TECHNICAL FIELD OF THE INVENTION The present invention relates to novel N- substituted piperazine NONOate compounds. In particular, the present invention relates to N- substituted piperazine compounds, bearing substituents such as acyl, sulfonyl, phosphoryl, alkyl, alkenyl, or the like and derivatives thereof to which are bound nitric oxide-releasing N ₂ 0 ₂ ^" .

BACKGROUND OF THE INVENTION

Endothelium-derived 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. Pharmacol. Toxicol. 24, 175-197, 1984.) Recently, Palmer et al., have shown that EDRF is identical to the simple molecule, nitric oxide, NO

(Nature 317i 524-526, 1987) . It has been hypothesized for years that many nitrovasodilators that mimic the effect of 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 Kruszyna et al., Tox. & Appl. Pharmacol., 91, 429-438, 1987; Ignarro, FASEB J. 3 , 31- 36, 1989 and Ignarro et al., J. Pharmacol. Exper. Therapeutics 218(3) , 739-749, 1981.)

BRIEF SUMMARY OF THE INVENTION

It has now been discovered that certain N- substituted piperazine NONOate compounds, having the structure:

♦ X

R—N N-N.0 ₂ ^" M (I)

wherein M is a pharmaceutically acceptable cation, x is the valence of the cation, and R is selected from the group consisting of: an unsubstituted or substituted C,- C ₂₀ straight chain alkyl, an unsubstituted or substituted C ₃ -C ₂₀ branched chain alkyl,an unsubstituted or substituted C ₂ -C ₂₀ straight chain alkenyl, an unsubstituted or substituted C ₃ -C ₂₀ branched chain 0 alkenyl, a group of formula R 1—C 11— , a group of formula

0

R 2—P 11— , a group of formula R —S0 ₂ —, or a group of

_R 3

formula R ⁵ —O—N=N(0)—, are potent nitric oxide releasing compounds. Nitric oxide releasing compounds, among other aspects, act as vasodilatory anti-hypertensives and thus are useful for treating cardiovascular disorders in which lowering the blood pressure has a beneficial result. It is believed that such nitric oxide releasing compounds function by releasing NO in the blood after injection; however the invention should not be limited by this hypothesis. In addition to having potent nitric oxide releasing capabilities, the N-substituted piperazine NONOates are effective at tagging polypeptides and proteins, thus creating potent NO releasing proteins.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides N-substituted piperazine NONOate compounds, having the structure:

R-N Nh-N.O. ^" M ^*X (I) x

wherein M is a pharmaceutically acceptable cation, x is the valence of the cation, and R is selected from the group consisting of: an unsubstituted or substituted C,- C ₂₀ straight chain alkyl, an unsubstituted or substituted C ₃ -C ₂₀ branched chain alkyl,an unsubstituted or substituted C ₂ -C ₂₀ straight chain alkenyl, an unsubstituted or substituted C ₃ -C ₂₀ branched chain

0 alkenyl, a group of formula R^-C- , a group of formula 0

R 2—P ii— , a group of formula R—S0 ₂ —, or a group of formula R ⁵ - -N=N(0)-.

R ¹ may be any of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, alkylthio, arylthio, alkoxy, aryloxy, amino, a mono- or di-substituted amino, and succinimidoxy.

R ² and R ³ may be the same or different and may be any of aryl, substituted aryl, heteroaryl, alkylthio, arylthio, mercapto, hydroxy, alkoxy, halo, aryloxy, amino, a mono- or di-substituted amino, phosphate, a mono- or di-substituted phosphate, an unsubstituted or substituted C,-C ₂₀ straight chain alkyl, an unsubstituted or substituted C ₃ -C ₂₀ branched chain alkyl, an unsubstituted or substituted C ₂ -C ₂₀ straight chain

alkenyl, and an unsubstituted or substituted C ₃ -C ₂₀ branched chain alkenyl.

R ⁴ may be any of an unsubstituted or substituted C,- C ₂₀ straight chain alkyl, an unsubstituted or substituted ^c ₃ ~ ^c ₂₀ branched chain alkyl, an unsubstituted or substituted C ₂ -C ₂₀ straight chain alkenyl, an unsubstituted or substituted C ₃ -C ₂₀ branched chain alkenyl, aryl, substituted aryl, and heteroaryl.

R ⁵ may be any of an unsubstituted or substituted C^ c ₂₀ straight chain alkyl, an unsubstituted or substituted C ₃ -C ₂₀ branched chain alkyl, an unsubstituted or substituted C ₂ -C ₂₀ straight chain alkenyl, an unsubstituted or substituted C ₃ -C ₂ o branched chain alkenyl, and a noncovalently bound cation. The substituted C,-C ₂₀ straight chain alkyl may be substituted with one or more substituents selected from the group consisting of: aryl, substituted aryl, heteroaromatic aryl, alkylthio, arylthio, acylthio, mercapto, hydroxy, aryloxy, alkoxy, halo, carboxy and esters thereof, phosphoryloxy, a mono- or di-substituted phosphoryloxy, phosphonyloxy, a mono- or di-substituted phosphonyloxy, phosphonyl, carbonyl, acyl, aroyl, carboxamido, cyano, nitro, oximino, acyloxy, with the proviso that there be no hydroxy, halo, mercapto, or phosphate substituent on the carbon attached to the piperazine ring.

The substituted C ₃ -C ₂₀ branched chain alkyl may be substituted with one or more substituents selected from the group consisting of: aryl, substituted aryl, heteroaryl, alkylthio, arylthio, acylthio, mercapto, hydroxy, aryloxy, alkoxy, halo, carboxy and esters thereof, amino, a mono- or di-substituted amino, phosphoryloxy, a mono- or di-substituted phosphoryloxy, phosphonyloxy, a mono- or di-substituted phosphonyloxy, phosphonyl, carbonyl, acyl, aroyl, carboxamido, cyano,

nitro, oximino, acyloxy, with the proviso that there be no hydroxy, halo, mercapto, or phosphate substituent on the carbon attached to the piperazine ring.

The substituted C ₂ -C ₂₀ straight chain alkenyl may be substituted with one or more substituents selected from the group consisting of: aryl, substituted aryl, heteroaryl, alkylthio, arylthio, acylthio, mercapto, hydroxy, aryloxy, alkoxy, halo, carboxy and esters thereof, amino, a mono- or di-substituted amino, phosphoryloxy, a mono- or di-substituted phosphoryloxy, phosphonyloxy, a mono- or di-substituted phosphonyloxy, phosphonyl, carbonyl, acyl, aroyl, carboxamido, cyano, nitro, oximino, acyloxy, with the proviso that there be no hydroxy, halo, mercapto, or phosphate substituent on the carbon attached to the piperazine ring.

The substituted C ₃ -C ₂₀ branched chain alkenyl may be substituted with one or more substituents selected from the group consisting of: aryl, substituted aryl, heteroaryl, alkylthio, arylthio, acylthio, mercapto, hydroxy, aryloxy, alkoxy, halo, carboxy and esters thereof, amino, a mono- or di-substituted amino, phosphoryloxy, a mono- or di-substituted phosphoryloxy, phosphonyloxy, a mono- or di-substituted phosphonyloxy, phosphonyl, carbonyl, acyl, aroyl, carboxamido, cyano, nitro, oximino, acyloxy, with the proviso that there be no hydroxy, halo, mercapto, or phosphate substituent on the carbon attached to the piperazine ring.

Any of R ¹ , R ² , R ³ , R ⁴ and R ⁵ may be substituted with a Cι-C ₂₀ straight chain alkyl, a substituted C ₃ -C ₂₀ branched chain alkyl, a substituted C ₂ -C ₂₀ straight chain alkenyl, or a substituted C ₃ -C ₂₀ branched chain alkenyl, with or without one or more substituents selected from the group consisting of: aryl, substituted aryl, heteroaryl, alkylthio, arylthio, acylthio, mercapto, hydroxy, aryloxy, alkoxy, halo, carboxy and esters thereof, amino, a mono- or di-substituted amino,

phosphoryloxy, a mono- or di-substituted phosphoryloxy, phosphonyloxy, a mono- or di-substituted phosphonyloxy, phosphonyl, carbonyl, acyl, aroyl, carboxamido, cyano, nitro, oximino, and acyloxy. 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. The most preferred cations are Na ⁺ , K ⁺ , Ca ⁺² , and Mg ^4"2 .

The disclosed compounds are potent nitric oxide releasing compounds. Nitric oxide releasing compounds, among other aspects, act as vasodilators and anti- hypertensives and thus are useful for treating cardiovascular disorders in which lowering the blood pressure has a beneficial result. It is believed that such nitric oxide releasing compounds function by releasing NO in the blood after injection; however the invention should not be limited by this hypothesis. In addition to having potent nitric oxide releasing capabilities, the N-substituted piperazine NONOates are effective for tagging polypeptides and proteins, thus creating potent NO releasing proteins. N-substituted piperazine NONOates may be covalently attached to a polypeptide chain at either terminus or within the chain itself, such that the resulting protein has a potent NO releasing compound attached to it and may be used as an anti-hypertensive agent or any other suitable use. The compounds of the present invention may be included in pharmaceutical compositions for administration to a mammal, including humans. The pharmaceutical compositions may comprise one or more of the compounds described herein and a suitable pharmaceutical carrier, such as those presently known to those skilled in the art.

EXAMPLES The preparation and characterization of N- substituted piperazines containing the nitric oxid- releasing N ₂ 0 ₂ ^" functional group are illustrated in the following examples:

Example I This example illustrates the preparation of Ethyl- 1-Piperazine Carboxylate-4-Nitric Oxide Complex/Sodium salt, as shown schematically as follows:

A solution of 20g (0.126 mol) of carboethoxy piperazine in 60 ml of methanol was placed in a Parr bottle. The solution was treated with 27.4 ml (0.126 mol) of 25% sodium methoxide in methanol, the system was evacuated, charged with 40 psi of nitric oxide and kept at 25°C for 48 hr. The white crystalline product was collected by filtration and washed with cold methanol as well as with copious amounts of ether. The product was dried under vacuum to give 14.5g(48%) yield of ethyl-1 piperazine carboxylate-4-nitric oxide complex/sodium salt: mp: 184- 5°C; uv(0.01N NaOH) λmax(e) , 252nm (10,396); NMR (D-O) ; 1.254 (t,3H), 3.107(m,2H), 3,677(m,2H), 2.147(q,2H). Anal calcd. for C ₆ H ₁₃ N ₄ 0 ₄ Na: C 35.00% H 5.42%, N 23.33%, Na 9.58%. Found: C 34.87%, H 5.53%, N 23.26%, Na 9.69%. The halflife of this compound at pH 7 and 25°C was assessed at 5 minutes. This measurement was based on the loss of the 252 nm chromophore in the ultraviolet spectrum.

Example II

This example demonstrates the attachment of a nucleophilic center to a protein that does not contain a nucleophilic center that will readily react with NO, yielding the compound l-[4-N-acetyl-L- methiony1)piperazin-l-yl]-l-oxo-2-hydroxydiazine, sodium salt shown schematically as follows:

α +-

H

CF ₃ COOH CH ₂ C1 ₂

Me n ^L - - ι ^H C-ιPC- O- r MeS , ^H _| p / \ N _K - _K NaOMe. NO NΉ N Na MeOH / Ac H Ac H

A solution of 4.78 g (0.025 mol) of Iv ^' -acetyl-L- methionine in CH ₂ C1 ₂ : acetonitrile (120 ml) was cooled to 0°C. To this solution was added 5.36 g (0.025 mol) of dicyclohexylcarbodiimide (DCC) followed by the rapid addition of 3.90 g (0.021 mol) of N-t- butoxycarbonylpiperazine in 6 ml of dichloromethane.

The progress of the reaction was followed on silica gel TLC plates developed with 4:1 acetonitrile: tetrahydrofuran and visualized with either iodine or ninhydrin spray. The reaction was complete within 2 h. A few drops of glacial acetic acid were added to the

reaction mixture and the solvent was removed on a rotary evaporator. The residue was taken up in ether and filtered. The clear filtrate was washed with dilute acid followed by dilute base. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and evaporated to give 8.2 g of l-(t- butoxycarbonyl)-4-(W-acetyl-L-methionyl)piperazine, a colorless oil which required no further purification: IR (film) 3304, 3058, 2973, 2931, 2868, 1701, 1645, 1539, 1420, 1237, 1173 cm ^-1 ; NMR (CDCl ₃ ) δ 1.47 (s, 9 H) , 1.80 (m, 2 H) , 2.02 (s, 3 H) , 2.10 (s, 3 H) , 2.46 (m, 2 H) , 3.53 (m, 8 H) , 5.10 (M, 1 H) , 6.35 (b, 0.5 H) , 6.43 (b, 0.5 H) .

To a solution of 8.6 g (0.024 mol) of l-(t- butoxycarbonyl)-4-(_7-acetyl-L-methionyl)piperazine in 60 ml of dichloromethane was added 10 ml of trifluoroacetic acid and the mixture was stirred at room temperature overnight. The solution was extracted with water and the resulting aqueous solution was made basic with sodium hydroxide. The product was extracted with dichloromethane, dried over sodium sulfate, and filtered. Evaporation of the solvent gave 2.1 g of 1- (ΛT-acetyl-L-methionyl)piperazine, as an oil: IR (film) 3304, 3051, 2917, 2861, 1645, 1546, 1448, 1377 cm ^-1 ; NMR (CDC1 ₃ ) δ 1.95 (m, 2 H) , 2.02 (s, 3 H) , 2.10 (s, 3 H) ,

2.54 (m, 2 H) , 2.98 (m, 4 H) , 3.74 (m, 4 H) , 5.10 (m, 1 H) , 6.40 (b, 0.5 H) , 6.48 (b, 0.5 H) .

To a solution of 510 mg (1.97 mmol) of 1-(_7-acetyl- L-methionyl)piperazine in 1 ml of methanol was added 428 μl (1.97 mmol) of 25% sodium methoxide in methanol. The system was degassed and charged with 40 psi of nitric oxide. After exposure of the solution to NO for 120 h, the pressure was released and the solid product was collected by filtration, washed with ether, and dried to give 27 mg of l-[4-(W-acetyl-L-methionyl)piperazin-l- yl]-l-oxo-2-hydroxydiazene, sodium salt, as a white

solid: UV λ _max (e) 252 nm (12.0 mM ^"1 cm ^"1 ). The product decomposed with a half-life of 6.9 min at pH 7 and 25°C to produce 1.72 moles of NO per mole of test agent.

EXAMPLE III This example illustrates the preparation of 1- Piperazine[2-methoxydiazene-l-oxide]-4-Nitric oxide complex sodium salt, as shown schematically as follows:

A solution of 271mg (1.69 mmol) of l-methoxy-2-oxo- piperazyl diazene in 1 ml of methanol was placed in a micro Parr bottle and treated with 0.368 ml (1.69 mmol) of 25% sodium methoxide in methanol. The solution was flushed with nitrogen and charged with 40 psi of nitric oxide at 25°C forming a solid mass within 24 h. The pressure was released, the solid suspended in ether and collected by filtration to give 156 mg (38%) of product: mp 165-6°C; UV in 0.01N NaOH, λmax (e) nm (18,984). The halflife in pH 7.4 buffer at ambient temperature was measured at 8 minutes for the ionic side, and stable for the protected side; NMR (D ₂ 0) , 3.327 (m,4H), 3,659 ( , 4H) , 4.090 (s, 3H) .

Example IV This example illustrates the preparation of GLO/NO (Dansylpiperazine NONOate) , as shown schematically as follows: Dansylpiperazine was prepared by refluxing a solution of 7.98 g piperazine (9.27 mmol) and 5.00 g dansyl chloride (18.5 mmol) in lOOmL toluene for 6 h. The product was isolated by washing the solution with 5% NaOH and then water and concentrating in vacuo to yield

5.0 g (84%) dansylpiperazine as yellow-green powder. *H NMR (200 MHz, CDC1 ₃ ) 2.7-2.9 (4H, multiplet), 2.9 (6H, singlet), 3.1-3.3 (4H, multiplet), 7.1-7.6 (3H, multiplet), 8.2-8.6 (3H, multiplet). To prepare GLO/NO, a solution of 3.08 g (9.64 mmol) dansyliperazine and 2.20 mL (9.64 mmol) sodium methoxide 25% solution in methanol in 25 mL N,N-dimethylformamide was treated with NO gas at 80 psig for 2 days. After flushing with argon, 150 ML ether was added and the product isolated by filtration. Yield 2.0 g (52%) mp 158-160°C dec. *H NMR (200 MHz, D ₂ 0) 2.8 (6H, singlet), 3.1-3.2 (4H, multiplet), 3.4-3.5 94H, multiplet), 7.3- 7.7 (3H, multiplet), 8.2-8.5 (3H, multiplet).