N-nitroso-N-substituted hydroxylamines as nitric oxide donors

Nitric oxide has proved to mediate many important physiological processes. The nitric oxide donors of the present invention have a NONOate anion linked to an ortho-substituted aryl, a heteroaromatic substituent, asteroid, or a catecholamine. Preferred ortho substituents are alkoxy, halo, and alkyl. The cation of the salt is an alkali metal, an alkaline-earth metal, an ammonium or substituted ammonium group. Nitric oxide donors provided herein are more stable than that of nitrogen-bonded NONOates described previously. The by product left after release of NO, and the nitric oxide donors themselves, are very probably less carcinogenic than the corresponding nitrogen-bonded NONOates.

The present invention relates generally to methods for treatment of a 
variety of medical conditions with previous or new nitric oxide donors 
such as, for example, N-nitroso-N-substituted hydroxylamine or salts 
thereof. 
BACKGROUND OF THE INVENTION 
The concept that NO regulates many biological functions dates back only 15 
years. In 1980, Furchgott and Zawadski first showed the endothelium must 
be intact for acetylcholine to produce vascular relaxation. Subsequently, 
numerous studies have shown that neurohumoral or pharmacological agents 
mediate vasodilation via the endothelium. It is now recognized that the 
endothelium releases a potent, labile, nonprostanoid vasodilating agent in 
response to various stimuli that either cause vasodilation or modulate 
vasoconstriction. This factor, originally termed endothelium-derived 
relaxing factor (EDRF), has been shown to be nitric oxide (NO) or a 
compound with a nitric oxide moiety. 
NO is synthesized by the oxidative deamination of a guanidino nitrogen of 
L-arginine by at least three different isoforms of a flavin-containing 
enzyme, nitric oxide synthase (NOS) (Moncada, Palmer and Higgs, 1991). 
Three distinct isoforms have been purified, cloned (Bredt et al., 1990; 
Stuehr et al., 1991) and expressed, and there is evidence for the presence 
of NOS in almost every tissue of the mammalian body, albeit at widely 
different levels. 
NO is an ideal local transcellular messenger because of its small size, 
lipophilic nature, and short duration of action. Commonly used chemical 
nitro-vasodilators, such as nitroglycerin and nitroprusside, appear to act 
by releasing NO. 
Biological effects of NO. Nitric oxide elevates levels of cGMP 
(1,3,5-cyclic guanosine monophosphate) within vascular smooth muscle to 
produce relaxation and reduce the tone on blood vessels (Moncada, et al., 
1991). Nitric oxide binds to heme and thus activates soluble guanylate 
cyclase (ignarro, 1991) to increase cellular content of cGMP. It has long 
been recognized that nitrovasodilators, such as nitroprusside and 
nitroglycerin, inhibit vascular smooth muscle contractility to produce 
relaxation or reduce vascular tone. These agents have been used since the 
late 1800's as vasodilators. However, it has only been recently that the 
mechanism of action of these compounds has been realized. 
Nitrovasodilators are now classified as nitric oxide donors (Moncada, et 
al., 1991). The long-used nitrovasodilators may be regarded as 
substitution therapy for a failing physiological mechanism. Nitric oxide 
is also produced by macrophages and other immune cells (Stuehr, et al., 
1991). Stimulated macrophages produce nitric oxide from L-arginine and it 
is considered the first line of defense against invading pathogens. 
There is a substantial body of evidence from animal experiments that a 
deficiency in nitric oxide contributes to the pathogenesis of a number of 
diseases, including hypertension, atherosclerosis and diabetes (Moncada, 
Palmer and Higgs, 1991). There are many recent studies showing that 
inhibition of nitric oxide synthase dramatically increases blood pressure. 
Inhibition of nitric oxide synthesis with L-NMMA (L-N.sup.G methyl 
arginine), L-NA (L-N.sup.G nitroarginine), or L-NAME (L-N.sup.G 
nitroarginine methyl ester) causes long-lasting elevation in blood 
pressure and suggests that a reduction in the synthesis of nitric oxide 
may contribute to the pathogenesis of hypertension (Moncada, et al., 
1991). 
Further, in patients with pregnancy-induced hypertension, release of nitric 
oxide by umbilical vessels in lessened (Pinto et al., 1991) and the 
physiological decrease in blood pressure in pregnant spontaneous 
hypertensive rats was shown to depend on endothelial nitric oxide (Ahokas, 
et al., 1991). Additionally, infusion of L-NA increases blood pressure in 
pregnant rats and potentiates responses to vasopressors (Molnar, et al., 
1992). These studies suggest that impaired nitric oxide synthesis may be 
an important mechanism in the etiology of pregnancy-induced hypertension 
(preeclampsia). Indeed, inhibition of NO in pregnant rats produced 
symptoms identical to preeclampsia (Yallampalli, et al., 1993). It has 
been suggested that preeclampsia is an endothelial cell disorder (Roberts 
et al., 1989). Nitric oxide is also produced by the uterine wall and it 
effectively inhibits uterine contractility during pregnancy but not during 
delivery (Yallampalli, et al. 1993). On the other hand, steroid hormones 
seem to regulate the nitric oxide-cGMP relaxation mechanism in the uterus 
(Yallampalli, et al., 1993). 
Nitric oxide is also involved in the control of blood clotting. Nitric 
oxide is a very potent inhibitor of coagulation and this action may be 
extremely important in preventing clotting in the placental circulation. 
Previously, it has been suggested that prostacyclin regulates placental 
clotting. However, nitric oxide may be very important in this process 
either in conjunction with the inhibitory effects of prostacyclin or 
acting alone. Nitric oxide has been found to be synthesized in almost all 
tissues of the body including brain and peripheral nervous systems, smooth 
muscle vascular tissue, (see above), kidney, lung, uterus, etc. 
Nitric oxide modulates various biological phenomena including regulation of 
smooth muscle contractility of several tissues. In previous studies, rat 
uterine tissues in vitro were examined to determine whether a 
L-arginine-nitric oxide-cGMP system is present in the rat uterus (Garfield 
and Yallampalli, 1993; Yallampalli et al., 1993; Izumi, et al., 1993). 
These studies reported that (1) the substrate and a donor of nitric oxide 
produced uterine relaxation, (2) inhibitors of the nitric oxide--cGMP 
pathway blocked the relaxation responses, (3) nitric oxide synthase was 
localized to several uterine cell types, (4) nitric oxide was produced by 
the uterus during periods when L-arginine was consumed and citrulline 
levels increased, (5) effects of nitric oxide substrate on relaxation were 
mimicked by cGMP, (6) the responses to L-arginine and NO were decreased 
during term and preterm labor, and (7) the NO synthetase isoforms are 
present in the uterus and upregulated during pregnancy but decreased when 
labor begins. These studies indicate that NO may control uterine 
contractility during pregnancy. 
Present NO Donors: Presently, there are only a few nitric oxide donor 
compounds that are used clinically (e.g., nitroglycerin, sodium 
nitroprusside and amyl nitrite). Table 1 indicates various NO donor agents 
with clinical potential. 
In a recent patent, Keefer (1993) included, in addition to aminoNONOates 
that he had reviewed, and whose structure he had proved by X-ray 
crystallography (Saavedra et al., 1992), cupferron, some derivatives as 
potential hypotensive agents. In a more recent review, Keefer indicated 
that when the NONO group is attached to a carbon atom as in cupferron, the 
parent N-aryl-N-nitroso-hydroxylamine is stable under protonating 
conditions, implying that unlike aminoNONOates which release NO readily, 
cupferron and its derivatives would act more sluggishly or not at all 
(Keefer et al. 1994). 
TABLE 1 
__________________________________________________________________________ 
Present NO Donor Compounds 
__________________________________________________________________________ 
Trade name or 
Route of 
Generic name 
synonym 
administration 
Dose Onset of action 
Duration 
__________________________________________________________________________ 
Amyl nitrite Inhalation 
0.3 ml 30-60 sec 
3 min 
Nitroglycerin 
Glyceryl 
Sublingual 
gr 1/150; 0.4 mg 
trinitrate 
Oral 2.5-6.5 mg 
30-60 min 
8-12 hr 
Transdermal 
0.1-0.4 mg/hr 
2 hrs 12 hrs 
Pentaerythritol 
Peritrate 
Sublingual 
10 mg 10 min 30 min 
tetranitrate 
Pentritol 
Oral 60 mg 30 min 12 hr 
Pentafin 
Oral 10-20 mg 
30-60 min 
4-5 hr 
Vasitol 
Sustained- 
80 mg 30-60 min 
12 hr 
release 2-4 hr 
4 hr 
Erythrityle 
Cardilate 
Sublingual 
5-30 mg 5-10 min 
2-4 hr 
tetranitrate 
Tetranitrol 
Oral 5-30 mg 30 min 4 hr 
Erythrol 
Oral 5-30 mg 30 min 2-4 hr 
tetranitrate 
Isosorbide 
Isordil 
Sublingual, 
5-10 mg 2 min 1.5-2 hr 
dinitrate oral 5-30 mg 15-30 min 
4 hr 
Trolnitrate 
Metamine 
Oral 2-10 mg Slow, 3 days 
Up to 1 wk 
phosphate 
Dipyrimadol 
Persantin 
Oral 25-50 mg 
2-5 min 
20-30 min 
Nitroprusside 
Nipride 
i.v. 10 .mu.g/kg 
10 sec. 
5 min 
__________________________________________________________________________ 
Uses of Nitric Oxide Donors: Presently, nitric oxide donors (nitroglycerin 
or amyl nitrite) are used for angina pectoris due to coronary artery 
disease and control of blood pressure associated with myocardial 
infarction or surgical procedures (nitroglycerin or sodium nitroprusside). 
Nitric oxide (NO) donors presently in use consist either of substances 
which are nitrite or nitrite esters (e.g. amyl nitrate and 
glycerol-trinitrate), or inorganic nitroso complexes (e.g. sodium 
nitroprusside). In addition to these clinically used compounds, the 
so-called NONO-compounds which were prepared initially by Drago et al. 
(1961) from gaseous NO and secondary amines have been studied. Among the 
latter compounds, substances such as the diethylamine-nitric oxide 
addition compound have the drawback that they may decompose, leading to 
compounds that are proved carcinogens (dialkyl-nitrosoamines), along with 
the desired nitric oxide. 
Problems with present nitric oxide donor compounds include the following: 
a. Short duration of action 
b. Short half-life 
c. Lack of tissue specificity 
d. Development of tolerance 
e. Accumulation of toxic substances--e.g. cyanide from sodium nitroprusside 
f. Compounds with a chain of three nitrogen atoms (i.e. having the NONO 
group attached to a nitrogen atom), on releasing nitric oxide, leave a 
remaining nitrosoamine fragment which may be carcinogenic in some cases. 
Because of all of the above problems, known procedures are not completely 
satisfactory, and persons skilled in the art have searched for 
improvements. 
Desirable qualities of new nitric oxide donor compounds are: 
a. Long duration of action 
b. Ease of use-oral preparation 
c. Tissue selectivity 
d. Lack of tolerance 
e. Low toxicity 
One object of the present invention is to provide a method of use of 
N-nitroso-N-substituted hydroxylamines or salts thereof as nitric oxide 
(NO) donors having at least some of these qualities. 
SUMMARY OF THE INVENTION 
The present invention describes the use of certain N-nitroso-N-substituted 
hydroxylamines and salts thereof (1) as new nitric oxide (NO) donors. Also 
part of the present invention are uses of these new NO donors in a variety 
of medical disorders including uterine muscular disorders, hypertension 
and cardiovascular problems. Even prior NO donors have been found to have 
a new use in treating uterine muscular disorders. 
The present invention provides nitric oxide donor compounds having the 
following structure: 
##STR1## 
R is an ortho-substituted aryl, a heteroaromatic substituent, a steroid, 
or a catecholamine; and M is an alkali metal, an alkaline-earth metal, an 
ammonium or substituted ammonium cation, wherein the compound decomposes 
under physiological conditions to release nitric oxide. 
The ortho-substituted aryl may be 2-methylphenyl, 2-methoxyphenyl, 
2-ethylphenyl, 2-isopropylphenyl, 2,4-difluorophenyl, 2,5-difluorophenyl, 
2-chlorophenyl, 2,3-dichlorophenyl, 2,4-dichlorophenyl, 
2,5-dichlorophenyl, 2-bromophenyl, 5-fluoro-2-methylphenyl, 
4-fluoro-2-methylphenyl, 4-choro-2-methylphenyl, or 
3-chloro-2-methylphenyl. Preferably, the ortho-substituted aryl is 
2-chlorophenyl or 2-methoxyphenyl. 
The heteroaromatic substituent is thienyl, oxazolyl, thiazolyl, imidazolyl, 
pyrazolyl, a six-membered aza-aromatic, 2,2,5,5-tetramethylpiperidine, 
2,2,5,5-tetramethylpyrrolidine, proline, hydroxyproline, morpholine, or 
3-azabicyclo3.2.2!nonane or simple substituted derivatives of all such 
systems. 
In preferred embodiments, the ortho-substituted aryl is .alpha.-naphthyl 
bearing a halo, alkyl or alkoxy substituent; or phenyl or .alpha.-naphthyl 
bearing a sulfonate or carboxylate group. 
The substituents on the R group are chosen so as: (i) to modulate the NO 
releasing potency either by means of their electron releasing/accepting 
ability, or by means of their steric effects when situated in the 
proximity of the nitroso group; (ii) to modify the hydrophilic/lipophilic 
properties of the NO donors, or, (iii) to counteract their negative 
electric charge. 
The cation M+ is preferably an alkali metal cation (most preferably sodium 
or potassium) although other physiologically and pharmacologically 
acceptable cations may be used. Such cations include calcium, magnesium, 
ammonium or ammonium substituted with lower alkyl (C1-C4), cycloalkyl (5- 
or 6-membered), benzyl or phenyl. The ammonium salts of compound 1 where R 
is phenyl or naphthyl are commercially available. Compound 1 where 
R=phenyl and M.sup.+ is NH.sub.4 + is known as cupferron, and compound 1 
where R=.alpha.-naphthyl and M.sup.+ is NH.sub.4.sup.+ is known as 
neocupferron; these are used as reagents in analytical chemistry, but 
their ammonium cations may make them toxic to mammals. 
Preferred R groups are .alpha.-naphthyl, ortho-substituted phenyl or a 
heterocyclic aromatic ring. In a preferred embodiment, R is a biologically 
active moiety designed to target the NO releasing agent to a specific 
organ or tissue. Specific examples of biologically active moieties include 
steroids (such as progesterone and estrogen, e.g.) and epinephrine or 
other catecholamines and simple derivatives thereof. 
A preferred embodiment is a compound having the structure 
##STR2## 
where R is 2-methylphenyl, 2-methoxyphenyl, 2-ethylphenyl, 
2-isopropylphenyl, 2,4-difluorophenyl, 2,5-difluorophenyl, 2-chlorophenyl, 
2,3-dichlorophenyl, 2,4-dichlorophenyl, 2,5-dichlorophenyl, 2-bromophenyl, 
5-fluoro-2-methylphenyl, 4-fluoro-2-methylphenyl, 4-choro-2-methylphenyl, 
or 3-chloro-2-methylphenyl; and M is an alkali metal, an alkaline-earth 
metal, an ammonium or substituted ammonium cation, wherein the compound 
decomposes under physiological conditions to release nitric oxide. A 
further preferred embodiment is where R is 2-chlorophenyl or 
2-methoxyphenyl. 
Synthetic procedures for preparing compounds (1) include the steps of; 
reducing the corresponding nitro- or nitroso-compounds under conditions 
favoring the formation of N-aryl-hydroxylamines, followed by treatment 
with an alkyl nitrite and gaseous ammonia to obtain the corresponding 
ammonium salt, followed by ion exchange for preparing the alkali metal 
salts. Alternative methods for preparing compounds (1) involve (i) the 
reaction of aryl Grignard reagents with nitric oxide followed by treatment 
with ammonia or a reagent leading to the formation of alkali metal salt.; 
(ii) the spin trapping of nitric oxide with a nitroso derivative, yielding 
an isolable N-aryl-N-nitrosonitroxide, which can then be reduced to (1); 
(iii) the Sandmeyer reaction of aryldiazonium salts with nitric oxide (NO) 
in the presence of copper (II) and iron (II) salts, whereby an aryl 
radical reacts with NO leading to a nitroso derivative, followed by 
reaction with a second molecule of NO as in the preceding reaction. 
Nitric oxide (either an overabundance or deficiency) is involved in many 
pathological problems such as preterm labor, climacterium, 
pregnancy-induced diabetes, postpartum hemorrhage, coronary artery 
disease, cancer and behavioral and digestive problems. 
An embodiment of the present invention is a method of inhibiting uterine 
contractions in a subject comprising administering a therapeutically 
effective amount of a nitric oxide donor to the subject. A preferred 
nitric oxide donor is as described hereinabove. 
A method of supplying nitric oxide to a subject comprising administering to 
the subject a pharmacologically effective amount of a compound having the 
structure 
##STR3## 
is a further embodiment of the present invention. R is an 
ortho-substituted aryl, a heteroaromatic substituent, asteroid, or a 
catecholamine; and M is an alkali metal, an alkaline-earth metal, an 
ammonium or substituted ammonium cation, wherein the compound decomposes 
under physiological conditions to release nitric oxide. Preferred 
compounds for this method are as herein described. In addition, compounds 
for this method include those where R is a phenyl or naphthyl group with 
an ortho or other substituent sufficiently bulky to force the NONO group 
out of coplanarity with the phenyl or naphthyl ring substituent. 
Indications for NO compounds: 
Primary Indications: 
(a) uterine contractility disorders including dysmenorrhea, preterm labor 
and cervical incompetence 
(b) preeclampsia 
(c) hormone replacement therapy in women and men (i.e., estrogen and/or 
progesterone treatment for women, testosterone for men--used in elderly 
patients deficient in these hormones) NO donors could be used to replace 
estrogen and/or progesterone therapy in women and to replace testosterone 
in men. NO donors could also be used in combination with either estrogen 
and/or progesterone in women and in combination with testosterone in men. 
(d) cardiovascular disease--including hypertension and atherosclerosis 
Secondary (Potential) Indications: 
(e) behavior 
(f) ovulation and implantation--contraception 
(g) induction of labor by softening the cervix 
(h) blood clotting by inhibiting coagulation 
(i) impotence 
(j) infections 
(k) topical applications to improve wound healing, skin texture and hair 
growth 
(l) lung function to dilate bronchioles 
(m) cancer 
In one aspect, this invention relates to a method of administering 
N-nitroso-N-substituted hydroxylamine salt derivatives as nitric oxide 
donors for the purposes of regulating a variety of biological functions, 
especially inducing smooth muscle relaxation. The amount of the nitric 
oxide donor being equivalent to that amount required to lower blood 
pressure about 10 to 50 mm Hg pressure. Usually, treatment is continued 
indefinitely or until otherwise prescribed.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention provides N-aryl-substituted N-nitrosohydroxylamine 
salts based on the compound 1 (C1) as having superior ability to act as 
nitric oxide donors. 
##STR4## 
Table 2 includes a listing of these nitric oxide donors synthesized by the 
present inventors. They were characterized, inter alia, by infrared 
spectra, .sup.1 H-NMR and .sup.13 C-NMR spectra. 
TABLE 2 
______________________________________ 
Compounds synthesized having superior ability 
to act as donors of nitric oxide in vitro and in vivo 
______________________________________ 
N-Nitroso-N-(1-naphthyl)-hydroxylamine, ammonium salt (neocupferron) 
Idem, sodium salt (via ionic exchange, from the preceding compound). 
N-Nitroso-N-(2-methylphenyl)-hydroxylamine, salt 
N-Nitroso-N-(2-methoxyphenyl)-hydroxylamine, salt 
N-Nitroso-N-(2-ethylphenyl)-hydroxylamine, salt 
N-Nitroso-N-(2-isopropylphenyl)-hydroxylamine, salt 
N-Nitroso-N-(2,4-difluorophenyl)-hydroxylamine, salt 
N-Nitroso-N-(2,5-difluorophenyl)-hydroxylamine, salt 
N-Nitroso-N-(2-chlorophenyl)-hydroxylamine, salt 
N-Nitroso-N-(2,3-dichlorophenyl)-hydroxylamine, salt 
N-Nitroso-N-(2,4-dichlorophenyl)-hydroxylamine, salt 
N-Nitroso-N-(2,5-dichloropheny1)-hydroxylamine, salt 
N-Nitroso-N-(2-bromophenyl)-hydroxylamine, salt 
N-Nitroso-N-(5-fluoro-2-methylphenyl)-hydroxylamine, salt 
N-Nitroso-N-(4-fluoro-2-methylphenyl)-hydroxylamine, salt 
N-Nitroso-N-(4-choro-2-methylphenyl)-hydroxylamine, salt 
N-Nitroso-N-(3-choro-2-methylphenyl)-hydroxylamine, salt 
______________________________________ 
Many earlier NO donors involve compounds in which the nitric 
oxide-releasing moiety is attached either to an oxygen or to a nitrogen 
atom. By contrast, the N-aryl-N-nitrosohydroxylamine salts of the present 
invention, as represented by (1) have this moiety attached to a carbon 
atom. The present inventors show that these compounds also exhibit NO 
releasing ability and significant biological effects. This NO releasing 
ability can be adjusted by varying the nature of the aryl group and its 
substituents. 
A further advantage of the nitric oxide donor of the present invention is 
the fact that after release of NO, the by product is selected to be not 
carcinogenic. Structural variation permits fine tuning of the 
dose-response and releasing kinetics. Such variation also allows the 
attachment of the NO releasing moiety to biologically active groups which 
target specific organs or cells. Finally, the aryl group may interact with 
cell membranes, when adequately substituted, allowing control of 
penetration through biological barriers (e.g., blood brain barrier or 
placental barrier). 
In order to obtain these new nitric oxide donors with higher stability and 
possibly no carcinogenic effect from secondary amines, the present 
inventors use: (i) cyclic secondary amines with steric shielding around 
the nitrogen atom, such as 2,2,6,6-tetramethylpiperidine or 
2,2,5,5-tetramethylpyrrolidine; (ii) proline, hydroxyproline or their 
esters (either the natural L-isomer or the non-natural D-stereoisomer); in 
this case, the functionalized aminoacid carboxyl group may provide 
additional bioactive functions, as well as potential variability for 
introducing lipophilic esterified alcohol groups; (iii) diethanolamine or 
its intramolecular dehydration product (morpholine), which confers 
hydrophilic properties; (iv) bicyclic secondary amines such as 
3-azabicyclo 3.2.2! nonane, whose NONO adducts would have the property 
that any disproportionations after splitting NO are forbidden by Bredt's 
rule (this rule states that double bonds involving bridgehead atoms are 
energetically unfavorable). 
High levels of steroid hormones (mainly progesterone) during pregnancy 
appear to modulate either the production or action of nitric oxide. If 
nitric oxide is a transduction mechanism of steroid hormones, nitric oxide 
is expected to regulate other estrogen--and/or progesterone--dependent 
steps in reproduction and women's health, including ovulation, 
implantation, menstruation, climacterium, etc. In addition, some of the 
actions of the antihormones (e.g., antiprogestins) appear to be mediated 
through nitric oxide. 
Based upon these considerations, nitric oxide donors are uterine relaxants 
and nitric oxide inhibitors will increase uterine contractility. In 
addition, nitric oxide inhibition substantially improves the action of 
antiprogesterone compounds to induce premature birth in rats and the same 
compounds alone induce premature birth in guinea pigs. 
The existing data strongly indicate that the chronic steroid (estrogen 
and/or progesterone) effects on the blood vessels are mediated by nitric 
oxide. Inhibition of nitric oxide synthesis produces both atherosclerosis 
and osteoporosis in animal models (Moncada, et al., 1991). On the other 
hand, nitric oxide exhibits no direct effects on the endometrium in terms 
of proliferation and differentiation. Therefore, it will be possible for a 
suitable nitric oxide donor to replace steroids for hormone replacement 
therapy (HRT) in women. With this innovative strategy the major problems 
of HRT: endometrial hyperplasia and uterine bleeding can be avoided. The 
NO donors may be used, therefore, to prevent atherosclerosis and bone loss 
without inducing bleeding, (so called "no blood sector in HRT"). In 
addition, a suitable nitric oxide donor can by used for HRT, since these 
compounds do not exert hormone activities. 
The pharmacologically active nitric oxide donors employed in this invention 
can be administered in admixture with conventional excipients, i.e., 
pharmaceutically acceptable liquid, semi-liquid or solid organic or 
inorganic carriers suitable, e.g., for parenteral or enteral application 
and which do not deleteriously react with the active compound in admixture 
therewith. Suitable pharmaceutically acceptable carriers include but are 
not limited to water, salt solutions, alcohols, vegetable oils, 
polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, 
silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and 
diglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, 
polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be 
sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, 
preservatives, stabilizers, wetting agents, emulsifiers, slats for 
influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic 
substances and the like which do not deleteriously react with the active 
compounds. 
For parenteral application, particularly suitable are solutions, preferably 
oily or aqueous solutions, as well as suspensions, emulsions, or implants, 
intrauterine devices and suppositories. Ampoules are convenient unit 
dosages. In a preferred aspect, the composition of this invention is 
adapted for ingestion. 
For enteral application, particularly suitable are unit dosage forms, e.g., 
tablets, dragees or capsules having talc and/or a carbohydrate carrier or 
binder or the like, the carrier preferably being lactose and/or corn 
starch and/or potato starch; particulate solids, e.g., granules; and 
liquids and semi-liquids, e.g., syrups and elixirs or the like, wherein a 
sweetened vehicle is employed. Sustained release compositions can be 
formulated including those wherein the active compound is protected with 
differentially degradable coatings, e.g., by microencapsulation, multiple 
coating, etc. 
Suitable for oral administration are, inter alia, tablets, dragees, 
capsules, pills, granules, suspensions, and solutions. The active compound 
is also suitable for transdermal patches and topical application. Each 
unit dose, i.e., each tablespoon of liquid or each tablet, or dragee 
contains, for example, 5-5000 mg of each active agent. Solutions for 
parenteral administration contain, for example, 0.01-1% of each active 
agent in an aqueous or alcoholic solution. The agents or combination can 
be administered as an admixture with any other optional active agent or as 
a separate unit dosage form, either simultaneously therewith or at 
different times during the day from each other. 
The combination of active agents is preferably administered at least once 
daily (unless administered in a dosage form delivering the active agents 
continuously or sequentially). The typical dose is about 0.5 to 1000 mg of 
each active agent, although some less active agents may require much 
higher oral dosages, et., 500 to 10,000 mg, and others may require lower 
doses, e.g., 500-2,000 .mu.g/kg/day. Doses for nitroglycerine typically 
are orally 2.5 mg 2.times. daily; sublingually, 0.8 mg 1-4.times. daily; 
and transdermally, 0.2-0.4 mg/hr. Since the LD.sub.50 dosages of most of 
these active agents are known, a lower dosage regimen can be initiated and 
the dosage increased until a positive effect is achieved or a higher 
dosage regimen can initially be employed, e.g., in a crises situation, and 
the dosages regulated downward until the desired effect is achieved. 
The following examples are included to demonstrate preferred embodiments of 
the invention. It should be appreciated by those of skill in the art that 
the techniques disclosed in these examples represent techniques discovered 
by the inventors to function well in the practice of the invention, and 
thus can be considered to constitute sundry modes of practice. However, 
those of skill in the art should, in light of the present disclosure, 
appreciate that many changes can be made in the specific disclosed 
embodiments to obtain a like or similar result without departing from the 
spirit and scope of the invention. 
EXAMPLE 1 
Synthesis of N-nitroso-N-aryl-substituted Hydroxylamine Derivatives 
The synthetic approach for synthesizing the compounds of Table 2 consists 
in reducing an aromatic or heteroaromatic nitro or nitroso derivative to 
the corresponding hydroxylamine, either electrochemically, or with zinc 
powder and ammonium chloride in water or aqueous lower alcohols. The 
hydroxylamine is extracted with ethyl ether or another non-polar solvent 
and, after drying, is converted into the crystalline cupferron analog by 
treatment with gaseous ammonia and an alkyl nitrite. Finally, by means of 
an ion exchange column, an alkali metal or other desired cation may 
replace the ammonium cation. 
This method, starting with nitro derivatives, was used for preparing a 
variety of compounds 1, where the R group is phenyl, or phenyl with the 
following substituents: 2-methyl; 2,3-dimethyl; 2-ethyl; 2-methoxy; 
2-hydroxy; 2-fluoro; 2-chloro; 2,4-dichloro, 2,5-dichloro; 
4-chloro-2-methyl; 2-acetyl; 2-bromo; .alpha.-naphthyl, 
2-methyl-1-naphthyl, 2-hydroxy-1-naphthyl, or 1-hydroxy-2-naphthyl. 
The preparation of neo-cupferron from .alpha.-nitronaphthylene, ammonia and 
hydrogen sulfide in ethanol, followed by treatment with butyl nitrite and 
ammonia in ethyl ether, according to O. Baudisch, was described by Smith 
(1938). 
Alternative methods for obtaining compounds 1 or cupferron analogs 
(ammonium salts) are available: (1) the reaction of aryl Grignard reagents 
with nitric oxide followed by treatment with ammonia (Sand et al., 1903); 
(ii) the spin trapping of nitric oxide with a nitroso derivative, yielding 
an isolable N-aryl-N-nitrosonitroxide, which can then be reduced to 1 
(Balaban et al., 1971, 1972, 1973, 1987); in a later publication, it was 
reported that nitrosobenzene and nitric oxide afforded cupferron in the 
presence of ammonia, and that the yield was increased when a reducing 
agent such as hydroquinol was added (Iida et al., 1978); (iii) the 
Sandmeyer reaction of aryldiazonium salts with nitric oxide in the 
presence of copper(II) and iron(II) salts, whereby an aryl radical reacts 
with NO leading to a nitroso derivative, followed by reaction with a 
second molecule of NO as in the preceding reaction (Minisci et al., 1964). 
When the substituent is electron-donating (e.g methoxy, ethoxy, hydroxy, 
dimethylamino or diethylamino) the hydroxylamine and its salts are 
sensitive to air oxidation which converts them into deeply colored 
products (azo or azoxy derivatives), therefore they must be processed 
rapidly at lower temperatures, under inert atmosphere, and kept in the 
freezer. Such compounds with dialkylamino substituents may be considered 
as phenyl analogs of nitric oxide donors (Drago's NONOates) prepared from 
nitric oxide and secondary amines (Drago et al., 1961, 1962; Ragsdale et 
al., 1965; Longhi et al., 1962; Hansen et aI. 1982). With other 
substituents, the dry crystalline products are stable at room temperature 
and can be kept at temperatures below 0.degree. C. 
Methods. The nitro derivative (0.1 mole) is stirred in an aqueous solution 
of ammonium chloride (0.1 mole). When the nitro derivative is solid with a 
melting point above 85.degree. C. or has a very low water solubility, 50% 
percent aqueous ethanol may be used, and the initial temperature is raised 
to 60.degree.-70.degree.Zinc powder (0.2 mole) is added gradually under 
vigorous mechanical stirring so as to maintain the temperature around 
70.degree. C. due to the exothermicity of the reaction. After 60-90 
minutes, the mixture is cooled below 35.degree. C. and filtered with 
suction. The solid residue is thoroughly washed with 3-4 portions of 
diethyl ether; this ether is used for extracting the filtrate each time 
(Kamm and Marvel, 1941). 
The combined ethereal extracts are dried over sodium sulfate, and cooled 
under 0.degree. C. in an ice-salt mixture. A vigorous stream of gaseous 
ammonia is bubbled into the ethereal solution, and after 5-10 minutes, 
n-butyl nitrite is added in small portions during 15 minutes maintaining 
the cooling and the stream of NH.sub.3. The cupferron analog (ammonium 
salt corresponding to 1) precipitates. If it separates as a liquid, one 
induces crystallization by scratching with a glass rod. The product is 
filtered off after being kept at 0.degree. C. for 1-2 hrs, and washed 
thoroughly with diethyl ether (Marvel and Kamm, 1941). 
A column packed with cationite which has been soaked in aqueous sodium 
hydrogen carbonate for 24 hrs and then rinsed with distilled water is used 
for exchanging ammonium with sodium cations: a concentrated solution of 
the cupferron analog in water or 50% aqueous ethanol is passed through the 
column; elution is performed with the same solvent, monitoring the UV 
absorption at 280 nm. The eluates are combined and the solvent is removed 
by using a rotary evaporator under vacuum at 30.degree.-40.degree. C. or 
freeze drying techniques for heat-sensitive compounds. Overall yields vary 
between 20 and 85%. 
The most preferred NO donors related to cupferron have either 
ortho-substituents (e.g., 2-chloro and 2-methoxy), or have types of bulky 
groups forcing the NONO group out of coplanarity with the aromatic ring: 
examples thereof include neocupferron analogs 
(N-nitroso-.alpha.-naphthylhydroxylamine salts), and substituted 
heterocyclic analogs. 
During the preparation of such sterically hindered analogs of cupferron it 
was observed that the precipitation of ammonia salts on introducing 
gaseous ammonia and alkyl nitrite into the ethereal solution of the 
N-arylhydroxylamine occurs more slowly than in the absence of bulky 
ortho-substituents; therefore one has to filter off the products only 
after keeping the solution at 0.degree. for 4-12 hours. 
EXAMPLE 2 
Biological Effects of N-nitroso-N-substituted Hydroxylamines 
FIGS. 1 and 2 show release of nitric oxide from various derivatives of 
N-nitroso-N-substituted hydroxylamine as tested in vitro in comparison to 
DEA-NO and Na nitroprusside, two other nitric oxide donor compounds. These 
results show that some of the compounds slowly release NO while others 
quickly release it. 
Several N-nitroso-N-arylhydroxylamine derivatives were tested on 
contractions of uterine strips from rats and humans in vitro. The 
derivative produced substantial relaxation responses in a dose-dependent 
manner consistent with the evidence that the compound is a nitric oxide 
donor (FIGS. 3, 4 and 5). The response was quickly reversible following 
washing. 
The effects of the derivatives were also compared to DETA/NO, a known 
nitric oxide donor compound. The N-nitroso-N-arylhydroxylamine compounds 
were more potent than DETA/NO on the rat uterus but it was less potent on 
the human uterus (FIGS. 3, 4 and 5). However, the duration of action of 
the derivative was long with washing required to terminate an inhibitory 
action. In these experiments uterine strips (about 1 mm.times.0.5 
mm.times.15 mm) (FIGS. 3 to 5) from rats (FIG. 3) or humans (FIG. 4) were 
dissected from the whole uterine and the contraction studies i muscle 
baths. Each upward deflection in the figures (FIGS. 3 to 5) represents a 
contraction and downward movements are equal to relaxation phases. 
Addition of agents to the muscle baths are indicated by vertical lines. 
The compound was tested on intact animals to determine if the derivative 
inhibited uterine contractions. A catheter was placed in the uterine 
cavity to measure pressure. FIG. 6 shows the inhibitory responses on 
pressure signals from the uterus recorded from pregnant rats at day 20 
gestation. Rats were injected with the cupferron derivative (1 to 5 mg IP) 
and it produced a dramatic fall in intrauterine pressure suggesting an 
inhibition of uterine contractility. Similarly, all rats demonstrated 
signs of vasodilation and bronchodilation. 
A human female suffering from conditions of a nitric oxide deficiency, such 
as hypertension, preeclampsia, or uterine hypercontractility could be 
treated with N-nitroso-N-arylkydroxylamine compounds described herein to 
relieve such disorders. 
All of the compositions and methods disclosed and claimed herein can be 
made and executed without undue experimentation in light of the present 
disclosure. While the compositions and methods of this invention have been 
described in terms of preferred embodiments, it will be apparent to those 
of skill in the art that variations may be applied to the composition, 
methods and in the steps or in the sequence of steps of the method 
described herein without departing from the concept, spirit and scope of 
the invention. More specifically, it will be apparent that certain agents 
which are both chemically and physiologically related may be substituted 
for the agents described herein while the same or similar results would be 
achieved. All such similar substitutes and modifications apparent to those 
skilled in the art are deemed to be within the spirit, scope and concept 
of the invention as defined by the appended claims. 
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