-1(6H)-yl)ac-
etamido)-3-phenyl propanoate (5i): The crude compound was recyrstallized
from methylene chloride/hexane and the product was obtained as a white
powder, 0.45 g (85.1%), mp 57—58 °C; IR (KBr) cmꢁ1: 3313 (NH), 1731
(CꢂO ester), 1690 (CꢂO amide), 1659 (CꢂO amide); 1H-NMR (CDCl3) d:
1.38 (s, 9H, 3 CH3), 3.09 (m, 2H, CH2), 3.94 (s, 2H, CH2), 4.75 (dd, 1H, CH,
Jꢂ13.0, 6.1 Hz), 4.82, 4.91 (2d, 2H, CH2, Jꢂ14.5 Hz), 6.63 (br d, 1H, NH,
D2O exchangeable), 7.01 (dd, 1H, ThH-4, Jꢂ5.4, 3.9 Hz), 7.09 (s, 1H,
PyrH), 7.14—7.17, 7.20, 7.26—7.38 (m, d, m, 12H, ThH-3, H-5, ArH). MS
[EI]: m/e 474 (Mꢀꢁ55, 2%), 309 (11), 281 (10), 91 (26), 57 (100). Anal.
Calcd for C30H31N3O4S: C, 68.03; H, 5.90; N, 7.93. Found: C, 68.24; H,
6.15; N, 8.02.
6) Tetrud J. W., Koller W. C., Neurology, 63, S2—S6 (2004).
7) Riederer P., Danielczyk W., Grunblatt E., Neurotoxicology, 25, 271—
277 (2004).
Biology The newly synthesized compounds 2, 3, 5 were evaluated for
their MAO-A inhibitory activity in vitro by the method described by Unden-
friend et al.13) using serotonin (5HT) as substrate. The method depends on
determination of MAO-A activity of rat liver mitochondria.14) All com-
pounds under test were used at a final concentration of 1ꢄ10ꢁ4 M. The re-
sults are expressed as a percentage inhibition of the activity of MAO-A
(Table 1).
8) Hassan S. Y., Khattab S. N., Bekhit A. A., Amer A., Bioorg. Med.
Chem. Lett., 16, 1753—1756 (2006).
9) Vallejos G., Rezende M. C., Cassels B. K., J. Comput.-Aided Mol.
Des., 16, 95—103 (2002).
10) Di Santo R., Costi R., Roux A., Artico M., Befani O., Meninno T.,
Agostinelli E., Palmegiani P., Turini P., Girilli R., Ferretti R., Gallinella
B., La Torre F., J. Med. Chem., 48, 4220—4223 (2005).
Furthermore, they were tested to determine their activity toward MAO-A 11) Chimenti F., Secci D., Bolasco A., Chimenti P., Granese A., Befani O.,
and MAO-B selectivity in the presence of the specific substrate, serotonin or
benzylamine, respectively. Bovine brain mitochondria were isolated accord-
Turini P., Alcaro S., Ortuso F., Bioorg. Med. Chem. Lett., 14, 3697—
3703 (2004).
ing to Basford.15 The activity of MAO-A and MAO-B was determined using 12) Tao G., Irie Y., Li D.-J., Keung W. M., Bioorg. Med. Chem., 13,
a fluorimetric method described by Matsumoto et al.16) The mitochondrial
fractions were preincubated at 38 °C for 30 min before adding the specific 13) Undenfriend S., Weissbach H., Clark C. T., J. Biol. Chem., 215, 337—
4777—4788 (2005).
inhibitor, L-deprenyl (l0.5 mM) to determine MAO-A activity and clorgyline
(10.5 mM) to determine MAO-B activity. The incubation mixture contained
(0.1 ml, 0.25 M) phosphate buffer pH 7.4, mitochondrial suspension
(6 mg/1 ml), the specific substrate for MAO-A or MAO-B (0.1 mM) and test
compounds at four different concentrations ranging from 5 mM to 0.1 mM
344 (1955).
14) Schneider W. C., J. Biol. Chem., 176, 259—266 (1948).
15) BasfordR. E., Methods Enzymol., 10, 96—101 (1967).
16) Matsumoto T., Suzuki O., Furuta T., Asai M., Kurokawa Y., Rimura Y.,
Katsumata Y., Takahashi I., Clin. Biochem., 18, 126—129 (1985).
dissolved in propylene glycol. The mixture was incubated in a shaking 17) Bradford M. M., Anal. Biochem., 72, 248—254 (1976).
water-bath at 37 °C for 60 min. The reaction was quenched by adding per- 18) Verma M., Tripathi M., Saxena A. K., Shanker K., Eur. J. Med. Chem.,
chloric acid. The samples were centrifuged at 10000 g for 5 min and the su-
pernatant was completed to 2.7 ml using 1 N NaOH and measured with a
Perkin-Elmer Lf 45 Spectrofluorimeter. Protein concentration was deter-
29, 941—946 (1994).
19) Litchfield J. T., Wilcoxon F., J. Pharmacol. Exp. Ther., 96, 99—113
(1949).
mined according to a previously reported method.17) The MAO-A and MAO- 20) Bekhit A. A., Fahmy H. T. Y., Arch. Pharm. Pharm. Med. Chem., 336,
B results are expressed as IC50 (Table 1).
111—118 (2003).
The test compounds were further evaluated for their oral acute toxicity in 21) El Massry A. M., Amer A., Heterocycles, 29, 1907—1914 (1989).
male mice using a literature method.19,20) The results indicated that test com- 22) Jahine H., Zaher H. A., Sayed A., Seada M., Indian J. Chem., 15B,
pounds proved to be non-toxic and well tolerated by the experimental ani-
352—355 (1977).