Anti-HAV activity of some newly synthesized triazolo[4,3-b]pyridazines

Article abstract of DOI:10.1002/ardp.200700181

6-Phenyl-[1,2,4]triazolo[4,3-b]pyridazine-3(2H)-thione 2 was used as precursor for the preparation of some novel 3-S-substituted-6-phenyl-[1,2,4] triazolo[4,3-b]pyridazine derivatives 3-11. Furthermore, the preparation of 1-[2-(6-phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanyl)-acetyl] -1H-pyrazole derivative 13 and 5-(6-phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3- ylsulfanylmethyl)-[1,3,4]oxadiazole derivatives 15 and 17, are described. Some of the prepared products revealed a promising antiviral activity against hepatitis-A virus (HAV, MBB-cell culture adapted strain). Plaque reduction infectivity assay was used to determine virus count reduction as a result of treatment with the test compounds. Compound 15 showed the highest effect on HAV compared to the other tested compounds.

Full text of DOI:10.1002/ardp.200700181

Arch. Pharm. Chem. Life Sci. 2008, 341, 223–230
A. H. Shamroukh et al.
223
Full Paper
Anti-HAV Activity of Some Newly Synthesized
Triazolo[4,3-b]pyridazines
Ahmed H. Shamroukh1 and Mohamed. A. Ali2
1 Photochemistry Department, National Research Centre, Dokki, Cairo, Egypt
2 Virology Laboratory, National Research Centre, Dokki, Cairo, Egypt
6-Phenyl-[1,2,4]triazolo[4,3-b]pyridazine-3(2H)-thione 2 was used as precursor for the preparation
of some novel 3-S-substituted-6-phenyl-[1,2,4]triazolo[4,3-b]pyridazine derivatives 3–11. Further-
more, the preparation of 1-[2-(6-phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanyl)-acetyl]-1H-pyra-
zole derivative 13 and 5-(6-phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanylmethyl)-[1,3,4]oxadia-
zole derivatives 15 and 17, are described. Some of the prepared products revealed a promising
antiviral activity against hepatitis-A virus (HAV, MBB-cell culture adapted strain). Plaque reduc-
tion infectivity assay was used to determine virus count reduction as a result of treatment with
the test compounds. Compound 15 showed the highest effect on HAV compared to the other
tested compounds.
Keywords: Antiviral activities / Pyridazines / Triazolopyridazines /
Received: August 27, 2007; accepted: September 20, 2007
DOI 10.1002/ardp.200700181
other fused heterocyclic compounds, was previously syn-
thesized by Gafitanu et al. [20] through the reaction of (6-
phenylpyridazin-3-yl)-hydrazine 1 [21] with phenyl iso-
thiocyanate. Here, we prepared compound 2 via the reac-
tion of compound 1 with carbon disulfide in dry pyri-
dine.
Introduction
In the last few decades, the chemistry of triazolo[4,3-
b]pyridazine derivatives has received considerable atten-
tion owing to their variety of biological activities, espe-
cially as a potential antiviral [1], antitumoral [2, 3], seda-
tive/hypnotic [4], anti-anxiety [5–7], adenine receptor
ligand [8, 9], hypotensive and heart rate activity [10], as
herbicidal, pesticidal [11], and bactericidal [12]. In con-
nection with our research program for the synthesis of
different fused heterocyclic compounds with antiviral
[1, 13, 14] and antimicrobial interest [15–19], we describe
the syntheses of some triazolo[4,3-b]pyridazine deriv-
atives with promising anti-HAV activity.
Theoretically, the resulting compound 2 can exist in
two forms, the thiolactam or thiolactim form. The pro-
posed structure for the predominant isomer was estab-
lished on the basis of its spectral data, since the IR spec-
trum showed absorption bands assignable to the (NH)
1
and (C=S) groups and its H-NMR spectrum revealed sig-
nal at d 14.80 ppm for (NH) (D2O exchangeable). In addi-
tion, the 13C-NMR spectrum showed signal at
d
161.94 ppm accountable for the (C=S) group (Experimen-
tal). All these facts confirm the existence of this com-
pound in the thiolactam form, but this form does not
necessarily means that this compound owes its reactivity
to this form only. It has been found that the reaction
medium affects the nature of the reacting tautomer.
Thus, compound 2 was easily S-alkylated with ethyl chlor-
oacetate in the presence of sodium hydroxide to give
ethyl 2-(6-phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfa-
nyl)acetate 3 (Scheme 1). Carrying out the same reaction
under reflux temperature, hydrolysis of the ester group
Results and discussion
Chemistry
6-Phenyl-[1,2,4]triazolo[4,3-b]pyridazine-3(2H)-thione 2, as
key compound for this study and for further syntheses of
Correspondence: Ahmed H. Shamroukh, Photochemistry Department,
National Research Centre, Dokki, Cairo 12622, Egypt.
E-mail: ahshamroukh@yahoo.com
Fax: +20 20337-0931
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Arch. Pharm. Chem. Life Sci. 2008, 341, 223–230
Scheme 1. Synthesis route of compounds 1–10.
took place to give the corresponding acid 4. This finding
Similarly, ethylation of the sodium salt of compound 2
guided us to carry out the hydrolysis of compound 3 with with ethyl iodide gave, 3-ethylsulfanyl-6-phenyl-[1,2,4]tri-
sodium hydroxide in refluxing ethanol to prove this azolo[4,3-b]pyridazine 5 (Scheme 1). Oxidation of the lat-
assumption and to obtain compound 4. The latter com- ter compound with hydrogen peroxide in acetic acid
pound could be prepared directly by refluxing com- afforded the corresponding sulphone 6. The structures of
pound 2 with chloroacetic acid in alkaline medium. Ana- the aforementioned compounds were confirmed on the
lytical and spectral data of these compounds are in agree- basis of their elemental and spectral data. Their 1H-NMR
ment with the proposed structures (Experimental, Sec- spectra showed triplet and quartet signals of the CH3 and
tion 4). In particular, the IR spectra of compounds 3 and 4 CH2 protons, respectively, while the IR spectrum of com-
showed absorption bands assignable to (C=O) groups. The pound 6 revealed absorption bands due to (C-SO2). The MS
absence of the NH-signal and the presence of S-CH2 and gave the molecular ion peaks at m/z (%) = 256 (100) and
OCH2CH3 protons in the 1H-NMR spectrum of compound 288 (39.07) for compounds 5 and 6, respectively (cf. Exper-
3 confirmed its structure. The alkylation at C3-S-triazole imental).
was confirmed by the absence of (C=S) in the 13C-NMR
spectrum.
In addition, treatment of compound 2 with chloroace-
taldehyde dimethylacetal in alcoholic sodium hydroxide
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Arch. Pharm. Chem. Life Sci. 2008, 341, 223–230
Anti-HAV of Triazolo[4,3-b]pyridazines
225
Scheme 2. Synthesis route of compounds 11–17.
solution afforded 3-(2,2-dimethoxy-ethylsulfanyl)-6-phe- pound for further reactions, was prepared through the
1
nyl-[1,2,4]triazolo[4,3-b]pyridazine 7 (Scheme 1). The H- reaction of the ester 3 and hydrazine hydrate by reflux-
NMR spectrum of compound 7 showed signals at d 3.28 ing in ethanol. The products revealed absorption bands
(s, 6H, 2OCH3), 3.50 (d, 2H, CH2), and 4.69 (t, 1H, CH). Oxi- for NH2- and C=O-groups in the IR spectra, and their 1H-
dation of compound 2 by the action of sodium hydrogen NMR spectra showed signals at d 7.28 ppm due to NH2
sulfate and sodium nitrite [22], gave the corresponding (D2O exchangeable) for compound 9, and at d 4.32 and
disulfide derivative 8. The MS of compound 8 furnished 9.40 ppm due to NH2 and NH (D2O exchangeable) for com-
the molecular ion peak at m/z (%) = 454 (100) (Experimen- pound 10. The MS gave the molecular ion peaks at m/z (%)
tal).
= 285 (58.44), 300 (17.82) for compounds 9 and 10, respec-
On the other hand, the amide derivative 9 was pro- tively (Experimental).
duced by stirring the ester 3 and ammonia solution in
When the hydrazide 10 was condensed with acetylace-
ethanol while the hydrazide derivative 10, as key com- tone in ethanol under reflux temperature, it afforded
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Arch. Pharm. Chem. Life Sci. 2008, 341, 223–230
Schiff' base hydrazide 11 instead of the expected pyrazole
derivative 12 (Scheme 2). However, our attempts to
cyclize compound 11 to the pyrazole derivative 12 by stir-
ring in sodium ethoxide solution gave an unexpected
product identical in all aspects with compound 3.
While heating compound 10 in reflux with methyl bis-
(methylsulfanyl)methylene cyanoacetate it afford the
corresponding pyrazole derivative 13. The structure of
the latter compound was confirmed on the basis of its ele-
mental analysis and spectral data (Experimental,
Scheme 2). The IR spectrum of compound 13 showed
absorption bands assignable to the NH2- and CO-groups.
Figure 1. Effect of some novel [1,2,4]triazolo[4,3-b]pyridazine
derivatives on HAV (MBB cell culture strain) in comparison to
amantadine (C*) as control. Compound 15 showed a relatively
high effect on the HAV virus (81% at 50 lg/mL).
1
Its H-NMR spectrum showed signals at d 2.30 (s, 3H,
SCH3), 3.73 (s, 3H, OCH3), and 7.40 ppm for NH2, D2O
exchangeable, and the MS gave the molecular ion peak at
m/z (%): 456 [M+ + 1] (10.43).
The N-imido derivatives 14a, b were obtained by treat-
ing the hydrazide 10 with phthalic anhydride or tetra-
chlorophthalic anhydride, respectively. The presence of
NH and two CO-groups in the IR spectra and the NH-pro-
ity. The test was performed to include the three possibil-
ities for antiviral activity, virucidal effect, virus adsorp-
tion, and effect on virus replication for HAV. Only com-
pounds 2, 6, 7, 9, 13, 15, and 17 revealed promising anti-
HAV activity and the results are summarized in Fig. 1. It
was obvious that at concentration of 20 lg/mL, com-
pounds 2, 7, 13, and 15 revealed the highest antiviral
activity in comparison with amantadine (C*) as control.
While at a concentration of 50 lg/mL, compounds 2, 13,
and 15 revealed the highest antiviral activity in compari-
1
ton (D2O exchangeable) in the H-NMR spectra of com-
pounds 14a, b confirmed their structures. Also, the MS of
compound 14b gave fragments showing the isotopic pat-
tern due to the presence of chlorine atom (Experimen-
tal).
5-(6-Phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanyl-
methyl)[1,3,4]oxadiazole-2(3H)-thione 15 (Scheme 2) was
prepared according to Young et al. [23] by heating the
acid hydrazide 10 with carbon disulfide in the presence son with amantadine (C*) as control. In general, com-
pound 15 showed the highest effect on HAV compared to
the other tested compounds and the control (amanta-
dine), where its antiviral activity increased from 66% at
concentration of 20 lg/mL to 81% at concentration of
50 lg/mL.
of sodium hydroxide. Inspection of the IR spectrum of
the reaction product 15 revealed the absence of the C=O-
group and showed absorption band characteristic for NH
and C=S; its 1H-NMR spectrum revealed signals at d
4.68 ppm for CH2 and 14.45 ppm for NH (D2O exchange-
able). In addition, the 13C-NMR spectrum showed signal at
d 177.73 ppm accountable for the C=S-group (Experimen-
tal).
Conclusions
Treating compound 10 with p-chlorobenzaldehyde,
gave the corresponding Schiff' base hydrazide 16 which;
Some novel 3-S-substituted-6-phenyl-[1,2,4]triazolo[4,3-
subjected to 1,5-dipolar-cyclization using bromine in ace- b]pyridazine derivatives 3–11 were prepared. Further-
more, some pyrazole derivative 13 and [1,3,4]oxadiazole
derivatives 15 and 17 were described. Structure-activity
correlation of the obtained results revealed that com-
tic acid in the presence of anhydrous sodium acetate;
gave the oxadiazole derivative 17 (Experimental,
Scheme 2). Product 16 revealed absorption bands charac-
teristic for NH and C=O, and its 1H-NMR spectrum showed pounds 2, 6, 7, 9, 13, 15 and 17 revealed promising anti-
HAV activity. 6-Phenyl-[1,2,4]triazolo[4,3-b]pyridazine-
3(2H)-thione 2 showed more HAV activity than the S-alky-
lated compounds 6, 7, 9, and 17. While addition of other
rings e. g., a pyrazole ring in compound 13 or an oxadia-
zole ring in compound 15, increased the activity percent-
age of HAV reduction.
the presence of NH (D2O exchangeable) and azomethine
CH=N. While the absence of NH as well as the azomethine
1
(CH=N) in the H-NMR spectrum of compound 17 con-
firmed its structure.
Antiviral screening
The plaque-infectivity assay was carried out to test all the
prepared compounds 2–11 and 13–17 for antiviral activ-
The authors have declared no conflict of interest.
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Anti-HAV of Triazolo[4,3-b]pyridazines
227
(6-Phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanyl)-
acetic acid 4
Method A
Experimental
Chemistry
All melting points are uncorrected and were measured using
Electrothermal IA 9100 apparatus, (Electrothermal, Essex, U.K.).
IR spectra were recorded as potassium bromide pellets on a Per-
kin-Elmer 1650 spectrophotometer (Perkin-Elmer, Norwalk, CT,
USA). 1H-NMR and13C-NMR spectra were determined on a Jeol-Ex-
300 NMR spectrometer (JEOL, Tokyo, Japan) and chemical shifts
were expressed as part per million, ppm (d values) against TMS
as internal reference. Mass spectra were recorded on VG 2AM-3F
mass spectrometer (Thermo electron corporation, USA) Micro-
analyses were operated using Mario El Mentar apparatus,
Organic Microanalysis Unit, and the results were within the
accepted range (l 0.20) of the calculated values. Follow up of the
reactions and checking the purity of the compounds was made
A solution of compound 2 (2.28 g, 10 mmol) and sodium hydrox-
ide (1.2 g, 30 mmol) in ethanol (100 mL) was treated with ethyl
chloroacetate (1.3 mL, 10 mmol) and the reaction mixture was
refluxed for 20 min. The formed precipitate was filtered off, dis-
solved in water (50 mL) and acidified with hydrochloric acid
(10 mL, 10%). The separated solid was filtered off, dried and
recrystallized from dioxane to give compound 4 in 81% yield.
Method B
A mixture of compound 3 (0.3 g, 1 mmol) and sodium hydroxide
(0.08 g, 2 mmol) in ethanol (50 mL) was refluxed for 15 min. The
formed precipitate was filtered off, dissolved in water (25 mL),
and acidified with hydrochloric acid (3 mL, 10%). The separated
solid was filtered off, dried, and recrystallized from dioxane to
give a compound identical in all aspects with compound 4 in
89% yield.
by TLC on silica gel-precoated aluminum sheets (Type 60 F254
;
Merck, Darmstadt, Germany). Column chromatography was per-
formed on Silica gel 60 (particle size 0.06–0.20 mm; Merck).
Compound 1 was prepared according to a reported method [21].
Method C
A mixture of compound 2 (0.23 g, 1 mmol), chloroacetic acid
(0.1 g, 1 mmol) and sodium hydroxide (0.04 g, 1 mmol) in etha-
nol (50 mL) was refluxed for 2 h. The formed precipitate was fil-
tered off, dissolved in water (25 mL) and acidified with hydro-
chloric acid (3 mL, 10%). The separated solid was filtered off,
dried, and recrystallized from dioxane to give compound 4. Yield
75%, mp. 285–2868C. The obtained product was identified by
mp. and thin layer chromatography in comparison with authen-
tic samples from methods A and B. IR m 3429 (br, OH) and 1737
(C=O) cm– 1. 1H-NMR (DMSO-d6) d 4.20 (s, 2H, CH2), 7.50–7.70 (m,
3H, Ph-H), 8.00 (d, J = 10 Hz, 1H, C7-H), 8.05–8.20 (m, 2H, Ph-H),
8.50 (d, J = 10 Hz, 1H, C8-H) and 13.00 (s, 1H, OH, D2O exchange-
able). MS m/z (%): 286 [M+] (100). Anal. calcd. for C13H10N4O2S: C
54.53, H 3.52, N 19.57, S 11.20. Found: C 54.60, H 3.49, N 19.60, S
11.12.
6-Phenyl-[1,2,4]triazolo[4,3-b]pyridazine-3(2H)-thione 2
[20]
A mixture of compound 1 (1.86 g, 10 mmol) and carbon disulfide
(10 mL) in dry pyridine (30 mL) was refluxed for 10 h. The reac-
tion mixture was cooled and poured into ice water (100 mL) and
acidified with hydrochloric acid (3 mL, 10%). The precipitated
material was filtered off, washed with water, dried, and recrys-
tallized from ethanol to give compound 2. Yield 94%, mp. 273–
2758C. IR m 3443 (NH) and 1346 (C=S) cm– 1. 1H-NMR (DMSO-d6) d
7.50–7.65 (m, 3H, Ph-H), 7.97 (d, J = 10 Hz, 1H, C7-H), 8.10–8.15
(m, 2H, Ph-H), 8.30 (d, J = 10 Hz, 1H, C8-H) and 14.80 (s, 1H, NH,
D2O exchangeable). 13C-NMR (DMSO-d6) d 122.56 (C-7), 125.45 (C4-
Ph), 127.15 (C3-Ph), 129.03 (C2-Ph), 130.69 (C-8), 133.77 (C1-Ph),
141.45 (C-8a), 153.15 (C-6) and 161.94 (C=S). MS m/z (%): 228 [M+]
(100). Anal. calcd. for C11H8N4S: C 57.88, H 3.53, N 24.54, S 14.05.
Found: C 57.80, H 3.59, N 24.61, S 13.99.
3-Ethylsulfanyl-6-phenyl-[1,2,4]triazolo[4,3-b]pyridazine 5
A solution of compound 2 (0.23 g, 1 mmol) and sodium hydrox-
ide (0.04 g, 1 mmol) in ethanol (50 mL) was treated with ethyl
iodide (0.16 mL, 1 mmol) and the reaction mixture was warmed
on a steam bath at 708C for 2 h. The formed precipitate was fil-
tered off, dried, and recrystallized from ethanol to give com-
pound 5. Yield 97%, mp. 123–1258C. IR showed absence of the
NH group. 1H-NMR (DMSO-d6) d 1.39 (t, J = 7.5 Hz, 3H, SCH2CH3),
3.37 (q, J = 7.5 Hz, 2H, SCH2CH3), 7.55–7.70 (m, 3H, Ph-H), 7.92 (d,
J = 10 Hz, 1H, C7-H), 8.05–8.12 (m, 2H, Ph-H) and 8.40 (d, J =
10 Hz, 1H, C8-H). 13C-NMR (DMSO-d6) d 15.00 (CH3), 25.70 (CH2S),
119.86 (C-7), 125.14 (C4-Ph), 127.20 (C3-Ph), 129.09 (C2-Ph), 130.89
(C-8), 133.78 (C1-Ph), 144.57 (C-8a), 144.96 (C-3) and 153.16 (C-6).
MS m/z (%): 256 [M+] (100). Anal. calcd. for C13H12N4S: C 60.91, H
4.72, N 21.86, S 12.51. Found: C 60.86, H 4.77, N 21.81, S 12.55.
Ethyl-2-(6-phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3-
ylsulfanyl)acetate 3
A solution of compound 2 (2.28 g, 10 mmol) and sodium hydrox-
ide (0.4 g, 10 mmol) in ethanol (100 mL) was treated with ethyl
chloroacetate (1.3 mL, 10 mmol) and the reaction mixture was
stirred at room temperature for 3 h. The formed precipitate was
filtered off, dried, and recrystallized from ethanol to give com-
1
pound 3. Yield 99%, mp. 137-1388C. IR m 1741 (C=O) cm– 1. H-
NMR (DMSO-d6) d 1.05 (t, J = 9 Hz, 3H, OCH2CH3), 4.05 (q, J = 9 Hz,
2H, OCH2CH3), 4.23 (s, 2H, CH2), 7.50–7.60 (m, 3H, Ph-H), 7.90 (d,
J = 10 Hz, 1H, C7-H), 8.10–8.15 (m, 2H, Ph-H) and 8.35 (d, J = 10 Hz,
1H, C8-H). 13C-NMR (DMSO-d6) d 13.74 (CH3), 33.95 (CH2S), 61.12
(CH2O), 120.11 (C-7), 125.23 (C4-Ph), 127.26 (C3-Ph), 129.11 (C2-Ph),
131.00 (C-8), 133.65 (C1-Ph), 143.95 (C-8a), 144.72 (C-3), 153.33 (C-
6) and 168.11 (C=O). MS m/z (%): 314 [M+] (49.93). Anal. calcd. for
C15H14N4O2S: C 57.31, H 4.49, N 17.82, S 10.20. Found: C 57.25, H
4.52, N 17.90, S 10.10.
3-Ethanesulfonyl-6-phenyl-[1,2,4]triazolo[4,3-
b]pyridazine 6
To a solution of compound 5 (0.26 g, 1 mmol) in glacial acetic
acid (25 mL), hydrogen peroxide solution (1 mL, 30%) was added
dropwise with occasional shaking. The reaction mixture was set
aside at room temperature for 24 h. The formed precipitate was
filtered off, dried, and recrystallized from dioxane to give com-
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A. H. Shamroukh et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 223–230
pound 6. Yield 70%, mp. 188–2008C. IR m 1338, 1148 (C-SO2) cm–1.
1H-NMR (DMSO-d6) d 1.23 (t, J = 7.5 Hz, 3H, SCH2CH3), 3.73 (q, J =
7.5 Hz, 2H, SCH2CH3), 7.59–7.60 (m, 3H, Ph-H), 8.10–8.11 (m, 2H,
Ph-H) 8.18 (d, J = 10 Hz, 1H, C7-H) and 8.62 (d, J = 10 Hz, 1H, C8-H).
MS m/z (%): 288 [M+] (39.07). Anal. calcd. for C13H12N4O2S: C 54.15,
H 4.19, N 19.43, S 11.12. Found: C 54.24, H 4.15, N 19.40, S 11.09.
NH2, D2O exchangeable), 7.50–7.65 (m, 3H, Ph-H), 7.97 (d, J =
10 Hz, 1H, C7-H), 8.10–8.18 (m, 2H, Ph-H), 8.45 (d, J = 10 Hz, 1H,
C8-H) and 9.40 (s, 1H, NH, D2O exchangeable). MS m/z (%): 300 [M+]
(17.82). Anal. calcd. for C13H12N6OS: C 51.99, H 4.03, N 27.98, S
10.68. Found: C 51.92, H 4.02, N 28.02, S 10.70.
3-(2,2-Dimethoxy-ethylsulfanyl)-6-phenyl-
[1,2,4]triazolo[4,3-b]pyridazine 7
(6-Phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanyl)-
acetic acid(1-methyl-3-oxo-butylidene)-hydrazide 11
A solution of compound 10 (0.3 g, 1 mmol), acetylacetone
(0.15 mL, 1.5 mmol) in anhydrous ethanol (30 mL) was refluxed
for 5 h. The formed precipitate on cooling was filtered off, dried,
and recrystallized from ethanol to give compound 11. Yield 63%,
A solution of compound 2 (0.23 g, 1 mmol) and sodium hydrox-
ide (0.04 g, 1 mmol) in ethanol (50 mL) was treated with chloroa-
cetaldehyde dimethyl acetal (0.13 mL, 1 mmol) and the reaction
mixture was refluxed for 5 h, then poured into water, and the
organic material was extracted with diethyl ether. The solvent
was removed under reduced pressure and chromatographed on
a silica gel column with chloroform/petroleum ether (40–60)
(9 : 1) as an eluent to give compound 7 as an oil, yield 62%. IR
mp. 229–2308C. IR m 3140 (NH), 1705 (C=O) and 1652 (C=O) cm– 1
.
1H-NMR (DMSO-d6) d 1.70 (s, 3H, CH3), 1.89 (s, 3H, CH3), 2.81 (s, 2H,
CH2), 4.50 (s, 2H, CH2), 6.50 (s, 1H, NH, D2O exchangeable), 7.55–
7.65 (m, 3H, Ph-H), 8.00 (d, J = 10 Hz, 1H, C7-H), 8.10–8.18 (m, 2H,
Ph-H) and 8.47 (d, J = 10 Hz, 1H, C8-H). 13C-NMR (DMSO-d6) d 15.69
(CH3), 25.66 (CH3), 36.95 (CH2S), 52.02 (CH2), 90.47 (N=C), 119.92
(C-7), 125.20 (C4-Ph), 127.29 (C3-Ph), 129.13 (C2-Ph), 130.96 (C-8),
133.78 (C1-Ph), 144.52 (C-8a), 144.62 (C-3), 153.18 (C-6), 155.34
(C=O) and 164.28 (C=O). MS m/z (%): 382 [M+] (10.77).
1
showed absence of the NH group. H-NMR (DMSO-d6) d 3.28 (s,
6H, 2OCH3), 3.50 (d, J = 7 Hz, 2H, CH2), 4.69 (t, 1H, CH), 7.55–7.65
(m, 3H, Ph-H), 7.95 (d, J = 10 Hz, 1H, C7-H), 8.05-8.15 (m, 2H, Ph-H)
and 8.44 (d, J = 10 Hz, 1H, C8-H). MS m/z (%): 316 [M+] (17.96).
Bis-(6-phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3-
yl)disulphide 8
A suspension of compound 2 (0.23 g, 1 mmol) and sodium hydro-
gen sulfate (0.06 g, 0.5 mmol) in ethanol (30 mL) was treated por-
tionwise with an aqueous solution of sodium nitrite (0.05 g,
0.5 mmol) in water (5 mL). The reaction mixture was heated on a
boiling water bath for 10 min, diluted with water (10 mL), and
the formed precipitate was filtered off, dried, and recrystallized
from dioxane to give compound 8. Yield 93%, mp. 265–2668C. IR
showed absence of the NH and C=S groups. 1H-NMR (DMSO-d6) d
7.41–7.70 (m, 10H, 2 Ph-H), 7.73 (d, J = 10 Hz, 2H, C7-H) and 8.20
(d, J = 10 Hz, 2H, C8-H). MS m/z (%): 454 [M+] (100). Anal. calcd. for
C22H14N8S2: C 58.13, H 3.10, N 24.65, S 14.11. Found: C 58.04, H
3.14, N 24.70, S 14.09.
Attempted cyclization of compound 11
A mixture of compound 11 (0.15 g, 0.5 mmol) and sodium ethox-
ide solution (0.5 mmol of sodium metal in 25 mL anhydrous
ethanol) was stirred for 2 h. The mixture was evaporated under
reduced pressure and the solid material was recrystallized from
ethanol to give compound 3 (yield 52%) identical in all aspects
1
(mp., mixed mp., tlc, IR, MS, and H-NMR) with compound 3
which was obtained before.
5-Amino-3-methylsulfanyl-1-[2-(6-phenyl-
[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanyl)-acetyl]-1H-
pyrazole-4-carboxylic acid methylester 13
(6-Phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanyl)-
acetamide 9
To a solution of compound 10 (0.3 g, 1 mmol) in anhydrous etha-
nol (50 mL), methyl bis(methylsulfanyl)methylene cyanoacetate
was added and the reaction mixture was refluxed for 3 h. The
product, which separated on cooling, was filtered off and recrys-
tallized from ethanol to give compound 13. Yield 72%, mp. 132–
To a solution of compound 3 (0.32 g, 1 mmol) in ethanol (50 mL),
ammonium hydroxide solution (2 mL, 30%) was added and the
reaction mixture was stirred at room temperature for 2 h. The
formed precipitate was filtered off, dried, and recrystallized
from ethanol to give compound 9. Yield 95%, mp. 269–2708C. IR
1348C. IR m 3464, 3338 (NH2), 1709 (C=O) and 1666 (C=O) cm– 1
.
1
m 3402, 3368 (NH2) and 1673 (C=O) cm– 1. H-NMR (DMSO-d6) d
1H-NMR (DMSO-d6) d 2.30 (s, 3H, SCH3), 3.73 (s, 3H, OCH3), 4.70 (s,
2H, CH2), 7.40 (s, 2H, NH2, D2O exchangeable), 7.55-7.65 (m, 3H,
Ph-H), 8.00 (d, J = 10 Hz, 1H, C79-H), 8.05–8.15 (m, 2H, Ph-H) and
8.50 (d, J = 10 Hz, 1H, C8`-H). MS m/z (%): 456 [M+ + 1] (10.43). Anal.
calcd. for C19H17N7O3S2: C 50.10, H 3.76, N 21.52, S 14.08. Found:
C 50.17, H 3.69, N 21.58, S 14.00.
4.09 (s, 2H, CH2), 7.28 (s, 2H, NH2, D2O exchangeable), 7.61–7.71
(m, 3H, Ph-H), 7.99 (d, J = 10 Hz, 1H, C7-H), 8.13–8.15 (m, 2H, Ph-
H) and 8.47 (d, J = 10 Hz, 1H, C8-H). MS m/z (%): 285 [M+] (58.44).
Anal. calcd. for C13H11N5OS: C 54.72, H 3.89, N 24.55, S 11.24.
Found: C 54.64, H 3.85, N 24.60, S 11.29.
(6-Phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanyl)-
acetic acid hydrazide 10
Synthesis of compounds 14a and 14b
To a solution of compound 3 (3.14 g, 10 mmol) in ethanol
(150 mL), hydrazine hydrate (6 mL, 99%) was added and the reac-
tion mixture was heated on a water bath for 5 h. After cooling,
the precipitated material was filtered off, washed with water,
dried, and recrystallized from ethanol to give compound 10.
Yield 96%, mp. 299–3008C. IR m 3422–3245 (NH2, NH) and 1666
General procedure: A mixture of compound 10 (0.3 g, 1 mmol)
and phthalic anhydride (0.15 g, 1 mmol) or tetrachlorophthalic
anhydride (0.29 g, 1 mmol) in glacial acetic acid (50 mL) was
heated at 808C for 2 h. The formed precipitates were filtered off
hot, dried, and recrystallized from dioxane to give compounds
14a or 14b, respectively.
1
(C=O) cm– 1. H-NMR (DMSO-d6) d 4.00 (s, 2H, CH2), 4.32 (s, 2H,
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Arch. Pharm. Chem. Life Sci. 2008, 341, 223–230
Anti-HAV of Triazolo[4,3-b]pyridazines
229
bromine (0.08 mL, 0.5 mmol) was added with stirring. The reac-
tion mixture was refluxed for 1 h, then poured into water and
the precipitated solid was filtered off, washed with water, and
recrystallized from dioxane to give compound 17. Yield 69%,
N-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-2-(6-phenyl-
[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanyl)-acetamide
14a
Yield 92%, mp. 289-2918C. IR m 3265 (NH), 1698 (C=O) and 1652
(C=O) cm– 1. 1H-NMR (DMSO-d6) d 4.22 (s, 2H, CH2), 7.45–7.89 (m,
7H, Ph-H), 8.05 (d, J = 10 Hz, 1H, C7-H), 8.10–8.20 (m, 2H, Ph-H)
and 8.50 (d, J = 10 Hz, 1H, C8-H) and 10.50 (s, 1H, NH, D2O
exchangeable). 13C-NMR (DMSO-d6) d 36.95 (CH2S), 120.11 (C-7),
125.25–131.23 (C-Ph and C-8), 144.25 (C-8a), 144.80 (C-3), 153.32
(C-6), 165.87 (C=O) and 167.47 (C=O). MS m/z (%): 430 [M+] (64.18).
Anal. calcd. for C21H14N6O3S: C 58.60, H 3.28, N 19.52, S 7.45.
Found: C 58.53, H 3.22, N 19.60, S 7.49.
1
mp. 230–2328C. IR showed absence of NH and C=O groups. H-
NMR (DMSO-d6) d 4.70 (s, 2H, CH2), 7.27 (d, J = 8.7 Hz, 2H, Ar-H),
7.37 (d, J = 8.7 Hz, 2H, Ar-H), 7.52–7.65 (m, 3H, Ph-H), 8.02–8.19
(m, 3H, C7-H and Ph-H) and 8.30 (d, J = 10 Hz, 1H, C8-H). MS m/z (%):
422 [M+] (Cl37) (3.9), 420 [M+] (Cl35) (13.68). Anal. calcd. for
C20H13ClN6OS: C 57.07, H 3.11, Cl 8.42, N 19.97, S 7.62. Found: C
56.98, H 3.05, Cl 8.50, N 20.00, S 7.66.
Antiviral screening
Preparation of synthetic compounds for the bioassay
The tested compounds were dissolved with 100 mg each in 1 mL
of 10% DMSO in water. The final concentration was 100 lg/lL
(stock solution). The dissolved stock solutions were decontami-
nated by addition of 50 lg/mL antibiotic-antimycotic mixture
(10000 U penicillin G sodium, 10000 lg streptomycin sulfates,
and 250 lg amphotericin B; PAA Laboratories GmbH, Austria).
2-(6-Phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanyl)-
N-(4,5,6,7-tetrachloro-1,3-dioxo-1,3-dihydro-isoindol-2-
yl)-acetamide 14b
ield 89%, mp. 249–2508C. IR m 3178 (NH), 1723 (C=O) and 1654
(C=O) cm– 1. 1H-NMR (DMSO-d6) d 4.21 (s, 2H, CH2), 7.55–7.69 (m,
3H, Ph-H), 8.00 (d, J = 10 Hz, 1H, C7-H), 8.10–8.20 (m, 2H, Ph-H),
8.50 (d, J = 10 Hz, 1H, C8-H) and 10.80 (s, 1H, NH, D2O exchange-
able). MS m/z (%): 570 [M+] (Cl37) (9.48), 570 [M+] (Cl35) (11.87). Anal.
calcd. for C21H10Cl4N6O3S: C 44.39, H 1.77, Cl 24.96, N 14.79, S
5.64. Found: C 44.43, H 1.80, Cl 24.91, N 14.70, S 5.70.
Cell culture
The human hepatoma cell line (HepG2) was used. Cells were
propagated in Dulbeccos' Minimal Essential Medium (DMEM)
supplemented with 10% foetal bovine serum, 1% antibiotic-anti-
mycotic mixture. The pH was adjusted to 7.2–7.4 with 7.5%
sodium bicarbonate solution. The mixture was sterilized by fil-
tration through 0.2 lm pore size nitrocellulose membrane.
5-(6-Phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3-
ylsulfanylmethyl)-[1,3,4]oxadiazole-2(3H)-thione 15
A solution of compound 10 (0.3 g, 1 mmol) and sodium hydrox-
ide (0.04 g, 1 mmol) in ethanol (50 mL) was treated with carbon
disulfide (5 mL) and the reaction mixture was refluxed for 10 h.
After removal of solvent under reduced pressure, the residual
solid was dissolved in water (20 mL) and acidified with hydro-
chloric acid (2 mL, 10%). The separated solid was filtered off,
dried, and recrystallized from ethanol to give compound 15.
Yield 80%, mp. 236–2388C. IR m 3345 (NH) and 1350 (C=S)
cm–1.1H-NMR (DMSO-d6) d 4.68 (s, 2H, SCH2), 7.58–7.69 (m, 3H, Ph-
H), 8.00–8.20 (m, 3H, C79-H and Ph-H), 8.52 (d, J = 10 Hz, 1H, C89-H)
and 14.45 (s, 1H, NH, D2O exchangeable). 13C-NMR (DMSO-d6) d
25.78 (CH2S), 120.62 (C-7`), 125.32 (C4-Ph), 127.34 (C3-Ph), 129.13
(C2-Ph), 131.03 (C-89), 133.58 (C1-Ph), 142.33 (C-8a9), 145.07 (C-39),
153.72 (C-69), 160.30 (C-5) and 177.73 (C=S). MS m/z (%): 342 [M+]
(10.15). Anal. calcd. for C14H10N6OS2: C 49.11, H 2.94, N 24.54, S
18.73. Found: C 49.19, H 2.99, N 24.48, S 18.66.
Viruses
Hepatitis-A virus cell culture-adapted strain HAV-MBB was kindly
provided by Prof. Dr. Verena Gauss-Muller, Luebeck University of
Medicine, Institute of Molecular Virology, Germany.
Plaque reduction infectivity assay
A 6-well plate was cultivated with cell culture (105 cell/mL) and
incubated for two days at 378C. HAV was diluted to give 104 PFU/
mL final concentrations and mixed with the tested compound
at the previous concentration and incubated overnight at 48C.
The growth medium was removed from the multiwell plate and
the virus-compound mixture was inoculated (100 lL/well). After
1 h contact time, the inoculum was aspirated and 3 mL of MEM
with 1% agarose was overlaid the cell sheets. The plates were left
to solidify and incubated at 378C until the development of virus
plaques. Cell sheets were fixed in 10% formaline solution for 2 h
and stained with crystal violet stain. Control virus and cells were
treated identically without chemical compounds. Virus plaques
were counted and the percentage of reduction was calculated
[24].
(6-Phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanyl)-
acetic acid(4-chloro-benzylidene)-hydrazide 16
A mixture of compound 10 (0.3 g, 1 mmol) and p-chlorobenzal-
dehyde (0.15 g, 1 mmol) in ethanol (30 mL) was heated at 80 C
for 2 h. The formed precipitate was filtered off, dried, and recrys-
tallized from dioxane to give compound 16. Yield 81%, mp. 247–
2498C. IR m 3442 (NH) and 1671 (C=O) cm– 1. 1H-NMR (DMSO-d6) d
4.20 (s, 2H, CH2), 7.30–7.70 (m, 8H, Ph-H and CH=N), 7.90 (d, J =
10 Hz, 1H, C7-H), 8.07–8.20 (m, 2H, Ph-H), 8.40 (d, J = 10 Hz, 1H,
C8-H) and 11.60 (s, 1H, NH, D2O exchangeable). MS m/z (%): 424
[M+] (Cl37) (3.66), 422.47 [M+] (Cl35) (11.35).
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