2-alkynyladenosines as antihypertensive agents

A 2-alkynyladenosine represented by general formula [I]: ##STR1## wherein n is an integer of from 2 to 15, has been found to have excellent properties as an antihypertensive agent. On the basis of this finding, the present invention has provided an antihypertensive agent comprising an antihypertensive effective amount of a compound of the above formula [I] and a pharmaceutically acceptable carrier. The present invention also discloses a novel compound 2-alkynyladenosine of formula [I] shown above wherein n is 6 to 15.

BACKGROUND OF THE INVENTION 
The present invention relates to an antihypertensive agent comprising a 
2-alkynyladenosine as an active ingredient. 
Heretofore, adenosine has been used for the treatment of cardiac 
incompetency, myocardial infarction, arterial sclerosis and angina 
pectoris. This compound, however, exhibits a strong transient 
pharmacological effect when taken up into organisms and is known to be 
rapidly deaminated by adenosine deaminase or taken up into red blood cells 
and tissues to lose its activity. Adenosine also exhibits a strong 
suppressive effect on the heart as a side effect. 
In order to improve the pharmacological activity of adenosine, impart 
thereto resistance to adenosine deaminase and mitigate undesirable side 
effects, a variety of adenosine analogues have been prepared 
synthetically. 
With respect to 2-substituted adenosines, various compounds have been 
synthesized. For example, 2-alkylthioadenosines, 2-phenylaminoadenosines 
and the like exhibiting such physiological activities as adenosine 
deaminase inhibitor effect, coronary vasodilator effect, platelet 
aggregation inhibitor effect and antiviral effect have so far been 
obtained. 
We have previously synthesized 2-alkynyladenosines of the following formula 
as 2-substituted adenosine derivatives each having a substituent 
introduced by a carbon-carbon bond in the 2 position of adenosine: 
##STR2## 
wherein R is Si(CH.sub.3).sub.3, H, Ph, CH.sub.2 OH, CH.sub.2 CH.sub.2 OH, 
CH(OH)CH.sub.3, (CH.sub.2).sub.2 CH.sub.3, (CH.sub.2).sub.3 CH.sub.3, 
(CH.sub.2).sub.4 CH.sub.3 or (CH.sub.2).sub.5 CH.sub.3, and have found 
that these compounds have an inhibitory effect on the I.sub.g E-mediated 
48-hr. passive cutaneous anaphylaxis reaction in rats. (Nucleic Acids 
Research Symposium Series No.12, pp. 5.about.8 (1983) and Chem. Pharm. 
Bull. Vol. 33, pp. 1766.about.1769 (1985)). 
Numerous pharmaceutical preparations have been developed to date as 
antihypertensive agents. In view of the nature of the disease, 
pharmaceutical preparations must be administered over a long period of 
time in many cases. The intake of pharmaceutical preparations over a long 
period of time may sometimes induce drug resistance therefor or side 
effects. For this reason, stepwise pharmacotherapy is generally adopted so 
that a particular pharmaceutical is selected with due consideration for 
age, severity of hypertension, complications and the like and another 
pharmaceutical preparation having a different function mechanism is 
further used in combination depending upon the symptoms. 
At present, no pharmaceutical preparations comprising compounds having 
adenosine skeletons are used as antihypertensive agents, and it would thus 
be highly profitable for the treatment of hypertension to provide 
antihypertensive agents comprising adenosine derivatives. 
SUMMARY OF THE INVENTION 
As a result of our intensive research directed to the development of novel 
2-substituted adenosine derivatives useful as antihypertensive agents, we 
have found that 2-alkynyladenosines having an alkynyl group in the 2 
position exhibit excellent antihypertensive properties. On the basis of 
this finding, we have arrived at the present invention. 
More specifically, this invention provides an antihypertensive agent 
comprising an antihypertensive effective amount of a 2-alkynyladenosine 
represented by general formula [I]: 
##STR3## 
wherein n is an integer from 2 to 15, and a pharmaceutically acceptable 
carrier. 
Among the 2-alkynyladenosines of the above formula [I] which form the 
active ingredients of the pharmaceutical preparations of the present 
invention, a 2-alkynyladenosine with a short-chain alkynyl substituent 
represented by formula [I-A] (hereinafter referred to as "short-chain 
alkynyl substituent compound"): 
##STR4## 
wherein n is an integer of from 2 to 5, is per se a known compound (ibid.) 
but a 2-alkynyladenosine with a long-chain alkynyl substituent represented 
by formula [I-B] (hereinafter referred to as "long-chain alkynyl 
substituent compound"): 
##STR5## 
wherein n is an integer of from 6 to 15, is a novel compound. The present 
invention, therefore, also provides a novel 2-alkynyladenosine compound of 
the above formula [I-B].

DETAILED DESCRIPTION OF THE INVENTION 
The 2-alkynyladenosine of the formula [I] shown supra which forms an active 
ingredient of the pharmaceutical preparation of the present invention can 
be synthesized by reacting a 2-halogenoadenosine of general formula [II]: 
##STR6## 
wherein X is iodine or bromine, with an alkyne of general formula [III]: 
EQU CH.tbd.C[CH.sub.2 ].sub.n CH.sub.3 [III] 
wherein n is an integer of from 2 to 15, in a solvent in the presence of 
bis(triphenylphosphine) palladium dichloride and cuprous iodide. 
Depending upon the desired compound among those of the present invention, 
an alkyne having the corresponding "n" is selected. 
For the solvent, basic solvents such as a solvent mixture of triethylamine 
and N,N-dimethylformamide are used. Triethylamine can be replaced by a 
tertiary amine such as tributylamine, trioctylamine, 
N,N,N',N'-tetramethyl-1,8-naphthalenediamine, dimethylaniline, 
diethylaniline or pyridine, while N,N-dimethylformamide can be replaced by 
a non-proton polar solvent such as N,N-dimethylacetamide, dimethyl 
sulfoxide or acetonitrile. 
The reaction terminates in several hours at room temperature to solvent 
reflux temperature. 
The compounds of the present invention can be isolated by any conventional 
separation and purification method. For example, adsorption 
chromatography, ion exchange chromatography, extraction or 
recrystallization is applied for the isolation. 
The pharmaceutical preparations of the present invention are clinically 
utilized for the treatment of hypertension. 
The pharmaceutical preparations of this invention are administered to 
patients orally, by injection, intrarectally or via topical 
administration. These preparations, when administered, are made by a 
conventional method into dosage forms suited for the desired route of 
administration. For example, solid form preparations such as tablets, 
powders, dragees, granules, sublingual tablets and capsules or liquid form 
preparations such as syrups, suspensions and elixirs are suitable for oral 
administration, injections for administration by means of a syringe, 
suppositories and ointments for intrarectal administration, and poultices 
for topical administration. 
In the process of preparation, suitable additives such as binders, 
vehicles, lubricants, disintegrators, emulsifiers, suspending agents, 
antiseptics, stabilizers, solubilizing agents, taste conditioners, and 
sweeteners can be selected and used as necessary from a pharmaceutical 
point of view. 
The optimum doses of the pharmaceutical preparations are determined 
according, for example, to the dosage form, severity of diseases, age and 
body weight. 
For oral administration, for example, the pharmaceutical preparation 
comprising a short-chain 2-alkynyladenosine of the formula [I-A] as an 
active ingredient is administered to an adult generally at a dose level of 
the order of 0.1 to 5 mg/kg/day while that comprising a long-chain 
2-alkynyladenosine of the formula [I-B] as an active ingredient is 
administered at a dose level of the order of 0.1 to 10 mg/kg/day. 
The long-chain 2-alkynyladenosine, especially the compounds of the formula 
[I-B] wherein n is an integer of from 11 to 15, among the active 
ingredients of the pharmaceutical preparations of the present invention 
exhibits mild hypotensive effect of long duration coupled with relatively 
low toxicity and minimal side effects. 
As a result of animal tests with normotensive rats (NR) and spontaneously 
hypertensive rats (SHR), the pharmaceutical preparations were further 
found to act on SHR selectively and barely affect normotension. 
It has been found by us that also the known short-chain 2-alkynyladenosine 
has hypotensive effect, but shows more drastic hypotensive effect than the 
long-chain alkynyl substituent compound and acts equally on normotension 
while having higher toxicity. 
In view of the foregoing, the long-chain 2-alkynyladenosine of the present 
invention can be said to be endowed with excellent properties that the 
known short-chain alkynyl substituent compound does not possess. It can 
also be said that, preferably, the short-chain 2-alkynyladenosine is used 
at lower dose levels than those for the long-chain alkynyl substituent 
compound. 
EXAMPLES 
Preparation of the compound of the present invention 
6.0 g of 6-chloro-2-iodo-9-(2,3,5-tri-O-acetyl-.beta.-D-ribofuranosyl) 
purine was added to 60 ml of methanol plus ammonia (saturated at 0.degree. 
C.) to cause reaction at a temperature of 60.degree. C. for 17 hours in a 
sealed tube. The reaction solution was cooled, then degassed, and 
concentrated under reduced pressure. Crystallization of the residue from 
water afforded 3.94 g of 2-iodoadenosine (90% yield) having a melting 
point of 141.degree. C. to 144.degree. C. 
393 mg (1 mmole) of the 2-iodoadenosine was dissolved in 10 ml of 
dimethylformamide plus 3 ml of triethylamine, and to the solution obtained 
were added 21 mg of bis(triphenylphosphine) palladium dichloride and 12 mg 
of cuprous iodide. To the resulting solution was added an alkyne (1.1 
equivalent) in an argon stream, and the mixture was stirred under heat at 
80.degree. C. After the reaction solution was concentrated under reduced 
pressure, the residue was dissolved in methanol, and hydrogen sulfide was 
passed through the solution for one minute. The precipitate formed was 
filtered off, and the filtrate was concentrated to dryness under reduced 
pressure. The residue was purified by column chromatography on silica gel 
and recrystallized from methanol or methanol-water to obtain 
2-alkynyladenosine. 
The reaction time, yield, melting point and infrared absorption spectrum 
are shown in TABLE 1. 
TABLE 1 
__________________________________________________________________________ 
Compound Reaction Melting Point 
IR(KBr)cm.sup.-1 
No. Species of Alkynyl Group 
Time (hr) 
Yield (%) 
(.degree.C.) 
.nu.C.tbd.C 
__________________________________________________________________________ 
1 --C.tbd.C(CH.sub.2).sub.2 CH.sub.3 
1 90 129-132 
2230 
2 --C.tbd.C(CH.sub.2).sub.3 CH.sub.3 
1 85 121-125 
2230 
3 --C.tbd.C(CH.sub.2).sub.4 CH.sub.3 
1 93 113-115 
2230 
4 --C.tbd.C(CH.sub.2).sub.5 CH.sub.3 
1 84 101-103 
2230 
5 --C.tbd.C(CH.sub.2).sub.7 CH.sub.3 
1 88 121-123 
2230 
6 --C.tbd.C(CH.sub.2).sub.9 CH.sub.3 
1 97 128-130 
2230 
7 --C.tbd.C(CH.sub.2).sub.11 CH.sub.3 
1 93 131-134 
2230 
8 --C.tbd.C(CH.sub.2).sub.13 CH.sub.3 
3 98 134-136 
2230 
9 --C.tbd.C(CH.sub.2).sub.14 CH.sub.3 
24 80 138-139 
2230 
10 --C.tbd.C(CH.sub.2).sub.15 CH.sub.3 
24 72 131-134 
2230 
__________________________________________________________________________ 
TEST EXAMPLES 
Test 1: Effect of the test compounds on the blood pressure in SHR 
To twelve- to fifteen-week-old male SHRs were orally administered test 
compounds each suspended in 0.5% CMC/physiological saline at a rate of 2 
ml/kg. The blood pressure of the thus treated SHRs was measured by means 
of a tail artery sphygmomanometer (supplied by Nalco Co., Model PE-300) 
prior to the oral administration and at 2, 4, 6 and 8 hours thereafter. 
Reduction in blood pressure was calculated on the basis of the values thus 
obtained, and the maximum reduction levels attained by the respective 
compounds are summarized in TABLE 2. Each test compound was evaluated with 
a group of 4 rats. 
TABLE 2 
______________________________________ 
Reduction in blood 
pressure 
Compound No. Dose (mg/kg) 
(-.DELTA.max, mmHg) 
______________________________________ 
2(n = 3) 1 &gt;136 
4(n = 5) 1 69 
5(n = 7) 3 106 
6(n = 9) 3 65 
7(n = 11) 3 65 
8(n = 13) 3 48 
9(n = 14) 3 35 
10(n = 15) 3 46 
______________________________________ 
As is apparent from TABLE 2, all Of the 2-alkynyladenosines exhibited 
hypotensive effect: the shorter the alkynyl substituent chain, the more 
drastic was the hypotensive effect while the longer the chain, the milder 
was the effect. Further, sedative effect was observed in the groups of 
rats administered with Compounds 2 to 4. 
Test 2: Effect of the test compounds on the blood pressure in SHR 
Thirty-two-week-old male SHRs (divided into groups of three rats each) were 
anesthetized with urethane (1.1 g/kg, i.p.), and the blood pressure of 
each of the rats delivered from the common carotid artery was recorded on 
a polygraph through a pressure transducer (Model MPU-0.5). 
The pharmaceutical preparation was administered through a cannula inserted 
into the femoral vein of the rat at a rate of 0.5 ml/kg, and the blood 
pressure was measured continuously from before administration to 30 
minutes after administration. The maximum levels of reduction in blood 
pressure were as set forth in TABLE 3. 
TABLE 3 
______________________________________ 
Reduction in blood 
pressure 
Compound No. Dose (.mu.g/kg) 
(-.DELTA.max, mmHg) 
______________________________________ 
Hg) 
6(n = 9) 100 20 
7(n = 11) 100 21 
8(n = 13) 100 16 
______________________________________ 
Test 3: Effect of the test compounds on the blood pressure in NR 
The procedure of Test 2 was followed with 10- to 12-week-old male Wistar 
rats (divided into groups of three rats each) to measure the hypotensive 
effects of the respective pharmaceutical preparations (100 .mu.g/kg). 
Simultaneously, the change in heart rate was measured. The heart rate was 
measured by an ictometer based on the systolic blood pressure as a 
trigger. The results obtained were as shown in TABLE 4. 
TABLE 4 
______________________________________ 
Reduction in blood 
pressure Change in heart rate 
Compound No. 
(-.DELTA.max, mmHg) 
(.DELTA.HR) 
______________________________________ 
1(n = 2) 21 -48 
2(n = 3) 42 -36 
3(n = 4) 48 -180 
4(n = 5) 55 -288 
5(n = 7) 24 -18 
6(n = 9) 26 -12 
7(n = 11) 10 0 
8(n = 13) 5 0 
9(n = 14) 3 -6 
10(n = 15 10 -5 
______________________________________ 
From the data given in TABLE 4 it has been found that the long-chain 
alkynyl substituent compound wherein n is 11 or more has far less effect 
on the blood pressure of normotensive rats than the short-chain alkynyl 
substituent compound wherein n is 5 or less. It has also been noted that 
the long-chain compound has less influence on heart rate than the 
short-chain compound. 
The overall results set forth in TABLES 3 and 4 show that Compound 6(n=9) 
exhibited substantially equal hypotensive effect on SHR and NR while 
Compounds 7(n=11) and 8(n=13) acted selectively on SHR. 
Test 4: Effect of the test compounds on the blood pressure in SHR and NR 
Eighteen- to twenty-week-old male SHR (divided into groups of four rats 
each) and eighteen- to twenty-week-old male Wistar rats (divided into 
groups of four rats each) were orally administered with Compound 8(n=13) 
in the same manner as in Test 2 at a dose of 10 mg/kg, and the blood 
pressure of each of the rats was measured. The results obtained are 
incorporated in TABLE 5. 
TABLE 5 
______________________________________ 
Reduction in blood pressure 
(-.DELTA., mmHg) 
1 hr. after 
2 hrs. after 
administration 
administration 
______________________________________ 
SHR 41 42 
Wistar rat 16 24 
______________________________________ 
As is apparent from TABLE 5, the tendency of Compound 8(n=13) to act 
selectively on SHR was also observed in the case of oral administration. 
Test 5: Acute toxicity 
The acute toxicity of Compound 8(n=13) was tested with seven-week-old male 
Jcl:ICR mice. 
The physically allowable doses of Compound 8 for intraperitoneal 
administration and oral administration were 800 mg/kg and 1,300 mg/kg, 
respectively. 
None of the mice administered with the test compound at these maximum 
doses, divided into groups of five mice each, died, so that the LD.sub.50 
values for intraperitoneal administration and oral administration were 
estimated respectively at more than 800 mg/kg and more than 1,300 mg/kg. 
When Compound 6(n=9) was tested by a similar procedure, on the other hand, 
all the five mice forming one group died with intraperitoneal 
administration of 500 mg/kg of the test compound. 
As is noted from the above data, the effective amount of Compound 8 is 3 
mg/kg while the LD.sub.50 thereof is more than 1,300 mg/kg, indicating a 
400-fold or higher safety coefficient. The longer-chain alkynyl 
substituent compound such as Compound 8 was thus found to be less toxic 
than the long-chain alkynyl substituent compound such as Compound 6. 
FORMULATION EXAMPLES 
Formulation 1: Tablets 
A total of 150 mg of a mixture comprising 20 mg of a long-chain alkynyl 
substituent compound of the present invention, 90 mg of lactose, 24 mg of 
corn starch, 10 mg of hydroxypropyl cellulose, and 6 mg of magnesium 
stearate was kneaded with water, and the resulting mixture was granulated 
for tableting purposes by a conventional method. After drying, the 
granules thus obtained were mixed with magnesium stearate to make tablets 
which were then formed into desired shape. In the case of a short-chain 
alkynyl substituent compound, tablets were formed in the same manner 
except that the quantity of the compound used was 10 mg. 
Formulation 2: Granules 
40 mg of a long-chain alkynyl substituent compound of the present 
invention, 200 mg of mannitol and 50 mg of lactose were mixed. The mixture 
was kneaded with an aqueous solution containing 10 mg of polyvinyl 
alcohol, granulated and dried to obtain granules. In the case of a 
short-chain alkynyl substituent compound, granules were prepared in the 
same manner except that the quantity of the compound used was 20 mg. 
Formulation 3: Capsules 
Capsules were formulated by encapsulating the granules prepared for 
tableting purposes in Formulation 1.