Design, synthesis and antiviral activity of novel pyridazines

Article abstract of DOI:10.1016/j.ejmech.2012.04.020

A series of pyridazines were prepared and evaluated for their anti-HIV activity. The new synthetic route involving a novel rearrangement reaction provided a practical method for the preparation of 5-hydroxypyridazines. The primary bioassay results indicated that most of the pyridazines possess anti-HIV activity. It ought to been mentioned that the rearranged compounds 35 and 39 exhibited relatively higher HIV inhibitory effect. Most of the synthesized compounds were also found to possess good anti-TMV activity, of which compound 9 showed similar in vivo anti-TMV activity to commercial plant virucide Ribavirin. This work provides a new and efficient approach to evolve novel multi-functional antiviral agents by rational integration and optimization of previously reported antiviral agents.

Full text of DOI:10.1016/j.ejmech.2012.04.020

European Journal of Medicinal Chemistry 54 (2012) 33e41
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and antiviral activity of novel pyridazines
,
Ziwen Wang a, Mingxiao Wang b, Xue Yao b, Yue Li b, Juan Tan b, Lizhong Wang a, Wentao Qiao b
***
**
,
,
a
a,
*
Yunqi Geng b , Yuxiu Liu , Qingmin Wang
a State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry, Nankai University, 94 Weijing Street, Tianjin 300071,
People’s Republic of China
b Key Laboratory of Molecular Microbiology and Biotechnology (Ministry of Education), Nankai University, Tianjin 300071, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyridazines were prepared and evaluated for their anti-HIV activity. The new synthetic route
involving novel rearrangement reaction provided practical method for the preparation of
5-hydroxypyridazines. The primary bioassay results indicated that most of the pyridazines possess
anti-HIV activity. It ought to been mentioned that the rearranged compounds 35 and 39 exhibited
relatively higher HIV inhibitory effect. Most of the synthesized compounds were also found to possess
good anti-TMV activity, of which compound 9 showed similar in vivo anti-TMV activity to commercial
Received 18 February 2012
Received in revised form
9 April 2012
Accepted 12 April 2012
Available online 21 April 2012
a
a
plant virucide Ribavirin. This work provides
multi-functional antiviral agents by rational integration and optimization of previously reported antiviral
agents.
a new and efficient approach to evolve novel
Keywords:
Rearrangement
Synthesis
Ó 2012 Elsevier Masson SAS. All rights reserved.
Pyridazine
Antiviral activity
Anti-HIV activity
1. Introduction
for the anti-integrase activity: a diketo acid chain able to interact
with Mg2þ metal ions (marked in bold) and a properly oriented
The causative agent of acquired immune deficiency syndrome
(AIDS) is the human immunodeficiency virus type 1 (HIV-1).
Resistance to marketed anti-HIV drugs is increasing at an alarming
rate, thus there is a persistent need to improve existing agents and
develop new agents which work by different mechanisms. HIV-1
integrase (IN), the viral enzyme that catalyzes the integration of
proviral cDNA into the host cell genome, has emerged as an
attractive target for developing novel anti-AIDS agents [1e3].
Recently, raltegravir (RAL, marketed as IsentressÔ by Merck and
Co.) was approved for clinical use by the FDA as a new HIV inhibitor
targeting the viral integrase enzyme. Raltegravir disrupts the crit-
ical viral process of integration in which newly made viral DNA is
inserted into the host cell chromosomal DNA [4]. Raltegravir is the
first approved integrase inhibitor whereas other integrase inhibi-
tors GS-9137 and S/GSK1349572 have reached clinical development
(Fig. 1) [5,6]. Like other well-known diketo acid inhibitors, these
compounds share two common structural chemotypes essential
hydrophobic benzyl moiety (marked in dashed box) [7,8]. They
selectively inhibit strand transfer reaction, suggesting that they
bind at the IN/DNA interface, acting as “interfacial inhibitors” [9,10].
Despite the lack of detailed structural information about HIV-1 IN/
DNA interactions, this speculative mechanism of action tends to be
validated by the recent X-ray crystal structure of integrase from the
prototype foamy virus (PFV-1 IN) in complex with its cognate viral
DNA and strand transfer inhibitors [11,12].
In recent years, there has been increasing interest in the
chemistry of pyridazinone derivatives because of their biological
significance. Pyridazinones have been reported to possess variety of
biological activities such as anti-HIV [13], anti-TMV [14], anti-
depressant [15], anti-hypertensive [16], anti-thrombotic [17], anti-
convulsant [18], cardiotonic [19], anti-bacterial [20], diuretics [21]
and anti-cancer [22].
Taking into account the above findings, a series of new
compounds containing a pyridazine core (Pyridazines, Fig. 1) were
designed and synthesized as chelating agents for bivalent metal
ions. We propose that the free hydroxyl and two carbonyl groups on
the pyridazines might sufficiently bind to the two metal cofactors in
the IN active site, and the substituents on the amide portion could
provide the interactions with the hydrophobic pocket of the
enzyme. The obtained compounds were characterized and
* Corresponding author. Tel./fax: þ86 22 23503952.
** Corresponding author. Tel.: þ86 22 23504547; fax: þ86 22 23503091.
*** Corresponding author. Tel.: þ86 22 23504906; fax: þ86 22 23503091.
E-mail addresses: wentaoqiao@nankai.edu.cn (W. Qiao), gengyq@nankai.edu.cn
(Y. Geng), wangqm@nankai.edu.cn (Q. Wang).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.020

34
Z. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 33e41
O
OK
O
O
ONa O
CH3
O
F
O
F
N
H
O
Cl
HO
N
H
N
N
N
F
N
O
N
O
H
F
O
N
OH
N
H
H
MK-0518
S/GSK1349572
N
GS-9137
O
O
OH
5
6
O
N
4
H
N
1
F
F3C
N
2
R
3
Pyridazines
Fig. 1. Chemical structures of MK-0518, GS-9137, S/GSK1349572 and pyridazines.
evaluated for their antiviral activity against HIV. As the pyr-
idazinone derivatives have been reported to exhibit anti-TMV
activity [14], the synthesized new structural compounds also were
evaluated for their anti-TMV activity. Herein, we report the
synthesis, evaluation and SAR studies of these new structural
pyridazinones.
of 4 with 10% aq. HCl solution afforded hydroxypyridazinone 5.
Unfortunately, treatment of 5 with 4-fluorobenzylamine in methanol
afforded the decarboxylated compound 6. The decarboxylation of
compound 5 may be due to the effect of 4-hydroxyl on the pyr-
idazinone ring. So the hydroxyl protected compound 7 was prepared.
However, treatment of 7 with 4-fluorobenzylamine in methanol or
tetrahydrofuran only material was recovered. Interestingly, reacting
of 7 with neat 4-fluorobenzylamine, the designed compound 9 was
successfully obtained.
2. Results and discussion
The possible mechanism was shown in Scheme 2. Compound 7
reacted with 4-fluorobenzylamine to afford 10. Addition of water
to 10 gave intermediate 11, which underwent allylic rearrangement
to afford 12. Elimination the benzylamino group of 12 gave
compound 9. Similar results were also reported by Hegde and co-
workers [23].
According to similar procedure for preparation of compounds
4e7, compounds 13e16 were obtained (Scheme 3). Treatment of 16
with 4-fluorobenzylamine afforded compounds 17 and 18. The
obtainingof18 furthersupportedtheabovemechanism in Scheme2.
2.1. Chemistry
To test the feasibility of the new approach, the synthetic plan
depicted in Scheme 1 was initially conducted. First, compound 5 was
prepared according to Hegde and co-workers’ procedure [23]. Addi-
tionofisopropylamineto dimethyl acetylenedicarboxylate (DMAD,1)
gave 2 in 96% yield. Trifluoroacetylation of 2 with trifluoroacetic
anhydride in ether afforded 3 as mixture of Z and E isomers (1H NMR
analysis indicated that ZdCH3 3.74:Ed
with phenylhydrazine gave cyclic product 4 in 59% yield. Acidization
¼ 2.71:1). Treatment of 3
CH3 3.80
HN
NH2
HN
(CF3CO)2O
Z:E = 2.69:1
MeO2C
CO2Me
MeO2C
F3C
Ether
96%
MeO2C
CO2Me
1
by HNMR
CO2Me
94%
O
3
1
2
H3N
O
OH
O
OH
NH2
PhNHNH2
59%
10% HCl aq.
87%
O
MeO2C
F3C
MeO2C
F3C
O
F
MeOH
91%
N
N
N
N
F3C
N
N
4
5
6
CH3I
90%
K2CO3
O
OH
O
OMe
NH2
NH2
OMe
O
O
F
F
N
H
MeO2C
O
N
11 equv.
44%
H
N
N
N
THF/MeOH
F
F3C
N
N
F
F3C
F3C
N
9
7
8
Scheme 1. Synthesis of compound 9.

Z. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 33e41
35
F
F
O
HN
O
HN
OMe
4-fluorobenzyl
amine
H+
O
O
MeO2C
F3C
O
N
H
N
H
rt. to reflux
F
N
N
N
N
F3C
F3C
N
F
N
H
HO
10
11
7
F
O
OH
O
HN
HO
O
O
N
H
N
H
N
N
F
F3C
N
F3C
F
N
H
9
12
Scheme 2. Possible mechanism.
In order to evaluate the antiviral activity and SARs of the new
structural pyridazines, a series of pyridazines 19e40 were designed
and synthesized by repeating the above procedure (Scheme 4). It
ought to be mentioned that treatment of compound 38 with
4-fluorobenzylamine afforded compounds 39 and 40. Possible
mechanism was shown in Scheme 5. Compound 38 reacted with
4-fluorobenzylamine to give decarboxylated compound 39. Addi-
tion of water to 39 gave intermediate 52. Rearrangement of 52
afforded intermediate 53. Elimination the benzylamino group of 53
gave compound 40.
Compounds 41e44 (Fig. 2) are commercially available. The
preparation of substituted hydrazine compounds 45e51 are shown
in Scheme 6. tert-Butylcarbazate [24] was firstly coupled with
corresponding aldehydes, then the resulting hydrazones were
reduced by Pd/C H2 or NaBH3CN to give corresponding Boc-
protected hydrazines. After N-Boc deprotection, hydrazine hydro-
chlorides 45e51 were obtained.
microplate reader (Molecular Devices) with a reference wave
length at 690 nm.
2.2.2. Drug screening
The cell-based anti-HIV activity of the synthesized compounds
was tested using our previously reported method [26]. Compounds
were prepared from initial dimethyl sulfoxide (DMSO) stocks and
plated as described. TZM-bl cells were seeded at concentration of
104 cells/well. The following day, samples and controls (AZT) were
then added. Finally, the pseudotyped virus was added with final
concentration of DMSO in all wells was maintained at 2%. The plates
were incubated at 37 ꢀC in a humidified CO2 incubator for 48 h.
Steady-glow substrate (Promega) was added directly to each well
and cell lysis was allowed to proceed in the dark for 20 min.
Luciferase activity was measured using the Envision microplate
luminometer (PerkinElmer).
2.2.3. Biological evaluation of synthesized compounds against HIV
The first toxic testing toward TZM-bl cells indicated that all
the synthesized compounds showed no toxic to TZM-bl cells at
2.2. Anti-HIV activity
10
m
M.
2.2.1. Cellular cytotoxicity screening
The antiviral activity of the synthesized compounds at 10
m
M
The cytotoxicity of synthesized compounds was evaluated
by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay method [25]. Briefly, TZM-bl cells were plated in
96-well plates and incubated for 18 h at 37 ꢀC, followed by a second
incubation for 48 h in the presence of the test compounds. MTT
was evaluated by using the above cell-based method. The
commercial MK-0518 and AZT were used as positive control. As
shown in Table 1, most of the synthesized compounds exhibited
anti-HIV activity, of which the rearranged compound 35 with 53.5%
inhibitory effect emerged as new lead compound.
(20 mL at 5 mg/mL) was added to each well and incubated for 3 h at
37 ꢀC. The plates were placed on a shaker for 10 min at room
temperature and the OD550 was measured using a Spectra MAX340
The compounds with a hydroxyl at 5-position and with
hydrogen or ester group at 4-position showed nearly no activity
OH
H3N
O
O
OH
HN
NH2
O
10% HCl aq.
88%
42
MeO2C
F3C
MeO2C
O
MeO2C
F3C
F
CO2Me
11 equiv.
N
Ether
47%
N
N
F C
N
41%
3
N
F3C
N
O
F
F
F
3
13
14
15
K2CO3
CH3I
95%
O
OH
OMe
O
HN
F
O
MeO2C
F3C
O
O
N
H
NH2
N
H
N
F
N
N
N
F
F3C
N
F
F
F3C
N
29%
11 equiv.
41%
F
F
17
18
16
Scheme 3. Synthesis of compounds 17 and 18.

36
Z. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 33e41
OMe
O
OH
F
O
HN
N
MeO2C
1) RNHNH2 HCl
O
F
NH2
11 equiv.
R
OH
HN
O
NH
O
R
O
R
O
N
H
MeO2C
F3C
O
R
N
H
N
CO2Me
F
F3C
2) K2CO3, MeI
N
or
F
N
or
F3C
N
or
F
F3C
N
N
N
3
F3C
R
N
F3C
N
OH
O
OH
OMe
O
OMe
MeO2C
F3C
O
O
F
MeO2C
F3C
F
MeO2C
F3C
O
N
H
N
N
N
N
N
N
N
N
O
F
F3C
N
20
N
19
21
22
OH
OH
O
O
OMe
OMe
O
MeO2C
F3C
O
MeO2C
F3C
O
N
H
N
H
F3C
N
N
N
N
N
24
N
N
N
F
F3C
N
25
F
F
N
23
N
O
26
OMe
O
OH
O
OH
OMe
MeO2C
F3C
O
O
O
O
MeO2C
F3C
O
N
H
N
H
N
N
N
N
N
O
O
F
O
S
N
28
F
F3C
S
F3C
N
29
N
27
30
O
OH
O
OH
OMe
O
HN
F
O
MeO2C
F3C
O
O
N
H
N
H
N
N
H
N
N
N
33
N
F
F3C
N
F3C
N
S
F
F
F3C
N
32
OMe
31
34
O
HN
O
OH
MeO2C
F3C
O
O
O
N
H
F3C
N
H
N
N
N
N
F
N
F
F3C
N
35
36
37
NH
OMe
OH
F
O
MeO2C
F3C
O
O
N
N
N
N
N
F3C
F3C
N
38
39
40
Scheme 4. Synthesis of compounds 19e40.
against HIV (inhibitory effect: 5, 14, 21, 6, 15, 40 < 7%). Main-
taining ester group at 4-position and methoxyl at 5-position,
a series of aryl group, benzyl group and alkyl group were
respectively introduced to the 1-position of pyridazines in order
to promote the antiviral activity. The results showed that 1-
(furan-2-yl)methyl (28), methyl (33), cinnamal (36) or t-butyl
(38) pyridazines didn’t exhibit any antiviral activity, but 1-aryl
pyridazines 7 and 16 and 1-benzyl pyridazines 19, 22, 24 and
30 and 1-(tetrahydrofuran-2-yl)methyl pyridazine 26 showed
relatively higher antiviral activity. Maintaining amide group at 4-
position and hydroxyl at 5-position, a series of aryl group, benzyl
group and alkyl group were respectively introduced to the 1-
position of pyridazines in order to promote the antiviral
activity. The 1-aryl pyridazines 9 and 17 and 1-benzyl pyridazine
20 exhibited relatively higher antiviral activity. Interestly, the
rearranged compounds 18, 32, 35 and 39 with a benzylamino
group at 5-position showed good antiviral activity, which indi-
cates that the benzylamino group at 5-position is favorable for
antiviral activity.
2.3. Anti-TMV activity
As the pyridazinone derivatives also were reported to possess
anti-TMV activity. The synthesized new structural compounds also
were evaluated for their anti-TMV activity using our previously
reported method [27]. The commercial plant virucide Ribavirin was
used as the control.
The primary in vitro bioassay results indicated that most of the
tested compounds possess good in vitro anti-TMV activity (Table 2),
of which compound 9 displayed similar inhibitory effect to
commercial plant virucide Ribavirin. Therefore, these compounds
were further tested to investigate their antiviral activity in vivo.
As shown in Table 2, most of the tested compounds also
exhibited good in vivo anti-TMV activity. As the control, Ribavirin
showed 34.2% inactivation effect, 32.7% curative effect and 39.6%
protection effect. Compounds 9, 18 and 34 exhibited similar in vivo
activity to Ribavirin.
The compounds with
hydrogen or ester group at 4-position showed moderate anti-TMV
a hydroxyl at 5-position and with
F
F
OMe
OH
NH
HN
HN
HO
F
MeO2C
F3C
O
O
O
O
O
H+
rt. to reflux
N
N
N
N
N
N
F3C
HO
F3C
F3C
N
F3C
N
4- fluo robenzyl
amine
N
H
N
H
40
52
39
53
38
Scheme 5. Possible mechanism.

Z. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 33e41
37
H
N
million using the solvent peak. Elemental analyses were deter-
mined on a Yanaco C, H, N Corder MT-3 elemental analyzer.
High-resolution mass spectra were obtained with an FT-ICR MS
spectrometer (Ionspec, 7.0 T). All anhydrous solvents were dried
and purified by standard techniques just before use.
H
N
H
N
NH2
HCl
NH2
NH2
N
H
NH2H2SO4
F
41
42
43
44
Fig. 2. Structures of compounds 41e44.
4.2. Dimethyl 2-(isopropylamino)fumarate (2)
activity. Maintaining ester group at 4-position and methoxyl at
5-position, a series of aryl group, benzyl group and alkyl group
were respectively introduced to the 1-position of pyridazines. The
bioassay results indicated that most of these compounds exhibited
good anti-TMV activity except for compounds 22, 28 and 38, which
showed nearly no activity against TMV. Maintaining amide group at
4-position and hydroxyl at 5-position, a series of aryl group, ben-
zyl group and alkyl group were respectively introduced to the
1-position of pyridazines. These compounds exhibited good to
excellent anti-TMV activity. The rearranged compounds 18, 32, 35
and 40 also showed good in vivo anti-TMV activity.
To a stirred solution of dimethyl acetylenedicarboxylate (5.0 g,
35 mmol) in CH2Cl2 (30 mL) was added 2-propanamine (2.1 g,
36 mmol) at 0 ꢀC under N2. The mixture was allowed to warm to
room temperature for 6 h, and then concentrated in vacuo. The
residue was purified by column chromatography on silica gel
(CH2Cl2) to afford compound 2 (6.8 g, 96% yield) as a slight yellow
oil. 1H NMR (400 MHz, CDCl3):
d 7.96 (brs, 1H, NH), 5.01 (s, 1H, CCH),
3.88e3.92 (m, 1H, NHCH), 3.84 (s, 3H, OMe), 3.67 (s, 3H, OMe), 1.21
(s, 3H, CH(CH3)2), 1.20 (s, 3H, CH(CH3)2).
4.3. Dimethyl 2-[(1-Methylethyl)amino]-3-(trifluoroacetyl)-2-
butenedioate (3)
3. Conclusion
To a stirred solution of compound 2 (6.3 g, 31 mmol) in ether
(63 mL) was added dropwise trifluoroacetic anhydride (6.7 g,
32 mmol) at 0 ꢀC under N2. The mixture was allowed to warm to room
temperature for 5 h, then washed with H2O (50 mL) and brine
(50 mL), dried with anhydrous MgSO4 and concentrated in vacuo. The
residue was purified by column chromatography on silica gel (CH2Cl2
as eluent) to give compound 3 (8.7 g, 94% yield) as a slight yellow
solid. Mp 55e57 ꢀC, (lit. [23] mp 46e49 ꢀC); 1H NMR (400 MHz,
A series of novel structural pyridazines have been prepared as
HIV-1 inhibitors. The synthetic route involved a novel rearrange-
ment reaction. Bioassay results indicated that most of the pyr-
idazines possess anti-HIV activity, of which the rearranged
compounds 35 and 39 exhibited relatively higher HIV inhibition
rate. In addition, the synthesized compounds were also evaluated
for their anti-TMV activity. Most of the tested compounds exhibited
good anti-TMV activity, especially for compound 9 showed similar
in vivo anti-TMV activity to commercial plant virucide Ribavirin.
This work provides a novel approach to develop multi-functional
antiviral agents from previously reported agents.
CDCl3):
d 11.39 (brs, 0.7H, major isomer, NH), 10.21 (brs, 0.3H, minor
isomer, NH), 3.96 (s, 2.1H, major isomer, OMe), 3.95 (s, 0.9H, minor
isomer, OMe), 3.80 (s, 0.8H, minor isomer, OMe), 3.74 (s, 2.2H, major
isomer, OMe), 3.61e3.72 (m, 1H, NHCH), 1.31e1.34 (t, 6H, CH(CH3)2).
4. Experimental
4.4. General procedure for the preparation of compounds 4 and 13
4.1. General
To a stirred solution of compound 3 (3.0 g, 10 mmol) in ether
(50 mL) was added dropwise phenylhydrazine (41) or 4-
fluorophenylhydrazine (42) (10 mmol), and then the mixture was
stirred for 10 h. Then the mixture was stayed at 0 ꢀC for 30 min, and
filtered to afford compound 4 or 13.
The melting points were determined with an X-4 binocular
microscope melting-point apparatus (Beijing Tech Instruments Co.,
Beijing, China) and were uncorrected. 1H NMR spectra were
obtained by using Bruker AV 400, Bruker AV300 and a Varian
Mercury Plus 400 MHz spectrometer. Chemical shifts (
d) were given
4.4.1. Methyl 1,6-dihydro-5-hydroxy-6-oxo-1-phenyl-3-
in parts per million (ppm) and measured downfield from internal
tetramethylsilane. 13C NMR spectra were recorded by using Bruker
AV 400 (100 MHz) and Bruker AV300 (75 MHz) with CDCl3 or
(trifluoromethyl)-4-pyridazinecarboxylate, isopropylamine salt (4)
White solid, 59% yield; mp 167e168 ꢀC (lit. [23] mp 169e170 ꢀC);
1H NMR (400 MHz, D2O):
d
7.49 (s, 5H, ArH), 3.79 (s, 3H, CO2CH3),
DMSO-d6 as a solvent. Chemical shifts (d) are reported in parts per
3.35e3.42 (m, 1H, NCH(CH3)2), 1.19 (d, J ¼ 6.5 Hz, 6H, NCH(CH3)2).
NH2NH2 H2O
Boc2O
97%
BocNHNH2
H
N
H
N
Pd/C, H2
HCl
N
NH
2HCl
NHBoc
NHBoc
R
R
R
RCHO
or NaBH3CN
NH2
NH
NH
HCl
2 HCl
2 HCl
N
H
N
N
H
H
O
N
HN NH
2 HCl
F
N
45
46
47
48
H
N
O
HN NH
NH
2 HCl
S
2 HCl
HN NH
2 HCl
49
50
51
Scheme 6. Synthesis of compounds 45e51.

38
Z. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 33e41
Table 1
4.5.2. Methyl 1,6-dihydro-5-hydroxy-6-oxo-1-(4-fluorophenyl)-3-
(trifluoromethyl)-4-pyridazinecarboxylate (14)
Inhibition activity of synthesized compounds 5e7, 9 and 14e40 toward HIV.
White solid, 88% yield; mp 129e131 ꢀC; 1H NMR (400 MHz,
Compd. Inhibition(%) Compd. Inhibition(%) Compd. Inhibition(%)
CDCl3):
d
¼ 7.36 (dd, J ¼ 4.6, 9.0 Hz, 2H, ArH), 7.19 (t, J ¼ 8.1 Hz, 2H,
5
6
7
9
14
15
16
17
18
19
20
ꢁ0.75
6.33
5.90
27.95
1.30
21
22
23
24
25
26
27
28
29
30
31
5.41
20.16
2.99
19.50
7.31
9.30
ꢁ3.02
ꢁ3.39
13
32
33
34
35
36
37
38
39
12.7
ꢁ4
ArH), 4.11 (s, 3H, CO2CH3); Anal. Calcd. for C13H8F4N2O4: C, 47.00; H,
2.43; N, 8.43. Found: C, 47.11; H, 2.47; N, 8.37.
ꢁ3.5
53.5
ꢁ19.4
ꢁ27.2
ꢁ6.5
41.9
ꢁ11.6
95.07
100
4.5.3. Methyl 1,6-dihydro-5-hydroxy-6-oxo-1-(pyridin-2-
ylmethyl)-3-(trifluoromethyl)-4-pyridazinecarboxylate (21)
White solid, 47% yield; mp 151e153 ꢀC; 1H NMR (400 MHz,
ꢁ11.61
7.34
19.53
19.53
13.87
29.34
40
MK-0518
AZT
CDCl3):
d
10.12 (brs, 1H, OH), 8.57 (d, J ¼ 4.4 Hz, 1H, NCHCH),
37
ꢁ7.5
7.79e7.83 (m, 1H, NCCHCH), 7.33e7.38 (m, 2H, NCCHCHCH), 6.02 (s,
2H, NCH2), 4.00 (s, 3H, CO2CH3); HRMS (ESI) calcd for C13H9F3N3O4
(MeH)ꢁ: 328.0551, found: 328.0549.
Bold value represents the good activity of compound 35.
4.4.2. Methyl 1,6-dihydro-5-hydroxy-6-oxo-1-(4-fluorophenyl)-3-
(trifluoromethyl)-4-pyridazinecarboxylate, isopropylamine salt (13)
White solid, 47% yield; mp 182e184 ꢀC; 1H NMR (400 MHz,
4.6. 4-Hydroxy-2-phenyl-6-(trifluoromethyl)pyridazin-3(2H)-one (6)
The solution of compound
5 (0.3 g, 1 mmol) and 4-
D2O):
d
7.49 (dd, J ¼ 4.7, 8.8 Hz, 2H, ArH), 7.22 (t, J ¼ 8.6 Hz, 2H, ArH),
fluorobenzylamine (0.3 g, 3 mmol) in methanol (30 mL) was
refluxed for 5 h, then cooled to room temperature and concentrated
in vacuo. The residue was taken into 5% KOH solution (30 mL),
washed with ether (2 ꢂ 10 mL), then acidified with 10% HCl to about
pH ¼ 2e3, filtered to afford compound 6 as a white solid (0.2 g, 91%
3.78 (s, 3H, CO2CH3), 3.35e3.42 (m, 1H, NCH(CH3)2), 1.19 (d,
J ¼ 6.6 Hz, 6H, NCH(CH3)2).
4.5. General procedurefor the preparation of compounds 5,14 and 21
yield). Mp 215e216 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 7.58 (s, 5H,
ArH); HRMS (ESI) calcd for C11H6F3N2O2 (MeH)ꢁ: 255.0387, found:
The solution of corresponding isopropylamine salt in water was
acidified with 10% HCl to about pH ¼ 2e3, then stirred at 0 ꢀC for
1 h. The mixture was filtered to afford compounds 5, 14 and 21.
255.0387.
4.7. Generalprocedureforthe preparation ofcompounds 7,16 and 22
4.5.1. Methyl 1,6-dihydro-5-hydroxy-6-oxo-1-phenyl-3-
(trifluoromethyl)-4-pyridazinecarboxylate (5)
The solution of compounds 5, 14 or 21 (0.6 mmol) and anhy-
drous K2CO3 (0.4 g, 3 mmol) in acetone (40 mL) was stirred for
30 min, then iodomethane (0.2 g, 1.3 mmol) was added. The
mixture was refluxed for 36 h, then cooled to room temperature
and concentrated in vacuo. The residue was taken into ethyl acetate
(80 mL), and washed with saturated aq NaHCO3 solution (50 mL)
and brine (50 mL), then dried with anhydrous MgSO4 and
concentrated in vacuo. The residue was purified by column chro-
matography on silica gel to afford compounds 7, 16 and 22.
White solid, 87% yield; mp 126e127 ꢀC, (lit. [23] mp
127e128 ꢀC); 1H NMR (400 MHz, DMSO-d6):
(s, 3H, CO2CH3).
d 7.60 (d, 5H, ArH), 3.85
Table 2
Anti-TMV activity of synthesized compounds 5e7, 9, 16e23, 25e28 and 30e40.
Compd.
500 mg/mL
In vitro inhibitory
In vivo
effect (%)
Inactivation
effect (%)
Curative
effect (%)
Protection
effect (%)
4.7.1. Methyl 1,6-dihydro-5-methoxy-6-oxo-1-phenyl-3-
(trifluoromethyl)-4-pyridazinecarboxylate (7)
5
6
7
9
15.5
0
20
38.5
22.5
18.6
28.5
27.5
30
21.3
0
10
7.5
13.6
0
8.5
12.4
0
18.9
0
16.5
36.7
7.5
10.3
30.2
11.2
21.4
18.3
0
10.7
14.8
12.5
30.2
18.6
17.4
34
10.3
21.7
9.3
0
7.6
12.8
0
0
11.4
7.6
White solid, 90% yield; mp 81e83 ꢀC, (lit. [23] mp 64e65 ꢀC); 1H
NMR (400 MHz, CDCl3):
d 7.50 (s, 5H, ArH), 3.92 (s, 3H, OMe), 3.87
19.2
32.6
11.7
15.6
31.3
20.2
28.3
16.7
15.2
18.6
18.9
10.3
0
(s, 3H, CO2CH3).
16
17
18
19
20
21
22
23
25
26
27
28
30
31
32
33
34
35
36
37
38
39
40
Ribavirin
4.7.2. Methyl 1,6-dihydro-5-methoxy-6-oxo-1-(4-fluorophenyl)-3-
(trifluoromethyl)-4-pyridazinecarboxylate (16)
Whitesolid, 95%yield;mp 99e100 ꢀC; 1HNMR(400 MHz, CDCl3):
d
7.49 (dd, J ¼ 4.6, 9.0 Hz, 2H, ArH), 7.19 (t, J ¼ 8.8 Hz, 2H, ArH), 3.92 (s,
3H, OMe), 3.87 (s, 3H, CO2CH3); Anal. Calcd. for C14H10F4N2O4: C,
5.2
48.57; H, 2.91; N, 8.09. Found: C, 48.62; H, 2.92; N, 8.09.
18.2
19.7
13.2
0
15.1
0
19.5
27.3
25.4
23.9
23.7
0
4.7.3. Methyl 1,6-dihydro-5-methoxy-6-oxo-1-(pyridin-2-
7.2
0
10
0
ylmethyl)-3-(trifluoromethyl)-4-pyridazinecarboxylate (22)
Slight yellow oil, 25%; 1H NMR (400 MHz, CDCl3):
d 8.53 (d,
19.5
14.2
23.6
26.5
28.4
11.3
29.8
10.2
6.3
J ¼ 5.4 Hz, 1H, NCH), 7.64e7.68 (m, 1H, NCCHCH), 7.22 (dd, J ¼ 5.0,
6.9 Hz, 1H, NCHCHCH), 7.06 (d, J ¼ 7.8 Hz, 1H, NCCHCHCH), 5.80 (s,
2H, NCH2), 3.90 (s, 3H, OMe), 3.88 (s, 3H, CO2CH3); HRMS (ESI) calcd
for C14H13F3N3O4 (M þ H)þ: 344.0853, found: 344.0856.
30
25
24.7
20.5
32.8
17.5
22.9
12.2
5.7
30.2
23.1
39.6
19.6
8.6
20.3
18.4
0
26.2
31.7
38.5
4.8. General procedure for the preparation of compounds 19, 24, 26,
28, 30, 33, 36, and 38
0
15.3
18.6
34.2
20.2
27.2
32.7
To a stirred solution of compound 3 (17 mmol) and anhydrous
Bold value represents the good activity of compound 9.
K2CO3 (34 mmol) in ether (180 mL) was added corresponding

Z. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 33e41
39
hydrazine hydrochlorides 43e45 or 47e51 (17 mmol) respectively,
and stirred for 10 h, then extracted with water (150 mL). The
aqueous layer was acidified with 10% HCl to about pH ¼ 2e3 at 0 ꢀC,
then extracted with ethyl acetate (2 ꢂ 150 mL). The extract was
dried with anhydrous MgSO4 and concentrated in vacuo. The
residue was taken into acetone (200 mL), then anhydrous K2CO3
(84 mmol) was added. The mixture was stirred for 30 min, then
added iodomethane (34 mmol), and refluxed for 24 h, then cooled
to room temperature and concentrated in vacuo. The residue was
taken into ethyl acetate (180 mL), and washed with saturated aq
NaHCO3 solution (100 mL) and brine (100 mL), then dried with
anhydrous MgSO4 and concentrated in vacuo. The residue was
purified by column chromatography on silica gel to afford target
compounds 19, 24, 26, 28, 30, 33, 36, and 38.
(m, 1H, ArCHCH), 5.23 (d, J ¼ 7.5 Hz, 2H, NCH2), 3.91 (s, 3H, OMe),
3.89 (s 3H, CO2CH3); HRMS (ESI) calcd for C17H15F3N2NaO4
(M þ Na)þ: 391.0876, found: 391.0871.
4.8.8. Methyl 1-tert-butyl-5-methoxy-6-oxo-3-(trifluoromethyl)-
1,6-dihydropyridazine-4-carboxylate (38)
White solid, 57% yield; mp 70e71 ꢀC; 1H NMR (400 MHz, CDCl3):
d
3.99 (s, 3H, OMe), 3.85 (s, 3H, CO2CH3), 1.64 (s, 9H, C(CH3)3); Anal.
Calcd. for C12H15F3N2O4: C, 46.76; H, 4.90; N, 9.09. Found: C,46.46;
H,4.79; N,8.86.
4.9. General procedure for the preparation of compounds 9, 15, 17,
18, 20, 23, 25, 27, 29, 31, 32, 34, 35, 37, 39 and 40
The mixture of compounds 7, 14, 16, 19, 22, 24, 26, 28, 30, 33, 36
or 38 (2 mmol) and 4-fluorobenzylamine (20 mmol) was stirred at
room temperature for 10 h, then refluxed for 1 h. The mixture was
cooled to room temperature, and added 5% HCl (60 mL), then
extracted with ethyl acetate (3 ꢂ 50 mL). The extract was washed
with water (50 mL) and brine (50 mL), then dried with anhydrous
MgSO4 and concentrated in vacuo. The residue was taken into 10%
NaOH (180 mL), and extracted with CH2Cl2 (3 ꢂ 50 mL). The CH2Cl2
layer was dried with anhydrous MgSO4 and concentrated in vacuo,
then purified by column chromatography on silica gel to afford
compounds 18, 32, 35 and 39. The aqueous layer was acidified with
10% HCl to about pH ¼ 2e3 at 0 ꢀC, and filtered to give compounds
9, 15, 17, 20, 23, 25, 27, 29, 31, 34, 37 and 40.
4.8.1. Methyl 1-(4-fluorobenzyl)-5-methoxy-6-oxo-3-
(trifluoromethyl)-1,6-dihydropyridazine-4-carboxylate (19)
Slight yellow oil, 42% yield; 1H NMR (400 MHz, CDCl3):
d 7.21
(dd, J ¼ 5.4, 8.5 Hz, 2H, ArH), 6.94 (t, J ¼ 8.6 Hz, 2H, ArH), 5.54 (s, 2H,
ArCH2), 3.80 (s, 6H, OMe); HRMS (ESI) calcd for C15H12F4N2NaO4
(M þ Na)þ: 383.0625, found: 383.0618.
4.8.2. Methyl 5-methoxy-6-oxo-1-(pyridin-3-ylmethyl)-3-
(trifluoromethyl)-1,6-dihydropyridazine-4-carboxylate (24)
Slight red oil, 20% yield; 1H NMR (400 MHz, CDCl3):
d 8.63 (d,
J ¼ 2.1 Hz, 1H, NCHC), 8.58 (dd, J ¼ 1.3, 4.8 Hz, 1H, NCH), 7.63e7.68
(m, 1H, NCHCH), 7.29 (dd, J ¼ 4.8, 7.9 Hz, 1H, NCHCCH), 5.70 (s, 2H,
NCH2), 3.90 (s, 3H, OMe), 3.89 (s 3H, CO2CH3); HRMS (ESI) calcd for
C14H13F3N3O4 (M þ H)þ: 344.0853, found: 344.0852.
4.9.1. N-(4-Fluorobenzyl)-5-hydroxy-6-oxo-1-phenyl-3-
(trifluoromethyl)-1,6-dihydropyridazine-4-carboxamide (9)
White solid, 44% yield; mp 250e252 ꢀC; 1H NMR (400 MHz,
4.8.3. Methyl 5-methoxy-6-oxo-1-((tetrahydrofuran-2-yl)methyl)-
3-(trifluoromethyl)-1,6-dihydropyridazine-4-carboxylate (26)
DMSO-d6):
d 13.45 (brs, 1H, OH), 9.54 (brs, 1H, NH), 7.48e7.60 (m,
Slight yellow oil, 53% yield; 1H NMR (400 MHz, CDCl3):
d 4.53 (t,
5H, ArH), 7.14 (dd, J ¼ 5.7, 8.4 Hz, 2H, ArH), 7.06 (t, J ¼ 8.9 Hz, 2H,
J ¼ 2.7 Hz, 2H, OCH2), 4.21e4.27 (m, 1H, OCHCH2), 3.94 (s, 3H, OMe),
3.88 (s 3H, CO2CH3), 3.70e3.74 (m, 2H, NCH2), 1.98e2.07 (m, 1H,
OCHCH2), 1.81e1.91 (m, 1H, OCHCH2), 1.69e1.79 (m, 1H, OCH2CH2),
1.58e1.66 (m, 1H, OCH2CH2); HRMS (ESI) calcd for C13H16F3N2O5
(M þ H)þ: 337.1006, found: 337.1003.
ArH), 4.34 (d, J ¼ 5.5 Hz, 2H, ArCH2); 13C NMR (100 MHz, DMSO-d6):
d
162.4, 161.1, 160.0, 158.5, 143.7, 140.5, 140.2, 137.8, 134.1, 129.7,
129.4, 129.3, 129.3, 129.2, 124.4, 121.8, 119.1, 115.0, 114.8, 112.2, 41.7;
Anal. Calcd. for C19H13F4N3O3: C, 56.03; H, 3.22; N, 10.32. Found: C,
55.98; H, 3.20; N, 10.38.
4.8.4. Methyl 1-(furan-2-ylmethyl)-5-methoxy-6-oxo-3-
4.9.2. 2-(4-Fluorophenyl)-4-hydroxy-6-(trifluoromethyl)pyridazin-
3(2H)-one (15)
(trifluoromethyl)-1,6-dihydropyridazine-4-carboxylate (28)
Slight yellow oil, 61% yield; 1H NMR (400 MHz, CDCl3):
d 7.37 (s,
White solid, 41% yield; mp 193e194 ꢀC; 1H NMR (400 MHz,
1H, OCH), 6.40 (d, J ¼ 3.1 Hz, 1H, OCCH), 6.33 (dd, J ¼ 1.8, 3.1 Hz, 1H,
OCHCH), 5.67 (s, 2H, NCH2), 3.94 (s, 3H, OMe), 3.88 (s 3H, CO2CH3);
HRMS (ESI) calcd for C13H12F3N2O5 (M þ H)þ: 333.0693, found:
333.0686.
CDCl3):
d
8.50 (s, 1H, NCCHC), 7.72 (dd, J ¼ 4.4, 8.5 Hz, 2H, ArH), 7.23
(t, J ¼ 8.4 Hz, 2H, ArH); HRMS (ESI) calcd for C11H5F4N2O2 (MeH)ꢁ:
273.0293, found: 273.0290.
4.9.3. N-(4-Fluorobenzyl)-1-(4-fluorophenyl)-5-hydroxy-6-oxo-3-
(trifluoromethyl)-1,6-dihydropyridazine-4-carboxamide (17)
White solid, 41% yield; mp 260e262 ꢀC; 1H NMR (400 MHz,
4.8.5. Methyl 5-methoxy-6-oxo-1-(thiophen-2-ylmethyl)-3-
(trifluoromethyl)-1,6-dihydropyridazine-4-carboxylate (30)
Slight yellow oil, 54% yield; 1H NMR (400 MHz, CDCl3):
d 7.28
DMSO-d6):
7.34e7.37(m, 2H, ArH), 7.06e7.15(m, 4H, ArH), 4.33(s, 2H, ArCH2); 13
NMR (100 MHz, DMSO-d6): 163.8, 163.5, 162.4, 161.0, 160.6, 160.0,
d13.49(s,1H, OH), 9.50(s,1H, NH), 7.50e7.58(m,2H, ArH),
(dd, J ¼ 1.2, 5.1 Hz, 1H, SCH), 7.12 (d, J ¼ 4.3 Hz, 1H, SCCH), 6.95 (dd,
J ¼ 3.6, 5.1 Hz, 1H, SCHCH), 5.84 (s, 2H, NCH2), 3.94 (s, 3H, OMe),
3.88 (s 3H, CO2CH3); HRMS (ESI) calcd for C13H11F3N2NaO4S
(M þ Na)þ: 371.0284, found: 371.0278.
C
d
159.5, 158.3, 157.8, 156.0, 144.0, 134.1, 134.0, 129.3, 129.2, 127.1, 127.0,
116.5,116.3,115.0,114.8, 41.7; Anal. Calcd. for C19H12F5N3O3: C, 53.66;
H, 2.84; N, 9.88. Found: C, 53.84; H, 2.99; N, 10.11.
4.8.6. Methyl 5-methoxy-1-methyl-6-oxo-3-(trifluoromethyl)-1,6-
dihydropyridazine-4-carboxylate (33)
White solid, 49% yield; mp 45e47 ꢀC; 1H NMR (300 MHz,
4.9.4. N-(4-Fluorobenzyl)-5-(4-fluorobenzylamino)-1-(4-
fluorophenyl)-6-oxo-3-(trifluoromethyl)-1,6-dihydropyridazine-4-
carboxamide (18)
CDCl3):
d 4.16 (s, 3H, OMe), 3.96 (s, 3H, CO2CH3), 3.91 (s 3H, NMe);
Anal. Calcd. for C9H9F3N2O4: C, 40.61; H, 3.41; N, 10.52. Found: C,
40.55; H, 3.36; N, 10.63.
White solid, 29% yield; mp 245e247 ꢀC; 1H NMR (400 MHz,
CDCl3):
d
8.9 (d, J ¼ 0.9 Hz,1H, NH), 7.37 (dd, J ¼ 4.7, 7.9 Hz, 2H, ArH),
4.8.7. Methyl 1-cinnamyl-5-methoxy-6-oxo-3-(trifluoromethyl)-
1,6-dihydropyridazine-4-carboxylate (36)
7.21e7.29 (m, 4H, ArH), 7.14 (t, J ¼ 8.1 Hz, 2H, ArH), 6.97e7.06 (m,
4H, ArH), 6.56 (d, J ¼ 0.9 Hz, 1H, NH), 4.55 (d, J ¼ 5.4 Hz, 2H, ArCH2),
4.45 (d, J ¼ 5.6 Hz, 2H, ArCH2); HRMS (ESI) calcd for C26H17F6N4O2
(MeH)ꢁ: 531.1261, found: 531.1258.
Slight yellow oil, 53% yield; 1H NMR (400 MHz, CDCl3):
d
7.26e7.38 (m, 5H, ArH), 6.63 (d, J ¼ 15.8 Hz, 1H, ArCH), 6.26e6.33

40
Z. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 33e41
4.9.5. N,1-Bis(4-fluorobenzyl)-5-hydroxy-6-oxo-3-(trifluoromethyl)-
1,6-dihydropyridazine-4-carboxamide (20)
J ¼ 3.0 Hz, 1H, SCCH), 6.90e7.04 (m, 4H, ArH), 6.92 (t, J ¼ 4.5 Hz, 1H,
SCHCH), 6.39 (s, 1H, NH), 6.00 (s, 2H, NNCH2), 4.54 (d, J ¼ 5.8 Hz, 2H,
ArCH2), 4.51 (d, J ¼ 5.7 Hz, 2H, ArCH2); 13C NMR (100 MHz, CDCl3):
White solid, 39% yield; mp 218e219 ꢀC; 1H NMR (400 MHz,
DMSO-d6):
d
13.41 (brs, 1H, OH), 9.52 (s, 1H, NH), 7.35 (dd, J ¼ 5.7,
d 163.7, 163.4, 162.8, 161.2, 161.0, 158.1, 137.0, 137.0, 133.3, 132.2,
8.6 Hz, 2H, ArH), 7.27 (dd, J ¼ 5.6, 8.7 Hz, 2H, ArH), 7.17 (t, J ¼ 8.9 Hz,
2H, ArH), 7.13 (t, J ¼ 7.3 Hz, 2H, ArH), 5.38 (s, 2H, ArCH2), 4.43 (d,
132.2, 129.6, 129.6, 129.5, 129.4, 128.4, 126.8, 115.9, 115.7, 115.6,
115.4, 115.2, 50.9, 44.0, 42.9; HRMS (ESI) calcd for C25H18F5N4O2S
(MeH)ꢁ: 533.1076, found: 533.1070.
J ¼ 5.8 Hz, 2H, ArCH2); 13C NMR (100 MHz, DMSO-d6):
d 163.1,162.5,
161.2, 160.7, 160.1, 158.5, 143.0, 139.3, 134.2, 131.4, 130.2, 130.2,
129.5, 129.4, 121.7, 119.1, 115.6, 115.3, 115.0, 114.8, 111.5, 53.4, 42.1;
Anal. Calcd. for C20H14F5N3O3: C, 54.68; H, 3.21; N, 9.56. Found: C,
54.39; H, 3.27; N, 9.79.
4.9.12. N-(4-Fluorobenzyl)-5-hydroxy-1-methyl-6-oxo-3-
(trifluoromethyl)-1,6-dihydropyridazine-4-carboxamide (34)
White solid, 42% yield; mp 219e221 ꢀC; 1H NMR (400 MHz,
DMSO-d6):
d
13.31 (brs, 1H, OH), 9.49 (s, 1H, NH), 7.42 (dd, J ¼ 5.7,
4.9.6. N-(4-Fluorobenzyl)-5-hydroxy-6-oxo-1-(pyridin-2-ylmethyl)-
3-(trifluoromethyl)-1,6-dihydropyridazine-4-carboxamide (23)
White solid, 44% yield; mp 180e182 ꢀC; 1H NMR (400 MHz,
8.6 Hz, 2H, ArH), 7.16 (t, J ¼ 8.9 Hz, 2H, ArH), 4.47 (d, J ¼ 5.8 Hz, 2H,
ArCH2), 3.86 (s, 3H, NMe); HRMS (ESI) calcd for C14H10F4N3O3
(MeH)ꢁ: 344.0664, found: 344.0660.
DMSO-d6):
d
13.38, (brs,1H, OH), 9.64 (s,1H, NH), 8.50 (d, J ¼ 4.3 Hz,
1H, NCH), 7.80 (t, J ¼ 7.7 Hz, 1H, NCCH), 7.34 (dd, J ¼ 5.1, 6.9 Hz, 1H,
NCHCH), 7.28 (dd, J ¼ 5.8, 8.2 Hz, 2H, ArH), 7.20 (d, J ¼ 7.8 Hz, 1H,
NCCHCH), 7.08 (t, J ¼ 8.8 Hz, 2H, ArH), 5.60 (s, 2H, ArCH2), 4.38 (d,
J ¼ 5.5 Hz, 2H, NNCH2); HRMS (ESI) calcd for C19H13F4N4O3 (MeH)ꢁ:
421.0929, found: 421.0920.
4.9.13. N-(4-Fluorobenzyl)-5-(4-fluorobenzylamino)-1-methyl-6-
oxo-3-(trifluoromethyl)-1,6-dihydropyridazine-4-carboxamide (35)
White solid, 15% yield; mp 181e183 ꢀC; 1H NMR (400 MHz,
CDCl3): d 9.62 (s, 1H, NH), 7.21e7.32 (m, 4H, ArH), 7.00e7.05 (m, 4H,
ArH), 6.38 (s, 1H, NH), 4.53 (d, J ¼ 5.6 Hz, 4H, ArCH2), 4.23 (s, 3H,
NMe); Anal. Calcd. for C21H17F5N4O2: C, 55.76; H, 3.79; N, 12.38.
Found: C, 55.51; H, 3.89; N, 12.32.
4.9.7. N-(4-Fluorobenzyl)-5-hydroxy-6-oxo-1-(pyridin-3-ylmethyl)-
3-(trifluoromethyl)-1,6-dihydropyridazine-4-carboxamide (25)
White solid, 31% yield; mp 279e281 ꢀC; 1H NMR (400 MHz,
4.9.14. 1-Cinnamyl-N-(4-fluorobenzyl)-5-hydroxy-6-oxo-3-
(trifluoromethyl)-1,6-dihydropyridazine-4-carboxamide (37)
White solid, 47% yield; mp 210e212 ꢀC; 1H NMR (400 MHz,
DMSO-d6):
d 13.45, (brs, 1H, OH), 9.63 (s, 1H, NH), 8.54 (s, 1H,
NCHC), 8.47 (s, 1H, NCHCH), 7.62 (dd, J ¼ 1.7, 5.7 Hz, 1H, NCHCH),
7.33e7.43 (m, 3H, ArH, NCHCCH), 7.13 (s, 2H, ArH), 5.47 (s, 2H,
ArCH2), 4.44 (s, 2H, NNCH2); HRMS (ESI) calcd for C19H13F4N4O3
(MeH)ꢁ: 421.0929, found: 421.0928.
DMSO-d6): d 13.33 (brs, 1H, OH), 9.54 (s, 1H, NH), 7.27e7.41 (m, 7H,
ArH), 7.06 (t, J ¼ 8.9 Hz, 2H, ArH), 6.54 (d, J ¼ 15.9 Hz, 1H, ArCHCH),
6.30e6.38 (m, 1H, ArCHCH), 5.00 (d, J ¼ 6.2 Hz, 2H, ArCH2), 4.44 (d,
J ¼ 5.8 Hz, 2H, NNCH2); HRMS (ESI) calcd for C22H16F4N3O3
(MeH)ꢁ: 446.1133, found: 446.1130.
4.9.8. N-(4-Fluorobenzyl)-5-hydroxy-6-oxo-1-((tetrahydrofuran-2-yl)
methyl)-3-(trifluoromethyl)-1,6-dihydropyridazine-4-carboxamide (27)
White solid, 33% yield; mp 216e217 ꢀC; 1H NMR (400 MHz,
4.9.15. 2-Tert-butyl-4-(4-fluorobenzylamino)-6-(trifluoromethyl)
pyridazin-3(2H)-one (39)
DMSO-d6):
d
13.26, (brs, 1H, OH), 9.52 (s, 1H, NH), 7.42 (dd, J ¼ 5.7,
8.5 Hz, 2H, ArH), 7.16 (t, J ¼ 8.9 Hz, 2H, ArH), 4.44 (d, J ¼ 5.7 Hz, 2H,
ArCH2), 4.12e4.27 (m, 3H, OCH, OCH2), 3.53e3.63 (m, 2H, NNCH2),
1.85e1.93 (m 1H, OCHCH2), 1.66e1.79 (m, 2H, OCHCH2, OCH2CH2),
1.52e1.60 (m, 1H, OCH2CH2); HRMS (ESI) calcd for C18H16F4N3O4
(MeH)ꢁ: 414.1082, found: 414.1086.
White solid, 39% yield; mp 184e186 ꢀC; 1H NMR (400 MHz,
DMSO-d6):
d
8.70 (t, J ¼ 5.7 Hz, 1H, NH), 8.57 (s, 1H, CF3CCH), 7.35
(dd, J ¼ 5.7, 8.5 Hz, 2H, ArH), 7.16 (t, J ¼ 8.9 Hz, 2H, ArH), 4.39 (d,
J ¼ 5.8 Hz, 2H, ArCH2), 1.55 (s, 9H, C(CH3)3); Anal. Calcd. for
C16H17F4N3O: C, 55.97; H, 4.99; N, 12.24. Found: C, 55.87; H, 5.12; N,
12.20.
4.9.9. N-(4-Fluorobenzyl)-1-(furan-2-ylmethyl)-5-hydroxy-6-oxo-
3-(trifluoromethyl)-1,6-dihydropyridazine-4-carboxamide (29)
White solid, 56% yield; mp 233e235 ꢀC; 1H NMR (400 MHz,
4.9.16. 2-Tert-butyl-4-hydroxy-6-(trifluoromethyl)pyridazin-
3(2H)-one (40)
DMSO-d6):
d 13.35 (brs, 1H, OH), 9.51 (s, 1H, NH), 7.63 (d,
White solid, 26% yield; mp 148e150 ꢀC; 1H NMR (400 MHz,
J ¼ 1.7 Hz, 1H, OCH), 7.40 (dd, J ¼ 5.7, 8.5 Hz, 2H, ArH), 7.15 (t,
J ¼ 8.9 Hz, 2H, ArH), 6.43 (dd, J ¼ 1.9, 3.2 Hz, 1H, OCHCH), 6.36 (d,
J ¼ 3.1 Hz, 1H, OCCH), 5.46 (s, 2H, ArCH2), 4.45 (d, J ¼ 5.8 Hz, 2H,
NNCH2); HRMS (ESI) calcd for C18H12F4N3O4 (MeH)ꢁ: 410.0769,
found: 410.0776.
DMSO-d6):
d 12.90 (brs, 1H, OH), 8.53 (s, 1H, NCCH), 1.56 (s, 9H,
C(CH3)3); Anal. Calcd. for C9H11F3N2O2: C, 45.77; H, 4.69; N, 11.86.
Found: C, 45.54; H, 4.45; N, 12.06.
4.10. General procedure for the preparation of compounds 45e48
4.9.10. N-(4-Fluorobenzyl)-5-hydroxy-6-oxo-1-(thiophen-2-ylmethyl)-
3-(trifluoromethyl)-1,6-dihydropyridazine-4-carboxamide (31)
White solid, 64% yield; mp 233e235 ꢀC; 1H NMR (400 MHz,
The solution of BocNHNH2 [24] (25 mmol) and corresponding
aldehydes (24 mmol) in methanol (60 mL) was refluxed for 1 h,
then cooled to room temperature, then added 10% Pd/C (0.8 g)
under H2. The mixture was stirred at room temperature for 10 h,
then filtered and concentrated in vacuo. The residue was taken into
ethanol (160 mL), and stirred at HCl atmosphere for 8 h, then
concentrated to give compounds 45e48.
DMSO-d6):
d
13.38 (brs, 1H, OH), 9.56 (s, 1H, NH), 7.52 (dd, J ¼ 1.2,
5.0 Hz, 1H, SCH), 7.39 (dd, J ¼ 5.8, 8.2 Hz, 2H, ArH), 7.15 (t, J ¼ 8.9 Hz,
2H, ArH), 6.91e7.02 (m, 2H, SCCHCH), 5.61 (s, 2H, ArCH2), 4.47 (d,
J ¼ 5.7 Hz, 2H, NNCH2); HRMS (ESI) calcd for C18H12F4N3O3S
(MeH)ꢁ: 426.0541, found: 426.0535.
4.9.11. N-(4-Fluorobenzyl)-5-(4-fluorobenzylamino)-6-oxo-1-
(thiophen-2-ylmethyl)-3-(trifluoromethyl)-1,6-dihydropyridazine-
4-carboxamide (32)
4.10.1. (4-Fluorobenzyl)hydrazine hydrochloride (45)
White solid, 60% yield; mp 149e150 ꢀC; 1H NMR (400 MHz,
White solid, 11% yield; mp 195e197 ꢀC; 1H NMR (400 MHz,
D2O):
d
7.39 (dd, J ¼ 5.6, 8.4 Hz, 2H, ArH), 7.11 (t, J ¼ 8.8 Hz, 2H, ArH),
CDCl3):
d 9.51 (s, 1H, NH), 7.21e7.27 (m, 5H, ArH, SCH), 7.12 (d,
4.17 (s, 2H, ArCH2).

Z. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 33e41
41
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4.10.2. 2-(Hydrazinylmethyl)pyridine hydrochloride (46)
White solid, 88% yield; mp 180e182 ꢀC; 1H NMR (400 MHz,
DMSO-d6):
d
9.24, (brs, 4H, HCl, NH, NH2), 8.81 (d, J ¼ 5.3 Hz, 1H, 6-
H), 8.46 (t, J ¼ 7.8 Hz, 1H, 5-H), 7.99 (d, J ¼ 7.9 Hz, 1H, 3-H), 7.88 (t,
J ¼ 6.6 Hz, 1H, 4-H), 4.48 (s, 2H, CCH2).
a
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White solid, 88% yield; mp 176e178 ꢀC; 1H NMR (400 MHz,
DMSO-d6):
d 8.50e10.00, (brs, 4H, HCl, NH, NH2), 8.95 (s, 1H, 2-H),
8.90 (d, J ¼ 5.5 Hz, 1H, 6-H), 8.62 (d, J ¼ 7.8 Hz, 1H, 4-H), 8.09 (t,
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4.10.4. ((Tetrahydrofuran-2-yl)methyl)hydrazine hydrochloride (48)
White solid, 74% yield; mp 149e151 ꢀC; 1H NMR (400 MHz,
D2O):
d 4.05e4.13 (m, 1H, OCH(CH2)2), 3.64e3.75 (m, 2H, OCH2),
2.94e3.10 (m, 2H, NHCH2), 1.93e2.01 (m, 1H, 3-H), 1.73e1.83 (m,
2H, 2-H), 1.48e1.55 (m, 1H, 3-H).
4.11. General procedure for the preparation of compounds 49e51
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hydes (29 mmol) in methanol (70 mL) was refluxed for 1 h, then
cooled to room temperature and concentrated in vacuo. The residue
was taken intoTHF (120 mL), then added NaBH3CN (43 mmol) under
N2, and then added dropwise acetic acid (45 mL). The mixture was
stirred at room temperature for 10 h, then added water (240 mL) and
ethyl acetate (240 mL), then basified with NaHCO3 to about
pH ¼ 9e10, extracted with ethyl acetate (2 ꢂ 150 mL). The organic
layer was dried with anhydrous MgSO4 and concentrated in vacuo.
The residue was taken into ethanol (180 mL), and stirred at HCl
atmosphere for 8 h, then concentrated to give compounds 49e51.
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4.11.1. (Furan-2-ylmethyl)hydrazine hydrochloride (49)
White solid, 92% yield; mp 109e111 ꢀC; 1H NMR (400 MHz,
D2O):
d
7.48 (d, J ¼ 1.7 Hz, 1H, 5-H), 6.50 (d, J ¼ 3.3 Hz, 1H, 3-H), 6.39
(dd, J ¼ 1.9, 3.0 Hz, 1H, 4-H), 4.19 (s, 2H, CCH2NH).
4.11.2. (Thiophen-2-ylmethyl)hydrazine hydrochloride (50)
White solid, 87% yield; mp 113e116 ꢀC; 1H NMR (400 MHz,
D2O):
d
7.47 (d, J ¼ 5.1 Hz, 1H, 5-H), 7.18 (d, J ¼ 3.4 Hz, 1H, 3-H), 7.04
(dd, J ¼ 3.7, 5.0 Hz, 1H, 4-H), 4.41 (s, 2H, CCH2NH).
4.11.3. Cinnamylhydrazine hydrochloride (51)
White solid, 67% yield; mp 143e145 ꢀC; 1H NMR (400 MHz,
DMSO-d6):
d 8.07, (brs, 4H, HCl, NH, NH2), 7.27e7.46 (m, 5H, ArH),
6.70 (d, J ¼ 16.0 Hz, 1H, ArCHCH), 6.27e6.35 (m, 1H, ArCHCHCH2),
3.70 (d, J ¼ 6.7 Hz, 2H, ArCHCHCH2).
Acknowledgments
We gratefully acknowledge assistance from the Chinese
Ministry of Health (2008ZX 10001-002), the National Natural
Science Foundation of China (21132003) and the China Postdoctoral
Science Foundation (2011M500519).
Appendix A. Supplementary material
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Synthesis and antiviral activities of phenanthroindolizidine alkaloids and their
derivatives, J. Agric. Food Chem. 58 (2010) 2703e2709.
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2012.04.020.
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