5-apylheteroarylalkyl-1,3,5-trisubstituted-1,2,4-triazole compounds for treatment of circulatory disorders

The following Example is a detailed description of the methods of 
preparation of compounds of Formula I. This detailed preparation falls 
within the scope of, and serves to exemplify, the above described General 
Synthetic Procedures which form part of the invention. This Example is 
presented for illustrative purposes only and is not intended as a 
restriction on the scope of the invention. All parts are by weight unless 
otherwise indicated. 
EXAMPLE 1 
##STR66## 
5-[2-[5-[(1,3-dibutyl-1H-1,2,4-triazol-5-yl)methyl]-2-pyridinyl]phenyl]-1H- 
tetrazole 
Step 1: Preparation of 2-(N-triphenylmethyltetrazol-5-yl)phenylboronic 
acid. 
A 64 g (350 mmol) sample of 2-bromobenzonitrile (Aldrich) was dissolved in 
650 mL of xylene and treated with 22.75 g (350 mmol) of sodium azide and 
95 mL (350 mmol) of tributyltin chloride at reflux for 48 h. The reaction 
was filtered; the filtrate was treated with 50 mL of anhydrous 
tetrahydrofuran (THF) and 20 g (550 mmol) of hydrogen chloride. The 
reaction was stirred for 2 h; filtration gave 59.6 g (76%) of 
5-(2-bromophenyl)-1H-tetrazole: mp 178.degree.-180.degree. C.; NMR 
(DMSO-d.sub.6).delta.7.50-7.64 (m, 2 H), 7.67-7.74 (m, 1H), 7.83-7.91 (m, 
1 H). A 41.8 g (187 mmol) sample of this material was dissolved in 650 mL 
of methylene chloride and treated with 55.5 g (193 mmol) of 
triphenylmethyl chloride and 30 mL (220 mmol) of anhydrous triethylamine. 
The reaction was allowed to stir overnight at reflux, cooled to ambient 
temperature, washed with water, dried (MgSO.sub.4), and concentrated in 
vacuo. Recrystallization from toluene/hexane gave 80.7 g (92%) of 
N-triphenylmethyl-5-(2-bromophenyl)-1H-tetrazole: mp 160.degree.- 
162.degree. C.; NMR (CDCl.sub.3).delta.7.14-7.21 (m, 6 H), 7.26-7.45 (m, 
11 H), 7.70 (dd, J=8 and 1.5 Hz, 1 H), 7.89 (dd, J=7.5 and 2 Hz, 1 H). A 
34.05 g (73.0 mmol) sample of this material was dissolved in 1700 mL of 
THF under a nitrogen atmosphere and treated with 73 mmol of n-butyllithium 
in hexane. The reaction was allowed to stir for 17 min and then was 
treated with 24.9 mL (220 mmol) of trimethyl borate. The reaction was 
allowed to come to ambient temperature overnight while stirring, quenched 
with 10 mL of methanol, and concentrated in vacuo. The residue was 
dissolved in 1M NaOH and extracted with toluene to remove any unreacted 
starting material. The pH was adjusted to 6 with 6M HCl and the product 
extracted with toluene and dried (MgSO.sub.4). Hexane was added and the 
solution kept in the freezer overnight. Filtration provided 31.3 g (99%) 
of 2-(N-triphenylmethyl-tetrazol-5-yl)phenylboronic acid: NMR 
(CDCl.sub.3).delta.7.13-7.21 (m, 7 H), 7.33-7.42 (m, 8 H), 7.49-7.55 (m, 2 
H), 8.15-8.19 (m, 1 H), 8.21-8.26 (m, 1 H). 
Step 2: Preparation of N.sup.2 -butyl valeric acid hydrazide. 
To a solution of 400 g (3.44 mol) of valeric acid hydrazide (Lancaster 
Synthesis) in 3000 mL of dichloromethane under a nitrogen atmosphere, was 
added 250 g (2 mol) of anhydrous magnesium sulfate and 310 g (3.88 mol) of 
butyraldehyde. The reaction was stirred at ambient temperature for 17 h, 
filtered, and concentrated in vacuo providing 613 g of nearly colorless 
solid: mp 66.5.degree.-68.0.degree. C.; NMR (CDCl.sub.3).delta.0.97 (t, 
J=7 Hz, 3 H), 0.92 (t, J=7 Hz, 3 H), 1.31-1.46 (m, 2 H), 1.48-1.73 (m, 4 
H), 2.19-2.28 (m, 2 H), 2.62 (t, J=7 Hz, 2 H), 7.14 (t, J=6 Hz, 1 H), 9.18 
(br s, 1 H). This material was dissolved in 3000 mL of ethanol and cooled 
to 0.degree. C. in an ice bath prior to the addition of 130.1 g (3.44 mol) 
of sodium borohydride. The reaction was maintained at 0.degree. C. for 3 h 
and then allowd to slowly warm to ambient temperature overnight. The 
volatiles were removed in vacuo and the residue dissolved in 1500 mL of 
water and continuously extracted with ether/dichloromethane (1:1) 
overnight. The extracts were dried (Na.sub.2 SO.sub.4) and concentrated in 
vacuo to give 549 g (93%) of colorless N.sup.2 -butyl valeric acid 
hydrazide: mp 66.0.degree.-67.5.degree. C.; NMR (CDCl.sub.3).delta.0.92 
(t, J=7 Hz, 6 H), 1.17-1.52 (m, 6 H), 1.56-1.69 (m, 2 H), 2.14 (t, J=7 Hz, 
2 H), 2.82 (t, J=7 Hz, 2 H). 
Step 3: Preparation of 2-bromo-5-picoline. 
A solution of 1500 mL (14 mol) of 48% hydrobromic acid was cooled to 
10.degree. C. and 300 g (2.8 mol) of 2-amino-5-picoline (Aldrich) was 
added slowly. The solution was maintained at or below 0.degree. C. while 
450 mL (8.8 mol) of bromine was added dropwise. After the bromine addition 
was complete, a solution of 500 g (7.3 mol) of sodium nitrite in 1000 mL 
of water was added slowly over 6 h. The reaction pH was adjusted by the 
careful addition of 1500 mL (56 mol) of 50% sodium hydroxide at such a 
rate that the temperature was maintained below 30.degree. C. The product 
precipitated from the nearly colorless reaction mixture; filtration gave 
450 g (94%) of 2-bromo-5-picoline as a yellow powder: mp 
38.degree.-40.degree. C.; NMR 7.27 (s, 1 H), 7.28 (s, 1 H), 7.12 (br s, 1 
H). 
Step 4: Preparation of 2-bromo-5-romomethylpyridine. 
A solution of 296.3 g (1.72 mol) of 2-bromo-5-picoline from step 3 in 6 L 
of carbon tetrachloride was treated with 306.5 g (1.72 mol) of 
N-bromosuccinimide (NBS) and 28.3 g 173 mmol) of azobisisobutyronitrile 
(AIBN). The reaction was stirred at reflux under nitrogen for 3 h, 
filtered, and concentrated in vacuo providing 476 g of crude 
2-bromo-5-bromomethylpyridine as a brownish yellow solid (NMR indicates 
that this material is only 60% monobromomethyl product): NMR 
(CDCl.sub.3).delta.4.42 (s, 2 H), 7.48 (d, J=9 Hz, 1 H), 7.60 (dd, J=9 and 
3 Hz, 1 H), 8.37 (d, J=3 Hz, 1 H). 
Step 5: Preparation of 2-bromo-5-cyanomethyl-pyridine. 
The 476 g of crude 2-bromo-5-bromomethylpyridine from step 4 was dissolved 
in 4000 mL of dimethylformamide (DMF)/water (7:1) and treated with 168 g 
(2.58 mol) of potassium cyanide. The reaction was allowed to stir at 
ambient temperature for 72 h, concentrated in vacuo, and partitioned 
between ethyl acetate and water; the organic layer was washed with water, 
washed with brine, dried (MgSO.sub.4), and reconcentrated in vacuo to 
provide the crude nitrile. Purification by silica gel chromatography 
(Waters Prep-500A) using ethyl acetate/hexane (25:75) gave 109 g (32% from 
2-bromo-5-picoline) of 2-bromo-5-cyanomethylpyridine as a yellowish orange 
solid: mp 55.5.degree.-57.5.degree. C.; NMR (CDCl.sub.3).delta.3.74 (s, 2 
H), 7.54 (d, J=8 Hz, 1 H), 7.59 (dd, J=8 and 2 HZ, 1 H), 8.35 (d, J=2 Hz, 
1 H); MS (FAB) m/e (rel intensity) 199 (85), 197 (100). 
Step 6: Preparation of ethyl imino(2-bromopyridin-5-yl)acetate. 
Under nitrogen, 299 mL (4.20 mol) of acetyl chloride was added dropwise to 
a solution of 299 mL (5.11 mol) of absolute ethanol and 400 mL of 
chloroform at 0.degree. C. A solution of 72.0 g (370 mmol) of 
2-bromo-5-cyanomethylpyridine from step 5 in 1100 mL of chloroform was 
added dropwise with stirring. Stirring was continued at 0.degree. C. for 4 
h and the reaction was allowed to come to ambient temperature overnight. 
The reaction was diluted with 2500 mL of anhydrous ether and stirring was 
continued for an additional 2 h. The precipitated imidate hydrochloride 
salt was collected by filtration in a glove bag under nitrogen and washed 
with anhydrous ether. Under nitrogen, a mechanically stirred suspension of 
this material in 3500 mL of anhydrous ether at -78.degree. C. was treated 
with 36 g (2.1 mol) of anhydrous ammonia. The reaction was allowed to 
slowly warm to ambient temperature overnight and the ammonium chloride 
removed by filtration. The filtrate was concentrated in vacuo giving 78.7 
g (89%) of crude ethyl imino(2-bromopyridin-5-yl)acetate as a brown oil: 
NMR (CDCl.sub.3).delta.1.29 (t, J=7 Hz, 3 H), 3.51 (s, 2 H), 4.13 (q, J=7 
Hz, 2 H), 7.43 (dd, J=8 and 2 Hz, 1 H), 7.48 (d, J=8 Hz, 1 H), 8.26 (d, 
J=2 Hz, 1 H); MS (FAB) m/e (rel intensity) 245 (100), 243 (90), 217 (35), 
215 (38), 200 (5), 198 (5). 
Step 7: Preparation of 
2-bromo-5-[(1,3-dibutyl-1H-1,2,4-triazol-5yl)methyl]pyridine. 
Under nitrogen, a solution of 1.2 g 5 mmol) of imidate ester from step 6 
and 850 mg (5 mmol) of N.sup.2 -butyl valeric acid hydrazide from step 2 
in 25 mL of absolute methanol was stirred for 24 h at ambient temperature. 
The methanol was removed in vacuo and replaced with 25 mL of anhydrous 
toluene. The reaction vessel was equipped with a Dean-Stark trap and the 
reaction stirred at reflux for 48 h. Concentration in vacuo produced the 
crude product residue. Purification by silica gel chromatography (Waters 
Prep-500A) using ethyl acetate/hexane (60:40) gave 700 mg (40%) of 
2-bromo-5-[(1,3-dibutyl-1H-1,2,4-triazol-5-yl)methyl]pyridine as an oil: 
NMR (CDCl.sub.3).delta.0.88 (t, J=7 Hz, 3 H), 0.92 (t, J=7 Hz, 3 H), 
1.25-1.75 (m, 8 H), 2.67 (t, J=8 Hz, 2 H), 3.93 (t, J=7 Hz, 2 H), 4.05 
(s, 2 H), 7.43 (s, 1 H), 7.44 (s, 1 H), 8.27 (s, 1 H); MS (FAB) m/e (rel 
intensity) 353 (50), 351 (50), 297 (100), 295 (100), 215 (40), 160 (90), 
158 (90), 138 (70). 
Step 8: Preparation of 
5-[2-[5-[(1,3-dibutyl-1H-1,2,4-triazol-5-yl)methyl]-2-pyridinyl]phenyl-1H- 
tetrazole. 
Under nitrogen, a solution of 700 mg (2.0 mmol) of 
2-bromo-5-[(1,3-dibutyl-1H-1,2,4-triazol-5-yl)methyl]pyridine from step 7 
and 180 mg (0.16 mmol) of tetrahis(tri-phenylphosphine)palladium in 12 mL 
of toluene and 3 mL of 2M sodium carbonate was treated with a solution of 
1.12 g (2.6 mmol) of 2-(N-triphenylmethyltetrazol-5 -yl)phenylboronic acid 
from step 1 in 3 mL of ethanol. The reaction was vigorously stirred at 
reflux for 17 h. The reaction was cooled to ambient temperature and three 
immiscible phase separated; the middle phase contained the deprotected 
tetrazole product. Purification by reverse phase chromatography (Waters 
Deltaprep-3000) using isocratic acetonitrile/water (27:73) (0.05% TFA) 
gave 300 mg (28%) of colorless 
5-[2-[5-[(1,3-dibutyl-1H-1,2,4-triazol-5-yl)methyl]-2-pyridinyl]phenyl-1H- 
tetrazole as the trifluoroacetic acid (TFA) salt after lyophilization: NMR 
(CDCl.sub.3).delta.0.93 (t, J=7 Hz, 6 H), 1.26-1.46 (m, 4 H), 1.67-1.89 
(m, 4 H), 2.74 (t, J=8 Hz, 2 H), 4.10 (t, J=7 Hz, 2 H), 4.30 (s, 2 H), 
7.43 (d, J=8 Hz, 1 H), 7.53-7.62 (m, 3 H), 7.74 (dd, J=8 and 2 Hz, 1 H), 
8.02-8.05 (m, 1 H), 8.52 (d, J=2 Hz, 1 H); MS (FAB) m/e (rel intensity) 
417 (100), 389 (75), 361 (20); HRMS. Calc'd for M+H: 417.2515. Found: 
417.2516. 
BIOLOGICAL EVALUATION 
Assay A: Angiotensin II Binding Activity 
Compounds of the invention were tested for ability to bind to the smooth 
muscle angiotensin II receptor using a rat uterine membrane preparation. 
Angiotensin II (AII) was purchased from Peninsula Labs. .sup.125 
I-angiotensin II (specific activity of 2200 Ci/mmol) was purchased from Du 
Pont-New England Nuclear. Other chemicals were obtained from Sigma 
Chemical Co. This assay was carried out according to the method of Douglas 
et al [Endocrinology, 106, 120-124 (1980)]. Rat uterine membranes were 
prepared from fresh tissue. All procedures were carried out at 4.degree. 
C. Uteri were stripped of fat and homogenized in phosphate-buffered saline 
at pH 7.4 containing 5 mM EDTA. The homogenate was centrifuged at 
1500.times.g for 20 min., and the supernatant was recentrifuged at 
100,000.times.g for 60 min. The pellet was resuspended in buffer 
consisting of 2 mM EDTA. and 50 mM Tris-HCl (pH 7.5) to a final protein 
concentration of 4 mg/ml. Assay tubes were charged with 0.25 ml of a 
solution containing 5 mM MgCl.sub.2, 2 mM EDTA, 0.5% bovine serum albumin, 
50 mM Tris-HCl, pH 7.5 and .sup.125 I-AII (approximately 10.sup.5 cpm) in 
the absence or in the presence of unlabelled ligand. The reaction was 
initiated by the addition of membrane protein and the mixture was 
incubated at 25.degree. C. for 60 min. The incubation was terminated with 
ice-cold 50 mM Tris-HCl (pH 7.5) and the mixture was filtered to separate 
membrane-bound labelled peptide from the free ligand. The incubation tube 
and filter were washed with ice-cold buffer. Filters were assayed for 
radioactivity in a Micromedic gamma counter. Nonspecific binding was 
defined as binding in the presence of 10 .mu.M of unlabelled AII. Specific 
binding was calculated as total binding minus nonspecific binding. The 
receptor binding affinity of an AII antagonist compound was indicated by 
the concentration (IC.sub.50) of the tested AII antagonist which gives 50% 
displacement of the total specifically bound .sup.125 I-AII from the high 
affinity AII receptor. Binding data were analyzed by a nonlinear 
least-squares curve fitting program. Results are reported in Table I. 
Assay B: In Vitro Vascular Smooth Muscle-Response for AII 
The compounds of the invention were tested for antagonist activity in 
rabbit aortic rings. Male New Zealand white rabbits (2-2.5 kg) were 
sacrificed using an overdose of pentobarbital and exsanguinated via the 
carotid arteries. The thoracic aorta was removed, cleaned of adherent fat 
and connective tissue and then cut into 3-mm ring segments. The 
endothelium was removed from the rings by gently sliding a rolled-up piece 
of filter paper into the vessel lumen. The rings were then mounted in a 
water-jacketed tissue bath, maintained at 37.degree. C., between moveable 
and fixed ends of a stainless steel wire with the moveable end attached to 
an FT03 Grass transducer coupled to a Model 7D Grass Polygraph for 
recording isometric force responses. The bath was filled with 20 ml of 
oxygenated (95% oxygen/5% carbon dioxide) Krebs solution of the following 
composition (mM): 130 NaCl, 15 NaHCO.sub.3, 15 KCl, 1.2 NaH.sub.2 
PO.sub.4, 1.2 MgSO.sub.4, 2.5 CaCl.sub.2, and 11.4 glucose. The 
preparations were equilibrated for one hour before approximately one gram 
of passive tension was placed on the rings. Angiotensin II 
concentration-response curves were then recorded (3.times.10.sup.-10 to 
1.times.10.sup.-5 M). Each concentration elicit its maximal contraction, 
and then AII was washed out repeatedly for 30 minutes before rechallenging 
with a higher concentration of AII. Aorta rings were exposed to the test 
antagonist at 10.sup.-5 M for 5 minutes before challenging with AII. 
Adjacent segments of the same aorta ring were used for all 
concentration-response curves in the presence or absence of the test 
antagonist. The effectiveness of the test compound was expressed in terms 
of pA.sub.2 values and were calculated according to H. O. Schild [Br. J. 
Pharmacol. Chemother., 2, 189-206 (1947)]. The pA.sub.2 value is the 
concentration of the antagonist which increases the EC.sub.50 value for 
AII by a factor of two. Each test antagonist was evaluated in aorta rings 
from two rabbits. Results are reported in Table I. 
TABLE I 
______________________________________ 
In Vitro Angiotensin II 
Activity of Compounds of the Invention 
.sup.1 Assay A 
Test Compound IC.sub.50 
.sup.2 Assay B 
Example # (nM) pA.sub.2 
______________________________________ 
1 31 7.80/7.91 
______________________________________ 
.sup.1 Assay A: Angiotensin II Binding Activity 
.sup.2 Assay B: In Vitro Vascular Smooth Muscle Response 
Also embraced within this invention is a class of pharmaceutical 
compositions comprising one or more compounds of Formula I in association 
with one or more non-toxic, pharmaceutically acceptable carriers and/or 
diluents and/or adjuvants (collectively referred to herein as "carrier" 
materials) and, if desired, other active ingredients. The compounds of the 
present invention may be administered by any suitable route, preferably in 
the form of a pharmaceutical composition adapted to such a route, and in a 
dose effective for the treatment intended. Therapeutically effective doses 
of the compounds of the present invention required to prevent or arrest 
the progress of the medical condition are readily ascertained by one of 
ordinary skill in the art. The compounds and composition may, for example, 
be administered intravascularly, intraperitoneally, subcutaneously, 
intramuscularly or topically. 
For oral administration, the pharmaceutical composition may be in the form 
of, for example, a tablet, capsule, suspension or liquid. The 
pharmaceutical composition is preferably made in the form of a dosage unit 
containing a particular amount of the active ingredient. Examples of such 
dosage units are tablets or capsules. These may with advantage contain an 
amount of active ingredient from about 1 to 250 mg, preferably from about 
25 to 150 mg. A suitable daily dose for a mammal may vary widely depending 
on the condition of the patient and other factors. However, a dose of from 
about 0.1 to 3000 mg/kg body weight, particularly from about 1 to 100 
mg/kg body weight, may be appropriate. 
The active ingredient may also be administered by injection as a 
composition wherein, for example, saline, dextrose or water may be used as 
a suitable carrier. A suitable daily dose is from about 0.1 to 100 mg/kg 
body weight injected per day in multiple doses depending on the disease 
being treated. A preferred daily dose would be from about 1 to 30 mg/kg 
body weight. Compounds indicated for prophylactic therapy will preferably 
be administered in a daily dose generally in a range from about 0.1 mg to 
about 100 mg per kilogram of body weight per day. A more preferred dosage 
will be a range from about 1 mg to about 100 mg per kilogram of body 
weight. Most preferred is a dosage in a range from about 1 to about 50 mg 
per kilogram of body weight per day. A suitable dose can be administered, 
in multiple sub-doses per day. These sub-doses may be administered in unit 
dosage forms. Typically, a dose or sub-dose may contain from about 1 mg to 
about 100 mg of active compound per unit dosage form. A more preferred 
dosage will contain from about 2 mg to about 50 mg of active compound per 
unit dosage form. Most preferred is a dosage form containing from about 3 
mg to about 25 mg of active compound per unit dose. 
The dosage regimen for treating a disease condition with the compounds 
and/or compositions of this invention is selected in accordance with a 
variety of factors, including the type, age, weight, sex and medical 
condition of the patient, the severity of the disease, the route of 
administration, and the particular compound employed, and thus may vary 
widely. 
For therapeutic purposes, the compounds of this invention are ordinarily 
combined with one or more adjuvants appropriate to the indicated route of 
administration. If administered per os, the compounds may be admixed with 
lactose, sucrose, starch powder, cellulose esters of alkanoic acids, 
cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium 
oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, 
acacia gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl 
alcohol, and then tableted or encapsulated for convenient administration. 
Such capsules or tablets may contain a controlled-release formulation as 
may be provided in a dispersion of active compound in hydroxypropylmethyl 
cellulose. Formulations for parenteral administration may be in the form 
of aqueous or non-aqueous isotonic sterile injection solutions or 
suspensions. These solutions and suspensions may be prepared from sterile 
powders or granules having one or more of the carriers or diluents 
mentioned for use in the formulations for oral administration. The 
compounds may be dissolved in water, polyethylene glycol, propylene 
glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl 
alcohol, sodium chloride, and/or various buffers. Other adjuvants and 
modes of administration are well and widely known in the pharmaceutical 
art. 
Although this invention has been described with respect to specific 
embodiments, the details of these embodiments are not to be construed as 
limitations.