Synthesis of some novel 2,6-disubstituted pyridazin-3-ones as TMC120 analogues

Article abstract of DOI:10.1002/jhet.5570440618

(Chemical Equation Presented) Different analogues of TMC120 derived from pyridazin-3(2H)-one rings were synthesized by coupling of 3,6-dichloropyridazine with arylacetonitriles, phenols and/or aniline derivative followed by hydrolysis and alkylation with different benzyl bromide derivatives.

Full text of DOI:10.1002/jhet.5570440618

Nov-Dec 2007
Synthesis of Some Novel 2,6-Disubstituted pyridazin-3-ones
as TMC120 Analogues
1351
Yasser M. Loksha and Erik B. Pedersen*
Nucleic Acid Center†, Department of Chemistry and Physics, University of Southern Denmark, Campusvej 55,
DK-5230 Odense M, Denmark
Paolo La Colla and Roberta Loddo
Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia e Virologia Generale e
Biotecnologie Microbiche, Universitá di Cagliari, Cittadella Universitaria, 09042 Monserrato, Italy
Received December 6, 2006
O
N
N
HN
N
NH
N
NH
Cl
N
N
Cl
CN
Dapivirine (TMC120).
CN
Pyridazinone analogue
Different analogues of TMC120 derived from pyridazin-3(2H)-one rings were synthesized by coupling
of 3,6-dichloropyridazine with arylacetonitriles, phenols and/or aniline derivative followed by hydrolysis
and alkylation with different benzyl bromide derivatives.
J. Heterocyclic Chem., 44, 1351 (2007).
NH2
INTRODUCTION
Br
O
N
N
The non-nucleoside reverse transcriptase inhibitors
(NNRTIs) represent a class of structurally diverse, potent
and highly-selective anti-HIV agents that were first
discovered in the early nineties as a result of large scale
compound library screening in antiviral and/or
biochemical assays, followed by extensive chemical lead
optimization [1–4]. Different chemical classes have been
reported to inhibit the reverse transcriptase (RT) enzyme,
such as tetrahydroimidazo[4,5,1-jk][1,4]-benzodiazepine-
2(1H)-one (TIBO) [1], 1-[(2-hydroxyethoxy)methyl]-6-
(phenylthio)thymine (HEPT) [5,6], α-anilinophenyl-
acetamide (α-APA) [7], pyridones [3], imdidazoles [8],
imidoyl thiourea (ITU) [9], and finally the diaryltriazine
(DATA) and diarylpyrimidine (DAPY) series [10,11] of
which dapivirine (TMC120, Figure 1) and etravirine
(TMC125, Figure 1) are representatives. TMC125
[12,13], was the first NNRTI demonstrating a beneficial
effect on HIV-infected patients with NNRTI-resistant
viruses. TMC125 is currently in phase IIB clinical trials.
Heeres et al [14] have reported novel analogues of
etravirine derived from N-methylated pyrazinone rings
which are different from the pyrimidine ring of etravirine
as shown in Figure 1. Pyrazinone analogues have also
shown a significant activity against HIV-1, both wild type
and mutants resistant to nucleoside and non-nucleoside
RT inhibitors [14].
HN
N
NH
HN
N
CN
CN
CN
Etravirine (TMC125).
Dapivirine (TMC120).
O
O
N
N
N
N
NH
HN
O
CN
Pyridazinone analogue
CN
Pyrazinone analogue
Figure 1
RESULTS AND DISCUSSION
On treatment of the potassium salt of the commercially
available pyridazine-3,6-dione (1) in dimethylformamide
with 3-cyanobenzyl bromide, O-monoalkylated product 2
and O,N-bisalkylated product 3 were isolated from the
reaction by column chromatography. Compound 3 was
used as a reference to determine O- versus N-alkylation.
The 13C nmr spectrum showed an N-substituted benzylic
carbon at 52.86 ppm and an O-substituted benzylic
From this point of view, we report a study on the
synthesis and biological evaluation of some pyridazinone
derivatives of etravirine and dapivirine.
13
carbon at 66.96 ppm which is in agreement with the C

1352
Y. M. Loksha, E. B. Pedersen, P. La Colla and R. Loddo
Vol 44
nmr spectrum of 2-benzyl-4,5-diiodopyridazin-3(2H)-one
[15]. The DBU salt of compound 2 was coupled with 2,6-
diflourobenzyl bromide in dry acetonitrile as a solvent to
afford another O,N-bisalkylated derivative (4) (Scheme I).
3,6-Dichloropyridazine was reacted with sodium phen-
oxide derivatives in DMF to afford the 3-aryloxy-6-
chloropyridazines (9a,b) which were hydrolyzed under
mild conditions in refluxing glacial acetic acid to give 6-
aryloxypyridazin-3(2H)-ones (10a,b). Coupling of com-
pounds 10a,b with benzyl halide derivatives furnished 2-
arylmethyl-6-aryloxypyridazin-3(2H)-ones (11a-e) which
are considered to be TMC120 analogues.
Scheme I
O
O
Br
HN
N
N
O
O
N
O
Scheme III
CN
+
HN
N
H
O
DMF, K2CO3
OH
Cl
CN
CN
CN
R1
N
AcOH, reflux
O
N
3
2
1
5
NaH, DMF
R1
O
Br
N
F
a, R1 = 2,4,6-(Me)3
b, R1 = 4-CN
F
N
F
O
9a,b
F
Br
2
O
DBU, CH3CN
O
R2
CN
HN
N
O
N
4
O
N
DBU, CH3CN
R1
R2
R1
3,6-Dichloropyridazine (5) was treated with the
sodium salt of benzyl cyanide derivatives to afford
(aryl)(3-chloropyridazin-6-yl)acetonitriles (6a,b)
which were hydrolyzed in a mixture of acetic acid and
7 M HCl to furnish 6-arylmethylpyridazin-3(2H)-ones
(7a,b). Alkylation of the DBU salt of compounds 7a,b
in acetonitrile with benzyl bromide derivatives
afforded 2,6-bis(arylmethyl)pyridzin-3(2H)-ones (8a-
f) (Scheme II).
a, R1 = 2,4,6-(Me)3
b, R1 = 4-CN
11a-f
10a,b
R1
R2
R1
R2
11
11
2,4,6-(Me)3
2,4,6-(Me)3
2,4,6-(Me)3
3-CN
4-CN
2,6-F2
2,4,6-(Me)3
4-CN
2,4,6-(Me)3
2,4,6-(Me)3
a
b
c
d
e
3,6-Dichloropyridazine was fused under nitrogen with
2,4,6-trimethylaniline to afford 6-chloro-N-mesityl-pyri-
dazin-3-amine (12) which was hydrolyzed by refluxing in
glacial acetic acid to give 6-(mesitylamino)-pyridazin-
3(2H)-one (13). 4-{[3-(Mesitylamino)-6-oxo-pyridazin-
Scheme II
Cl
CN
N
CN
N
Cl
R1
Scheme IV
7M HCl, AcOH
N
R1
NH2
Cl
N
NaH, DMF
Cl
a, R1 = 3,5-(Me)2
b, R1 = 2,6-F2
5
6a,b
N
AcOH, reflux
Br
NH
N
O
5
O
Fusion, 150o
HN
R2
N
N
N
12
DBU, CH3CN
O
R1
R2
R1
O
N
Br
NH
N
a, R1 = 3,5-(Me)2
b, R1 = 2,6-F2
HN
8a-f
NC
7a,b
NH
N
DBU, CH3CN
8
a
b
c
R1
3,5-(Me)2
3,5-(Me)2
3,5-(Me)2
R2
3-CN
4-CN
2,6-F2
8
d
e
f
R1
2,6-F2
2,6-F2
2,6-F2
R2
3-CN
4-CN
CN
14
2,4,6-(Me)3
13

Nov-Dec 2007
Novel 2,6-Disubstituted pyridazin-3(2H)-ones as TMC120 Analogues
1353
mixture was stirred at room temperature for 1 hour then poured
on ice-cold water (50 ml) and the solid product formed was
collected by filtration, dried to give 240 mg of the pure
1(6H)-yl]methyl}benzonitrile (14, TMC120 analogue)
was prepared by reaction of the DBU salt of compound
13 in acetonitrile with 4-cyanobenzyl bromide.
All the compounds were evaluated for their activity
against HIV-1 wild type and its NNRTI-resistant mutants.
Only compound 4 showed moderate activity against HIV-
1 wild type (EC50 = 8 µM; CC50 = 40 µM). All other
compounds were inactive at the maximum concentration
tested (100 µM).
1
compound 4; yield 68%; mp 133-135°; H nmr (DMSO-d6) δ:
5.09 (s, 2H, CH2N), 5.21 (s, 2H, CH2O), 7.01-7.80 (m, 9H,
H
arom, H-4 and H5); 13C nmr (DMSO-d6) δ: 40.93 (CH2N), 66.59
(CH2O), 111.36 (dd, J = 7.9, 16.9 Hz, Carom), 118.49 (CN),
112.82 (t, J = 20.1 Hz, Carom), 111.42, 126.91, 129.47, 131.17,
131.67, 137.58 (Carom), 130.57 (t, J = 10.3 Hz, Carom), 132.48
(C4), 132.79 (C5), 151.14 (C3), 157.65 (C6), 161.11 (dd, J =
7.6, 234.7 Hz, Carom); EI ms: m/z 353 (39%, M+), 127 (100%).
Anal. Calcd. for C19H13F2N3O2 (353.32): C, 64.59; H, 3.71; N,
11.89. Found: C, 64.18; H, 4.10; N, 11.25.
EXPERIMENTAL
General Procedure for the Synthesis of (aryl)(3-chloro-
pyridazin-6-yl)acetonitriles 6a,b. To a stirred solution of 3,6-
dichloropyridazine (5, 1.49 g, 0.01 mole) and benzyl cyanide
derivatives (3,5-dimethylbenzyl cyanide and/or 2,6-diflouro-
benzyl cyanide) (0.01 mole) in dry DMF (25 ml) sodium hydride
(0.92 g, 0.021 mole, 55% susp. in paraffin oil) was added
portionwise at 0º. The reaction mixture is allowed to reach room
temperature gradually with stirring, then poured on ice-cold
water (100 ml) with stirring and the precipitated material was
collected by filtration, dried to give compounds 6a,b.
Nmr Spectra were recorded on a Varian Gemini 2000 nmr
spectrometer at 300 MHz for 1H and 75 MHz for 13C with TMS
as an internal standard. EI mass spectra were recorded on a
Finnigan MAT SSQ 710. MALDI spectra were recorded on a
4.7 T Ultima Fourier transform Mass spectrometer (IonSpec,
Irvine, CA). Melting points were determined in a Büchi melting
point apparatus. The silica gel (0.040–0.063 mm) used for
column chromatography was purchased from Merck.
Microanalyses were carried out at Chemical Laboratory II at
University of Copenhagen, Denmark.
Synthesis of Compounds 2 and 3. Pyridazine-3,6-dione (1,
5.6 g, 0.05 mole) was added portionwise to a solution of K2CO3
(7 g, 0.05 mole) in dry DMF (50 ml) and the solution was stirred
for 0.5 hour. 3-Cyanobenzyl bromide (9.8 g, 0.05 mole) was
added to the reaction mixture and stirred for 12 hours. The
reaction mixture was poured on ice-cold water (150 ml) with
stirring and the solid product formed was filtered off, dried and
chromatographed on a column of silica gel using CH2Cl2:
petroleum ether (1:1, v/v) as an eluent to give 2 and 3.
3-{[(6-Oxo-1,6-dihydropyridazin-3-yl)oxy]methyl}benzo-
nitrile (2). Yield 2.1 g (19%); mp 198-200°; 1H nmr (DMSO-d6)
δ: 5.21 (s, 2H, CH2), 6.92 (d, 1H, J = 9.9 Hz, H5), 7.26 (d, 1H, J
= 9.9 Hz, H-4), 7.62 (t, 1H, J = 7.8 Hz, Harom), 7.79-7.84 (m,
2H, Harom), 7.93 (s, 1H, Harom), 12.26 (s, 1H, NH); 13C nmr
(DMSO-d6) δ: 66.72 (CH2), 118.54 (CN), 111.33, 127.50,
129.57, 131.25, 132.57, 137.98 (Carom), 131.66 (C4), 133.16
(C5), 152.11 (C3), 159.54 (C6); EI ms: m/z 227 (30%, M+), 116
(100%). Anal. Calcd. for C12H9N3O2 (227.22): C, 63.43; H,
3.99; N, 18.49. Found: C, 63.12; H, 3.85; N, 18.31.
3-({[1-(3-Cyanobenzyl)-6-oxo-1,6-dihydropyridazin-3-yl]-
oxy}methyl)benzonitrile (3). Yield 3.6 g (21%); mp 122-124°;
1H nmr (DMSO-d6) δ: 5.16 (s, 2H, CH2N), 5.24 (s, 2H, CH2O),
7.00 (d, 1H, J = 9.7 Hz, H5), 7.32 (d, 1H, J = 9.7 Hz, H-4),
7.50-7.90 (m, 8H, Harom); 13C nmr (DMSO-d6) δ: 52.86 (CH2N),
66.96 (CH2O), 118.49 (2CN), 111.31, 111.37, 127.28, 129.48,
129.61, 131.18, 131.30, 131.38, 132.62, 132.68, 137.77, 138.17
(Carom), 131.71 (C4), 133.06 (C5), 151.67 (C3), 158.03
(C6); EI ms: m/z 342 (36%, M+), 116 (100%). Anal.
Calcd. for C20H14N4O2·0.8H2O (357.78): C, 67.14; H, 4.68;
N, 15.66. Found: C, 67.17; H, 4.23; N, 15.59.
(6-Chloropyridazin-3-yl)(3,5-dimethylphenyl)acetonitrile
1
(6a). Yield 2.5 g (97%); mp 108-110°; H nmr (DMSO-d6) δ:
2.27 (s, 6H, (CH3)2Ar), 6.11 (s, 1H, CH-CN), 7.01 (s, 1H, Harom),
7.08 (s, 2H, Harom), 7.81 (d, 1H, J = 9.0 Hz, H-4), 7.95 (d, 1H, J =
9.0 Hz, H5); 13C nmr (DMSO-d6) δ: 20.73 (2 x CH3), 41.27 (CH-
CN), 118.51 (CN), 125.46 (C4), 129.49, 130.07, 138.73 (Carom),
134.08 (C5), 156.17 (C3), 158.21 (C6). HRms (MALDI, peak
matching): m/z calcd. for C14H13ClN3 (MH+) 258.0793, found
258.0789. Anal. Calcd. for C14H12ClN3·0.3 H2O (263.13): C,
63.91; H, 4.83; N, 15.97. Found: C, 63.81; H 4.47; N, 15.65.
(6-Chloropyridazin-3-yl)(2,6-difluorophenyl)acetonitrile
1
(6b). Yield 2.1 g (80%); as red crystals; mp 126-128°; H nmr
(DMSO-d6) δ: 6.61 (CH-CN), 7.13-7.65 (m, 3H, Harom), 7.88 (d,
1H, J = 9.0 Hz, H5), 8.03 (d, 1H, J = 9.0 Hz, H-4); 13C nmr
(DMSO-d6) δ: 31.16 (CH-CN), 111.98-112.63 (m, Carom),
119.68 (CN), 128.72 (C4), 130.05 (C5), 132.30 (t, J = 10.6 Hz,
C
arom), 156.12 (C3), 156.29 (C6), 159.92 (dd, J = 6.7, 251.3 Hz,
arom); HRms (MALDI, peak matching): m/z calcd. for
C
C12H7ClF2N3 (MH+) 266.0297, found 266.0293. Anal. Calcd. for
C12H6ClF2N3 (265.65): C, 54.26; H, 2.28; N, 15.82. Found: C,
54.44; H, 2.06; N, 15.49.
Synthesis of 6-arylmethylpyridazin-3(2H)-ones 7a,b.
Compound 6a,b (5 mmole) was refluxed in a solution of conc.
HCl (20 ml), acetic acid (10 ml), water (10 ml) for 30 hours.
The solvents were evaporated under reduced pressure, and
then water (20 ml) was added to the residual material and
neutralized with saturated solution of sodium bicarbonate. The
solid product formed was collected by filtration and dried to
give compounds 7a,b.
6-(3,5-Dimethylbenzyl)pyridazin-3(2H)-one (7a). Yield
1
660 mg (62%); mp 120-122°; H nmr (DMSO-d6) δ: 2.23 (s,
Synthesis of 3-({[1-(2,6-difluorobenzyl)-6-oxo-1,6-dihydro-
pyridazin-3-yl]oxy}methyl) benzonitrile (4). Sodium hydride
(0.05 g, 1 mmole, 55% susp. in paraffin oil) was added portion
wise at room temperature to a stirred solution of compound 2
(0.23 g, 1 mmole) and 2,6-diflourobenzyl bromide (0.21 g, 1
mmole) in dry dimethylformamide (10 ml). The reaction
6H, (CH3)2Ar), 3.78 (s, 2H, CH2Ar), 6.80 (d, 1H, J = 9.6 Hz,
H5), 6.85 (s, 3H, Harom), 7.28 (d, 1H, J = 9.6 Hz, H-4), 12.83
(s, 1H, NH); 13C nmr (DMSO-d6) δ: 20.81 ((CH3)2Ar), 39.61
(CH2Ar), 126.49, 127.99, 137.58, 137.85 (Carom), 129.95
(C4), 134.05 (C5), 146.87 (C3), 160.17 (C6); HRms
(MALDI, peak matching): m/z calcd. for C13H14NaN2O

1354
Y. M. Loksha, E. B. Pedersen, P. La Colla and R. Loddo
Vol 44
(MNa+) 237.0998, found 237.0999. Anal. Calcd. for
C13H14N2O·0.1 H2O (216.07): C, 72.27; H, 6.62; N, 12.96.
Found: C, 72.12; H, 6.71; N, 12.57.
3-{[3-(2,6-Difluorobenzyl)-6-oxopyridazin-1(6H)-yl]methyl}-
benzonitrile (8d). Yield 280 mg (83%); mp 108-110°; H nmr
1
(CDCl3) δ: 3.99 (s, 2H, CH2-C3), 5.24 (s, 2H, CH2-N), 6.86 (d, 1H, J
= 9.5 Hz, H5), 6.94 (t, 2H, J = 7.7 Hz, Harom), 7.14 (d, 1H, J = 9.5
Hz, H-4), 7.22-7.65 (m, 5H, Harom); 13C nmr (CDCl3) δ: 27.57 (CH2-
C3), 54.05 (CH2-N), 118.56 (CN), 112.61, 129.27, 131.52, 132.39,
133.35, 137.48 (Carom), 111.40 (dd, J = 7.8, 17.3 Hz, Carom), 112.62
(t, J = 19.5 Hz, Carom), 129.13 (t, J = 10.3 Hz, Carom), 145.12 (C3),
159.33 (C6), 161.31 (d, J = 7.8, 256.6 Hz, Carom); HRms (MALDI,
peak matching): m/z calcd. for C19H14F2N3O (MH+) 338.1099, found
338.1083. Anal. Calcd. for C19H13F2N3O·0.5 H2O (346.34): C, 65.89;
H, 4.07; N, 12.13. Found: C, 65.44; H, 3.46; N, 11.91.
6-(2,6-Difluorobenzyl)pyridazin-3(2H)-one (7b). Yield 1.1
g (98%); mp 130-132°; 1H nmr (DMSO-d6) δ: 3.98 (s, 2H, CH2),
6.87 (d, 1H, J = 9.9 Hz, H5), 7.11 (t, 2H, J = 8.0 Hz, Harom),
7.34-7.44 (m, 2H, Harom and H-4); 13C nmr (DMSO-d6) δ: 26.61
(CH2), 111.47 (dd, J = 7.8, 17.4 Hz, Carom), 113.13 (t, J = 20.1
Hz, Carom), 129.31 (t, J = 10.2 Hz, Carom), 130.09 (C4), 133.63
(C5), 144.44 (C3), 160.80 (d, J = 8.5, 246.0 Hz, Carom), 160.04
(C6); HRms (MALDI, peak matching): m/z calcd. for
C11H8F2N2NaO (MNa+) 245.0497, found 245.0502.
Synthesis of 2,6-bis(arylmethyl)pyridzin-3(2H)-ones 8a-f.
Compound 7a,b (1 mmole) was added to a solution of DBU (0.2
ml, 1.3 mmole) in dry acetonitrile (10 ml) and stirred for 15
min., then benzyl halide derivative (3-cyanobenzyl bromide, 4-
cyanobenzyl bromide, 2,6-diflourobenzyl bromide and/or 2,4,6-
trimethylbenzyl chloride) (1.1 mmole) was added to the reaction
mixture at room temperature and stirred for 5 hours. The
solvents were removed under reduced pressure, water (15 ml)
was added to the residual material and the solid product formed
was collected by filtration, washed with water, and dried to give
compounds 8a-f.
4-{[3-(2,6-Difluorobenzyl)-6-oxopyridazin-1(6H)-yl]methyl}-
benzonitrile (8e). Yield 300 mg (88%); mp 134-136°; H nmr
1
(DMSO-d6) δ: 4.00 (s, 2H, CH2-C3), 5.21 (s, 2H, CH2-N), 6.97
(d, 1H, J = 10.2 Hz, H5), 7.12 (t, 2H, J = 8.0 Hz, Harom), 7.33-
7.43 (m, 4H, Harom and H-4), 7.78 (d, 2H, J = 8.1 Hz, Harom); 13
C
nmr (DMSO-d6) δ: 26.85 (CH2-C3), 53.57 (CH2-N), 118.56
(CN), 110.26, 128.47, 133.11, 142.06 (Carom), 111.45 (dd, J =
7.5, 17.4 Hz, Carom), 112.81 (t, J = 20.1 Hz, Carom), 129.44 (t, J =
10.5 Hz, Carom), 145.03 (C3), 158.60 (C6), 160.75 (d, J = 8.5,
246.0 Hz, Carom); HRms (MALDI, peak matching): m/z calcd.
for C19H14F2N3O (MH+) 338.1099, found 338.1091.
3-{[3-(3,5-Dimethylbenzyl)-6-oxopyridazin-1(6H)-yl]methyl}-
benzonitrile (8a). Yield 240 mg (72%); mp 74-76°; 1H nmr
(DMSO-d6) δ: 2.22 (s, 6H, 2 x CH3), 3.82 (s, 2H, CH2-C3), 5.29
(s, 2H, CH2-N), 6.83 (s, 2H, Harom), 6.85 (s, 1H, Harom), 6.93 (d,
1H, J = 9.6 Hz, H5), 7.33 (d, 1H, J = 9.5 Hz, H-4), 7.57-7.79 (m,
4H, Harom); 13C nmr (DMSO-d6) δ: 20.76 (2 x CH3), 39.57 (CH2-
C3), 53.11 (CH2-N), 118.46 (CN), 130.00 (C4), 132.63 (C5),
111.38, 126.34, 128.02, 129.70, 131.14, 131.30, 133.64, 137.49,
137.56, 138.41 (Carom), 147.48 (C3), 158.69 (C6); HRms (MALDI,
peak matching): m/z calcd. for C21H20N3O (MH+) 330.1601, found
330.1611. Anal. Calcd. for C21H19N3O·0.5 H2O (338.41): C, 74.53;
H, 5.96; N, 12.58. Found: C, 74.46; H, 5.70; N, 12.37.
6-(2,6-Difluorobenzyl)-2-(mesitylmethyl)pyridazin-3(2H)-
one (8f). Yield 300 mg (85%); mp 110-112°; H nmr (CDCl3) δ:
1
2.19 (s, 6H, 2 x CH3), 2.26 (s, 3H, CH3), 3.83 (s, 2H, CH3-C3),
5.23 (s, 2H, CH2-N), 6.78-6.87 (m, 5H, Harom and H5), 7.08 (d,
1H, J = 9.3 Hz, H-4), 7.15-7.21 (m, 1H, Harom); 13C nmr (CDCl3)
δ: 19.94 (2 × CH3), 20.95 (CH3), 27.49 (CH2-C3), 47.62 (CH2-N),
111.06 (dd, J = 7.8, 17.3 Hz, Carom), 112.90 (t, J = 19.9 Hz, Carom),
128.51 (t, J = 10.7 Hz, Carom), 128.68, 129.51, 137.16, 138.39
(Carom), 129.73 (C4), 131.48 (C5), 144.06 (C3), 160.82 (dd, J =
8.1, 249.8 Hz, Carom), 159.72 (C6); HRms (MALDI, peak
matching): m/z calcd. for C21H20F2NaN2O (MNa+) 377.1436,
found 377.1420. Anal. Calcd. for C21H20F2N2O·0.7 H2O (367.01):
C, 68.73; H, 5.88; N, 7.63. Found: C, 68.53; H, 5.57; N, 7.73.
Synthesis of 3-aryloxy-6-chloropyridazines 9a,b. Sodium
hydride (0.48 g, 11 mmole, 55% susp. in paraffin oil) was added
portionwise to a solution of compound 5 (1.49 g, 10 mmole) and
phenol derivative (2,4,6-trimethylphenol and/or 4-cyanophenol)
(10 mmole) in dry DMF (15 ml). The reaction mixture was
stirred at room temperature for 3 hours, poured on ice-cold
water (40 ml) and the solid product formed was filtered off,
washed with water and dried to give compound 9a,b.
4-{[3-(3,5-Dimethylbenzyl)-6-oxopyridazin-1(6H)-yl]methyl}-
1
benzonitrile (8b). Yield 220 mg (67%); mp 83-85°; H nmr
(DMSO-d6) δ: 2.21 (s, 6H, 2 x CH3), 3.81 (s, 2H, CH2-C3), 5.32
(CH2-N), 6.81 (s, 2H, Harom), 6.85 (s, 1H, Harom), 6.93 (d, 1H, J
= 9.6 Hz, H5), 7.33 (d, 1H, J = 9.6 Hz, H-4), 7.45 (d, 2H, J =
8.1 Hz, Harom), 7.82 (d, 2H, J = 8.1 Hz, Harom); 13C nmr (DMSO-
d6) δ: 20.76 (2 x CH3), 39.56 (CH2-C3), 53.50 (CH2-N), 118.58
(CN), 110.24, 126.36, 128.04, 128.45, 132.37, 137.44, 137.58,
142.46 (Carom), 129.98 (C4), 133.67 (C5), 147.50 (C3), 158.73
(C6); EI ms: m/z 329 (100%, M+). Anal. Calcd. for
C21H19N3O·0.6 H2O (340.21): C, 74.14; H, 5.98; H, 12.35.
Found: C, 73.93; H, 5.68; H, 12.32.
2-(2,6-Difluorobenzyl)-6-(3,5-dimethylbenzyl)pyridazin-
3(2H)-one (8c). Yield 320 mg (95%); mp 88-90°; 1H nmr
(CDCl3) δ: 2.25 (s, 6H, (CH3)2Ar)), 3.73 (s, 2H, CH2-C3), 5.42
(s, 2H, CH2-N), 6.72-7.00 (m, 7H, Harom, H-4 and H5), 7.23-7.33
(m, 1H, Harom); 13C nmr (CDCl3) δ: 21.19 [(CH3)2Ar], 40.75
(CH2-C3), 43.04 (CH2-N), 111.20 (dd, J = 7.7, 17.9 Hz, Carom),
111.84 (t, J = 19.2 Hz, Carom), 126.59, 128.52, 137.15, 138.30
(Carom), 129.95 (C4), 132.57 (C5), 129.78 (t, J = 10.1 Hz, Carom),
146.94 (C3), 159.51 (C6), 161.92 (dd, J = 7.5, 250.5 Hz, Carom);
HRms (MALDI, peak matching): m/z calcd. for C20H19F2N2O
(MH+) 341.1460, found 341.1449. Anal. Calcd. for
C20H18F2N2O·0.6 H2O (351.19): C, 68.40; H, 5.51; N, 7.98.
Found: C, 68.43; H, 5.14; N, 7.93.
3-Chloro-6-(mesityloxy)pyridazine (9a). Yield 2.4 g (97%);
1
mp 114-116°; H nmr (CDCl3) δ: 2.08 (s, 6H, (CH3)2Ar), 2.29 (s,
3H, CH3Ar), 6.90 (s, 2H, Harom), 7.09 (d, 2H, J = 9.0 Hz, H5), 7.45
(d, J = 9.0 Hz, H4); 13C nmr (CDCl3) δ: 16.34 ((CH3)2Ar), 20.77
(CH3Ar), 118.54 (C4), 129.60, 129.85, 131.45, 151.55 (Carom),
135.44 (C6), 164.30 (C3); HRms (MALDI, peak matching): m/z
calcd. for C13H14ClN2O (MH+) 249.0789, found 249.0788.
4-[(6-Chloropyridazin-3-yl)oxy]benzonitrile (9b). Yield
460 mg (20%); mp 138-140°; 1H nmr (CDCl3) δ: 7.26 (s, 1 H, J =
9.0 Hz, H5), 7.35 (d, 2H, J = 8.7 Hz, Harom), 7.57 (d, J = 9.0 Hz,
H4), 7.73 (d, 2H, J = 8.7 Hz, Harom); 13C nmr (CDCl3) δ: 118.25
(CN), 109.32, 121.92, 132.00, 151.53 (Carom), 130.30 (C4), 134.02
(C5), 153.07 (C6), 156.32 (C3); HRms (MALDI, peak matching):
m/z calcd. for C11H7ClN3O (MH+) 232.0272, Found 232.0280.
Synthesis of 6-aryloxypyridazin-3(2H)-ones 10a,b.
Compound 9a,b (3.3 mmole) was refluxed in acetic acid (20 ml)

Nov-Dec 2007
Novel 2,6-Disubstituted pyridazin-3(2H)-ones as TMC120 Analogues
1355
for 6 hours. The solvent was removed under reduced pressure,
water (30 ml) was added and the solid product formed was
collected by filtration, washed with water and dried to give
compound 10a,b.
m/z calcd. for C20H19F2N2O2 (MH+) 357.1409, found 357.1399.
Anal. Calcd. for C20H18F2N2O2·0.25H2O (360.88): C, 66.57; H,
5.24; N, 7.76. Found: C, 66.84; H, 5.21; N, 7.39.
2-(2,4,6-Trimethylbenzyl)-6-(2,4,6-trimethylphenoxy)-2H-
pyridazin-3-one (11d). Yield 280 mg (77%); mp 86-88°; 1H nmr
(CDCl3) δ: 1.72 (s, 6H, 2 x CH3), 1.92 (s, 6H, 2 x CH3), 2.26 (s,
3H, CH3), 2.30 (s, 3H, CH3), 5.04 (CH2), 6.64 (s, 2H, Harom), 6.69
(s, 2H, Harom), 6.98 (d, 1H, J = 9.8 Hz, H5), 7.08 (d, 1H, J = 9.8
Hz, H4); 13C nmr (CDCl3) δ: 15.77 (2 x CH3), 19.56 (2 x CH3),
20.71 (CH3), 20.90 (CH3), 46.25 (CH2), 124.83, 128.40, 128.80,
128.91, 133.18, 137.03, 138.31, 151.02 (Carom), 129.92 (C4),
134.50 (C5), 147.55 (C3), 158.79 (C6); HRms (MALDI, peak
matching): m/z calcd. for C23H26NaN2O2 (MNa+) 385.1887, found
385.1880. Anal. Calcd. for C23H26N2O2 (362.46): C, 73.30; H,
7.38; N, 7.43. Found: C, 73.25; H, 7.41; N, 7.31.
6-(Mesityloxy)pyridazin-3(2H)-one (10a). Yield 720 mg
1
(95%); mp 166-168°; H nmr (DMSO-d6) δ: 2.04 (s, 6H, 2 ×
CH3), 2.23 (s, 3H, CH3), 6.91 (s, 2H, Harom), 7.00 (d, 1H, J =
9.9 Hz, H5), 7.45 (d, 1H, J = 9.9 Hz, H4), 12.03 (bs, 1H, NH);
13C nmr (DMSO-d6) δ: 15.84 (2 x CH3), 20.24 (CH3), 126.89,
129.08, 133.91, 151.80 (Carom), 129.53 (C4), 134.36 (C5),
147.34 (C3), 159.53 (C6); HRms (MALDI, peak matching): m/z
calcd. for C13H15N2O2 (MH+) 231.1128, found 231.1130.
4-[(6-Oxo-1,6-dihydropyridazin-3-yl)oxy]benzonitrile (10b).
1
Yield 400 mg (56%); mp 162-164°; H nmr (DMSO-d6) δ: 7.05
(d, 1H, J = 9.9 Hz, H5), 7.40 (d, 2H, J = 8.7 Hz, Harom), 7.45 (d,
1H, J = 9.9 Hz, H4), 7.91 (d, J =8.7 Hz, Harom); 13C nmr
(DMSO-d6) δ: 118.38 (CN), 107.24, 120.97, 133.98, 151.45
(Carom), 128.06 (C4), 134.33 (C5), 157.28 (C6), 159.75 (C3);
HRms (MALDI, peak matching): m/z calcd. for C11H7N3O2
(MH+) 214.0611, found 214.0616.
Synthesis of 2-arylmethyl-6-aryloxypyridazin-3(2H)-ones
11a-e. Compound 10a,b (1 mmole) was added to a solution of
DBU (0.2 ml, 1.3 mmole) in dry acetonitrile (10 ml) and stirred
for 15 min., then benzyl halide derivative (3-cyanobenzyl
bromide, 4-cyanobenzyl bromide, 2,6-diflourobenzyl bromide
and/or 2,4,6-trimethylbenzyl chloride) (1.1 mmole) was added
to the reaction mixture at room temperature and stirred for 5
hours. The solvents were removed under reduced pressure,
water (15 ml) was added to the residual material and the solid
product formed was collected by filtration, washed with water,
and dried to give compounds 11a-e.
3-{[3-(Mesityloxy)-6-oxopyridazin-1(6H)-yl]methyl}benzo-
nitrile (11a). Yield 280 mg (80%); mp 90-92°; 1H nmr (CDCl3)
δ: 2.01 (s, 6H, 2 x CH3), 2.33 (s, 3H, CH3), 5.00 (s, 2H, CH2),
6.92 (s, 2H, Harom), 6.99 (d, 1H, J = 9.9 Hz, H5), 7.14 (d, 1H, J
= 9.9 Hz, H4), 7.34-7.56 (m, 4H, Harom); 13C nmr (CDCl3) δ:
16.13 ((CH3)2Ar), 20.76 (CH3Ar), 52.93 (CH2), 118.40 (CN),
112.47, 125.93, 129.19, 129.38, 129.70, 132.56, 133.52, 135.29,
137.36, 151.80 (Carom), 131.49 (C4), 133.73 (C5), 147.45 (C3),
158.61 (C6); HRms (MALDI, peak matching): m/z calcd. for
C21H20N3O2 (MH+) 346.1550, found 346.1540.
4-{[3-(Mesityloxy)-6-oxopyridazin-1(6H)-yl]methyl}benzo-
nitrile (11b). Yield 260 mg (75%); mp 100-102°; 1H nmr
(CDCl3) δ: 2.01 (s, 6H, 2 x CH3), 2.31 (s, 3H, CH3), 5.03 (CH2),
6.88 (s, 2H, Harom), 6.99 (d, 1H, J = 9.8 Hz, H5), 7.14 (s, 1H, J =
9.8 Hz, H4), 7.31 (d, 2H, J = 8.1 Hz, Harom), 7.5 (d, 2H, J = 8.1 Hz,
Harom); 13C nmr (CDCl3) δ: 16.18 (2 x CH3), 20.74 (CH3), 53.54
(CH2), 118.57 (CN), 111.66, 125.92, 129.24, 129.50, 132.10,
135.15, 141.08, 151.77 (Carom), 129.80 (C4), 133.72 (C5), 147.47
(C3), 158.66 (C6); HRms (MALDI, peak matching): m/z calcd. for
C21H20N3O2 (MH+) 346.1550, found 346.1540.
4-{[1-(Mesitylmethyl)-6-oxo-1,6-dihydropyridazin-3-yl]-
1
oxy}benzonitrile (11e). Yield 260 mg (75%); mp 108-110°; H
nmr (CDCl3) δ: 2.10 (s, 6H, 2 x CH3), 2.32 (s, 3H, CH3), 5.15 (s,
2H, CH2), 6.80 (s, 2H, Harom), 6.91 (d, 2H, J = 8.7 Hz, Harom),
7.02-7.10 (m, 2H, H4 and H5), 7.46 (d, 2H, J = 8.7 Hz, Harom);
13C nmr (CDCl3) δ: 19.81 (2 x CH3), 20.93 (CH3), 46.71 (CH2),
118.36 (CN), 108.16, 121.22, 125.67, 129.39, 133.35, 137.65,
138.22, 150.66 (Carom), 128.76 (C4), 133.44 (C5), 156.13 (C6),
158.75 (C3); HRms (MALDI, peak matching): m/z calcd. for
C21H19NaN3O2 (MNa+) 368.1370, found 368.1373.
Synthesis of 6-chloro-N-mesitylpyridazin-3-amine (12).
Under stream of nitrogen, compound 5 (1.49 g, 10 mmole) was
fused with 2,4,6-trimethylaniline (1.48 g, 11 mmole) at 150 °C
for 2.5 hours. The reaction mixture was left to reach 70 °C, then
ethanol (10 ml) was added and the mixture was stirred with a
spatula until obtaining a suspension. The solid product formed
was collected by filtration, dried and chromatographed on a
column of silica gel using CH2Cl2:EtOAc (10:1, v/v) as an
eluent to give 2 g of the pure compound 12; yield 81%; mp 180-
1
182°; H nmr (CDCl3) δ: 2.17 (s, 6H, 2 x CH3), 2.31 (s, 3H,
CH3), 6.25 (d, 1H, J = 9.3 Hz, H4), 6.77 (bs, 1H, NH), 7.12 (d,
1H, J = 9.3 Hz, H5); 13C nmr (CDCl3) δ: 18.16 (2 x CH3),
220.91 (CH3), 113.91 (C4), 129.50, 129.55, 136.57, 137.52
(Carom), 131.88 (C5), 146.97 (C6), 159.00 (C3); EI ms: m/z 247
(17%, M+), 232 (100%). Anal. Calcd. for C13H14ClN3 (247.72):
C, 63.03; H, 5.70; N, 16.96. Found: C, 63.20; H, 5.46; N, 16.75.
Synthesis of 6-(mesitylamino)pyridazin-3(2H)-one (13).
Compound 12 (5 mmole) was refluxed in acetic acid (20 ml) for
6 hours. The solvent was removed under reduced pressure, water
(30 ml) was added and the solid product formed was collected
by filtration, washed with water and dried to give compound 13;
1
yield 460 mg (40%); mp 212-214°; H nmr (DMSO-d6) δ: 2.10
(s, 6H, 2 x CH3), 2.21 (s, 3H, CH3), 6.76 (d, 1H, J = 9.6 Hz,
H5), 6.86 (s, 2H, Harom), 7.12 (d, 1H, J = 9.6 Hz, H4), 7.69 (s,
1H, NH), 11.63 (bs, 1H, NH); 13C nmr (DMSO-d6) δ: 18.03 (2 x
CH3), 20.41 (CH3), 127.38, 128.40, 134.32, 134.53 (Carom),
131.25 (C4), 134.10 (C5), 146.29 (C3), 158.72 (C6); HRms
(MALDI, peak matching): m/z calcd. for C13H16N3O (MH+)
230.1288, found 230.1286.
Synthesis of 4-{[3-(mesitylamino)-6-oxopyridazin-1(6H)-yl]-
methyl}benzonitrile (14). Compound 13 (0.23 g, 1 mmole) was
added to a solution of DBU (0.2 ml, 1.3 mmole) in dry acetonitrile
(10 ml) and stirred for 15 min., then 4-cyanobenzyl bromide
(0.215 g, 1.1 mmole) was added to the reaction mixture at room
temperature and stirred for 5 hours. The solvents were removed
under reduced pressure, water (15 ml) was added to the residual
2-(2,6-Difluorobenzyl)-6-(mesityloxy)pyridazin-3(2H)-one
(11c). Yield 280 mg (79%); mp 78-80°; 1H nmr (CDCl3) δ: 1.90
(s, 6H, 2 x CH3), 2.27 (s, 3H, CH3), 5.12 (s, 2H, CH2), 6.68 (t,
2H, J = 7.8 Hz, Harom), 6.73 (s, 2H, Harom), 6.98 (d, 1H, J = 9.9
Hz, H5), 7.10-7.19 (m, 2H, H4 and Harom); 13C nmr (CDCl3) δ:
15.92 (2CH3), 20.71 (CH3), 41.98 (CH2), 110.75 (dd, J = 7.9,
17.5 Hz, Carom), 111.39 (C5), 129.54 (t, J = 10.3 Hz, Carom),
125.51, 128.93, 133.25, 151.21, (Carom), 129.80 (C4), 134.55
(C5), 147.49 (C3), 158.77 (C6), 160.07 (d, J = 7.4 Hz, Carom),
163.39 (d, J = 7.6 Hz, Carom); HRms (MALDI, peak matching):

1356
Y. M. Loksha, E. B. Pedersen, P. La Colla and R. Loddo
Vol 44
REFERENCES AND NOTES
material and the solid product formed was collected by filtration,
dried and purified by column chromatography of silica gel using
CH2Cl2:EtOAc (10:1, v/v) as an eluent to give 100 mg of
[†] A research center funded by the Danish National Research
Foundation for studies on nucleic acid chemical biology.
[*] To whom correspondence should be addressed. Fax: +45
66158780; e-mail: ebp@ifk.sdu.dk.
[1] Pauwels, R.; Andries, K.; Desmijter, J.; Schols, D.; Kukla, M.
J.; Breslin, H. J.; Raeymaeckers, A.; Van Gelder, J.; Woestenborghs, R.;
Heykants, J.; Schellekens, K.; Janssen, M. A. C.; De Clercq, E.; Janssen,
P.A. Nature, 1990, 343, 470.
[2] Merluzzi, V. J.; Hargrave, K. D.; Labadia, M.; Grozinger, K.;
Skoog, M.; Wu, J. C.; Shih, C.-K.; Eckner, K.; Hattox, S.; Adams, J.
Rosenhtal, A. S.; Faanes, R.; Eckner, R. J.; Koup, R. A.; Sullivan, J. L.
Science, 1990, 250, 1411.
1
compound 14; yield 30%; mp 154-156°; H nmr (CDCl3) δ: 2.12
(s, 6H, 2 x CH3), 2.30 (s, 3H, CH3), 5.14 (s, 2H, CH2Ar), 5.70 (s,
1H, NH), 6.66 (d, 1H, J = 9.9 Hz, H5), 6.81 (d, 1H, J = 9.9 Hz,
H4), 6.91 (s, 2H, Harom), 7.41 (d, 2H, J = 8.4 Hz, Harom), 7.56 (d,
2H, J = 8.4 Hz, Harom); 13C nmr (CDCl3) δ: 18.29 (2 x CH3), 20.86
(CH3), 53.82 (CH2), 118.73 (CN), 111.35, 124.68, 129.23, 132.17,
132.71, 136.08, 136.83, 141.95 (Carom), 129.09 (C4), 131.98 (C5),
146.98 (C3), 158.21 (C6); HRms (MALDI, peak matching): m/z
calcd. for C21H21N4O (MH+) 345.1710, found 345.1723.
Antiviral Assay Procedures
[3] Goldman, M. E.; Nunberg, J. H.; O’Brien, J. A.; Quintero, J.
C.; Schleif, W. A.; Freund, K. F.; Lee Gaul, S.; Saari, W. S.; Wai, J. S.;
Hoffman, J. M.; Anderson, P. S.; Hupe, D. J.; Emini, E. A.; Stern, A. M.;
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Compounds were dissolved in DMSO at 100 mM and then
diluted in culture medium.
Cells and Viruses. MT-4, C8166, and H9/IIIB cells were
grown at 37 ° in a 5% CO2 atmosphere in RPMI 1640 medium
supplemented with 10% fetal calf serum (FCS), 100 IU/ml
penicillin G, and 100 μg/ml streptomycin. Cell cultures were
checked periodically for the absence of mycoplasma contam-
ination with a MycoTect Kit (Gibco). Human immunodeficiency
viruses type 1 (HIV-1, IIIB strain) was obtained from supernatants
of persistently infected H9/IIIB cells. The HIV-1 stock solutions
had titers of 4.5 x 106 50% cell culture infectious dose
(CCID50)/ml. The K103R+V179D+P225H mutant (EFVR) was
derived from an IIIB strain passaged in MT-4 cells in the presence
of efavirenz (up to 2 μM). The Y181C mutant (NIH N119) was
derived from an AZT-sensitive clinical isolate passaged initially in
CEM and then in MT-4 cells in the presence of nevirapine (10
μM). The double mutant K103N+Y181C (NIH A17) was derived
from the IIIB strain passaged in H9 cells in the presence of BI-RG
587 (1 μM). EFVR, N119, and A17 stock solutions had titers of
4.0 x 107, 1.2 x 108, and 2.1 x 107 CCID50/ml, respectively.
HIV Titration. Titration of HIV was performed in C8166
cells by the standard limiting dilution method (dilution 1:2,
four replica wells per dilution) in 96-well plates. The
infectious virus titer was determined by light microscope
scoring of syncytia after 4 days of incubation. Virus titers
were expressed as CCID50/ml.
Anti-HIV Assays. The activity of test compounds against
multiplication of wild type HIV-1, EFVR, N119, and A17 in
acutely infected cells was based on inhibition of virus-induced
cytopathicity in MT-4 cells. Briefly, an amount of 50 μl of
culture medium containing 1 x 104 cells was added to each well
of flat-bottom microtiter trays containing 50 μl of culture
medium with or without various concentrations of test
compounds. Then an amount of 20 μl of HIV suspensions
(containing the appropriate amount of CCID50 to cause complete
cytopathicity at day 4) was added. After incubation at 37ο, cell
viability was determined by the 3-(4,5-dimethyl-thiazol-1-yl)-
2,5-diphenyltetrazolium bromide (MTT) method [16]. The
cytotoxicity of test compounds was evaluated in parallel with
their antiviral activity and was based on the viability of mock-
infected cells, as monitored by the MTT method.
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R.; Moereels, H. E. L.; Heeres, J.; Koymans, L. M. H.; De Jonge, M. R.;
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P.; Azijn, H.; Pauwels, R.; Lewi, P. J.; Heeres, J.; Koymans, L. M. H.;
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D. W.M.; Kukla, J.; De Corte, B.; Kavash, R. W.; Ho, C. Y.; Ye, H.;
Lichtenstein, M. A.; Andries, K.; Pauwels, R.; De Béthune, M.-P.;
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F. D.; Vinkers, M.; Van Aken, K. J. A.; Arnold, E.; Das, K.; Kilonda, A.;
G. J. Hoornaert, F. Compernolle, M. Cegla, R. A. Azzam, K. Andries,
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[17] The information in this document is provided as is and no
guarantee or warranty is given that the information is fit for any
particular purpose. The user thereof uses the information as its sole risk
and liability.
Acknowledgement. This work received funding from the
European Community’s Sixth Framework Programme under
contract number LSHP-CT-2004-503162 (Selection and
development of microbicides for mucosal use to prevent sexual
HIV transmission/acquisition) [17].