Coupling reactions of hydralazine with amino acids and their adducts for antihypertensive activities

Full text of DOI:10.1002/jhet.5570410313

May-Jun 2004
387
Coupling Reactions of Hydralazine with Amino Acids and their
Adducts for Antihypertensive Activities.
Sherine N. Khattab [a], Abdel Moneim M. El Massry [a], Ayman El-Faham [a],
Adnan A. Bekhit [b] and Adel Amer [a][1]
[a] Department of Chemistry, Faculty of Science, Alexandria University, Egypt
[b] Department of Pharmaceutical Chemistry, College of Pharmacy, Alexandria University, Egypt
Received October 13, 2003
Dedicated to the memory of Prof. Hans Zimmer
J. Heterocyclic Chem., 41, 387 (2004).
Despite the fact that hydralazine (phthalazin-1-yl-
hydrazine - Apresoline) 1 was introduced in hyperten-
sion therapy for more than 50 years, the mechanism of its
action is still obscure [2]. The hydrazine group being a
reactive pharmacophore might actively react with any of
the C=O of the biological receptors to exert its effect [3,4].
It is notable that the so called N-acetyl derivative of 1
which was found to be among the major metabolites, was
reassigned as the hydrate form of the stable triazolophtha-
lazine system, namely, 3-methyl-s-triazolo[3,4-a]phthalazine
(3-MeTP) [5-7]. Consequently, an extensive acylation of 1
was studied using a variety of acids and derivatives and
attempts were made to fuse six, seven and eight-membered
rings to the phthalazine ring system for better understanding
of this molecule chemical behavior [8-18]. Furthermore, up
to our knowledge no trial was carried out to deliver
hydralazine supported on amino acids or peptide moiety as a
pro-drug concept for improving its bioavailability and
decreasing its undesirable side effects. Spurred by the afore-
mentioned finding, it became interesting enough to assign a
synthetic program to obtain such triazolophthalazines sup-
ported on amino acid or peptide moiety. The open-chain
counter-parts were also not far from our attention.
On the other hand, molecular modeling supported the
idea that the introduction of a bulky R group might provide
an avenue to stabilize the open intermediate structure by
keeping distance between the two active sites (N-2 of the
phthalazine ring and the amide carbonyl side chain), with
the hope that such designed structure comply with the
enhanced characters or pro-drug requirements. Indeed it
was the case. Thus, utilizing the N-Boc-L-(O-benzyl)-tyro-
sine 2f (R= CH2-[4-(OBn)C6H4]) afforded compound in
1
the open structure and this was also supported by ir, H
nmr, 13C nmr and elemental analysis. The infrared spec-
trum of 4 showed an absorption peak for the amide car-
bonyl at 1663 cm-1, an urethane carbonyl at 1710 cm-1 and
the absorption peak of the N-H at 3442 cm-1. The 13C nmr
showed also a peak corresponding to the amide carbonyl at
172.16 ppm in addition to the urethane carbonyl peak at
166.88 ppm.
Sterric hindrance may prevented ring closure in com-
pound 4 as the distance between the N-2 nitrogen atom and
the amide carbonyl carbon atom is larger. The N-C dis-
tance was compared for the intermediate open structure 4
w i t h d i fferent R group utilizing PM3/MOPAC [19]
(Table 1, Figure 2 and 3).
Results and Discussion.
Chemistry.
Table 1
The N-C Bond Distance of Compound 4
The reaction of phthalazin-1-yl-hydrazine hydrochloride
1. HCl with different N-Boc-L-amino acids 2a-2e in
dimethylformamide in the presence of diisopropylethy-
lamine (DIEA) as a base and N-[(Dimethylamino)-1H-
1, 2, 3-triazolo[4, 5-b]pyridin-1-ylmethylene]- N- m e t h y l-
methanaminium hexafluorophsphate N-oxide (HATU) as
coupling reagent at 0 °C gave smoothly the triazolophtha-
lazine derivatives 3a-3e. The structures of compounds 3a-
R
N-C Bond Distance
CH
CH(CH )
CH(CH )CH -CH
CH -OBn
CH -CH -COOBn
CH -[4-(OBn)C H ]
3.460 Aº
3.503 Aº
3.546 Aº
3.461 Aº
3.376 Aº
4.035 Aº
3
3 2
3
2
3
2
2
2
2
6 4
1
3e were established by mean of ir, H nmr, 13C nmr and
Unfortunately, it was found that this intermediate was
very sensitive to acidic medium. Thus, deprotection of the
Boc group by passing HCl gas into a solution of com-
pounds 3a-3e and compound 4 as well as in methylene
chloride led to the formation of the corresponding tria-
zolophthalazine 5 as stable white hydrochloride salts in
good yield. Deprotection of the Boc group from com-
pounds 3a-3e and 4 was carried out with p-toluenesul-
phonic acid (2 equivalent) in acetonitrile, and gave the
elemental analyses. The infrared spectra of these com-
pounds showed absorption peak of the urethane carbonyls
in the range 1690-1711 cm-1 and the absorption peak of the
N-H in the range 3234-3359 cm-1. The H nmr showed
1
only one exchangeable signal for NH and the structure of 3
was secured by 13C nmr where a peak corresponding to the
amide carbonyl was detected and only the urethane car-
bonyls were observed at 148.10-155.94 ppm.

388
S. N. Khattab, A. M. M. El Massry, A. El-Faham, A. A. Bekhit and A. Amer
Vol. 41
The N-C distance measured for the intermediate of structure 3e as a model
Figure 2
Biological Results.
Antihypertensive Activity.
Compounds 5b, 5e and 5f showed significant hypoten-
sive activity comparable to or higher than that of the refer-
ence standard hydralazine, whereas other compounds
showed lower activity (Table 2). The Mean change in arte-
rial pressure (MAP) of compound 5b was -36±2.57
mmHg, while that of compound 5 f was -39±2.32.
Hydralazine showed MAP of -32±2.44. Moreover 5e
showed hypotensive activity of -31.74±0.96 mmHg. It is
noteworthy, that the hypotensive effect of compounds 5b,
5e and 5f were sustained for about 3 hours. This result
speculates that the free amino group of 5b, 5e and 5f is
essential for their hypotenssive activity.
corresponding tosylate as moisture sensitive salts 6. The
structure of compounds 5 and 6 were confirmed by spec-
troscopic measurements. Our attempt to remove the pro-
tecting group (Boc) from compounds 3a-3e and com-
pound 4 with acetic acid as a weaker acid, led to the for-
mation of the N-acetyl triazolophthalazines 7. The struc-
1
ture of compounds 7 were established by ir, H nmr and
13C nmr as well as elemental analyses.
Our approach to build up a peptide chain on the tria-
zolophthalazine structure 6 using N-Boc-amino acid and
HATU as a coupling reagent in the presence of DIEA has
met with success and compounds 8a and 8b were prepared
as a prototype (Figure 1). Further synthetic work is in
progress to furnish more compounds for SAR.
The N-C distance measured for structure 4
Figure 3
Acute Toxicity.
Compounds 5b, 5f and 5e were further evaluated for
their approximate LD50 in male mice using the method
described in the literature [20,21]. The results indicated
that most of the tested compounds proved to be non-toxic
and well tolerated by the experimental animals as evi-
denced by their LD50 values (> 90 mg/Kg). Moreover,
these compounds were tested for their toxicity through

May-Jun 2004
Coupling Reactions of Hydralazine
389
sodium bicarbonate solution and finally with water, dried and
recrystallized.
parenteral route [22]. The results revealed that all the test
compounds were non-toxic up to 50 mg/Kg.
(1-[1,2,4]Triazolo[3, 4-a]phthalazin-3-yl-ethyl)-carbamic Acid
tert-Butyl Ester (3a).
Table 2
Hypotensive Activity of the Test Compounds
This compound was obtained as yellow crystals (methylene
chloride-hexane), 0.13 g (83 %) yield, mp 128-130 °C; ir (KBr):
Compound
Change in MAP mmHg as mean ± SE
-1
3353 (NH), 1696 (C=O, urethane) cm ; H nmr (DMSO-d ): δ
1
6
1.37 (s, 9H, 3 CH ), 1.57 (d, 3H, CH ), 5.29 (m, 1H, CH), 7.65
Control
Hydralazine
1.0 ± 0.62
-32±2.44
3
(d, 1H, NH), 7.91, 8.03 (2 t, 2H, H , H aromatic), 8.19, 8.47 (2d,
3
8
2H, H , H aromatic), 9.06 (s, 1H, H aromatic); C nmr
9
13
5b
5f
-36±2.57
-39±2.32
7
10
6
(DMSO-d ): δ 19.3, 28.5, 42.2, 76.5, 120.1, 120.6, 120.9, 127.1,
6
129.0, 132.3, 140.3, 146.1, 149.7, 153.0.
5e
3b
4
3d
3c
3a
-31.74±0.96
-11.12±1.35
-11.85.25±0.67
-16.35±1.36
-12.32±1.56
-7.86±2.39
Anal. Calcd. for C
H N O : C, 61.33; H, 6.11; N, 22.35.
16 19 5 2
Found: C, 61.08; H, 6.21; N, 22.11.
(2-Methyl-1-[1,2,4]triazolo[3,4-a]phthalazin-3-yl-propyl)-car -
bamic Acid tert-Butyl Ester (3b).
This compound was obtained as yellow needles (methylene
chloride-hexane), 0.09 g (52.8 %) yield, mp 186-188 °C; ir
Post-hoc Gabriel test between control and the rest of the groups
P<0.001
-1
1
(KBr): 3234 (NH), 1710 (C=O, urethane) cm ; H nmr (DMSO-
d ): δ 0.8, 0.9 (2d, 6H, 2 CH ), 1.29 (s, 9H, 3 CH ), 2.31 (m, 1H,
CH), 5.08 (m, 1H, CH), 7.50 (d, 1H, NH), 7.70, 7.83 (2 t, 2H, H ,
6
3
3
EXPERIMENTAL
8
H aromatic), 7.96, 8.23 (2d, 2H, H , H aromatic), 9.10 (s, 1H,
13
9
H aromatic); C nmr (DMSO-d ): δ 19.18, 19.44, 28.5, 31.7,
7
10
Chemistry.
6
6
50.14, 76.79, 120.8, 121.4, 121.6, 127.8, 129.7, 133.1, 140.6,
146.8, 149.6, 154.2.
Melting points were determined with a Mel-Temp apparatus
1
and are uncorrected. Magnetic resonance spectra ( H nmr and
13
C nmr spectra) were recorded on a Brucker 300 MHz spec-
Anal. Calcd for C
H N O : C, 63.32; H, 6.79; N, 20.51.
18 23 5 2
Found: C, 63.22; H, 6.83; N, 20.55.
trometer with chemical shift values reported in δ (part per mil-
lion) relative to an internal standard (tetramethylsilane). Infrared
data were obtained on a Perkin-Elmer 1600 series Fourier trans-
form instrument as KBr pellets. Geometry optimizations were
performed initially by PM3/MOPAC in Alchemy 2000 and sub-
sequently the N-C distances were measured. The compounds
were named using Chem. Draw Ultra version 6, Cambridgesoft
Corporation. The abbreviations: HATU, N-[(Dimethylamino)-
1H-1, 2, 3-triazolo[4, 5-b]pyridin-1-ylmethylene]-N-methylmeth -
anaminium hexafluorophsphate N-oxide; Boc, t-butyloxycar-
bonyl; DIEA, diisopropylethylamine; DMF, N,N-dimethyl-
formamide; PTSA, p-toluenesulphonic acid were used through
out the manuscript. Elemental analyses were performed in the
Chemistry Department, Faculty of Science, Cairo University.
(2-Methyl-1-[1, 2, 4]triazolo[3, 4-a]phthalazin-3-yl-butyl)-car-
bamic Acid tert-Butyl Ester (3c).
This compound was obtained as yellow crystals (methylene
chloride-hexane), 0.171 g (96.3 %) yield, mp 11 4 - 115 °C; ir
-1
(KBr): 3269 (NH), 1710 (C=O, urethane) cm ; H nmr (CDCl ):
1
3
δ 0.95 (m, 6H, 2 CH ), 1.2, 1.6 (2 m, 2H, CH ), 1.35 (s, 9H, 3
3
CH ), 2.13 (m, 1H, CH), 5.5 (m, 1H, CH), 5.65 (d, 1H, NH),
2
3
7.75, 7.90, 8.70 (3m, 5H, aromatic); C nmr (CDCl ): δ 11.72,
13
3
15.79, 25.33, 28.72, 39.80, 50.94, 80.12, 123.54, 123.70, 123.86,
128.51, 131.28, 134.43, 142.97, 147.98, 151.37, 155.82.
Anal. Calcd. for C
H N O : C, 64.20; H, 7.09; N, 19.70.
19 25 5 2
Found: C, 64.30; H, 7.15; N, 19.82.
(2-Benzyloxy-1-[1,2,4]triazolo[3,4-a]phthalazin-3-yl-ethyl)-car-
bamic Acid tert-Butyl Ester (3d).
General Procedure for the Reaction of N-Boc-L-Amino Acids
with Phthalazin-1-yl-hydrazine Hydrochloride (1.HCl).
This compound was obtained as yellow crystals (methylene
chloride-hexane), 0.17 g (81.2 %) yield, mp 138-139 °C; ir
A mixture of (0.5 mmol) of N-Boc-L-amino acid (2), 0.19 g
(0.5 mmol) of HATU and 0.17 mL (1 mmol) of DIEA was stirred
at 0 °C for 3 min in 2 mL dimethylformamide. This solution was
then added to a solution of hydralazine hydrochloride (1.HCl)
(0.098 g, 0.5 mmol) and (0.09 mL, 0.5 mmol) DIEA in 2 mL of
dimethylformamide at 0 °C. The reaction mixture was stirred at 0
°C for 1 hour and left overnight at room temperature. The reac-
tion mixture was diluted with 80 mL ethyl acetate, and the mix-
ture was washed with 5% aqueous citric acid solution (2×10 mL),
saturated sodium bicarbonate solution (2×10 mL) and saturated
sodium chloride solution (2×10 mL). The organic layer was dried
over anhydrous sodium sulphate, filtered and the solvent was
removed in vacuo. The crude product was crystallized from
methylene chloride-hexane. Only in the case of N-Boc L-(O-ben-
zyl)-tyrosine (2f) the reaction mixture was poured into ice water,
filtered, washed with 5% aqueous citric acid solution, saturated
-1
(KBr): 3303 (NH), 1711 (C=O, urethane) cm ; H nmr (CDCl ):
1
3
δ 1.39 (s, 9H, 3 CH ), 3.91 (m, 2H, CH ), 4.46 (m, 2H, CH ),
3
2
2
5.65 (m, 1H, CH), 6.20 (d, 1H, NH), 7.08 (s, 5H, aromatic), 7.82
(d, 1 H, H aromatic), 7.90 (t, 2H, H , H aromatic), 8.5 (d, 1H,
13
10
H aromatic) 8.6 (s, 1H, H aromatic); C nmr (CDCl ): δ 27.3,
8
9
7
6
3
45.6, 69.0, 71.9, 78.8, 121.9, 122.1, 122.2, 126.48, 126.51,
126.75, 127.1, 127.35, 130.0, 133.1, 136.6, 142.0, 146.7, 148.1.
Anal. Calcd. for C
H N O : C, 65.86; H, 6.01; N, 16.70.
23 25 5 3
Found: C, 65.78; H, 6.03; N, 16.55.
4-t e rt-Butoxycarbonylamino-4-[1,2,4]triazolo[3,4-a]phthalazin-
3-yl-butyric Acid Benzyl Ester (3e).
This compound was obtained as pale beige crystals (methylene
chloride-hexane), 0.17 g (73.8 %) yield, mp 143-144 °C; ir

390
S. N. Khattab, A. M. M. El Massry, A. El-Faham, A. A. Bekhit and A. Amer
Vol. 41
(KBr): 3359 (NH), 1733 (C=O, ester), 1690 (C=O, urethane),
2-Amino-3-(4-benzyloxy-phenyl)-propionic Acid N'-Phthalazin-
1-yl-hydrazide Dihydrochloride Salt (5f).
-1
cm ; H nmr (DMSO-d ): δ 1.37 (s, 9H, 3 CH ), 2.06 (m, 2H,
1
6
3
CH ), 3.2 (m, 2H, CH -CO), 4.13 (m, 1H, CH), 5.13 (m, 2H,
2
CH -O), 7.34 (s, 5H, aromatic), 7.5 (d, 1H, NH), 7.92, 8.08 (2 t,
2
Compound 2f (0.094 g (0.18 mmol) was dissolved in a mixture
of 4 mL of methylene chloride and 4 mL of ether. Gaseous
hydrochloric acid was passed through the solution for 2 hours. A
white precipitate was formed, collected by filtration and washed
with ether. The crude product was recrystallized from methyl
alcohol-ether to give white crystals, 0.07 g (76.1 %), mp 194 °C;
2
2H, H , H aromatic), 8.21, 8.48 (2 d, 2H, H , H aromatic),
13
8
9
7
10
9.05 (s, 1H, H aromatic); C nmr (DMSO-d ): δ 20.8, 27.7,
6
6
28.5, 53.4, 66.3, 78.70, 122.38, 123.07, 123.28, 128.05, 128.33,
128.70, 129.40, 131.30, 134.63, 136.27, 142.48, 148.37, 149.90,
155.94, 172.48.
-1
1
ir (KBr): 3031-2892 (br., NH), 1720 (C=O) cm ; H nmr
(DMSO-d ): δ 3.1, 3.27 (2 m, 2H, CH ), 4.40 (m, 1H, CH), 4.95,
5.1 (2 s, 2H, CH -O), 6.81-9.1 (m, 16H, aromatic + 3NH), 9.15
Anal. Calcd. for C
H N O : C, 65.06; H, 5.90; N, 15.17.
25 27 5 4
Found: C, 65.15; H, 6.03: N, 15.30.
6
2
2
13
(s, 1H, H aromatic); C nmr (DMSO-d ): δ 37.0, 46.45, 53.65,
6
[2-(4-Benzyloxy-phenyl)-1-(N'- phthalazin-1-yl-hydrazinocar-
bonyl)-ethyl]-carbamic Acid tert-butyl Ester (4).
6
69.40, 115.08, 115.28, 122.48, 122.63, 123.39, 127.21, 127.93,
128.05, 128.11, 128.19, 128.69, 128.80, 129.72, 130.69, 131.19,
131,96, 135.12, 137.29, 137.47, 142.97, 147.21, 148.98, 157.72,
157.98.
This compound was obtained as pale yellow crystals (methylene
chloride-hexane), 0.23 g (89.5 %) yield, mp 85 °C; ir (KBr): 3442
-1
(NH), 1710 (C=O, urethane), 1663 (C=O, amide) cm ; H nmr
1
Anal. Calcd. for C
N, 13.89. Found: C, 57.34; H, 5.61; N, 14.18.
H N O•2HCl•2H O: C, 57.10; H, 5.36;
24 21 5 2
(DMSO-d ): δ 1.32 (s, 9H, 3 CH ), 2.8, 3.0 (2 m, 2H, CH ), 4.15
6
(m, 1H, CH), 5.07 (2brs, 2H, CH -O), 6.92-9.09 (m, 15H, aromatic
3
2
2
+ NH), 9.89, 11.15 (2 br.d, 2NH); C nmr (DMSO-d ): δ 27.7,
13
1 - [ 1 , 2 , 4 ] Tr i a z o l o [ 3 , 4 - a]phthalazin-3-yl-ethylamine p-
Toluenesulphonate Salt (6a).
6
35.99, 47.4, 68.6, 77.50, 113.87, 113.94, 122.32, 125.94, 127.08,
127.13, 127.23, 127.87, 128.58, 129.58, 129.78, 130.55, 130.80,
133.85, 136.72, 136.86, 154.85, 155.08, 156.41, 166.88, 172.16.
Compound 3a (0.166 g, 0.4 mmol) was dissolved in 3 mL ace-
tonitrile. p-Toluene sulfonic acid 0.154 g (0.8 mmol) was added
to the reaction mixture, and the reaction mixture was allowed to
be stirred for 3 hours. Then the solvent was evaporated in vacuo
and the crude product obtained was recrystallized from methyl
alcohol-ether to give white crystals, 0.17 g (57.63 %), mp 225-
Anal. Calcd. for C
H N O : C, 67.82; H, 6.08; N, 13.64.
29 31 5 4
Found: C, 67.99; H, 6.18; N, 13.49.
2-Methyl-1-[1,2,4]triazolo[3,4-a]phthalazin-3-yl-propylamine
Dihydrochloride Salt (5b).
-1
1
226 °C; ir (KBr): 2951 (NH) cm ; H nmr (CDCl ): δ1.82 (d,
3
3H, CH ), 2.24 (s, 6H, 2CH ), 5.19 (m, 1H, CH), 7.06, 7.59 (2d,
Compound 2b (0.237 g, 0.69 mmol) was dissolved in a mix-
ture of 4 mL of methylene chloride and 4 mL of ether. Gaseous
hydrochloric acid was passed through the solution for 2 hours. A
white precipitate was formed, was collected by filtration and
washed with ether. The crude product was recrystallized from
3
8H, aromatic), 7.73-8.00 (m, 3H, H , H , H aromatic), 8.6 (d,
3
8
9
7
1H, H aromatic), 8.73 (s, 1H, H aromatic), 9.05 (brs, 3H,
10
13
6
3NH); C nmr (CDCl ): δ18.00, 21.56, 42.73, 123.80, 126.30,
3
129.00, 129.10, 132.30, 134.90, 141.50, 141.90, 149.10.
methyl alcohol-ether to give white crystals, 0.17 g (71.7 %), mp
1
229 °C; ir (KBr): 3083-2890 (NH); H nmr (DMSO-d ): δ 0.92,
2-Methyl-1-[1,2,4]triazolo[3,4-a]phthalazin-3-yl-propylamine p-
Toluenesulphonate Salt (6b).
6
1.05 (2d, 6H, 2 CH ), 2.20 (m, 1H, CH), 4.83 (m, 1H, CH), 7.99,
3
8.10 (2 t, 2H, H , H aromatic), 8.25, 8.57 (2d, 2H, H , H aro-
Compound 3b (0.137 g, 0.4 mmol) was dissolved in 3 mL ace-
tonitrile. p-Toluene sulfonic acid 0.154 g (0.8 mmol) was added
to the reaction mixture and the reaction mixture was allowed to
be stirred for 3 hours. Then the solvent was evaporated in vacuo
and the crude product obtained was recrystallized from methyl
alcohol-ether to give white crystals, 0.171 g (74.35 %), mp 244-
8
9
7
matic), 9.07 (br s, 3H, 3NH), 9.23 (s, 1H, H aromatic); C nmr
10
13
6
(DMSO-d ): δ 18.07, 18.79, 31.02, 50.07, 118.46, 122.45,
6
123.34, 129.52, 131.73, 134.88, 142.77, 147.00, 149.04.
Anal. Calcd. for C
N, 20.51. Found: C, 45.43; H, 6.14; N, 20.21.
H N 2HCl 1.5H O: C, 45.72; H, 5.86;
13 15 5 2
-1
1
245 °C; ir (KBr): 2923 (NH) cm ; H nmr (DMSO-d ): δ 0.93,
6
1.03 (2d, 6H, 2 CH ), 2.28 (s, 6H, 2CH ), 2.50 (m, H, CH), 4.90
4-Amino-4-[1, 2, 4]triazolo[3, 4-a]phthalazin-3-yl-butyric Acid
Benzyl Ester Dihydrochloride Salt (5e).
3
(m, H, CH), 7.10, 7.47 (2d, 4H, aromatic), 7.98, 8.12 (2t, 2H, H ,
3
8
Compound 2e (0.239 g, 0.52 mmol) was dissolved in a mixture
of 4 mL of methylene chloride and 4 mL of ether. Gaseous
hydrochloric acid was passed through the solution for 2 hours. A
white precipitate was formed, collected by filtration and washed
with ether. The crude product was recrystallized from methyl
alcohol-ether to give white crystals, 0.186 g (76.2 %), mp 149-
H aromatic), 8.29, 8.57 (2d, 2H, H , H aromatic), 8.79 (br.s,
9
7
10
13
3H, 3NH), 9.21 (s, 1H, H aromatic); C nmr (DMSO-d ): δ
6
6
18.28, 18.76, 21.13, 31.35, 50.54, 122.68, 122.71, 123.55,
125.82, 128.46, 129.76, 131.97, 135.14, 138.15, 143.15, 145.73,
147.22, 149.33.
2-Methyl-1-[1,2,4]triazolo[3,4-a]phthalazin-3-yl-butylamine p-
Toluenesulphonate Salt (6c).
1
150 °C; ir (KBr): 3100-2884 (NH), 1720 (C=O); H nmr
(DMSO-d ): δ 2.49 (m, 2H, CH ), 3.32 (m, 2H, CH -CO), 4.35
6
(m, 1H, CH), 5.23 (s, 2H, CH -O), 7.39 (m, 5H, aromatic), 7.99,
2
2
Compound 6c (0.142 g, 0.4 mmol) was dissolved in 3 mL ace-
tonitrile. p-Toluene sulfonic acid 0.154 g (0.8 mmol) was added
to the reaction mixture. The reaction mixture was allowed to be
stirred for 3 hours. Then the solvent was evaporated in vacuo and
the crude product obtained was recrystallized from methyl alco-
hol-ether to give white crystals, 0.13 g (54.4 %), mp 228-229 °C;
2
8.10 (2 m, 2H, H , H aromatic), 8.28, 8.60 (2 m, 2H, H , H
8
aromatic), 8.93 (br s, 3H, 3NH), 9.19 (s, 1H, H aromatic);
9
7
10
13
C
nmr (DMSO-d ): δ 20.20, 26.83, 51.65, 67.58, 122.01, 122.97,
6
6
123.73, 128.45, 128.63, 128.77, 129.63, 132.17, 134.93, 135.39,
142.34, 149.14, 149.49, 169.49.
Anal. Calcd. for C H N O 2HCl 2H O: C, 50.91; H, 5.52;
20 20 5 2 2
N, 14.85. Found: C, 51.12; H, 5.26; N, 15.08.
-1
1
ir (KBr): 2953 (NH) cm ; H nmr (DMSO-d ): δ 0.91 (m, 6H,
6
2CH ), 1.04 (d, 2H, CH ), 1.56 (m, 1H, CH), 2.27 (s, 6H, 2CH ),
3
2
3

May-Jun 2004
Coupling Reactions of Hydralazine
391
5.00 (m, H, CH), 7.11, 7.48 (2d, 8H, aromatic), 8.00, 8.12 (2t,
2H, H , H aromatic), 8.28, 8.44 (m, 2H, H , H aromatic), 8.56,
13
3-yl-ethyl]-acetamide (7f).
8
8.64, 8.80 (3br.d, 3H, 3NH), 9.21 (s, 1H, H aromatic); C nmr
9
7
10
Compound 4 (0.1 g, 0.19 mmol) was dissolved in 3 mL acetic
acid. The reaction mixture was allowed to undergo reflux for 8
hours. The reaction mixture was poured over a cold solution of
saturated sodium carbonate. A white precipitate was formed, fil-
tered and washed with water. The crude product was recrystal-
lized from methyl alcohol to give white crystals, 0.074 g (83.8
6
(DMSO-d ): δ 11.40, 14.90, 21.23, 25.06, 37.60, 49.24, 122.68,
6
123.54, 124.43, 125.81, 127.92, 128.49, 129.76, 131.96, 135.23,
138.20, 143.80, 145.50, 147.04, 149.32, 168.60.
2-(4-Benzyloxy-phenyl)-1-[1,2,4]Triazolo[3,4-a]phthalazin-3-
yl-ethylamine p-Toluenesulphonate Salt (6f).
-1
%), mp 215 °C; ir (KBr): 3291 (NH), 1651 (C=O, amide) cm ;
1
H nmr (DMSO-d ): δ 1.87 (s, 3H, CH ), 3.2 (m, 2H, CH ), 5.03
6
(s, 2H, CH ), 5.77 (m, 1H, CH), 6.89, 7.15 (2d, 4H, aromatic),
3
2
Compound 4 (0.21 g, 0.4 mmol) was dissolved in 3 mL ace-
tonitrile. p-Toluene sulfonic acid 0.154 g (0.8 mmol) was added
to the reaction mixture, and the reaction mixture was allowed to
be stirred for 3 hours. Then the solvent was evaporated in vacuo
and the crude product obtained was recrystallized from methyl
alcohol-ether to give white crystals, 0.195 g (65 %), mp 180-182
2
7.38 (s, 5H, aromatic), 7.95-8.23 (m, 3H, NH + 2H aromatic),
8.51, 8.80 (2 d, 2H, H , H aromatic), 9.07 (s, 1H, H aromatic);
13
7
C nmr (DMSO-d ): δ 22.75, 38.4, 46.15, 69.4, 114.80, 115.18,
10
6
6
122.41, 122.86, 123.25, 127.98, 128.09, 128.70, 129.43, 129.90,
130.49, 131.44, 134.71, 137.44, 142.44, 148.54, 150.80, 157.33,
169.30.
-1
1
°C; ir (KBr): 2921 (NH) cm ; H nmr (DMSO-d ): δ2.28 (s, 6H,
6
2CH ), 3.40 (d, 2H, CH ), 4.90 (s, 2H, CH ), 5.20(m, 1H, CH),
3
2
2
Anal. Calcd. for C
H N O : C, 71.50; H, 6.00; N, 15.44.
27 27 5 2
Found: C, 71.32; H, 6.16; N, 15.18.
6.80, 7.00 (2d, 4H, aromatic), 7.1, 7.5 (2d, 8H, aromatic), 7.35
(m, 5H, aromatic), 8.00, 8.10 (2t, 2H, H , H aromatic), 8.20,
8
9
[2-(4-Benzyloxy-phenyl)-1-(2-methyl-1-[1, 2, 4]triazolo[3, 4-
a]phthalazin-3-yl-propylcarbamoyl)-ethyl]-carbamic Acid t e rt-
Butyl Ester (8a).
8.50 (2d, 2H, H , H aromatic), 8.90 (brs, 3H, 3NH), 9.10 (s,
7
10
13
1H, H aromatic); C nmr (DMSO-d ): δ 21.13, 37.15, 46.60,
6
6
69.40, 115.10, 122.44, 122.65, 123.4, 125.82, 127.04, 127.92,
128.11, 128.51, 128.70, 129.74, 130.76, 132.00, 135.13, 137.30,
138.30, 143.03, 145.50, 147.21, 149.10, 157.80.
A mixture of N-Boc L-(O-benzyl)-tyrosine (2f) 0.186 g (0.5
mmol), HATU 0.19 g (0.5 mmol) and DIEA 0.17 mL (1 mmol)
was stirred in 2 mL dimethylformamide at 0 °C for 3 minutes and
then the solution was added to a solution of (6b) 0.293 g (0.5
mmol) and 0.09 mL (0.5 mmol) DIEA in 2 mL of dimethylfor-
mamide at 0 °C. The reaction mixture was stirred at 0 °C for 1
hour and then overnight at room temperature. The reaction mix-
ture was poured into ice water, the solid was collected by filtra-
tion, washed with 5% citric acid solution, saturated sodium bicar-
bonate solution then with water, dried and then recrystallized.
The crude product was recrystallized from methylene chloride-
hexane to give white crystals, 0.291 g (97.65 %), mp 84-85 °C; ir
(KBr): 3428, 3351 (NH), 1710 (C=O, urethane), 1677 (C=O,
N-(1-[1, 2, 4]Triazolo[3, 4-a]phthalazin-3-yl-ethyl)-acetamide
(7a).
Compound 3a (0.17 g, 0.41 mmol) was dissolved in 3 mL
acetic acid. The reaction mixture was allowed to undergo reflux
for 8 hours. The reaction mixture was then poured over a cold
solution of saturated sodium carbonate. A white precipitate was
formed, which was collected by filtration and washed with water.
The crude product was recrystallized from methyl alcohol to give
white crystals, 0.125 g (85.03 %), mp 230 °C; ir (KBr): 3274
-1
1
(NH), 1655 (CO, amide) cm ; H nmr (DMSO-d ): δ 1.68 (d,
6
3H, CH ), 1.91 (s, 3H, CH ), 5.65 (m, 1H, CH), 7.99, 8.09 (2 t,
3
2H, H , H aromatic), 8.27, 8.53 (2 d, 2H, H , H aromatic),
3
-1
1
amide) cm ; H nmr (CDCl ): δ 0.8, 0.95 (2d, 6H, 2CH ), 1.39
3
(s, 9H, 3CH ), 2.25 (m, 1H, CH), 2.8, 2.9 (2m, 2H, CH ), 4.2 (m,
3
8
8.69 (d, 1H, NH)-9.15 (s, 1H, H aromatic); C nmr (DMSO-
9
7
10
3
1H, CH), 4.7 (s, 2H, CH ), 5.1 (m, 1H, CH), 6.5 (brs, 2H, 2NH),
2
13
6
d ): δ 19.35, 22.78, 40.4, 122.43, 123.19, 129.45, 131.46, 134.72,
2
6.9 (m, 4H, aromatic), 7.2 (m, 5H, aromatic), 7.8-8.00 (m, 3H,
6
142.66, 148.56, 151.72, 169.09.
13
H , H , H aromatic), 8.5 (m, 2H, H , H aromatic); C-NMR
8
(CDCl ): δ 19.08, 19.47, 28.69, 33.24, 38.48, 50.96, 56.97,
9
7
10
6
Anal. Calcd. for C
Found: C, 61.71; H, 6.59; N, 26.09.
H N O: C, 61.98; H, 6.32; N, 25.81.
14 17 5
3
69.77, 80.46, 114.78, 115.21, 123.40, 123.73, 127.50, 127.86,
128.11, 128.51, 128.78, 128.94, 129.11, 130.58, 131.33, 134.54,
137.34, 142.99, 147.87, 155.74, 157.60, 171.21.
N-(2-Benzyloxy-1-[1,2,4]triazolo[3,4-a]phthalazin-3-yl-ethyl)-
acetamide (7d).
Anal. Calcd. for C
H N O : C, 68.67; H, 6.44; N, 14.13.
34 38 6 4
Found: C, 68.42; H, 6.15; N, 14.41.
Compound 3d (0.1 g, 0.32 mmol) was dissolved in 3 mL acetic
acid. The reaction mixture was allowed to undergo reflux for 8
hours. The reaction mixture was then poured over a cold solution
of saturated sodium carbonate. A white precipitate was formed,
whcih was collected by filtration and washed with water. The
crude product was recrystallized from methyl alcohol to give
white crystals, 0.078 g (90.5 %), mp 163-164 °C; ir (KBr): 3247
{1-[2-(4-Benzyloxy-phenyl)-1-[1,2,4]triazolo[3,4- a]phthalazin-
3-yl-ethylcarbamo-yl]-ethyl}-carbamic Acid tert-Butyl Ester
(8b).
A mixture of N-Boc-L-alanine (2a) 0.095 g (0.5 mmol), HATU
0.19 g (0.5 mmol) and DIEA 0.17 mL (1 mmol) was stirred at 0
°C for 3 minutes in 2 mL of dimethylformamide and then the
solution was added to a solution of (6f) 0.37 g (0.5 mmol) and
0.09 mL (0.5 mmol) DIEA in 2 mL of dimethylformamide at 0
°C. The reaction mixture was stirred at 0 °C for 1 hour and then
overnight at room temperature. The reaction mixture was poured
into ice water, the solid was collected by filtration, washed with
5% citric acid solution, saturated sodium bicarbonate solution
then with water, dried and then recrystallized. The crude product
was recrystallized from methylene chloride-hexane to give white
-1
1
(NH), 1659 (C=O, amide) cm ; H nmr (CDCl ): δ 2.02 (s, 3H,
3
CH ), 3.94 (dd, 2H, CH ), 4.42 (m, 2H, CH ), 6.02 (m, 1H, CH),
3
7.02 (br.s, 6H, 5H aromatic + NH), 8.64 (m, 5H, aromatic);
2
2
13
C
nmr (CDCl ): δ 23.65, 45.70, 69.97, 73.39, 123.48, 123.62,
3
123.96, 127.96, 128.05, 128.59, 128.62, 131.69, 134.69, 137.88,
143, 33, 148.28, 149.35, 170.35.
Anal. Calcd for C
H N O : C, 66.47; H, 5.30; N, 19.38.
20 19 5 2
Found: C, 66.21; H, 5.57; N, 19.62.
N-[2-(4-Benzyloxy-phenyl)-1-[1,2,4]triazolo[3,4-a]phthalazin-

392
S. N. Khattab, A. M. M. El Massry, A. El-Faham, A. A. Bekhit and A. Amer
Vol. 41
crystals, 0.25 g (87.9 %), mp 90-91 °C; ir (KBr): 3303 (NH),
-1
mals were used [22]. The compounds or their vehicle, propylene
glycol (control) were given by intrapretonial injection in doses of
10, 25, 50, 75, 100 mg / kg, respectively. Survival was followed up
to 7 days.
1
; H nmr
1710 (C=O, urethane), 1674 (C=O, amide) cm
(CDCl ): δ 1.25 (d, 3H, CH ), 1.37 (s, 9H, 3CH ), 3.40 (m, 2H,
3
CH ), 4.15 (m, 1H, CH), 4.90 (s, 2H, CH ), 5.00 (m, 1H, CH),
3
3
2
2
6.10 (m, 1H, NH), 6.70, 7.00 (2d, 4H, aromatic), 7.3 (m, 5H, aro-
matic), 7.84 (t, 1H, H aromatic), 7.90-8.03 (m, 2H, H , H aro-
13
8
matic), 8.56 (s, 1H, H aromatic), 8.67 (d, 1H, H aromatic;
9
7
REFERENCES AND NOTES
C
nmr (CDCl ): δ 18.91, 28.71, 39.69, 46.49, 50.59, 70.25, 80.43,
6
10
3
[1] Part 17 of the series, Synthetic Reactions and Structural
Studies of Heterocycles Containing Nitrogens; for part 16 see: H.
Zimmer, C. P. Librera, S. Hausner, J. Bauer and A. Amer, Molecules, 8,
735 (2003).
115.13, 123.53, 123.87, 127.83, 128.30, 128.56, 128.61, 128.93,
130.90, 131.54, 134.60, 137.35, 143.15, 148.14, 150.45, 158.08,
172.53.
Anal. Calcd. for for C H N O : C, 67.83; H, 6.05; N, 14.83.
32 34 6 4
Found: C, 67.66; H, 5.81; N, 15.13.
[2] B. L. Pegram, S. Ishize and E. D. Frohlich, Cadiovasc. Res.,
45, 77 (2000).
[3] G. R. Echevarria-Gorostidi, A. Basagoitia, E. Pizarro, R.
Goldsmid, J. G. Santos Blanco and F. Garcia Blanco, Helv. Chim. Acta,
81, 837 (1998).
Antihypertensive Activity.
Rats were divided into groups, each of six animals. Each group
was separately anaesthesized with thiopental sodium (50 mg/kg,
i.p.) and fixed in supine position. Hair of the upper part of the left
hind limb was shaved then 1 cm incision was made in the skin of
the groin and underlying muscles were cut. The femoral artery
and vein were exposed. The femoral artery was tied closed to its
connection with the pelvic artery and an open tie was made about
1 cm apart near its connection with the internal iliac artery. An
artery clamp was placed in the front of the open tie to prevent
hemorrhage, then by means of a fine scissors a very small inci-
sion was made in the artery through which a polyethylene
catheter filled with heparin (100IU/mL saline) was introduced.
Then the artery clamp was removed and catheter was connected
to the pressure transducer the arterial blood pressure was dis-
placed on a Grass polygraph. The catheter was tunneled subcuta-
neously and exteriorized at the back of the neck between the
scapulae the each rat was housed separately. Blood pressure was
recorded at 0 hour then 2 hours after giving 6 mg of the
drug/kg/p.o., suspended in olive oil. The control group received
olive oil / p.o. The change in MAP was calculated [23].
[4] M. Sanchez-Salvatori, C. Rios and H. Vidrio, Cardiovascular
Research, 37, 748 (1998).
[5] S. Edwards and F. H. Marquardt, Hoppe-Seylers’s Z. Physiol.
Chem., 350, 85 (1969).
[6] H. Zimmer, J. McManus, T Novinson, E. V. Hess, and A. H.
Litwin, Arzneimittel-Forsch (Drug Research), 20, 1586 (1970).
[7] H. Zimmer, J. Kokosa and D. A. Garteiz, Arzneimittel-
Forschung (Drug Research), 23, 1028 (1973).
[8] J. Druey and B. H. Ringier, Helv. Chim. Acta, 34, 195 (1951).
[9] P. A. Reece, Med. Res. Rev., 1, 73 (1981).
[10] K. T. Potts and Lovelette, J. Org. Chem., 34, 3221 (1969).
[11] H. Zimmer, J. Kokosa and K. J. Shah, J. Org. Chem., 40, 2901
(1975).
[12] A. Amer and H. Zimmer, J. Heterocyclic Chem., 18 ,1625
(1981).
[13] H. Zimmer, A. Amer and L. Baldwin, Pharmazie, 3 7, 451
(1982).
[14] A. Amer and H. Zimmer, J. Heterocyclic Chem., 20, 1231
(1983).
[15] A. Amer and H. Zimmer, Heterocycles, 26,1177 (1987).
[16] A. Amer, K. Weisz and H. Zimmer, Heterocycles, 26, 1853
(1987).
[17] A. Amer, A. M. El-Massry, S. El-Sadany and H. Zimmer, J.
Chem. Research (S), 298, (1988).
Acute Toxicity.
The same biologically significant compounds were further
investigated for their approximate LD in male mice [20,21]
[18] H. Zimmer, A. R. Safwat, D. Ho, A. Amer and M. Badawi, J.
Org. Chem., 60, 1908 (1995).
50
[19] D. C. Young, Computational Chemistry, John Wiley & Sons,
New York, NY, 2001, pp 37, pp 342.
[20] M. Verma, M. Tripathi, A. K. Saxena and K. Shanker, Eur. J.
Med. Chem., 29, 941 (1994).
[21] J. T. Litchfield and F. Wilcoxon, J. Pharmacol. Exp. Ther., 96,
99 (1949).
[22] A. A. Bekhit, and H. T. Y. Fahmy, Archiv Pharm. Pharm.
Med. Chem., 336, 111 (2003).
(each 20 g, supplied by Medical Research Institute, Alexandria
University). Groups of mice each consisting of six animals were
used. The compounds were given orally suspended in 1% gum
acacia, in doses of 1, 10, 100, 200, 250, 500 mg / kg, respectively.
Twenty-four hours later, the % mortality in each group and for
each compound was recorded. The LD values were calculated
50
using the method described by Litchfield and Wilcoxon [21] .
Moreover, these compounds were tested for their toxicity
through parenteral route. Groups of mice each consisting of six ani-
[23] M. M. El-Mas and A. A. Abdel-Rahman, J. Pharmacol. Exp.
Ther., 266, 602 (1993).