ADDUCTS AND USE THEREOF

Introduction

This invention concerns novel pharmaceutical compositions and a method of treatinc hypertension. Related compositions and methods are described ir. US patent application SN 07/316,958, filed 2/28/89 and in SN -tdZ T?, filed /θ//$/γ ^< ϊ

Background of the Invention

Endothelium-derivec. relaxing factor (EDRF) is a labile ^• humoral agent which is part of a cascade of interacting agents involved in the relaxation of vascular smooth muscle. EDRF is thus important in the control of vascular resistance to blood flow and in the control of blood pressure. Some vasodilators act by causing EDRF to be released from endothelial cells. (See Furchgott, Ann.Rev.Pharnacol.Toxicol. 2_i _. 175-197, 1984.) Recently, Palmer et al. have shown that EDRF is identical to the simple molecule, nitric oxide, NO. (Nature 3T7, 524-526, 1987.) It has been hypothesized for years that many nitrovasodilators that mimic the effect o~ EDRF, like glyceryl trinitrate, amyl nitrite, NaN0 ₂ and sodium nitroprusside (SNP) , do so by virtue of their conversion to a common moiety, namely NO, which is also a vasodilator. (See Kruszvna et al., Tox.S Appl. Pharmacol..91. 429-438, 1987; Ignarro, FAΞEB J. 3 , 31-36, 1989

an _d Ignarro et al., J. P _h armacol. Exper. Theraputics 2 ¹⁸ 1_ L. 7 ³⁹ - ₇₄₉ , ₁₉₈₁ . ₎ It has now _be en discovered that a class of compoun ^d s t- of the structure: /. - /vj - O M l

Ai -O wherein A is a secondary amino group, are potent anti- hypertensives and thus are useful for treating cardiovascular _d isor _d ers in which lowering the blood pressure has a ^b eneficial result. It is _b elieved t.iat these compounds function by releasing _NO in the _b loo _d after injection; however the invention shoul ^d not • _b e limite _{d b} y this hypot _h esis. While these compounds are, for the most part, known, there is no suggestion in the prior art that they are anti-hypertensive, indeed, there is no suggestion in the prior art that these corpounds have any pharmaceutical use. They are _d escri _b e _{d b} y Drago in "Free Radicals in Inorganic Chemistry", _N um _b er ₃₆ , Advances in Chemistry Series, American Chemical _S ociety, _W ash. D _C , 1 ₉₆₂ , pages ^' 143-149 and Drago et al. ^J . Amer. _C hem. _S oc. ₈ 2, 1 ₈ 1 ₉ -1 ₈ 2 ₂ , 1961. These two articles by Drago are incorporate _d by reference, in their entirety. The references are of a theoretical nature and mention no utility whatsoever. Reilly, U _S Patent ₃ ,15 ₃ , ₀₉ 4, discloses these compounds on columns ₁ 1-13, but does not teach any biological activity.

Description of the Invention The present invention provides pharmaceutical compositions comprising: a compound of the following formula

wherein R ₁ and ₂ are independently chosen from straight chain and branched chain alky] groups of one to twelve carbon atoms or benzyl, with the proviso that no branch occur on the alpha carbon of the alkyl groups, or R ₁ and R ₂ together with the nitrogen atom they are bonded to form a pyrrolidino, piperidino, piperazino or morpholino ring, M ^+x is c. pharmaceutically acceptable cation, wherein x is the valencε of the cation (its charge) ; and a

-pharmaceutically acceptable carrier. By straight chain alkyl is meant the non-branched r.ethyl, ethyl, n-propyl, n-butyl, n-decyl, and etc. groups. By brar.ched chain alkyl is meant groups like 3-

. methylpentyl, 2-ethylprcpyl, and etc. The proviso means that groups like isopropyl or 1-methylbutyl are excluded. The C ₂ to C ₆ alkyls are preferred. Or; the R.,R ₂ N- heterocyclic groups, morpholino is preferred. By a pharmaceutically acceptable cation is meant any non-toxic cation; these are well known to one of ordinary skill in the art. The cation should not render the compound unstable or insoluble in water. Generally the cation will be a group 1 or group 2 cation, such as sodium, potassium, magnesium or calcium ions, or NR ₃ RR ₅ R ₆ ^' , wherein R ₃ , R ₄ , R ₅ , and R ₆ are independently chosen from H, C-,-C ₁₂ alkyl, C ₃ - C ₇ cycloalkyl or benzyl, more preferably H, C,-C alkyl or C ₆ cycloalkyl. The method of synthesis discussed below first results in the cation

being R ₁ R-,H ₂ N ⁺ , these cations work well. The most preferred cations are Na ⁺ ,K ⁺ ,Ca ^+z , and R_,R ₂ H ₂ N ⁺ .

The compositions ar>_ potent anti-hypertensives. They are useful for lowering the blood pressure and treating any cardiovascular disorder ._n which lowering the blood pressure will have a beneficial effect. The invention provides an effective method of lowering the b:_ood pressure by administering the composition to a mammal.

The methods of synthesis are the same as disclosed by Drago et al., J. Amer. Chem. S c, 83 _. , 1819-1822,1961. Generally, the secondary amine (R.,R ₂ HN) is dissolved in anhydrous ether, oxygen is removed from the system, the temperature is lowered to -78°C, and dry NO is bubbled through the ether solution. The reaction can be run at high press ire (100 psi) or at atmospheric pressure. The same product -is obtained, but the yields are higher using the high pressure method. Th ≥ same method is used to make all the compounds, the only diffarence being the starting secondary amine. Example 1 gives the details of how the diethylamine complex was made. Example la

Anhydrous diethylamine (100ml) was dissolved in 100ml of anhydrous diethyl ether and was placed in a three-necked flask. Two of the necks served as inlets for N ₂ and NO, and the

third was an outlet. T _h e flask was flushed with N ₂ and the mixture cooled to - ₇₈ ° _C using an acetone-dry ice bath. Commercial grade NO was bubbled through 10M NaOH and dried by passing it through a column containing NaOH pellets, and then bubbled for 3 h ^' r through the diethylair.ine/diethyl ether solution. The mixture was allowed to warm to room temperature overnight (18 hr) . The product precipitated frcrα solution. The product was filtered and washed with diethyl ether. Three grams (3% yield) of crude product was o _b taine _d . T _h e product was purified by suspending it in diethyl ether, adding sufficient neutralized chloroform dropwise to just dissolve the product, and placing the mixture in the freezer. The resultant crystals were then filtered, washed with diethyl ether and cried by vacuum.

¹ H NMR (200MHz): S 3.07(q, 4H, J=7.2 Hz),2.94(q, 4H,J=7.2Hz) ,1.27.(t, 6H, J=7.2Hz), 0.96(q, 6H, J=7.2 Hz ⁾ .

¹³ C NMR (50MHZ): S 51.3, 45.2, 13.8, 13.5.

Calculated: C=46.56%, N=27.17%, H=10.75%

Found: C=46.49%, N=26.29, H=11.20% The above physico-chemical characteristics correspond to the structure:

Example lb

In the high"pressure method a Parr shaker was used. NO was added to the' reservoir which makes it possible to fill the

reaction vessel without opening it directly to the NO tank. The mixture of diethylamine (100ml) and diethyl ether (100 ml) was added to the reaction vessel. The reaction vessel was cooled to -78°C and subjected to several evacuation/nitrogen flush cycles to remove as much of the oxygen as possible. NO was then introduced at a pressure of about 100 psi. The reaction vessel was allowed to slowly warm to room temperature overnight (18 hr) , with shaking. ^' The excess NO was flushed off with N ₂ and the product was filtered and washed with diethyl ether. The crude y-ield was 5 grams. Purification and analysis were the same as in example la.

The other secondary amine-NO complexes are made using the same methods as examples la and lb, the only difference being the identity of the starting secondary amine. The product made in each case has the formula R ₁ R ₂ NN ₂ 0 ₂ ^' R _l R ₂ H ₂ N ⁺ . The synthesis of the salts containing the other cations is done by conventional methods, most particularly by a metathesis reaction, a method well known to one of ordinary skill in the art. (See Drago et al.) To make the sodium salt of the compound produced in example 1, the diethylammonium salt is dissolved in ethyl alcohol and reacted with sodium etho.<ide according to the following reaction:

I^R _g NN _j O _j ^' R^H _g N ^* + NaOEt - > EtOH + R^NH + R^NN^Na". The product is precipitated by flooding the reaction mixture with ether and then wasned with neutralized chloroform. The other salts can be made by similar metathesis reactions.

Pharmacological Properties

The effect on the ean arterial blood pressure and heart rate of male Sprague-Daw ^' .ey rats of the compositions of the invention was measured using a standard technique. A pressure transducer (Bell and How_.ll, type 4-327-1) was connected to the right carotid artery via a catheter containing heparinized saline. The mean arterial pressure and heart rate were recorded on a Gould (Model 2800) 3-channel recorder. The rats were anesthetized with nembutal at an initial dose of 35 g/kg body •weight and recurrent smaller injections as needed. The compounds were dissolved in a pharmaceutical carrier and injected into the rats via a catheter in the right femoral vein. Table 1 shows the results.

TABLE 1

Compound Dose Maan Arterial Pressure Heart Rate

(^mole/kg) Initial - Post Change Initial Post (mm Hg) (beats/min)

In Table 1, the pharmaceutical carrier was Abbott's 5% dextrose USP. SNP, NaN0 ₂ ,. and NaNC ₃ were used as controls. SNP and NaN0 ₂ are known vasodilators, ..aN0 ₃ is the oxidative breakdown product

of NaN0 ₂ and has little vasodilatory effect. The results show that (Et) ₂ NN ₂ 0 ₂ ^" (Et) ₂ H ₂ N ⁺ is a potent anti-hypertensive, decreasing the blood pressure significantly. The peak value of the blood pressure decrease, showr. in Table 1, takes only about 30 sec to 1 min to occur, after injection, and the blood pressure starts to rise again soon after ar.d has totally recovered within 10-15 min.

The compositions of this invention are useful for treating any cardiovascular disorder that will respond favorably to a .decrease in blood pressure. These disorders include chronic hypertension, hypertensive crisis (an acute hypertensive emergency) , acute congestive heart failure, angina, acute yocardial infarction, left ventricular failure, cerebrovascular insufficiency and intracranial haemorrhage. Because of the fast response upon intravenous injection the compositions are particularly advantageous for treating acute disorders such as hypertensive crisis, toyemia of pregnancy and acute congestive heart failure.The preferred method of administration is by injection into the blooc. system, most preferably by intravenous injection. The chronic disorders can be treated by continuous intravenous infusion. A suitable dosage for intravenous administration is about 0.01 to 10.0 mg/kg per day.

The pharmaceutical compositions of the invention are comprised of the'compounds of formula I and a pharmaceutical

carrier. The carrier car. be any of those conventionally used and is limited only by chemico-physical considerations such as solubility and lack of reactivity with the compound and by the route of administration. For intravenous administration, the carrier will be aqueous and may contain solubilizing agents, buffers,preservatives, c.ntioxidants, chelating agents, and agents to control the tonicity , such as dextrose or sodium chloride. The requirements for effective pharmaceutical carriers for injectable compositions are well known by one of ordinary skill in this art. (See "Pharr.aceutics and Pharmacy Practice" , J.B. Lippincott Company, Philadelphia, 1982,edited by Banker and Chalmers, pages 238-250.. which are incorporated by reference, also see ASHP "Handbook on Injectable Drugs" 4th edition by Trissel, pages 622-630, which lists commercially available intravenous infusion-.sol.utions, these pages are incorporated by reference.) The compounds may also be formulated as inclusion complexes, such as, for example, cyclodextrin inclusion complexes; or the compounds may be carried within liposomes. Preferred pharmaceutical carriers for injection are PBS (phosphate buffc'.d saline), 5% dextrose and sterile water. Since the compou.___.s of formula I are subject to being oxidized by oxygen, an antioxidant, such as ascorbate, can be added to the carrier to increase' the shelf-life.