Design, synthesis and antihypertensive screening of novel pyridazine substituted s-triazin-2-imine/one/thione derivatives

Article abstract of DOI:10.3109/14756366.2012.656623

Some new 7-substituted-phenyl-3,4,8,9-tetrahydro-2H-pyridazino[1,6-a][1,3, 5]triazin-2-imine/one/thione derivatives were synthesized by a sequence of reactions starting from appropriate aryl hydrocarbons. The final compounds were screened for antihypertensive activities by non-invasive method using Tail Cuff method. All the test compounds showed significant antihypertensive activity; 7-(biphenyl-4-yl)-3,4,8,9-tetrahydro-2H-pyridazino[1,6-a][1,3,5]triazin-2-imine (4p) exhibited antihypertensive activity more than the reference standard drugs.

Full text of DOI:10.3109/14756366.2012.656623

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(3): 552–559
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.656623
ReseaRch aRtIcle
Design, synthesis and antihypertensive screening of novel
pyridazine substituted s-triazin-2-imine/one/thione
derivatives
Ravinesh Mishra1,2, Anees A. Siddiqui1, Asif Husain1, Mohd. Rashid1, and Chirag Goda2
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), Hamdard
Nagar, New Delhi, (India) and 2Institute of Pharmacy and Emerging Sciences, Baddi University of Emerging Science and
Technology, Makhnumajra, Baddi, Dist. Solan, Himanchal Pradesh, India
abstract
Some new 7-substituted-phenyl-3,4,8,9-tetrahydro-2H-pyridazino[1,6-a][1,3,5]triazin-2-imine/one/thione derivatives
were synthesized by a sequence of reactions starting from appropriate aryl hydrocarbons. The final compounds were
screened for antihypertensive activities by non-invasive method using Tail Cuff method. All the test compounds
showed significant antihypertensive activity; 7-(biphenyl-4-yl)-3,4,8,9-tetrahydro-2H-pyridazino[1,6-a][1,3,5]triazin-
2-imine (4p) exhibited antihypertensive activity more than the reference standard drugs.
Keywords: Pyridazine, 1,3,5-triazine, antihypertensive activity, non-invasive method
Introduction
antidepressant8, antibacterial9, anticancer10, nephrotopic
drugs11, antithrombic12, diuretic13 and anti-HIV14 activities.
Some pyridazine derivatives, such as bemoradan15, indoli-
dan16, levosimendan17, pimobendan18, zardaverine19 and
imazodan20, have already appeared in the clinical market.
Nitrogen containing heterocycles play an important
role not only for life science industry but also in many
other industrial fields related to special and fine chemis-
try. Among them s-triazines represent a widely used lead
structure with multitude of interesting applications in
numerous fields21. In the present work, some new 7-sub-
stituted-phenyl-3,4,8,9-tetrahydro-2H-pyridazino[1,6-a]
[1,3,5]triazin-2-imine/one/thione derivatives were
synthesized and evaluated for antihypertensive activity
by non-invasive Tail Cuff method. is work is based
on the intensive literature survey on biologically active
pyridazine in relation to antihypertensive activities; it
has been observed that various pyridazine derivatives
possess antihypertensive activity due to their vasorelax-
ant activity considering the pyridazine nucleus residue
as the pharmacophoric group for the activity.
Hypertension is a disease, which if left untreated affects
all important organs of human body. It is known as silent
killer as without showing significant symptoms, it may
quietly lead to stroke, brain haemorrhage, cardiac dis-
orders, renal failure and vision loss. Hypertension has
affected 10–15% of global population and has killed a
large number of human race in every region of world1.
e interesting pharmacological activity displayed by
pyridazine derivatives has been demonstrated in recent
years not only by the growing number of papers and pat-
ents describing them but also by the development of sev-
eral pyridazine-based drugs and pharmacological tools.
Nowadays, pyridazine derivatives have received special
attention in the search for drugs acting on the cardiovas-
cular system2 as shown in Figure 1.
e quite noticeable efforts have been made on obtain-
ing novel antihypertensive agents acting on different
mechanisms to control blood pressure3. Pyridazines are
important biologically active scaffolds, possessing antihy-
pertensive4, cardiotonic5, analgesic6, anti-inflammatory7,
Address for Correspondence: Dr. Ravinesh Mishra, Assistant Professor, Institute of Pharmacy and Emerging Sciences, Baddi University of
Emerging Science and Technology, Makhnumajra, Baddi, Dist. Solan, Himachal Pradesh-173205, India. Tel: +91-9990031907.
Fax: +91-1795-247352. E-mail: ravi_kcp@rediffmail.com
(Received 09 January 2012; accepted 09 January 2012)
552

Design, synthesis and antihypertensive screening of novel pyridazine substituted s-triazin-2-imine/one/thione derivatives 553
Figure 1. Structures of positive inotropic agents possessing pyridazine nucleus.
the residue poured into crushed ice to separate out
the 2-(hydroxymethyl)-6-(substituted-phenyl)-4,5-di-
experimental protocols
Chemistry
hydropyridazin-3(2H)-one derivatives 3. e solid,
Melting points were determined by open tube capillary
which separated out, was filtered and crystallized from
method and are uncorrected. Purity of the compounds
methanol.
was checked by thin layer chromatography (TLC)
method. e FT-IR spectra were recorded on Bio-rad
General procedure for the synthesis of 7-substituted-phenyl-
3,4,8,9-tetrahydro-2H-pyridazino[1,6-a][1,3,5]triazin-2-
imine/one/thione derivatives (4a–4u)
A mixture of 2-(hydroxymethyl)-6-(substituted-phenyl)-
FTS-135 spectrophotometer using KBr pellets; υmax val-
1
ues are given in cm−1. e H and 13C NMR spectra were
recorded on Bruker Spectrospin DPX 300MHz using
CDCl3 as solvent and tetramethylsilane (TMS) as an inter-
4,5-dihydropyridazin-3(2H)-one derivatives
3 (0.001
nal standard. Chemical shifts are given in δ (ppm) scale
and coupling constants (J values) are expressed in Hz.
e FAB Mass spectra were obtained on JEOL-JMS-DX
303 system, equipped with direct inlet probe system.
Solvent system for TLC was toluene: ethyl acetate: formic
acid (5: 4: 1, v/v/v). Elemental analysis was carried out
on CHNS Elementar (Vario EL III) using sulphanilic acid
as a standard and tungsten (VI) oxide as a combusting
agent and analyses for C, H, N were within 0.4% of the
theoretical values.
mole) and guanidine hydrochloride/urea/thiourea
(0.001 mole) was heated in an oil bath at 130°C for 3h,
cooled and triturated with ethanol. e whole content
was refluxed on water bath for 8 h. After completion of the
reaction, ethanol was distilled off and the residue poured
into crushed ice to separate out the title compound
(4a–4g). e solid, which separated out, was filtered and
crystallized from ethanol.
7-phenyl-3,4,8,9-tetrahydro-2H-pyridazino[1,6-a]
[1,3,5]triazin-2-imine (4a). Yield: 30%; m.p. 240°C–242°C;
Rf 0.45; IR (KBr) υmax (cm−1): 3218 (NH), 3014 (CH), 1425
(C=N); 1H-NMR (δ) CDCl3: 2.17 (s, 1H, NH), 2.63 (t, 2H,
CH2), 2.96 (t, 2H, CH2), 5.90 (s, 2H, CH2), 7.38-7.52 (m, 3H,
Ar-H), 7.58–7.73 (m, 2H, Ar-H), 8.50 (s, 1H, = NH); 13C-
NMR (δ) CDCl3: 23.6, 26.6, 67.7, 87.8, 128.2, 128.8, 131,
136.4, 146.5, 157.2, 158.9 (C=NH); Mass (m/z): 227/228
(M+/M++1); Anal. Calc. for C12H13N5 (mol. wt. 227.26):
C: 63.42, H: 5.77, N: 30.82. Found: C: 63.38, H: 5.75, N:
30.78.
General procedure for the synthesis of substituted β-aroyl
propionic acids (1a–1g)
e substituted β-aroyl propionic acids (1a–1g) were
synthesized from respective aromatic hydrocarbon and
characterized on the basis of spectral data as per reported
procedure22.
General procedure for the synthesis of 6-substituted-phenyl-
4,5-dihydropyridazin-3(2H)-one (2a–2g)
7-phenyl-3,4,8,9-tetrahydro-2H-pyridazino[1,6-a]
[1,3,5]triazin-2-one (4b). Yield: 82%; m.p. 236°C–238°C;
Rf 0.49; IR (KBr) υmax (cm−1): 3256 (NH), 2986 (CH), 1665
(C=O), 1475 (C=N); 1H-NMR (δ) CDCl3: 2.62 (t, 2H, CH2),
2.92 (t, 2H, CH2), 5.93 (s, 2H, CH2), 7.46–7.86 (m, 5H,
Ar-H), 8.2 (s, 1H, NHCO); 13C-NMR (δ) CDCl3: 23.2, 25.8,
63.8, 102.2, 128.2, 128.8, 131, 136.4, 146.5, 164.1, 165.7
(C=O); Mass (m/z): 228/229 (M+/M++1); Anal. Calc. for
C12H12N4O (mol. wt. 228.24): C: 63.15, H: 5.30, N: 24.55.
Found: C: 63.18, H: 5.25, N: 24.68.
e appropriate substituted β-aroyl propionic acids were
reacted with hydrazine hydrate to get corresponding
pyridazinone and characterized on the basis of spectral
data as per earlier reported procedure23.
General procedure for the synthesis of 2-(hydroxymethyl)-
6-(substituted-phenyl)-4,5-dihydropyridazin-3(2H)-one
derivatives (3a–3g).
To a solution of 6-(substituted-phenyl)-4,5-dihydro-
pyridazine-3(2H)-one (0.001 mole) derivatives 2 in
methanol (30 mL) formaldehyde (37–41%) (2.5 mL) was
added and the contents refluxed for 6 h. After comple-
tion of the reaction, methanol was distilled off and
7-phenyl-3,4,8,9-tetrahydro-2H-pyridazino[1,6-a]
[1,3,5]triazin-2-thione (4c). Yield: 72%; m.p. 252°C–254°C;
Rf 0.42; IR (KBr) υmax (cm−1): 3278 (NH), 2964 (CH), 1232
© 2013 Informa UK, Ltd.

554 R. Mishra et al.
1
(C=S), 1458 (C=N), 770; 1H-NMR (δ) CDCl3: 2.61 (t, 2H,
CH2), 2.88 (t, 2H, CH2), 5.94 (s, 2H, CH2), 7.48–7.86 (m,
5H, Ar-H), 8.22 (s, 1H, NHCS); 13C-NMR (δ) CDCl3: 24.2,
26.5, 72.4, 106, 128.1, 128.8, 131.2, 136.4, 146.6, 165.3,
191.7 (C=S); Mass (m/z): 244/245 (M+/M++1); Anal. Calc.
for C12H12N4S (mol. wt. 244.31): C: 58.99, H: 4.95, N: 22.93.
Found: C: 58.88, H: 5.05, N: 22.88.
(NH), 2986 (CH), 1664 (C=O), 1586 (C=N); H-NMR (δ)
CDCl3: 2.22 (s, 1H, NH), 2.63 (t, 2H, CH2), 2.91 (t, 2H,
CH2), 3.81 (s, 3H, CH3O), 4.92 (m, 2H, CH2), 7.32 (dd, 2H,
J = 8.8, H-3′, H-5′), 7.74 (dd, 2H, J = 8.8, H-2′, H-6′), 8.58
(s, 1H, NHCO); 13C-NMR (δ) CDCl3: 23.2, 25.8, 55.8, 63.8,
102.2, 114.4, 128.7, 146.5, 164.1, 165.2 (C=O); Mass (m/z):
258/259 (M+/M++1); Anal. Calc. for C13H14N4O2 (mol. wt.
258.34): C: 60.45, H: 5.46, N: 21.69. Found: C: 60.38, H:
5.42, N: 21.58.
7-(4-methylphenyl)-3, 4, 8, 9-tetrahydro-2H-
pyridazino[1,6-a][1,3,5]triazin-2-imine (4d). Yield: 58%;
m.p. 232°C–234°C; Rf 0.58; IR (KBr) υmax (cm−1): 3324
(NH), 2922 (CH), 1517 (C=N); 1H-NMR (δ) CDCl3: 2.19 (s,
1H, NH), 2.38 (s, 3H, CH3), 2.60 (t, 2H, CH2), 2.98 (t, 2H,
CH2), 4.90 (m, 2H, CH2), 7.23 (d, 2H, J = 8.1, H-3′, H-5′),
7.26 (d, 2H, J = 8.1, H-2′, H-6′), 8.45 (s, 1H, = NH); 13C-NMR
(δ) CDCl3: 21.6, 23.5, 26.1, 65.2, 104.3, 127.8, 130.1, 132.8,
146.4, 158.3, 159.2 (C=NH); Mass (m/z): 241/242 (M+/
M++1); Anal. Calc. for C13H15N5 (mol. wt. 241.29): C: 64.71,
H: 6.27, N: 29.02. Found: C: 64.68, H: 6.25, N: 28.96.
7-(4-methylphenyl)-3, 4, 8, 9-tetrahydro-2H-
pyridazino[1,6-a][1,3,5]triazin-2-one (4e). Yield: 88%;
m.p. 226°C–228°C; Rf 0.60; IR (KBr) υmax (cm−1): 3320 (NH),
7-(4-methoxyphenyl)-3,4,8,9-tetrahydro-2H-
pyridazino[1,6-a][1,3,5]triazin-2-thione (4i). Yield: 86%;
m.p. 238°C–240°C, Rf 0.52; IR (KBr) υmax (cm−1): 3336
1
(NH), 2992(CH), 1228 (C=S), 1578 (C=N); H-NMR (δ)
CDCl3: 2.19 (s, 1H, NH), 2.62 (t, 2H, CH2), 2.91 (t, 2H,
CH2), 3.80 (s, 3H, CH3O), 4.94 (m, 2H, CH2), 7.32 (dd, 2H,
J = 8.8, H-3′, H-5′), 7.74 (dd, 2H, J= 8.8, H-2′, H-6′), 8.45 (s,
1H, NHCS); 13C-NMR (δ) CDCl3: 24.2, 26.5, 55.8, 72.4, 106,
114.4, 128.7, 146.5, 162.9, 165.3, 191.6 (C=S); Mass (m/z):
274/275 (M+/M++1); Anal. Calc. for C13H14N4OS (mol. wt.
274.34): C: 56.91, H: 5.14, N: 20.42. Found: C: 56.88, H:
5.08, N: 20.36.
1
2944 (CH), 1672 (C=O), 1524 (C=N); H-NMR (δ) CDCl3:
7 - ( 4 - e t h y l p h e n y l ) - 3 , 4 , 8 , 9 - t e t r a h y d r o - 2 H-
pyridazino[1,6-a][1,3,5]triazin-2-imine (4j). Yield: 64%;
m.p. 226°C–228°C; Rf 0.62 IR (KBr) υmax (cm−1): 3308
2.18 (s, 1H, NH), 2.37 (s, 3H, CH3), 2.61 (t, 2H, CH2), 2.97
(t, 2H, CH2), 4.93 (m, 2H, CH2), 7.22 (d, 2H, J = 8.1, H-3′,
H-5′), 7.26 (d, 2H, J = 8.1, H-2′, H-6′), 8.38 (s, 1H, NHCO);
13C-NMR (δ) CDCl3: 21.3, 23.2, 25.8, 63.8, 102.2, 127,
129.1, 133.4, 140.7, 146.5, 164.1, 165.8 (C=O); Mass (m/z):
242/243 (M+/M++1); Anal. Calc. for C13H14N4O (mol. wt.
242.27): C: 64.45, H: 5.82, N: 23.13. Found: C: 64.48, H:
6.02, N: 22.96.
1
(NH), 2965 (CH), 1598 (C=N), 1069, 762; H-NMR (δ)
CDCl3: 0.93 (t, 3H, CH3), 2.17 (s, 1H, NH), 2.52 (q, 2H,
CH2), 2.59 (t, 2H, C-CH2), 2.63 (t, 2H, CH2), 2.92 (t, 2H,
CH2), 3.96 (m, 2H, CH2), 7.33 (dd, 2H, J = 8.8, H-3′, H-5′),
7.74 (dd, 2H, J = 8.8, H-2′, H-6′), 8.48 (s, 1H, = NH); 13C-
NMR (δ) CDCl3: 14.3, 22.8, 26.2, 28.7, 67.1, 99.2, 126.7,
128.6, 131.5, 146.6, 148.2, 157.6, 158.4 (C=NH); Mass
(m/z): 255/256 (M+/M++1); Anal. Calc. for C14H17N5
(mol. wt. 255.31): C: 65.86, H: 6.71, N: 27.43. Found: C:
65.82, H: 6.66, N: 27.42.
7-(4-methylphenyl)-3, 4, 8, 9-tetrahydro-2H-
pyridazino[1,6-a][1,3,5]triazin-2-thione (4f). Yield: 77%;
m.p. 238°C–240°C; Rf 0.62; IR (KBr) υmax (cm−1): 3326 (NH),
1
2968 (CH), 1224 (C=S), 1546 (C=N); H-NMR (δ) CDCl3:
2.16 (s, 1H, NH), 2.38 (s, 3H, CH3), 2.60 (t, 2H, CH2), 2.98
(t, 2H, CH2), 4.92 (m, 2H, CH2), 7.23 (d, 2H, J = 8.1, H-3′,
H-5′), 7.26 (d, 2H, J = 8.1, H-2′, H-6′), 8.45 (s, 1H, NHCS);
13C-NMR (δ) CDCl3: 21.3, 24.2, 26.5, 72.4, 106, 127, 129,
129.1, 133.4, 146.5, 165.3, 191.8 (C=S); Mass (m/z):
258/259 (M+/M++1); Anal. Calc. for C13H14N4S (mol. wt.
258.34): C: 60.44, H: 5.46, N: 21.69. Found: C: 60.40, H:
5.38, N: 21.72.
7 - ( 4 - e t h y l p h e n y l ) - 3 , 4 , 8 , 9 - t e t r a h y d r o - 2 H-
pyridazino[1,6-a][1,3,5]triazin-2-one (4k). Yield: 92%;
m.p. 234°C–236°C; Rf 0.64 IR (KBr) υmax (cm−1): 3212
1
(NH), 2974 (CH), 1672 (C=O), 1542 (C=N); H-NMR (δ)
CDCl3: 0.95 (t, 3H, CH3), 2.16 (s, 1H, NH), 2.52 (q, 2H,
CH2), 2.60 (t, 2H, C-CH2), 2.64 (t, 2H, CH2), 2.91 (t, 2H,
CH2), 3.93 (m, 2H, CH2), 7.32 (dd, 2H, J = 8.8, H-3′, H-5′),
7.74 (dd, 2H, J = 8.8, H-2′, H-6′), 8.54 (s, 1H, NHCO); 13C-
NMR (δ) CDCl3: 14.5, 23.2, 25.8, 28.2, 63.8, 107.2, 127,
127.8, 133.6, 146.5, 148.2, 164.1, 166 (C=O); Mass (m/z):
256/257 (M+/M++1); Anal. Calc. for C14H16N4O (mol. wt.
256.30): C: 65.61, H: 6.29, N: 21.86. Found: C: 65.46, H:
6.18, N: 21.76.
7-[4-(2-methylpropyl)phenyl]-3,4,8,9-tetrahydro-2-
H-pyridazino[1,6-a][1,3,5]triazin-2-imine (4m). Yield:
60%; m.p. 204°C–206°C; Rf 0.56; IR (KBr) υmax (cm−1): 3412
(NH), 3021 (CH), 1516 (C=N); 1H-NMR (δ) CDCl3: 0.91 (d,
6H, 2xCH3), 1.73 (m, H, C-CH2), 2.18 (s, 1H, NH), 2.53 (t,
2H, CH2), 2.90 (t, 2H, CH2), 4.95 (m, 2H, CH2), 7.33 (dd,
2H, J = 8.2, H-3′, H-5′), 7.66 (dd, 2H, J = 8.8, H-2′, H-6′),
8.51 (s, 1H, = NH); 13C-NMR (δ) CDCl3: 22.6, 23.6, 26.4,
29, 45.1, 66.3, 101.8, 126.1, 128.8, 131, 143.5, 146.8, 156.6,
159.2 (C=NH); Mass (m/z): 283/284 (M+/M++1); Anal.
7-(4-methoxyphenyl)-3,4,8,9-tetrahydro-2H-
pyridazino[1,6-a][1,3,5]triazin-2-imine (4g). Yield: 45%;
m.p. 252°C–254°C, Rf 0.55; IR (KBr) υmax (cm−1): 3350
1
(NH), 3016 (CH), 1608 (C=N); H-NMR (δ) CDCl3: 2.20
(s, 1H, NH), 2.61 (t, 2H, CH2), 2.91 (t, 2H, CH2), 3.78 (s,
3H, CH3O), 4.94 (m, 2H, CH2), 7.32 (dd, 2H, J = 8.8, H-3′,
H-5′), 7.74 (dd, 2H, J = 8.8, H-2′, H-6′), 8.45 (s, 1H, = NH);
13C-NMR (δ) CDCl3: 23.5, 26.8, 56.3, 65.7, 101.8, 112.6,
128.8, 144.9, 157.1, 159.8 (C=NH); Mass (m/z): 257/258
(M+/M++1); Anal. Calc. for C13H15N5O (mol. wt. 257.29):
C: 60.09, H: 5.88, N: 27.22. Found: C: 60.02, H: 5.78, N:
27.12.
7-(4-methoxyphenyl)-3,4,8,9-tetrahydro-2H-
pyridazino[1,6-a][1,3,5]triazin-2-one (4h). Yield: 88%;
m.p. 246°C–248°C, Rf 0.58; IR (KBr) υmax (cm−1): 3342
Journal of Enzyme Inhibition and Medicinal Chemistry

Design, synthesis and antihypertensive screening of novel pyridazine substituted s-triazin-2-imine/one/thione derivatives 555
Calc. for C16H21N5 (mol. wt. 283.37): C: 67.82, H: 7.47, N:
24.71. Found: C: 67.80, H: 7.38, N: 24.62.
light/dark cycle at 25°C 2°C. Animals were provided
with pellet diet (Lipton, Calcutta, India) and water
ad libitum. ey were marked for easy identifica-
tion. All the experimental protocols were carried out
with the permission from Institutional Animal Ethics
Committee (IAEC), Form number 563 and the guide-
lines provided by the Committee for the Purpose of
Control and Supervision of Experiments in Animal
(CPCSEA). Registration number and date of registra-
tion is 173/CPCSEA, 28 January 2000.
7-[4-(2-methylpropyl)phenyl]-3,4,8,9-tetrahydro-2H-
pyridazino[1,6-a][1,3,5]triazin-2-one (4n). Yield: 84%;
m.p. 212°C–214°C; Rf 0.58; IR (KBr) υmax (cm−1): 3216
1
(NH), 3019 (CH), 1674 (C=O), 1509 (C=N); H-NMR (δ)
CDCl3: 0.90 (d, 6H, 2xCH3), 1.75 (m, H, C-CH2), 2.16 (s,
1H, NH), 2.52 (t, 2H, CH2), 2.92 (t, 2H, CH2), 4.94 (m,
2H, CH2), 7.34 (dd, 2H, J = 8.2, H-3′, H-5′), 7.68 (dd, 2H,
J = 8.8, H-2′, H-6′), 8.52 (s, 1H, NHCO); 13C-NMR (δ)
CDCl3: 22.8, 23.2, 25.8, 29, 44.5, 63.8, 102.2, 126.2, 128.6,
133.2, 143.7, 146.5, 164.1, 165.2 (C=O); Mass (m/z):
284/285 (M+/M++1); Anal. Calc. for C16H20N4O (mol. wt.
284.35): C: 67.58, H: 7.09, N: 19.70. Found: C: 67.54, H:
7.04, N: 19.68.
7 - ( b i p h e n y l - 4 - y l ) - 3 , 4 , 8 , 9 - t e t r a h y d r o - 2 H-
pyridazino[1,6-a][1,3,5]triazin-2-imine (4p). Yield: 50%;
m.p. 228°C–230°C; Rf 0.48; IR (KBr) υmax (cm−1): 3204
(NH), 3076 (CH), 1518 (C=N); 1H-NMR (δ) CDCl3: 2.16 (s,
1H, NH), 2.48 (t, 2H, CH2), 2.96 (t, 2H, CH2), 4.94 (m, 2H,
CH2), 7.50 (m, 5H, H-3′, H-5′), 7.60-7.80 (dd, 4H, J = 8.8,
H-2′, H-6′, H-3′, H-5′), 8.56 (s, 1H, = NH); 13C-NMR (δ)
CDCl3: 22.9, 26.3, 63.4, 105.1, 127.9, 128.3, 128.8, 129.5,
133.7, 140.2, 144.6, 145.8, 157.6, 159.3 (C=NH); Mass
(m/z): 303/304 (M+/M++1); Anal. Calc. for C18H17N5 (mol.
wt. 303.36): C: 71.27, H: 5.65, N: 23.09. Found: C: 71.22, H:
5.61, N: 23.04.
Conditioning/training of animals
e non-invasive blood pressure system was designed
to reduce the animal′s stress and enhance blood flow to
the tail. Every rat in each group was given training in the
restrainer for a period of 15 min every day at least 10–15
days prior to the day of measurement of the haemody-
namic parameters. is exercise was done to avoid the
fluctuation in blood pressure due to aggressive behaviour
of animal while keeping into the restrainer for measuring
the activity. e room temperature was maintained at or
above 26°C.
Induction of hypertension in normotensive rats
After recording the initial blood pressure of rats, the ani-
mals were divided into groups of five animals each. One
group was taken as control. Hypertension was induced
in the remaining groups by subcutaneous injection of
methyl prednisolone acetate (20 mg/kg/week) for 2
weeks24.
7 - ( b i p h e n y l - 4 - y l ) - 3 , 4 , 8 , 9 - t e t r a h y d r o - 2 H-
pyridazino[1,6-a][1,3,5]triazin-2-one (4q). Yield: 86%;
m.p. 240°C–242°C; Rf 0.50; IR (KBr) υmax (cm−1): 3208 (NH),
1
3066 (CH), 1685 (C=O), 1488 (C=N); H-NMR (δ) CDCl3:
2.15 (s, 1H, NH), 2.46 (t, 2H, CH2), 2.98 (t, 2H, CH2), 4.93
(m, 2H, CH2), 7.53 (m, 5H, H-3′, H-5′), 7.62-7.80 (dd,
4H, J = 8.8, H-2′, H-6′, H-3′, H-5′), 8.58 (s, 1H, NHCO);
13C-NMR (δ) CDCl3: 23.2, 25.8, 63.8, 102.2, 127.6, 127.9,
128, 129.2, 129.7, 135.3, 140.8, 143.1, 146.5, 164.1, 165.4
(C=O); Mass (m/z): 304/305 (M+/M++1); Anal. Calc. for
C18H16N4O (mol. wt. 304.34): C: 71.04, H: 5.30, N: 18.41.
Found: C: 70.96, H: 5.26, N: 18.36.
Animal preparation
e animals were placed in the holder 10–15 min prior
to obtaining pressure measurements. Acclimated ani-
mals provided faster blood pressure measurements then
non-acclimated animals. A nervous, stressed animal may
have diminished circulation in the tail.
Measurement of mean blood pressure of rats
7 - ( 4 - c h l o ro ph e ny l ) - 3 , 4 , 8 , 9 - t e t rahy d ro - 2 H-
pyridazino[1,6-a][1,3,5]triazin-2-imine (4s). Yield: 40%;
m.p. 188°C–190°C; Rf 0.55; IR (KBr) υmax (cm−1): 3218
Mean arterial blood pressure (MABP) was measured
in conscious rats using CODA Non-Invasive Blood
Pressure Recorder by Tail-Cuff method (Kent Scientific
Corporation, USA). e restrainer carrying the rat was
placed in the blood pressure instrument with tail pro-
truding out. e tail was gently placed in contact with
a transducer membrane, which was connected to the
digital blood pressure display panel. e instrument
was then turned on and allowed to stabilize until steady
pulse rate was observed. Once the ‘pulse level ready′ sig-
nal appeared, the blood pressure recording button was
pressed and the MABP was recorded. Albino rats (body
weight 200–250 g) were used in present study. Rats were
assigned to groups of four animals in each. Each com-
pound (20 mg/kg body weight) was injected intraperi-
toneally after suspending in 1% carboxymethyl cellulose
(CMC) solution. e MABP was recorded after 1, 3, 6, 12,
24, 48, 72, 96, 120 h.
1
(NH), 3014 (CH), 1425 (C=N); H-NMR (δ) CDCl3: 2.52
(t, 2H, CH2), 2.91 (brs, 1H, NH), 2.95 (t, 2H, CH2), 3.88
(s, 1H, CH2), 7.32 (d, 2H, J = 8.6, H-3′, H-5′), 7.62 (d, 2H,
J = 8.6, H-2′, H-6′), 8.49 (s, 1H, = NH); 13C-NMR (δ) CDCl3:
22.5, 25.6, 63.4, 102.8, 127.6, 128.3, 135.1, 136.7, 147.4,
156.6, 158.9(C=NH); Mass (m/z): 261/263 (M+/M++2);
Anal. Calc. for C12H12ClN5 (mol. wt. 261.71): C: 55.07, H:
4.62, N: 26.76. Found: C: 55.02, H: 4.56, N: 26.74.
Pharmacology: antihypertensive activity
Procurement, identification and housing of animals
Albino rats (body weight 200–250 g) were supplied by
Central Animal House facility, Hamdard University
and kept under standard laboratory conditions in 12 h
© 2013 Informa UK, Ltd.

556 R. Mishra et al.
hydrocarbons with succinic anhydride in the presence
of anhydrous aluminium chloride. e β-aroyl propi-
onic acid (1) was cyclized on hydrazinolysis to yield
6-(substituted-phenyl)-4,5-dihydropyridazine-3(2H)-
one (2a–2g). Pyridazinone (2) was treated with form-
aldehyde to give the 2-hydroxymethyl derivative, that
is, 2-(hydroxymethyl)-6-(substituted-phenyl)-4,5-dihy-
dropyridazin-3(2H)-one (3a–3g) which on cyclocon-
densation with guanidine hydrochloride/urea/thiourea
yielded 7-phenyl-3,4,8,9-tetrahydro-2H-pyridazino[1,6-
a][1,3,5]triazin-2-imine/one/thione (4a–4u).
e final compounds (4a–4u) were structurally
elucidated on the basis of spectral data, explained
with the example of compound 4a. In the initial step,
benzene was acylated using Friedel–Craft′s reaction to
yield β-benzoyl propionic acid 1a. It gave positive effer-
vescence test with sodium bicarbonate solution and
showed sharp melting point, 126°C. e IR spectrum
showed characteristic band at 3428, 1679 and 1235 for
hydroxyl, carbonyl and C-C-O moiety. e 1H-NMR
spectrum indicated the two triplets at δ 2.59 and 3.23
for CH2 and CH2CO- moieties, respectively. e aro-
matic protons were visualized in the form of doublet
Statistical analysis of data
e statistical analysis was performed using GRAPHPAD
INSTAT 3 software (Graph Pad Software Inc, San Diego,
CA, USA). Data obtained from animal experiments were
expressed as arithmetic mean SEM. e comparison
between various groups was performed by one-way
analysis of variance (ANOVA), and the effect in treat-
ment groups were compared with toxic control group by
Dunnet multiple comparison test. A p< 0.05 was consid-
ered to be significant [*p< 0.05; **p< 0.01]. e percent-
age reduction in MABP for all the treatment groups was
also calculated and compared.
Results and discussion
Chemistry
e synthesis of various 7-substituted-phenyl-3,4,8,9-
tetrahydro-2H-pyridazino[1,6-a][1,3,5]triazin-2-imine/
one/thione derivatives (4a–4u) have been carried out
according to the steps shown in the Scheme 1.
In the initial step, β-aroyl propionic acids (1a–1g) were
synthesized by Friedel–Crafts acylation of appropriate
Scheme 1. Synthesis protocol for various 7-substituted-phenyl-3,4,8,9-tetrahydro-2H-pyridazino[1,6-a][1,3,5]triazin-2-imine/one/
thione derivatives.
Journal of Enzyme Inhibition and Medicinal Chemistry

Design, synthesis and antihypertensive screening of novel pyridazine substituted s-triazin-2-imine/one/thione derivatives 557
Table 1. Percent reduction in mean arterial blood pressure (mm Hg) in rats.
% Reduction in mean arterial blood pressure
Compound (20mg/kg)
1 h
3h
6 h
12h
24 h
29.55
31.44
14.46
19.49
23.27
20.75
21.38
20.44
17.61
18.23
17.61
22.01
25.78
29.55
20.12
24.71
19.37
22.01
21.38
18.23
16.98
13.13
11.63
48 h
23.86
22.50
10.42
16.82
19.38
16.18
17.46
14.90
12.98
15.54
12.85
18.10
20.70
21.30
15.67
18.74
14.77
13.62
15.29
14.52
14.33
11.70
9.48
72 h
17.10
15.78
6057
13.15
13.81
12.50
11.84
10.13
6.57
96 h
10.66
12.00
4.66
8.00
9.33
8.66
9.20
7.60
4.00
7.33
6.66
7.00
12.00
11.25
8.53
7.73
7.46
5.33
7.20
6.53
6.93
5.24
4.46
120 h
4.72
5.40
2.70
4.78
4.72
4.72
6.08
4.05
1.35
3.37
4.05
3.37
5.00
6.08
4.05
2.70
2.02
3.37
4.18
3.78
1.35
2.36
2.22
Propranolol
41.40
40.76
23.85
28.65
34.12
36.30
32.46
36.14
24.50
26.48
28.52
40.88
36.82
38.06
40.98
37.92
32.64
42.04
30.15
31.64
24.12
26.17
18.52
41.40
40.73
22.98
27.96
3295
38.71
39.28
20.77
25.76
30.13
30.10
29.43
27.83
20.77
22.02
24.46
36.86
35.47
34.86
36.20
36.91
29.68
33.82
25.90
28.88
20.00
19.52
13.04
33.75
35.62
17.50
22.50
26.25
26.15
25.46
25.00
19.37
20.50
22.50
27.65
30.00
31.80
25.67
31.50
26.12
29.46
25.00
23.67
18.60
15.00
12.50
Hydralazine
4a
4b
4c
4d
4e
4f
4g
4h
4i
34.35
32.10
32.98
22.36
24.21
26.91
39.71
36.21
36.58
39.47
37.39
32.53
38.28
29.06
30.99
22.21
24.21
15.83
9.93
9.21
4j
4k
4l
11.71
15.78
15.13
11.18
12.76
10.26
7.89
4m
4n
4o
4p
4q
4r
4s
4t
10.03
9.86
11.05
7.50
4u
6.44
a Dose of Propranolol was taken as 14mg/kg. b Dose of Hydralazine was taken as 2.6mg/kg. All values were expressed as mean SEM
(*p≤0.05), each group comprised of four animals (i.e. n=4). Toxic control group was compared with control group. All the treatment
groups were compared with toxic control group and p<0.05 was considered to be significant. ** p<0.01; * p<0.05
with D2O) and CH2-N-O moiety, respectively. e mul-
tiplet at δ 7.42 was due to three protons of aromatic ring
attached at 6-position. e other aromatic two protons
appeared in downfield at δ 7.76 as multiplet. e mass
spectrum of intermediate 3a showed an M++1 peak at
m/z 205 according to molecular formula C11H12N2O2.
In final step, 2-(hydroxymethyl)-6-phenyl-4,5-dihydro-
pyridazin-3(2H)-one 3a on cyclocondensation with
guanidine hydrochloride yielded 7-phenyl-3,4,8,9-
tetrahydro-2H-pyridazino[1,6-a][1,3,5]triazin-2-imine
4a, which showed different Rf value and sharp melting
point at 242°C. e IR spectrum showed characteristic
band at 3218 cm−1, 3014 cm−1 and 1425 cm−1 for NH,
CH stretching and C=N functional group, respectively.
e singlet at δ 2.17 was due to NH group. e 1H-NMR
showed triplet at δ 2.63 and 2.96 for the cyclic CH2 group
at positions 5 and 4, respectively. e singlet at δ 5.90
was due to cyclic CH2 group of triazin-2-imine ring. e
aromatic protons appeared as multiplets around δ 7.38
and δ 7.73. e imine proton appeared as singlet δ 8.50.
e structure is also supported by elemental analysis
data and 13C NMR data. e 13C-NMR showed the peak
at δ 157.2 and 158.9 for tertiary carbon (C-6) and imine
moiety (C-8). e mass spectrum showed the peak at
m/z 228 for M++1 in accordance with molecular formula
C12H13N5. e other compounds are also identified in a
similar manner.
due to H-2′ and H-6′ protons at δ 7.72. e H-3′ to H-5′
protons appeared as multiplet at δ 7.53 to 7.62. e
characteristic peak of singlet at 12.17 showed the pres-
ence of COOH group. e β-benzoyl propionic acid 1a
was cyclized on hydrazinolysis to yield 6-phenyl-4,5-di-
hydropyridazine-3(2H)-one 2a. It showed different Rf
value and melting point while comparing with inter-
mediate 1a and did not react with sodium bicarbonate
solution. e IR spectrum showed characteristic band
at 3206 and 1678 cm−1 due to cyclic amide. e band due
1
to C=N appeared at 1619 cm−1. e H-NMR spectrum
indicated the two triplets at δ 2.45 and 2.93 for C-CH2-
and -CH2CO- moiety, respectively. e aromatic pro-
tons appeared as multiplets around δ 7.41 due to H-3′
to H-5′ protons and doublet at δ 7.74 for H-2′ and H-6′
protons. e amide proton appeared as singlet δ 10.94.
e mass spectrum of compound 2a exhibited molecu-
lar ion peak at m/z 174 according to molecular formula
C10H10N2O. In third step, the above pyridazinone 2a was
treated with formaldehyde to give 2-(hydroxymethyl)-
6-phenyl-4,5-dihydropyridazin-3(2H)-one 3a. e IR
spectrum showed characteristic band at 3340, 1659 and
1605 for hydroxyl, carbonyl and C=N moiety, respec-
tively. e 1H-NMR spectrum indicated the two trip-
lets at δ 2.63 and δ 3.0 for CH2 and CH2CO- moieties,
respectively. e multiplets appeared at δ 3.30 and at
δ 5.29 due to presence of −OH group (exchangeable
© 2013 Informa UK, Ltd.

558 R. Mishra et al.
Pharmacology
Declaration of interest
e newly synthesized 7-substituted-phenyl-3,4,8,9-
tetrahydro-2H-pyridazino[1,6-a][1,3,5]triazin-2-imine/
one/thione derivatives and standard drugs hydralazine25
and propranolol were screened for antihypertensive
activity by non-invasive Tail Cuff method. e pharma-
cological effects observed contribute to give information
about therapeutic interest of pyridazine derivatives in
hypertension.
e final compounds (4a–4u) were evaluated for anti-
hypertensive activity by non-invasive method using Tail
Cuff method. Compound 4p was found to show highly
significant reduction in MABP in comparison to standard
drugs (Table 1). On the basis of activity reported, it can
be concluded that groups like p-C2H5, p-CH2CH(CH3)2,
p-C6H5 in phenyl ring at 6-position increases the activ-
ity as shown by the compound 4j, 4m and 4p with per-
centage reduction in MABP 40.88%, 40.98% and 42.04%,
respectively.
All compounds possessing substituted aryl moiety
at C-7 of pyridazino[1,6-a]-s-triazin-2-imine/one/
thione ring showed significant percentage reduc-
tion in comparison to the compounds possessing
unsubstituted aryl ring. It indicated that substitution
on aromatic ring is essential for antihypertensive
activity. The compounds substituted with groups
like ethyl, chloro at para position of phenyl ring on
pyridazinone moiety did not show appreciable activ-
ity. Substitution with isobutyl group at para position
of phenyl ring on pyridazinone moiety increases the
antihypertensive activity significantly. Substitution
by methoxy group at para position of phenyl ring
on pyridazinone moiety did not produce significant
activity. Maximum activity was also shown by the
biphenyl substituent at C-6 position of pyridazine
ring.
Ravinesh Mishra expresses sincere thanks to the
University Grant Commission (UGC), New Delhi, India,
for the award of Research Fellowship in Science for
Meritorious Students (RFSMS).
References
1. Williams AD, Lemke LT. Foye’s Principles of Medicinal Chemistry,
in: David Troy (Ed.), fifth ed. Lippincott Williams & Wilkins,
Baltimore, 2002, pp. 824–1195.
2. Frank H, Heinisch G. In Pharmacological Active Pyridazines. Part
2. Progress in Medicinal Chemistry; Ellis, GP, Luscombe, DK, Eds.;
Elsevier: Amsterdam, 1992; Vol. 29, p 141–168.
3. Bristol JA, Sircar I, Moss WH, Evans DB, Weishmaan E.
Cardiotonic agents. 1. 4,5-Dihydro-6-[4-(1H-imidazol-1-yl)
phenyl]-3(2H)-pyridazinones: novel positive inotropic agents
for the treatment of congestive heart failure. J. Med. Chem.
1984;27:1099–1101.
4. Demirayak S, Karaburun AC, Beis R. Some pyrrole substituted
aryl pyridazinone and phthalazinone derivatives and their
antihypertensive activities. Eur. J. Med. Chem. 2004;39:1089–1095.
5. Smolyar NN, Yutilov YM, Gresko SV. Synthesis of 4-amino-
6-(hetaryl)pyridazin-3-ones as analogs of pyridazine-based
cardiotonic agents. Pharm. Chem. J. 2009;43:87–88.
6. Unsal-Tan O, Ozden K, Rauk A, Balkan A. Synthesis and
cyclooxygenase inhibitory activities of some N-acylhydrazone
derivatives of isoxazolo[4,5-d]pyridazin-4(5H)-ones. Eur. J. Med.
Chem. 2010;45(6):2345–2352.
7. Mirzoeva S, Sawkar A, Zasadzki M, Guo L, Velentza AV, Dunlap
V, Bourguignon JJ. Discovery of a 3-amino-6-phenyl-pyridazine
derivative as a new synthetic antineuroinflammatory compound.
J. Med. Chem. 2002;45:563–566.
8. Sotelo E, Coelho A, Ravina E., Pyridazines. Part 34: Retro-ene-
assisted palladium-catalyzed synthesis of 4,5-disubstituted-3(2H)-
pyridazinones. Tetrahedron Lett. 2003;44:4459–4462.
9. Foks H, Wisterowicz K, Miszke A, Brozewicz K, Wisnlewska K,
Szponar MD. Synthesis, fungicidal and antibacterial activity of
new pyridazine derivatives. Heterocycles. 2009;78:961–975.
10. Kaizerman J, Lucas B, McMinn DL, Zamboni R. Annelated
pyridazines for the treatment of tumors driven by inappropriate
hedgehog signaling. PCT Int. Appl. 2009, WO 2009035568.
11. Ishida A, Honma K, Yato M, Nishiyama S, Okumura F. e syntheses
of several heterocyclic skeletons of pyridazine. Eur. Pat. Appl. 1995,
EP 661273.
conclusion
The 7-substituted-phenyl-3,4,8,9-tetrahydro-2H-pyri-
dazino[1,6-a][1,3,5]triazin-2-imine/one/thione
derivatives (4a–4u) can be further modified to exhibit
better potency than the standard drugs. Further stud-
ies to acquire more information about quantitative
structure activity relationships are in progress in the
laboratory. The pyridazine derivatives discovered in
this study may provide valuable therapeutic inter-
vention for the treatment of hypertension because
it is one of the most serious health problems of the
modern world with a continuous rise in the number
of patients.
12. Monge A, Parrado P, Font M, Alvarez EF. Selective thromboxane
synthetase inhibitors and antihypertensive agents. New
derivatives of 4-hydrazino-5H-pyridazino[4,5-b]indole, 4-hydrazi-
notriazino[4,5-a]indole, and related compounds. J. Med. Chem.
1987;30(6):1029–1035.
13. Akahane A, Katayama H, Mitsunaga T, Kato T, Kinoshita T,
Kita Y, Kusunoki T, Terai T, Yoshida K, Shiokawa Y. Discovery
of
6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-
pyridazinebutanoic acid (FK 838): A novel non-xanthine adenosine
A1 receptor antagonist with potent diuretic activity. J. Med. Chem.
1999;42(5):779–783.
14. Livermone DGH, Bethell RC, Cammack N, Hancock AP, Hann
MM, Green DVS, Lamont RB, Noble SA, Orr DC. Synthesis and
anti-HIV-1 activity of a series of imidazo[1,5-b]pyridazines. J. Med.
Chem. 1993;36:3784–3794.
15. Combs DW, Rampulla MS, Bell SC, Klaubert DH, Tobia AJ, Falotico
R, Haertlein B, Weiss CL, Moore JB. 6-Benzoxazinylpyridazin-
3-ones: potent, long-acting positive inotrope and peripheral
vasodilator agents. J. Med. Chem. 1990;33(1):380–386.
16. Robertson DW, Jones ND, Krushinski JH, Pollock GD,
Swartzendruber JK, Hayes JS. Molecular structure of the
acknowledgements
e authors are also thankful to Jamia Hamdard, New
Delhi, India for providing facility for the research
work.
Journal of Enzyme Inhibition and Medicinal Chemistry

Design, synthesis and antihypertensive screening of novel pyridazine substituted s-triazin-2-imine/one/thione derivatives 559
dihydropyridazinone cardiotonic 1,3-dihydro-3,3-dimethyl-
5-(1,4,5,6-tetrahydro-6-oxo-3-pyridazinyl)-2H-indol-2-one,
potent inhibitor of cyclic AMP phosphodiesterase. J. Med. Chem.
1987;30(4):623–627.
21. Gajare AS, Shingare MS. Synthesis of 2,4-Diarylamino-6-(3′,5′,6′-
trichloropyridin-2′-yl)oxy Triazine and Its Herbicidal Activity. Ind.
J. Chem. 1998;37B:510–513.
22. Khan MSY, Siddiqui AA. Pyridazinone derivatives: A potent anti-
inflammatory agents. Ind. J. Chem. 2000;39B:614–620.
23. Siddiqui AA, Ahamad SR, Mira MS, Hussain SA, Raish M, Kaur
R. Synthesis and in-vitro antifungal activity of 6-substituted-
phenyl-2- {[(4’-substituted phenyl-5’-thioxo)-1,2,4-triazol-3-yl]-
methyl}-2,3,4,5-tetrahydropyridazin-3-one derivatives. Acta. Pol.
Pharm. 2008;65(2):223–228.
24. Krakoff LR, Selvadurai R, Sytter E. Effect of methylprednisolone
upon arterial pressure and the renin angiotensin system in the rat.
Am. J. Physiol. 1975;228:613–617.
25. Borchard RE, Barnes CD, Eltherington LG. Drug Dosage in
Laboratory Animals: A Handbook, third ed. e Telford Press Inc.,
New Jersey, 1991.
a
17. Archan S, Toller W. Levosimendan: current status and future
prospects. Curr. Opin. Anesthesiol 2008;21(1):78–84.
18. ota S, Bansal R. Synthesis of new pyridazinone derivatives as
platelet aggregation inhibitors. Med. Chem. Res. 2010;19:808-816.
DOI: 10.1007/s00044-009-9232-6.
19. SchadeFU,SchudtC.especifictypeIIIandIVphosphodiesterase
inhibitor zardaverine suppresses formation of tumor necrosis
factor by macrophages. Eur. J. Pharmacol. 1993;230(1):9–14.
20. Sotelo E, Fraiz N, Yanez M, Terrades V, Laguna R, Cano E, Ravina E.,
Pyridazines. Part XXIX: synthesis and platelet aggregation inhibition
activityof5-substituted-6-phenyl-3(2H)-pyridazinones.Novelaspects
of their biological actions. Bioorg. Med. Chem. 2002;10:2873–2882.
© 2013 Informa UK, Ltd.