Substituted guanidines having high binding to the sigma receptor and the use thereof

The invention relates to a method for the treatment or prophylaxis of psychosis, depression, hypertension, or anxiety in an animal by administering an effective amount of an N,N'-disubstituted guanidine or 2-iminoimadazolidine having an affinity for the sigma receptor. The invention also relates to the novel guanidines of the invention as well as pharmaceutical compositions thereof.

FIELD OF THE INVENTION 
The invention is in the field of medicinal chemistry. In particular, the 
invention relates to N,N'-disubstituted guanidines and N,N'-disubstituted 
2-iminoimidazolidines which have high binding to the sigma receptor, 
pharmaceutical compositions thereof, and methods for treating or 
preventing psychotic mental illness, depression, hypertension, and anxiety 
in animals. 
BACKGROUND OF THE INVENTION 
Recently, the inventors have described a series of di-arylguanidines which 
are potent ligands for brain sigma receptors (Weber, et al., PNAS (USA) 
83:8784-8788 (1986); Campbell et al., J. Neurosci. 9:3380-3391 (1989); 
U.S. Pat. No. 4,709,094). Brain sigma receptors bind many psychotropic 
drugs (Sonders et al., Trends Neurosci. 11:37-40 (1988)). The 
physiological function of sigma receptors in the nervous system is subject 
to intense investigations (Sonders et al., Trends Neurosci. 11:37-40 
(1988)) because certain sigma receptor selective compounds have known 
antipsychotic activity suggesting that sigma receptor active compounds can 
be used for the treatment of schizophrenia (Largent et al., Eur. J. 
Pharmacol., 11:345-347 (1988)). 
A wide variety of substituted guanidines are disclosed in the patent 
literature. For example: 
U.S. Pat. Nos. 1,411,731 and 1,422,506 discloses diphenylguanidine as a 
rubber accelerator; 
U.S. Pat. No. 1,597,233 discloses N-o-tolyl-N'-phenyl-guanidine as a rubber 
accelerator; 
U.S. Pat. No. 1,672,431 discloses N,N'-di-o-methoxyphenyl-guanidine as 
being useful for therapeutic purposes, especially in the form of 
water-soluble salts; 
U.S. Pat. No. 1,730,338 discloses 
N-p-dimethyl-amino-phenyl-N'-phenylguanidine as a rubber accelerator; 
U.S. Pat. No. 1,795,738 discloses a process for the production of 
N,N'-dialkyl-di-substituted guanidines, including 
N-di-ethyl-N'-phenyl-guanidine, N-diethyl-N-isoamylguanidine, 
N-dimethyl-N'-isoamylguanidine and N-dimethyl-N'-ethylguanidine; 
U.S. Pat. No. 1,850,682 discloses a process for the preparation of 
disubstituted guanidine rubber accelerators bearing an additional 
substituent on the imine nitrogen atom; 
U.S. Pat. No. 2,145,214 discloses the use of disubstituted guanidines, 
e.g., diarylguanidines especially dixylylguanidine, as parasiticides; 
U.S. Pat. No. 2,254,009 discloses sym-di-2-octyl-guanidine and U.S. Pat. 
Nos. 2,274,476 and 2,289,542 disclose sym-dicyclohexylguanidine as 
insecticides and moth larvae repellents; 
U.S. Pat. No. 2,633,474 discloses 1,3-bis(o-ethylphenyl)guanidine and 
1,3-bis(p-ethylphenyl)guanidine as rubber accelerators; 
U.S. Pat. No. 3,117,994 discloses N,N',N"-trisubstituted guanidines and 
their salts as bacteriostatic compounds; 
U.S. Pat. No. 3,140,231 discloses N-methyl- and N-ethyl-N'-octylguanidines 
and their salts as antihypertensive agents; 
U.S. Pat. No. 3,248,246 describes (Example 5) a 1,3-disubstituted guanidine 
whose substituents are hydrophobic hydrocarbon groups, one of which is 
naphthylmethyl and the other is n-butyl; 
U.S. Pat. No. 3,252,816 discloses various N-substituted and unsubstituted 
cinnamyl-guanidines and generically the corresponding N'- and N"-alkyl 
substituted compounds and their salts as antihypertensive agents; 
U.S. Pat. No. 3,270,054 discloses N-2-adamant-1-yl- and 
N-2-homoadamant-1-yl-oxy-ethyl-thioethyl- and aminoethyl-guanidine 
derivatives bearing at most two lower alkyl groups on the N'- and/or 
N"-nitrogen atom as sympathicolytic and anti-viral agents; 
U.S. Pat. No. 3,301,755 discloses N-ethylenically 
unsubstituted-alkyl-guanidines and the corresponding N'- and/or N"-lower 
alkyl compounds as hypoglycemic and antihypertensive agents; 
U.S. Pat. No. 3,409,669 discloses 
N-cyclohexylamino-(3,3-dialkyl-substituted-propyl)-guanidines and the 
corresponding N'-alkyl- and/or N"-alkyl-substituted compounds as 
hypotensive agents; 
U.S. Pat. No. 3,547,951 discloses 1,3-dioxolan-4-yl-alkyl-substituted 
guanidines which have anti-hypertensive activity and discloses lower 
alkyl, including n-butyl, as a possible substituent on the other amino 
group; 
U.S. Pat. No. 3,639,477 discloses propoxylguanidine compounds as having 
anorectic properties; 
U.S. Pat. Nos. 3,681,459; 3,769,427; 3,803,324; 3,908,013; 3,976,787; and 
4,014,934 disclose aromatic substituted guanidine derivatives wherein the 
phenyl ring can contain hydroxy and/or halogen substituents for use in 
vasoconstrictive therapy; 
U.S. Pat. No. 3,804,898 discloses N-benzylcyclobutenyl and 
N-benzylcyclo-butenyl-alkyl-guanidines and the corresponding N-alkyl 
and/or N"-alkyl-substituted compounds as hypotensive agents; 
U.S. Pat. No. 3,968,243 discloses N-axalkyl substituted guanidines and the 
corresponding N'-alkyl-n"alkyl and N',N'-aralkyl compounds as being useful 
in the treatment of cardiac arrhythmias; 
U.S. Pat. No. 3,795,533 discloses o-halo-benzylidene-amino-guanidines and 
their use as anti-depressants for overcoming psychic depression; 
U.S. Pat. No. 4,007,181 discloses various N,N'-disubstituted guanidines 
substituted on the imine nitrogen atom by adamantyl as possessing 
antiarrhythmic and diuretic activities; 
U.S. Pat. No. 4,051,256 discloses N-phenyl- and 
N-pyridyl-N'-cycloalkylguanidines as antiviral agents; 
U.S. Pat. Nos. 4,052,455 and 4,130,663 disclose styrylamidines, as 
analgesics agents or for the prevention of blood platelet aggregation; 
U.S. Pat. No. 4,109,014 discloses N-hydroxysubstituted guanidines and the 
corresponding N-methyl disubstituted guanidines as vasoconstrictor agents; 
U.S. Pat. No. 4,169,154 discloses the use of guanidines in the treatment of 
depression; 
U.S. Pat. No. 4,393,007 discloses N-substituted and unsubstituted, 
N-substituted methyl-N'-unsubstituted, monosubstituted and 
disubstituted-N"-unsubstituted and substituted guanidines as ganglionic 
blocking agents; and 
U.S. Pat. No. 4,471,137 discloses N,N,N'N"-tetraalkyl guanidines as being 
sterically hindered bases useful in chemical synthesis. 
U.S. Pat. No. 4,709,094 discloses 1,3-disubstituted-guanidines, e.g., 
1-3-dibutylguanidine and 1,3 di-o-tolyl-quinidine, as sigma brain receptor 
ligands. 
For examples of other substituted guanidines, see, e.g., U.S. Pat. Nos. 
1,422,506; 1,642,180; 1,756,315; 3,159,676; 3,228,975; 3,248,426; 
3,283,003; 3,320,229; 3,479,437; 3,547,951; 3,639,477; 3,784,643; 
3,949,089; 3,975,533; 4,060,640 and 4,161,541. 
Geluk, H. W., et al., J. Med. Chem., 12, 712 (1969) describe the synthesis 
of a variety of adamantyl disubstituted guanidines as possible antiviral 
agents, including N,N'-di-(adamantan-1-yl)-guanidine hydrochloride, 
N-(adamantan-1-yl)-N'-cyclohexyl-guanidine hydrochloride and 
N-(adamantan-1-yl)-N'-benzyl-guanidine hydrochloride. 
U.S. Pat. No. 4,709,094 (1987), discloses N,N'-disubstituted guanidine 
derivatives which exhibit high binding activity with respect to the sigma 
receptor having the Formula (I): 
##STR1## 
wherein R and R' are an alkyl group of at least 4 carbon atoms, a 
cycloalkyl group of 3-12 carbon atoms, or carbocyclic or aryl, of at least 
6 carbon atoms. 
Two of the novel N,N'-disubstituted guanidines disclosed therein are also 
claimed therein viz., 1,3-di-(4-halo-2-methylphenyl)-guanidine and 
1,3-di-(4-.sup.3 H]-(2-methylphenyl)-guanidine. 
Also claimed therein is a method of determining the relationship of 
abnormal psychotic-like behavior in a mammal displaying such behavior to 
sigma receptor system dysfunction, which comprises administering to the 
mammal displaying such behavior a water-soluble 
N,N'-disubstituted-guanidine which displaces in vitro N,N'-di-(4-[.sup.3 
H]-2-methylphenyl)-guanidine bound to mammalian brain membrane, in an 
amount effective to alter the sigma brain receptor-modulated mental 
activity of the mammal; a method of treating a human being suffering from 
a psychotic mental illness associated with hallucinations, which comprises 
administering thereto a water-soluble N,N'-disubstituted guanidine which 
is an antagonist to the sigma receptor binding activity of a 
hallucinogenic benzomorphan, in an amount effective to ameliorate the 
hallucinations. 
In U.S. Pat. No. 4,709,094 is further disclosed a method of determining the 
sigma brain receptor binding activity of an organic compound which 
comprises the steps of a) contacting in an aqueous medium a known mount of 
isolated mammalian brain membrane which has sigma receptor-like binding 
activity, with a mixture of (i) a tritium labeled N,N'-disubstituted 
guanidine which selectively binds sigma brain receptors, in a known mount 
capable of being bound to the sigma receptors of that brain membrane; and 
(ii) varying known amounts of a water soluble organic compound to be 
assayed for sigma receptor binding activity; b) separating the brain 
membrane from the tritium labeled compound which is not bound to the brain 
membrane in step a); and c) determining, from the molar relationship of 
the proportion of bound tritium-labeled compound which is separated in 
step b) to the molar amount of the organic compound employed in step a), 
the sigma receptor binding activity of that organic compound. 
Certain benzomorphan opiates, such as N-allyl-normetazocine (SKF 10,047) 
and cyclazocine, in addition to analgesia, cause hallucinations, 
depersonalization, drunkenness and other psychotomimetic effects in man. 
In monkeys, dogs and rodents the psychotomimetic opiates cause behavioral 
and autonomic effects that are unlike those observed with administration 
of classical opiates such as morphine or the opioid peptides. Specific 
sigma "opioid" receptors in the brain are believed to mediate such 
atypical effects. Martin et al., J. Pharmacol. Exp. Ther. 197:517-532 
(1976). It is believed that the sigma receptors also mediate some of the 
psychotomimetic effects of phencyclidine [PCP, angel dust], or 
alternatively, that psychotomimetic opiates act at specific PCP receptors. 
Zukin, R. S., et al., Mol. Pharmacol. 20:246-254 (1981); Shannon, H. E., 
J. Pharmacol. Exp Ther. 225:144-152 (1983); White, J. M., et al., 
Psycho-pharmacology 80:1-9 (1983); and Zukin et al., J. Neurochem. 
46:1032-1041 (1986). PCP is a drug of abuse that causes a behavioral 
syndrome in man similar to that which is observed in schizophrenic 
psychosis. Aniline, O., et al., CRC Critical Rev. Toxicol. 10:145-177 
(1982). Because of the potent psychotomimetic effects of sigma opiates and 
PCP, it is believed that sigma (and/or PCP) receptors play a role in 
mental illness, particularly schizophrenia. 
A systematic investigation of the role of sigma receptors in normal and 
abnormal brain function has been hindered by a lack of specific sigma 
receptor binding assays and bioassays. Development of such specific assays 
requires well-characterized, highly selective and potent sigma receptor 
ligands. Recent studies have shown that brain membrane receptors can be 
labeled in vitro with (.+-.)[.sup.3 H]SKF 10,047, Su, T. P., J. Pharmacol. 
Exp. Ther. 223:284-290 (1982); (+)[.sup.3 H]SKF 10,047, Tam, S. W., et 
al., Proc. Natl. Acad. Sci. U.S.A. 81:5618-5621 (1984); Martin et al., J. 
Pharmacol. Exp. Ther. 231:539-544 (1984); and Mickelson, M. M., et al., 
Res. Commun. Chem. Pathol. Pharmacol. 47:255-263 (1985), although not 
selectively, Gundlach et al., Eur. J. Pharmacol. 113:465-466 (1985); and 
Largent, B. L., et al., J. Pharmacol. Exp. Ther. 238:739-748 (1986), and 
with (+)[.sup.3 H]3-(3-hydroxyphenyl)-N-(1-propyl)-piperidine ((+)[.sup.3 
H]3-PPP), Largent et al., Proc. Natl. Acad. Sci. U.S.A. 81:4983-4987 
(1984), which is apparently more selective for sigma receptors than the 
others. 
After the initial in vitro studies by Martin et al., (1976) supra, Keats 
and Telford (Keats, A. S., et al., "Analgesics: Clinical Aspects." In 
Molecular Modification in Drug Design, R. F. Gould (ed.), Advances in 
Chemistry Series #45 Amer. Chem. Soc., Wash. D.C. (1964)), and Haertzen 
(Haertzen, C. A. Cyclazocine and Nalorphine on the Addiction Research 
Center Inventory (ARCI), Psychopharmacologia (Berl.) 18:355-377 (1970)), 
numerous investigators set out to biochemically characterize the different 
opiate receptors (mu receptors, kappa receptors and sigma receptors) in 
vitro. 
The first evidence for the existence of a separate sigma receptor in test 
tube experiments was provided by Su (1982) supra in a paper describing an 
etorphine-inaccessible binding site in guinea pig brain membranes which 
was apparently selectively labeled by tritium-labeled SKF-10,047. To 
overcome the fact that SKF10,047 could label multiple opioid receptors in 
the brain, Su performed his receptor binding assay using tritium labeled 
SKF-10,047 in the presence of excess unlabeled etorphine. Etorphine is a 
very strong opiate agonist drug which is known to bind to delta receptors, 
mu receptors and kappa receptors with almost equal potency. Su used 
etorphine to saturate all mu, kappa and delta receptors in a brain 
membrane preparation and then added tritium labeled SKF-10,047. This 
enabled him to detect a sigma binding site that was apparently different 
from mu, kappa and delta receptors. 
A major breakthrough in identifying the sigma receptor as a separate entity 
occurred when Tam et al. (1984), supra, demonstrated that the previous 
problems in selectively labeling the sigma receptor were caused by the 
fact that in all previous experiments a racemic SKF-10,047 preparation was 
used. Tam showed that using a tritium labeled (+)-SKF-10,047 isomer one 
could selectively label a sigma receptor that was different from the mu, 
delta and kappa opioid receptors. On the other hand, Tam showed that 
(-)-SKF-10,047 apparently labeled the mu and kappa receptors but not the 
sigma receptors. Tam, S. W., Eur. J. Pharm. 109:33-41 (1985). This finding 
has now been confirmed. (Martin et al., 1984, supra). Moreover, there is 
evidence from behavioral experiments, Khazan et al., Neuropharm. 
23:983-987 (1984); Brady et al., Science 215:178-180 (1981), that it is 
the (+)-SKF-10,047 isomer that is solely responsible for the 
psychotomimetic effects of SKF-10,047. 
One of the most important findings of the biochemical characterization of 
the sigma receptor has been that this receptor binds all synthetic opiate 
drugs that are known to have hallucinogenic and psychotomimetic effects. 
Opiates that do not have psychotomimetic effects in vivo do not bind to 
this receptor. Most importantly, it has been shown that besides 
hallucinogenic opiate drugs, the sigma receptor also binds many 
antipsychotic drugs that are used clinically to treat hallucinations in 
schizophrenic patients. (Tam and Cook, 1984). The initial observations 
with regard to antipsychotic drug binding to the sigma receptor (Su, 1982) 
were subsequently extensively confirmed and extended by Tam et al. (1984), 
supra, also showed that when one used radioactively labeled haloperidol, 
one of the most potent antipsychotic drugs that is used clinically, about 
half of the binding sites in brain membrane preparations are actually 
sigma receptors whereas the other half of the binding sites are apparently 
dopamine receptors. It has long been known that most antipsychotic drugs 
are also dopamine receptor antagonists. Previously the beneficial actions 
of antipsychotic drugs in psychotic patients have been attributed to the 
dopamine receptor-blocking effect of these drugs. It is clear from the 
work by Tam, however, that numerous clinically used antipsychotic drugs 
also bind to the sigma site. All antipsychotic drugs that bind to the 
sigma receptor may in part cause the beneficial effect of alleviating 
hallucinations through the sigma receptor. Taken together all these 
observations suggest the sigma receptor as a prime candidate to be 
involved in the pathogenesis of mental illness, particularly schizophrenia 
in which hallucinations are a major clinical symptom. 
Deutsch, S. I., et al. (Clinical Neuropharmacology, Vol. 11, No. 2, pp. 
105-119 (1988)) provided a review of the literature which implicates the 
sigma receptor site in psychosis and anti-drug efficacy. According to 
Deutsch et al., certain benzomorphans which possess analgesic potency in 
humans are also associated with a high incidence of psychotomimetic 
effects. It has now been concluded that the analgesic action is associated 
with the levorotatory isomers of racemic mixtures of the benzomorphans, 
while the psychotomimetic effects are attributable to the dextrorotatory 
isomers in the racemic mixtures. See Haertzen, C. A., Psychopharmacologia 
18:366-77 (1970), and Manallack, D. T., et al., Pharmacol. Sci. 7:448-51 
(1986). Coupled with the fact that many of the in vivo effects of these 
dextrorotatory enantiomers and the binding of dextrorotatory tritiated 
SKF-10,047 are not antagonized by naloxone or naltrexone, these data 
strongly support the concept that the psychotomimetic effects of the 
dextrorotatory enantiomers are associated with the sigma receptor binding 
site. 
Further, Su, T. P., et al. (Life Sci. 38:2199-210 (1986)), and Contreras, 
P. C., et al. (Synapse 1:57-61 (1987)), have established the existence of 
endogenous ligands for the sigma receptor, suggesting that the 
dysregulation of the synthesis, release, or degradation of these natural 
ligands may be a naturally occurring mechanism of psychosis. Accordingly, 
sigma receptor antagonism provides the potential for an effective 
antipsychotic therapeutic treatment. See Ferris, R. M., et al., Life Sci. 
38:2329-37 (1986), and Su, T. P., Neurosci. Let. 71:224-8 (1986). 
As further evidence of the role of the sigma receptor in psychosis, the 
substituted carbazole 
cis-9-[3-(3,5-dimethyl-1-piperazinyl)propyl]carbazole dihydrochloride 
(rimcazole) was identified as a potential antipsychotic agent based on its 
ability to antagonize apomorphine-induced mesolimbic behaviors selectively 
without altering the intensity of stereotypic behaviors. Further, the 
compound does not accelerate the rate of dopamine synthesis and does not 
affect dopamine-stimulated production of cAMP in homogenates of rat 
striatum and olfactory tubercle, thus establishing that rimcazole does not 
exert its action at the level of post-synaptic dopamine receptors in the 
mesolimbic area. 
Rimcazole is able to competitively inhibit the specific binding of 
dextrorotatory tritiated SKF-10,047, the prototype sigma receptor agonist, 
suggesting that rimcazole acts at the sigma receptor site. Rimcazole, 
therefore, shows potential antipsychotic activity in humans, without 
extrapyramidal effects, pharmacological behavior which is consistent with 
its role as a competitive antagonist of the sigma-receptor. 
Another compound, BMY 14802, has demonstrated many properties in 
preclinical behavioral tests which suggest its efficacy as a potential 
antipsychotic agent which is devoid of extrapyramidal side effects. The 
compound (1) did not cause catalepsy in rats; (2) does not inhibit the 
binding of [.sup.3 H]spiperone to the D.sub.2 class of striatal dopamine 
receptors in rats; (3) did not increase the maximal density of the [.sup.3 
H]spiperone-labeled D.sub.2 site in striatum even following chronic 
administration (20 days) to rats; (4) does not appear to interact with the 
D.sub.1 subclass of dopamine receptors; and (5) does not inhibit 
dopamine-stimulated cAMP production or the binding of [.sup.3 H]SCH 23390 
in vitro. These data suggest that BMY 14802 has a low potential for 
production of tardive dyskinesia and further suggests that the 
antipsychotic effects would be mediated by a nondopaminergic site. 
Further, BMY 14802 binds with relatively high affinity to the sigma 
receptor, with the binding being stereoselective (the dextrorotatory 
enantiomer being 10 times more potent at inhibiting binding than the 
levorotatory enantiomer). BMY 14802 does not bind to the adrenergic, 
muscarinic, cholinergic, or histaminergic sites, suggesting that the 
compound would not be associated with unpleasant sedative and autonomic 
side effects. 
Accordingly, compounds which bind selectively to the sigma receptor site 
and which antagonize this site may be expected to be useful antipsychotic 
drugs which are devoid of extrapyramidal effects. 
The antipsychotic and anti-schizophrenia drugs that are currently in use 
have very strong side effects that are mainly due to their action on 
dopamine receptors. The side effects often involve irreversible damage to 
the extrapyramidal nervous system which controls movement functions of the 
brain. Patients under long term antischizophrenic drug treatment often 
develop a syndrome that involves permanent damage of their ability to 
control coordinated movement. 
The foregoing studies have shown that the sigma binding site has the 
characteristics of 1) stereo-selectivity towards dextrorotatory 
benzomorphan opiates and insensitivity for naloxone; 2) high affinity for 
haloperidol and moderate to high affinity for phenothiazine antipsychotic 
drugs which are also known to be potent dopamine receptor blockers; and 3) 
insensitivity for dopamine and apomorphine. This intriguing drug 
selectivity profile calls for a thorough analysis of the role of sigma 
receptors in normal and abnormal brain function. In order to do so, it is 
essential that a spectrum of highly selective and potent sigma receptor 
active compounds be available. This invention provides such compounds and 
methods for identifying other drugs having such activity. 
Fear, or apprehension, is characterized by the anticipation of a known 
danger or event. In contrast, neurotic anxiety is characterized by an 
apprehension with no known cause, or a maladaptive response to a trivial 
danger. In recent years, generalized anxiety disorder (GAD) has been 
characterized by psychiatrists as being chronic (continually present for 
at least 1 month) and exemplified by three of four psychomotor symptoms: 
motor tension, autonomic hyperactivity, apprehensive expectation, 
vigilance and scanning. Before this characterization was adopted, clinical 
trials of anxiolytic agents in the U.S. occurred in patients which were 
described variously as suffering from anxiety neurosis, anxiety with 
associated depression, and other such terms. Anxiety disorders affect 2-3% 
of the general population (the 67 million prescriptions written in 1977 
for just two popular anxiolytics confirm this projected incidence). The 
popularity of anxiolytics attests to their ability to ameliorate the 
debilitating symptoms of the disease. Taylor, D. P., FASEB J. 2: 2445-2452 
(1988). 
Historically, anxiety has been treated by agents including alcohol opiates, 
and belladonna, which have a sedative component to their action. In the 
20th century novel chemical entities were discovered which are safer for 
the treatment of anxiety including barbiturates, propanediol carbamates, 
and benzodiazepines. The pharmacological profiles of these drugs have 
suggested that their actions are mediated by receptors for 
.gamma.-aminobutyric acid (GABA). Although the benzodiazepines present a 
safer alternative than meprobamate and phenobarbital, they also are 
sedatives. In addition, the benzodiazepines control convulsions and 
produce muscle relaxation, properties that are unneeded or undesirable in 
the treatment of anxiety. Furthermore, these drugs can interact with 
alcohol, with potentially disastrous consequences. Recently, it has been 
appreciated that the benzodiazepines produce habituation and possess a 
pronounced liability, for example, withdrawal symptoms after chronic use. 
The need for anxioselective drugs that are more selective, have fewer side 
effects, and present a profile consistent with safety during protracted 
treatment has resulted in a continuing search for such drugs. This search 
has led to the synthesis and evaluation of agents that possess no obvious 
homology with the benzodiazepines. Taylor et al., supra. 
Buspirone (Buspar) was the first novel anxiolytic to be approved for 
clinical use in the U.S. since the benzodiazepines were introduced almost 
30 years ago. The introduction of buspirone into clinical trials for the 
treatment of anxiety was a direct result of its efficacy in a predictive 
animal model for the disease--the taming of the aggressive response of 
rhesus monkeys to the introduction of foreign objects into their cages 
according to the protocol described by Tompkins, E. C. et al., Res Commun. 
Psychol. Psychiatry Behav. 5: 337-352 (1980). See Taylor et al., supra. 
The preclinical screening of putative anxiolytics is dependent upon animal 
tests. Most of the laboratory data on new putative anxiolytics come from 
animal tests form two main classes. The first group of tests are based on 
conflict or conditioned fear. The second group of tests are based upon 
anxiety generated by novel situations. Although these tests differ in the 
way anxiety is produced, there has been surprising agreement amongst them 
in the classification of drugs as anxiolytic or anxiogenic. See File, S. 
E., TINS 10:461-463 (1987). 
In two particular tests for anxiolytic activity, it is assumed that the 
anticipation of punishment causes a reduction in a response associated 
with the punishment. Conversely, anxiolytic agents that reduce anxiety 
result in an increased response rate. In the Geller-Seifter test, the rat 
receives food reward for pressing a lever, but also receives an electric 
footshock, which has the effect of suppressing the response. This punished 
schedule alternates with an unpunished schedule wherein electric 
footshocks are not administered. During this unpunished schedule, 
lever-pressing is still rewarded. In the Vogel test, a rat is allowed to 
drink water, but also receives an electric shock through the water spout 
or the bars of the floor. In both the Vogel and Geller-Seifter tests, a 
measure of unpunished response is obtained in order to allow assessment of 
any non-specific stimulant or sedative drug effects or any changes in food 
or water intake. In both of these tests, benzodiazepines enhance the 
response rate in the punished periods, without increasing the rate of 
response in the absence of shock. While these tests are valid tests of 
anxiety, the only means of assessing them has been pharmacological. Taylor 
et al., supra. 
A less widely used test utilizes punished locomotion, wherein a measure of 
unpunished crossing is obtained according to the rate at which a mouse 
crosses from one metal plate to another, wherein footshocks are 
administered whenever the mouse crosses. Although less widely utilized to 
test anxiolytic agents, this test has been able to detect drug-induced 
increases and decreases in anxiety by manipulating the shock level. File, 
S. E., J. Neurosci Methods 2:219-238 (1980). 
The social interaction test of anxiety (File, supra; Jones, B. J. et al., 
Br. J. Pharmacol. 93:985-993 (1988) exploits the uncertainty and anxiety 
generated by placing rats in an unfamiliar environment and in bright 
light. The dependent variable is the time that pairs of male rats spend in 
active social interaction (90% of the behaviors are investigatory in 
nature). Both the familiarity and the light level of the test arena may be 
manipulated. Undrugged rats show the highest level of social interaction 
when the test arena is familiar and is lit by low light. Social 
interaction declines if the arena is unfamiliar to the rats or is lit by 
bright light. Anxiolytic agents prevent this decline. The overall level of 
motor activity may also be measured to allow detection of drug effects 
specific to social behaviors. 
The social interaction test of anxiety is one of the few animal tests of 
anxiety that has been validated behaviorally. Other behavioral measures 
indicative of anxiety and stress (e.g. defecation, self-grooming and 
displacement activities) were correlated with the reductions in social 
interaction; and other causes of response change (e.g. exploration of the 
environment, odor changes) were excluded. In order to validate the test 
physiologically, ACTH and corticosterone levels and changes in 
hypothalamic noradrenaline also were measured. File, TINS 10: 461-463 
(1987). 
Another test of anxiety that exploits the anxiety generated by a novel 
situation is the "elevated plus maze." In this test, the anxiety is 
generated by placing the animals on an elevated open arm. Height, rather 
than the light level, is responsible for generating behavioral and 
physiological changes. The apparatus is in the shape of a plus with two 
open and two enclosed arms. The rat has free access to all arms on the 
apparatus. Anxiolytic activity may be measured by the percentage increase 
in the time that the test animal spends on the open arms and the number of 
entries onto the open arms. This test has also been validated behaviorally 
and physiologically. 
Agents which have been determined to have anxiolytic activity include the 
carbazole derivative 9-[3-(3,5-cis-dimethylpiperazino)propyl]carbazole 
having the Formula (II): 
##STR2## 
and pharmaceutical compositions thereof. See U.S. Pat. No. 4,400,383 
(1983). 
Serotonin receptor antagonists are also known to be useful for the 
treatment of anxiety. See Kahn, R. S. et al., J. Affective Disord. 
8:197-200 (1987); Westenberg, H. G. M. et al., Psychopharmacol. Bull. 
23:146-149 (1987). 
SUMMARY OF THE INVENTION 
The invention relates to novel N,N'-disubstituted guanidines which bind to 
sigma receptor sites, especially those which do so selectively. 
The invention also relates to a novel class of N,N'-disubstituted 
guanidines which are radioactively tagged and which are useful for 
assaying in vitro the sigma receptor binding activity of organic 
compounds. 
The invention also relates to pharmacological compositions comprising 
certain of the aforesaid N,N'-disubstituted guanidines having sigma 
receptor binding activity and the use thereof to treat or prevent 
psycosis, depression, hypertension or anxiety. 
The invention also relates to a method for determining the sigma receptor 
binding activity of organic compounds. 
The invention also relates to an in vitro screening method for assaying 
compounds having sigma receptor activity and utility as antipsychotic, 
antidepressant, antihypertensive and anxiolytic drugs. 
The invention also relates to a method of determining the relationship of 
abnormal psychotic-like behavior in a mammal displaying such behavior to 
sigma receptor system dysfunction. 
The substituted guanidines of the invention have the Formula (I): 
##STR3## 
wherein R and R' are an alkyl group of at least 4 carbon atoms, a 
cycloalkyl group of at least 3 carbon atoms, a carbocyclic aryl group of 
at least 6 carbon atoms, alkaryl or aralkyl of at least 6 carbon atoms and 
containing 1-3 separate or fused rings, a heterocyclic ring or a 
heteroaryl group, and wherein each of R and R' may be substituted in 1-3 
positions, or wherein R and R' together with the guanidine group to which 
they are attached form a saturated or unsaturated cyclic ring containing 
at least 2 carbon atoms exclusive of the guanidine carbon atom, and 
wherein said cyclic ring may be substituted with one or more alkyl groups 
of 1-6 carbon atoms, carbocyclic aryl groups of at least 6 carbon atoms, 
cycloalkyl groups of 3-12 carbon atoms, or 1-2 fused aromatic rings, and 
further wherein said N,N'-disubstituted guanidine exhibits a high affinity 
for the sigma receptor. Preferably, such N,N'-disubstituted guanidines 
also exhibit activity in one or more of the animal models disclosed 
herein. 
In particular, the invention relates to N,N'-disubstituted guanidines of 
the Formula (I), above, wherein R and R' each are adamantyl, cyclohexyl, 
coumarinyl, norbornyl, isobornyl, or a monocyclic carbocyclic aryl of at 
least 6 carbon atoms. 
The invention also relates to substituted guanidines having the Formula 
(III): 
##STR4## 
wherein X and Y are independently a branched or straight chain C.sub.1 
-C.sub.12 alkylene or a branched or straight chain C.sub.2 -C.sub.12 
unsaturated alkylene or wherein one of X and Y is a single bond; 
R and R' are independently hydrogen, a cycloalkyl group of at least 3 
carbon atoms, a carbocyclic aryl group of at least 6 carbon atoms, aralkyl 
of at least 6 carbon atoms and containing 1-3 separate or fused rings, a 
heterocyclic ring or a heteroaryl group, and wherein each of R and R' may 
be substituted in 1-3 positions, or wherein R and R' together with the 
guanidine group to which they are attached form a saturated or unsaturated 
cyclic ring containing at least 2 carbon atoms exclusive of the guanidine 
carbon atom, and wherein said cyclic ring may be substituted with one or 
more alkyl groups of 1-6 carbon atoms, carbocyclic aryl groups of at least 
6 carbon atoms, cycloalkyl groups of 3-12 carbon atoms, or 1-2 fused 
aromatic rings. Preferably, said N,N'-disubstituted guanidine exhibits a 
high affinity for the sigma receptor. 
Preferably, X and Y are C.sub.1 -C.sub.4 alkylene groups. Also contemplated 
for use in the claimed invention are the N,N'-disubstituted guanidines 
having Formulae (I) and (III), wherein one or two C.sub.1 -C.sub.6 lower 
alkyl groups are substituted on N and/or N'. 
The invention also relates to compounds having the Formula (IV): 
##STR5## 
wherein n is 2, 3, 4 or 5; 
X and Y are independently a single bond, a branched or straight chain 
C.sub.1 -C.sub.12 alkylene or a branched or straight chain C.sub.2 
-C.sub.12 alkylene; 
R and R' are independently hydrogen, a cycloalkyl group of at least 3 
carbon atoms, a carbocyclic aryl group of at least 6 carbon atoms, aralkyl 
of at least 6 carbon atoms and containing 1-3 separate or fused rings, a 
heterocyclic ring, and wherein each of R and R' may be substituted in 1-3 
positions, or wherein R and R' together with the guanidine group to which 
they are attached form a saturated or unsaturated cyclic ring containing 
at least 2 carbon atoms exclusive of the guanidine carbon atom, and 
wherein said cyclic ring may be substituted with one or more alkyl groups 
of 1-5 carbon atoms, carbocyclic aryl groups of at least 6 carbon atoms, 
cycloalkyl groups of 3-12 carbon atoms, or 1-2 fused aromatic rings. 
Preferably, said compound exhibits a high affinity for the sigma receptor. 
Also preferable is where R and R' are m-ethylphenyl, X and Y are single 
bonds and n is 2, e.g., N,N'-di(m-ethylphenyl)-2-iminoimidazolidine. 
The invention also relates to tritiated derivatives of the above-listed 
N,N'-disubstituted guanidines wherein at least one of the ring carbon 
atoms of R and R' bears at least one radioactive atom, preferably a 
tritium atom 
The invention also relates to a method of determining the sigma brain 
receptor binding activity of an organic compound which comprises the steps 
of: 
a) contacting in an aqueous medium a known amount of isolated mammalian 
brain membrane which has sigma receptor-type binding activity, with a 
mixture of (i) a radio-labeled N,N'-disubstituted guanidine which 
selectively binds sigma brain receptors, in a known amount capable of 
being bound to the sigma receptors of that brain membrane; and (ii) 
varying known amounts of a water soluble organic compound to be assayed 
for sigma receptor binding activity; 
b) separating the brain membrane from the radio-labeled compound which is 
not bound to the brain membrane in step a); 
c) determining, from the molar relationship of the proportion of bound 
radio-labeled compound which is separated in step b) to the molar amount 
of the organic compound employed in step a), the sigma receptor binding 
activity of that organic compound. 
The invention also relates to a method of determining the relationship of 
abnormal psychotic-like behavior in a mammal displaying such behavior to 
sigma receptor dysfunction, which comprises administering thereto a sigma 
brain receptor-modulating amount of a water-soluble N,N'-disubstituted 
guanidine which displaces in vitro N,N'-di-(4-[.sup.3 
H]-2-methylphenyl)-guanidine bound to mammalian brain membrane, effective 
to alter the sigma brain receptor-modulated mental activity of that 
mammal. 
By the present invention, there is provided a means for identifying 
compounds which bind competitively and selectively to the sigma receptor 
site. Accordingly, the invention provides compounds, pharmaceutical 
compositions, and the use of same for treatment of psychoses, depression, 
hypertension or anxiety. Selective sigma receptor binding is of particular 
value in reducing or eliminating undesirable extrapyramidal side effects 
associated with present antipsychotic medications. 
It has been discovered further that certain N,N'-disubstituted guanidines 
are potent anxiolytics, and at the same time, are substantially 
non-sedative in an animal model. Therefore, these N,N'-disubstituted 
guanidines are useful for the treatment or prophylaxis of anxiety in 
animals, i.e., humans.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
We have discovered that the disubstituted guanidines of this invention 
have-sigma receptor binding activity, as evidenced by their ability to 
displace from guinea pig membrane binding sites [.sup.3 
H]-1,3-di-ortho-tolyl-guanidine(N,N'-di-(4-[.sup.3 
H]-2-methylphenyl)-guanidine ([.sup.3 H]--DTG]) which has the Formula 
(IV): 
##STR6## 
This compound binds reversibly, saturably, selectively and with high 
affinity to sigma receptor binding sites in guinea pig brain membrane 
homogenates and slide-mounted rat and guinea pig brain sections. We have 
established that (+)-[.sup.3 H]3-PPP binds to the same sites. Availability 
of the selective sigma ligands of this invention facilitates 
characterization of sigma receptors in vivo and in vitro. 
The preferred N,N'-disubstituted guanidines of this invention are those of 
the Formula (I): 
##STR7## 
wherein R and R' each are an alkyl group of at least 4 carbon atoms or 
carbocyclic aryl groups of at least 6 carbon atoms, e.g., R and R', which 
can be the same or different, are alkyl of 4 or more carbon atoms, e.g., a 
4 to 12, preferably a straight chain alkyl and more preferably a 4 to 8 
carbon atom alkyl group, for example, butyl, isobutyl, tert-butyl, amyl, 
hexyl, octyl, nonyl and decyl; cycloalkyl of 3 to 12 carbon atoms, e.g., 
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 
1,4-methylene-cyclohexanyl, adamantyl, norbornyl, isobornyl, 
cyclopentylmethyl, cyclohexylmethyl, 1- or 2-cyclohexylethyl and 1-, 2- or 
3-cyclohexylpropyl; carbocyclic aryl, alkaryl or aralkyl, e.g., of up to 
18 carbon atoms and containing 1-3 separate or fused aromatic rings, e.g., 
phenyl, benzyl, 1- and 2-phenylethyl, 1-, 2-, or 3-phenylpropyl; o-, m- or 
p-tolyl, m,m'-dimethylphenyl, o-, m- or p-ethylphenyl, m,m'diethylphenyl, 
m-methyl-m'-ethylphenyl and o-propylphenyl, naphthyl, 2-naphthyl, 
biphenyl; and heterocyclic rings, e.g. 3-, 4-, 5-, 6-, 7-, and 
8-coumarinyl; 2- and 4-pyridyl, pyrrolyl especially 2- and 
3-N-methylpyrrolyl, pyrrolidinyl, 2- and 3-furanyl, 2- and 3-thiophenyl, 
2- and 3-benzofuranyl, 2-benzoxazolyl, pyrazinyl, piperazinyl, pyrimidyl, 
2-, 4- and 5- thiazolyl, 2-, 4- and 5- oxazolyl, 2-, 4- and 5-imidazolyl, 
2- and 3-indolyl, and 2-4-benzothiazolyl, thienyl, benzofuranyl and 
morpholino. 
Additionally, 1, 2, 3 or more substituents which do not adversely affect 
the activity of the N,N'-disubstituted guanidine moiety may be present on 
one or both of the R and R' hydrocarbon groups thereof, e.g., alkyl of 1-8 
carbon atoms, e.g., methyl, ethyl; hydroxyalkyl of 1-8 carbon atoms; halo, 
e.g., chloro, bromo, iodo, fluoro; hydroxy; nitro; azido, cyano; 
isocyanato; amino; lower-alkylamino; di-lower-alkylamino; trifluoromethyl; 
alkoxy of 1-8 carbon atoms, e.g., methoxy, ethoxy and propoxy; acyloxy, 
e.g., alkanoyloxy of 1-8 carbon atoms, e.g., acetoxy and benzoxy; amido, 
e.g., acetamido, N-ethylcetamido; carbamido, e.g., carbamyl, 
N-methylcarbamyl, N,N'-dimethylcarbamyl; etc. 
Especially preferred are compounds of Formula I wherein R and R' each are 
cyclohexyl, norbornyl, adamantan-1-yl, adamantan-2-yl, 8-coumarinyl, or 
phenyl groups. Such phenyl groups, which need not necessarily be 
identical, may be substituted with one or more of the foregoing 
substituents, for example, in the o-, m- or p- position or the 2,2-, 2,4- 
or 3,5-position, when the phenyl group is disubstituted or R is as herein 
defined and R' is adamantyl. Specific examples are those wherein R and R' 
both are phenyl or o-tolyl; R is o-tolyl and R' is p-bromo-o-tolyl, 
p-CF.sub.3 -o-tolyl, p-iodo-o-tolyl, o-iodo-phenyl, p-azido-o-tolyl, 
cyclohexyl or adamantyl; and R is phenyl and R' is p-bromo-o-tolyl, 
p-iodo-o-tolyl, m-nitro-phenyl or o-iodo-phenyl. 
The highly active disubstituted guanidines of this invention have 
substantially the same stereoconfiguration as (+)3-PPP with phenyl axial 
and with SKF 10,047 with the piperidine ring in a skew-boat form and 
N-allyl axial. This similarity in spacial configuration, i.e., as 
demonstrated by computer modeling, of compounds having sigma receptor 
binding activity provides a screening technique for predicting the 
probable level of sigma receptor binding activity of other 
N,N'-disubstituted guanidines. Computer assisted molecular modifying can 
be used to determine the molecular similarity of the various species in 
three dimensions. 
Examples of those compounds which have been isolated and/or prepared and 
found to possess the aforesaid in vitro [.sup.3 H]DTG displacement 
activity are N,N'-dibutylguanidine, N,N'-diphenylguanidine, 
N,N'-di-o-tolylguanidine, N,N'-di-(2-methyl-4-bromo-phenyl)guanidine, 
N,N'-di-(2-methyl-4-iodophenyl)guanidine, 
N-(2-methyl-azidophenyl)-N'-(2-methylphenyl)guanidine, 
N,N'-diadamantylguanidine, N-adamantyl-N'-(2-methylphenyl)guanidine, 
N-(2-iodophenyl)-N'-(2-methylphenyl)guanidine, 
N-(2-methyl-4-nitrophenyl)-N'-(2-methylphenyl)guanidine, 
N,N'-di-(2,6-dimethylphenyl)guanidine, 
N-(2,6-dimethylphenyl)-N'-N'-(2-methylphenyl)guanidine, 
N-(adamantyl)-N'-(cyclohexyl)guanidine, N,N'-di(cyclohexyl)guanidine, 
N-(2-iodophenyl)-N'-(adamantyl)guanidine, 
N-(2-methylphenyl)-N'-cyclohexyl-guanidine, 
N-adamantyl-N'-phenylguanidine, N,N'-di-(m-n-propylphenyl)guanidine, 
N,N'-di-(1-tetralinyl)guanidine, 
N-(3,5-dimethyl-1-adamantanyl)-N'-(o-tolyl)guanidine, 
N-(3,5-dimethyl-1-adamantanyl)-N'-(o-iodophenyl)guanidine, 
N-(1-adamantyl)-N'-(o-nitrophenyl)guanidine, 
N,N'-di-((.+-.)-endo-2-norbornyl)guanidine, 
N-(exo-2-isobornyl)-N'-(o-iodophenyl)guanidine, 
N,N'-di-(exo-2-norbornyl)guanidine, 
N-(exo-2-isobornyl)-N'-(o-tolyl)guanidine, 
N-(o-iodophenyl)-N'-(t-butyl)guanidine, N,N'-dibenzylguanidine, 
N-(adamant-1-yl)-N'-(o-isopropylphenyl)guanidine, 
N-(adamant-1-yl)-N'-(p-bromo-o-tolyl)guanidine, 
N-(cyclohexyl)-N'-(p-bromo-o-tolyl)guanidine, and 
N-(adamant-2-yl)-N'-(p-iodophenyl)guanidine. 
Among the compounds tested to date, some of those having the highest sigma 
receptor binding activity are those of Formula I wherein one of R and R' 
is adamantyl and the other is also adamantyl or o-substituted phenyl. 
Therefore, the preferred compounds of this invention include those wherein 
R and R.sup.1 have those values, i.e., wherein the other of R and R.sup.1 
is, e.g., o-lower alkyl phenyl, wherein alkyl is of 1-4 carbon atoms, 
e.g., CH.sub.3, C.sub.2 H.sub.5, i-C.sub.3 H.sub.7, o-halophenyl wherein 
halo is Cl, Br, I or F, o-nitro-o-amino, o-carbo-lower alkoxy, e.g., 
COOCH.sub.3, o-amino, e.g., --CONH.sub.2, o-sulfato, o-carboxy, o-acyl, 
e.g., acetyl, o-CF.sub.3, o-sulfamido and o-lower-alkoxy, e.g., o-methoxy, 
phenyl or another phenyl group ortho substituted by any other substituent 
of a molecular weight less than 150. 
Especially preferred N,N'-disubstituted guanidine compounds which have high 
binding to the sigma receptor include 
N-(2-iodophenyl)-N'-(adamant-1-yl)guanidine (AdIpG, IC.sub.50 =6.2 nM); 
N-(o-tolyl)-N'-(adamant-1-yl)guanidine (AdTG, IC.sub.50 =7.6 nM); 
N,N'-di-(adamant-1-yl)guanidine (DAG, IC.sub.50 =16.5 nM); 
N-cyclohexyl-N'-(2-methylphenyl)-guanidine (IC.sub.50 =13 nM); 
N-(adamant-1-yl)-N'-cyclohexylguanidine (IC.sub.50 =12.5 nM); 
N-(adamant-2-yl)-N'-(2-iodophenyl)guanidine (IC.sub.50 =3.5 nM); 
N-(adamant-2-yl)-N'-(2-methylphenyl)guanidine (IC.sub.50 =7.0 nM); 
N-(exo-2-norbornyl)-N'-(2-methylphenyl)guanidine (IC.sub.50 =7.0nM); 
N-((.+-.)-endo-2-norbornyl)-N'-(2-methylphenyl)guanidine (IC.sub.50 =6.0 
nM); N-(exo-2-norbornyl)-N'-(2-iodophenyl)guanidine (IC.sub.50 =4.0 nM); 
N-((.+-.)-endo-2-norbornyl)-N'-(2-iodophenyl)guanidine (IC.sub.50 =5.0 
nM); N,N'-di-(o-tolyl)guanidine (IC.sub.50 =32 nM); 
N-(o-tolyl)-N'-(o-iodophenyl)guanidine (IC.sub.50 =21 nM); 
N,N'-di-(p-bromo-o-methylphenyl)guanidine (IC.sub.50 =37 nM); 
N,N'-di-(m-n-propylphenyl)guanidine (IC.sub.50 =36 nM); 
N-(o-tolyl)-N'-(p-nitro-o-tolyl)guanidine (IC.sub.50 =37 nM); 
N,N'-di-(1-tetralinyl)guanidine (IC.sub.50 =58 nM); 
N-(o-tolyl)-N'-(o-xylyl)guanidine (IC.sub.50 =70 nM); 
N,N'-di-(o-xylyl)guanidine (IC.sub.50 =90 nM); 
N,N'-di-(cyclohexyl)guanidine (IC.sub.50 =71 nM); 
N-(3,5-dimethyladamantan-1-yl)-N'-(o-tolyl)guanidine (IC.sub.50 =15 nM); 
N-(3,5-dimethyl-1-adamantanyl)-N'-(o-iodophenyl)guanidine (IC.sub.50 =16 
nM); N-(1-adamantyl)-N'-(o-nitrophenyl)guanidine (IC.sub.50 =30 nM); 
N,N'-di-((.+-.)-endo-2-norbornyl)guanidine (IC.sub.50 =16 nM); 
N-(exo-2-isobornyl)-N'-(o-iodophenyl)guanidine (IC.sub.50 =18 nM); 
N,N'-di-(exo-2-norbornyl)guanidine (IC.sub.50 =22 nM); 
N-(exo-2-isobornyl)-N'-(o-tolyl)guanidine (IC.sub.50 =25 nM); 
N-(o-iodophenyl)-N'-(t-butyl)guanidine (IC.sub.50 =20 nM); 
N,N'-dibenzylguanidine (IC.sub.50 =90 nM); 
N-(adamant-1-yl)-N'-(o-isopropylphenyl)guanidine (IC.sub.50 =24 nM); 
N-(adamant-1-yl)-N'-(p-bromo-o-tolyl)guanidine (IC.sub.50 =2.7 nM); 
N-(cyclohexyl)-N'-(p-bromo-o-tolyl)guanidine (IC.sub.50 =5.5 nM); 
N-(4-azido-2-methylphenyl)-N'-(2-methylphenyl)guanidine (IC.sub.50 =20 
nM); N-(2-methyl-4-nitro-5-bromophenyl)-N'-(2-methyl-4,5-dibromophenyl)gua 
nidine; N,N'-di-(o-iodophenyl)guanidine (IC.sub.50 =13 nM); 
N,N'-di-(3-methylphenyl)guanidine (IC.sub.50 =43 nM); 
N,N'-di-(m-ethylphenyl)-2-imino-imidazolidine (IC.sub.50 =70 nM); 
N-(4-nitro-2-methylphenyl)-N'-(2-methylphenyl)guanidine (IC.sub.50 =37 
nM); N-(1-naphthyl)-N'-(2-iodophenyl)guanidine (IC.sub.50 =40.1 nM); 
N,N'-di-(4-indanyl)guanidine (IC.sub.50 =28.5 nM); 
N-(adamantan-1-yl)-N'-(2-trifluoromethylphenyl)guanidine (IC.sub.50 =7.44 
nM); N-(adamantan-1-yl)-N'-(2-methylphenyl)-N'-methylguanidine (IC.sub.50 
=22.6 nM); N-(adamantan-1-yl)-N'-(6-coumarinyl)guanidine (IC.sub.50 =21.9 
nM); N-(adamantan-1-yl)-N'-(2,4-difluorophenyl)guanidine (IC.sub.50 =8.92 
nM); and N-(adamantan-1-yl)-N'-(2-trifluoromethyl-4-fluorophenyl)guanidine 
(IC.sub.50 =4 nM). 
The level of sigma receptor activity of the disubstituted guanidines can 
also be determined in vivo in a discriminative stimulus property test 
employing rats trained to discriminate between intraperitoneal injections 
of cyclazocine (2.0 mg/kg) and saline in a discrete-trial avoidance 
paradigm with sessions of 20 trials each. For example, ditolylguanidine 
(DTG) and diphenylguanidine (DPG) were fully substitutable for cyclazocine 
at the same concentrations. (Holtzman, S. G., Emery University, Atlanta, 
Ga., private communication.) 
Although the discussion hereinafter of the experiments below relates to 
certain of these selective sigma receptor ligands, viz., the 
N,N'-disubstituted guanidines of Table I below, the activity and utility 
of that compound apply comparably to the other disubstituted guanidines 
which compete with and displace in vitro N,N'-di-(4-[.sup.3 
H]-2-methylphenyl)-guanidine bound in vitro to isolated guinea pig brain 
membrane. 
In carrying out the sigma receptor binding activity measurement method of 
this invention, a known amount of a mammalian brain membrane, e.g., human 
or other primate, porcine, rodent, e.g., rat or guinea pig, which has SKF 
10,047 and like psychotomimetic benzomorphan binding activity is contacted 
in a suitable aqueous vehicle, e.g., physiological saline solution, with a 
mixture, usually in a solution in a suitable aqueous vehicle of (i) a 
tritium-labeled N,N'-disubstituted guanidine of this invention having 
sigma receptor binding activity, in an mount capable of being fully bound 
to the above said amount of membrane and (ii) a water soluble organic 
compound whose sigma receptor activity is to be assayed, in known mounts, 
sufficiently varied to obtain a dose-response curve. The techniques for 
obtaining a dose-response curve are standard and well known to those 
skilled in the art. Typically, one could employ molar amounts varying as 
much as from 10.sup.-4 to 10.sup.4 of the molar amount of the tritium 
labeled compound present in the mixture, e.g., employing from 10 to 120 
and preferably 30 to 90 such mixtures. 
If the organic compound being assayed has sigma receptor binding activity, 
a portion of the tritium labeled compound which, in the absence of the 
organic compound would bind to the membrane remains unbound and is thus 
separable from the membrane. The amount which remains unbound is 
proportional to the sigma receptor binding activity of the organic 
compound and the molar ratio thereof in the mixture to the tritium labeled 
compound. 
The two compounds can be employed at any convenient collective 
concentration, e.g., from 10.sup.-8 to 10.sup.3 nM. 
In the next step, the membrane is separated from and washed until free of 
the solution in which step (a) is conducted. In the next step, the amount 
of tritium labeled compound which is thus separated from the membrane is 
determined, e.g., by measuring the collective radioactivity level of the 
separated solution and wash water and comparing that radioactivity to that 
obtained when the foregoing steps are conducted with the same amount of 
tritium-labeled N,N'-disubstituted guanidine in the absence of the organic 
compound. 
In the next step of the method, the activity of sigma receptor binding 
activity of the organic compound is determined from the dose response 
curve thus obtained. 
Although tritium radiolabels are preferred, any radiolabel which can be 
substituted on the N,N'-disubstituted guanidines of the invention may be 
employed, e.g., .sup.11 C, .sup.14 C, .sup.18 F, .sup.125 I, .sup.131 I, 
.sup.15 N, .sup.35 S, and .sup.32 P. 
All of the foregoing steps are conventional and have been employed in the 
prior art with other types of .sup.3 H-labeled compounds having sigma 
receptor binding activity. The method of this invention is, however, 
unique in that the tritium-labeled N,N'-disubstituted guanidines of this 
invention are highly selective to binding by the sigma receptors and 
therefore will not compete with organic compounds which bind to other 
brain receptors. 
In carrying out the method of treatment aspect of this invention, e.g., 
treating a human being suffering from a psychotic mental illness 
associated with hallucinations, or suffering from depression, hypertension 
or anxiety, there is administered thereto an effective amount of a 
water-soluble N,N'-disubstituted guanidine which has high binding to the 
sigma receptor. When treating psychosis, the compound is an antagonist to 
the sigma receptor binding activity of a hallucinogenic benzomorphan. 
Preferably, the guanidine is a compound of Formulae I, III or IV wherein R 
and R' each is an alkyl group of at least 4 carbon atoms, a cycloalkyl 
group of 3 to 12 carbon atoms or a carbocyclic aryl group of at least 6 
carbon atoms. In preferred aspect, the human being is schizophrenic; in 
another preferred aspect, the compound is 
N,N'-di-(2-methylphenyl)-guanidine, 
N-(adamantyl)-N'-(cyclohexyl)guanidine, N-adamantyl-N'-(2-methylphenyl)gua 
nidine, N-(1-adamantyl)-N'-(o-iodophenyl)guanidine, 
N-(2-adamantyl)-N'-(o-iodophenyl)guanidine, 
N-cyclohexyl-N'-(2-methylphenyl)guanidine, N,N'-di-(cyclohexyl)guanidine, 
N,N'-di-(2-adamantyl)guanidine, N,N'-di-(m-n-propylphenyl)guanidine, 
N-(o-tolyl)-N'-(p-nitro-o-tolyl)guanidine, 
N,N'-di-(1-tetralinyl)guanidine, N-(o-tolyl)-N'-(o-xylyl)guanidine, 
N,N'-di-(o-xylyl)guanidine, 
N-(3,5-dimethyladmantan-1yl)-N'-(o-tolyl)guanidine, 
N-(3,5-dimethyladamantan-1-yl)-N'-(o-iodophenyl)guanidine, 
N-(1-adamantyl)-N'-(o-nitrophenyl)guanidine, (+) and 
(-)N-(exo-2-norbornyl)-N'-(2-iodophenyl)guanidine, (+) and 
(-)N-(endonorbornyl)-N'-(o-tolyl)guanidine, (+) and 
(-)N-(exonorbornyl)-N'-(o-tolyl)guanidine, (+) and 
(-)N,N'-di(endonorbornyl)guanidine, (+) and 
(-)N-(exoisobornyl)-N'-(o-iodophenyl)guanidine, (+) and 
(-)N,N'-di-(exonorbornyl)guanidine, 
N-(o-iodophenyl)-N'-(t-butyl)guanidine, N,N'-dibenzylguanidine, 
N-(adamant-1-yl)-N'-(o-isopropylphenyl)guanidine, 
N-(adamant-1-yl)-N'-(p-bromo-o-tolyl)guanidine, 
N-cyclohexyl)-N'-(p-bromo-o-tolyl)guanidine, 
N-(adamant-2-yl)-N'-(p-iodophenyl)guanidine, 
N,N'-di(o-methylbenzyl)guanidine, N,N'-di(1-adamantanemethyl)guanidine, 
N-(adamantan-1-yl)-N'-(2-trifluoromethylphenyl)-guanidine, 
N-(adamantan-1-yl)-N'-(2,4-difluoromethylphenyl)guanidine, and 
N-(adamantan-1-yl)-N'-(2-trifluoromethyl-4-fluorophenyl)guanidine; or a 
corresponding compound bearing 1, 2, 3 or more additional or other 
substituents on one or both hydrocarbon groups, e.g., alkyl of 1-8 carbon 
atoms, e.g., methyl-, ethyl; halo, e.g., chloro, bromo, iodo, fluoro; 
nitro; azido; cyano; isocyanato; amino; lower-alkylamino; 
di-lower-alkylamino; trifluoromethyl; alkoxy of 1-8 carbon atoms, e.g., 
methoxy, ethoxy and propoxy; acyloxy, e.g., alkanoyloxy of 1-8 carbon 
atoms, e.g., acetoxy and benzoyl; amido, e.g., acetamido, 
N-ethylacetamido; carbamido, e.g., carbamyl, N-methylcarbamyl, 
N,N'-dimethyl carbamyl; etc. 
N,N'-disubstituted guanidines, e.g., of Formulae I, III and IV, can act in 
an agonistic, antagonistic or inverse agonistic manner in relation to the 
prototypical sigma benzomorphans. Those which act as antagonists can 
therefore be expected to affect pupil size, heart rate and mentation in a 
direction opposite that caused by benzomorphans which can be determined by 
standard tests in laboratory animals. The type and level of activity for a 
given dosage of each compound can be conventionally determined by routine 
experimentation using well known pharmacological protocols for each of the 
activities; the corresponding indications treatable at that dosage will be 
well known to skilled workers based on the pharmacological results. The 
compounds of this invention are particularly noteworthy for their 
antipsychotic activity to treat psychotic conditions, e.g., schizophrenia, 
by analogy to the known agents prolixin and haloperidol and for diagnosing 
sigma receptor intoxicated conditions. 
The invention is also related to the discovery that N,N'-disubstituted 
guanidines having a high affinity for the sigma receptor are anxiolytic, 
and at the same time, are substantially non-sedative in an animal model. 
The term "high affinity to sigma receptor" means the compound exhibits an 
IC.sub.50 of less than 100 nM in a sigma receptor binding assay, 
preferably against .sup.3 H--DTG as disclosed in the examples, below. 
Alternatively, the compounds may be tested against (+)-[.sup.3 H]3-PPP as 
described by Largent, B. L., et al., Mol. Pharmacol. 32:772-784 (1987); 
Largent B. L., et al., Eur. J. Pharmacol. 155:345-347 (1988); and 
Wikstrom, H., et al., J. Med. Chem. 30:2169-2174 (1987). The values of 
IC.sub.50 obtained by screening against .sup.3 H--DTG and (+)-[.sup.3 
H]3-PPP are well correlated. See Weber, E. et al., Proc. Natl. Acad. Sci. 
USA 83: 8784-8788 (1986). 
These N,N'-disubstituted guanidines exhibit anxiolytic activities of 
generally 100-1000 times greater than that of benzodiazepines. However, 
unlike benzodiazepines, the N,N'-disubstituted guanidines employed in this 
invention are non-sedative. Therefore, these N,N'-disubstituted guanidines 
are particularly useful for the treatment or prevention of anxiety in 
animals. 
Recent work by the inventors has shown that sigma receptor active drugs 
including the diaryl-guanidines can block acetylcholine release induced by 
serotonin acting at 5HT.sub.3 receptors in the guinea pig ileum myenteric 
plexus (Campbell et al., J. Neurosci. 9:3380-3391 (1989)). The sigma 
receptor active drugs act in a non-competitive manner to block 
acetylcholine release stimulated by 5HT.sub.3 receptor activation. Work by 
others has shown that compounds acting as competitive antagonists at 
5HT.sub.3 receptors have anxiolytic activity (Jones et al., Br. J. 
Pharmacol. 93:985-993 (1988)). Therefore, the inventors reasoned that 
non-competitive antagonists of 5HT.sub.3 receptor-induced acetylcholine 
release might also be anxiolytic. This invention shows that certain sigma 
receptor active N,N'-disubstituted guanidines indeed have potent 
anxiolytic activity. 
Disubstituted guanidines are the subject of U.S. Pat. No. 4,709,094, whose 
disclosure is incorporated herein by reference. As a class, these 
compounds are described in this patent as exhibiting a highly selective 
binding activity to the sigma brain receptor. 
Certain specific members of this class of disubstituted guanidines, i.e., 
those demonstrating a high affinity for the sigma receptor, are useful for 
the treatment or prophylaxis of anxiety in an individual susceptible to 
anxiety. Individuals susceptible to anxiety are those who have experienced 
a plurality of prior episodes of GAD. 
The anxiolytic activity of any particular N,N'-disubstituted guanidine may 
be determined by use of any of the recognized animal models for anxiety. A 
preferred model is described by Jones, B. J. et al., Br. J. Pharmacol. 
93:985-993 (1988). This model involves administering the compound in 
question to mice which have a high basal level of anxiety. The test is 
based on the finding that such mice find it aversive when taken from a 
dark home environment in a dark testing room and placed in an area which 
is painted white and brightly it. The test box has two compartments, one 
white and brightly illuminated and one black and non-illuminated. The 
mouse has access to both compartments via an opening at floor level in the 
divider between the two compartments. The mice are placed in the center of 
the brightly illuminated area. After locating the opening to the dark 
area, the mice are free to pass back and forth between the two 
compartments. Control mice tend to spend a larger proportion of time in 
the dark compartment. When given an anxiolytic agent, the mice spend more 
time exploring the more novel brightly lit compartment and exhibit a 
delayed latency to move to the dark compartment. Moreover, the mice 
treated with the anxiolytic agent exhibit more behavior in the white 
compartment, as measured by exploratory rearings and line crossings. Since 
the mice can habituate to the test situation, naive mice should always be 
used in the test. Five parameters may be measured: the latency to entry 
into the dark compartment, the time spent in each area, the number of 
transitions between compartments, the number of lines crossed in each 
compartment, and the number of rears in each compartment. As disclosed 
more fully below in the examples, the administration of several 
N,N'-disubstituted guanidines has been found to result in the mice 
spending more time in the larger, brightly lit area of the test chamber. 
Unlike diazepam, the N,N'-disubstituted guanidines did not cause 
significant decreases in the numbers of line crossings and rears. Thus, 
these N,N'-disubstituted guanidines exhibit potent anxiolytic activity, 
and at the same time, are non-sedating. 
In the light/dark exploration model, the anxiolytic activity of a putative 
agent can be identified by the increase of the numbers of line crossings 
and rears in the light compartment at the expense of the numbers of line 
crossings and rears in the dark compartment, in comparison with control 
mice. 
A second preferred animal model is the rat social interaction test 
described by Jones, B. J. et al., supra, wherein the time that two mice 
spend in social interaction is quantified. The anxiolytic activity of a 
putative agent can be identified by the increase in the time that pairs of 
male rats spend in active social interaction (90% of the behaviors are 
investigatory in nature). Both the familiarity and the light level of the 
test arena may be manipulated. Undrugged rats show the highest level of 
social interaction when the test arena is familiar and is lit by low 
light. Social interaction declines if the arena is unfamiliar to the rats 
or is lit by bright light. Anxiolytic agents prevent this decline. The 
overall level of motor activity may also be measured to allow detection of 
drug effects specific to social behaviors. 
As noted above, the compounds of this invention are useful as 
anti-hypertensive agents and can be used in the same manner as known 
antihypertensive agents, e.g., methyldopa, metoprolol tartrate and 
hydralazine hydrochloride. 
Like guanidines generally and N,N'-diphenyl-guanidine specifically, the 
disubstituted guanidines of this invention, including those of Formulae I, 
III and IV, are accelerators for the vulcanization of rubbers, e.g., 
natural rubbers and epoxy group-containing acrylic rubber, and can be used 
for such purpose in the same manner as N,N'-diphenylguanidine. Thus 
[.sup.3 H]--DTG can be incorporated into a vulcanized rubber object, e.g., 
a tire tread, and rate of loss of rubber therefrom by water can be 
monitored by rate of loss of radioactivity. 
The N,N'-disubstituted guanidines can readily be prepared by conventional 
chemical reactions, e.g., when R and R' are the same, by reaction of the 
corresponding amine with cyanogen bromide. Other methods which can be 
employed include the reaction of an amine or amine salt with a preformed 
alkyl or aryl cyanamide. See Safer, S. R., et al., J. Org. Chem. 13:924 
(1948). This is the method of choice for producing N,N'-disubstituted 
guanidines in which the substituents are not identical. For a recent 
synthesis of unsymmetrical guanidines, see G. J. Durant et al., J. Med. 
Chem. 28:1414 (1985), and C. A. Maryanoff et al., J. Org. Chem. 51:1882 
(1986). 
Included as well in the present invention are the novel compounds disclosed 
herein as well as pharmaceutical compositions thereof comprising an 
effective mount of the N,N'-disubstituted guanidine in combination with a 
pharmaceutically acceptable carrier. 
The N,N'-disubstituted guanidines and the pharmaceutical compositions of 
the present invention may be administered by any means that achieve their 
intended purpose. For example, administration may be by parenteral 
subcutaneous, intravenous, intramuscular, intra-peritoneal, transdermal, 
or buccal routes. Alternatively, or concurrently, administration may be by 
the oral route. The dosage administered will be dependent upon the age, 
health, and weight of the recipient, kind of concurrent treatment, if any, 
frequency of treatment, and the nature of the effect desired. 
Compositions within the scope of this invention include all compositions 
wherein the N,N'-disubstituted guanidine is contained in an amount which 
is effective to achieve its intended purpose. While individual needs vary, 
determination of optimal ranges of effective mounts of each component is 
with the skill of the art. Typically, the compounds may be administered to 
mammals, e.g. humans, orally at a dose of 0.0025 to 15 mg/kg, or an 
equivalent amount of the pharmaceutically acceptable salt thereof, per day 
of the body weight of the mammal being treated for psychosis, depression, 
hypertension, or anxiety disorders, e.g., generalized anxiety disorder, 
phobic disorders, obsessional compulsive disorder, panic disorder, and 
post traumatic stress disorders. Preferably, about 0.01 to about 10 mg/kg 
is orally administered to treat or prevent such disorders. For 
intramuscular injection, the dose is generally about one-half of the oral 
dose. For example, for treatment or prevention of psychosis or anxiety, a 
suitable intramuscular dose would be about 0.0025 to about 15 mg/kg, and 
most preferably, from about 0.01 to about 10 mg/kg. 
The unit oral dose may comprise from about 0.25 to about 400 mg, preferably 
about 0.25 to about 100 mg of the compound. The unit dose may be 
administered one or more times daily as one or more tablets each 
containing from about 0.10 to about 300, conveniently about 0.25 to 50 mg 
of the anxiolytic compound or its solvates. 
In addition to administering the compound as a raw chemical, the compounds 
of the invention may be administered as part of a pharmaceutical 
preparation containing suitable pharmaceutically acceptable carriers 
comprising excipients and auxiliaries which facilitate processing of the 
compounds into preparations which can be used pharmaceutically. 
Preferably, the preparations, particularly those preparations which can be 
administered orally and which can be used for the preferred type of 
administration, such as tablets, dragees, and capsules, and also 
preparations which can be administered rectally, such as suppositories, as 
well as suitable solutions for administration by injection or orally, 
contain from about 0.01 to 99 percent, preferably from about 0.25 to 75 
percent of active compound(s), together with the excipient. 
The pharmaceutical preparations of the present invention are manufactured 
in a manner which is itself known, for example, by means of conventional 
mixing, granulating, dragee-making, dissolving, or lyophilizing processes. 
Thus, pharmaceutical preparations for oral use can be obtained by 
combining the active compounds with solid excipients, optionally grinding 
the resulting mixture and processing the mixture of granules, after adding 
suitable auxiliaries, if desired or necessary, to obtain tablets or dragee 
cores. 
Suitable excipients are, in particular, fillers such as saccharides, for 
example lactose or sucrose, mannitol or sorbitol, cellulose preparations 
and/or calcium phosphates, for example tricalcium phosphate or calcium 
hydrogen phosphate, as well as binders such as starch paste, using, for 
example, maize starch, wheat starch, rice starch, potato starch, gelatin, 
tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium 
carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, 
disintegrating agents may be added such as the above-mentioned starches 
and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, 
or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries 
are, above all, flow-regulating agents and lubricants, for example, 
silica, talc, steric acid or salts thereof, such as magnesium stearate or 
calcium stearate, and/or polyethylene glycol. Dragee cores are provided 
with suitable coatings which, if desired, are resistant to gastric juices. 
For this purpose, concentrated saccharide solutions may be used, which may 
optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene 
glycol and/or titanium dioxide, lacquer solutions and suitable organic 
solvents or solvent mixtures. In order to produce coatings resistant to 
gastric juices, solutions of suitable cellulose preparations such as 
acetylcellulose phthalate or hydroxypropymethylcellulose phthalate, are 
used. Dye stuffs or pigments may be added to the tablets or dragee 
coatings, for example, for identification or in order to characterize 
combinations of active compound doses. 
Other pharmaceutical preparations which can be used orally include push-fit 
capsules made of gelatin, as well as soft, sealed capsules made of gelatin 
and a plasticizer such as glycerol or sorbitol. The push-fit capsules can 
contain the active compounds in the form of granules which may be mixed 
with fillers such as lactose, binders such as starches, and/or lubricants 
such as talc or magnesium stearate and, optionally, stabilizers. In soft 
capsules, the active compounds are preferably dissolved or suspended in 
suitable liquids, such as fatty oils, or liquid paraffin. In addition, 
stabilizers may be added. 
Possible pharmaceutical preparations which can be used rectally include, 
for example, suppositories, which consist of a combination of the active 
compounds with a suppository base. Suitable suppository bases are, for 
example, natural or synthetic triglycerides, or paraffin hydrocarbons. In 
addition, it is also possible to use gelatin rectal capsules which consist 
of a combination of the active compounds with a base. Possible base 
materials include, for example, liquid triglycerides, polyethylene 
glycols, or paraffin hydrocarbons. 
Suitable formulations for parenteral administration include aqueous 
solutions of the active compounds in water-soluble form, for example, 
water-soluble salts. In addition, suspensions of the active compounds as 
appropriate oily injection suspensions may be administered. Suitable 
lipophilic solvents or vehicles include fatty oils, for example, sesame 
oil, or synthetic fatty acid esters, for example, ethyl oleate or 
triglycerides. Aqueous injection suspensions may contain substances which 
increase the viscosity of the suspension include, for example, sodium 
carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the 
suspension may also contain stabilizers. 
The characterization of sigma receptors in vitro has been difficult because 
of the lack of selective drug ligands. Most benzomorphan opiates 
cross-react with other (mu, delta, kappa), opioid receptors and are 
therefore of only limited value for characterizing and isolating 
receptors. Pasternak et al., J. Pharmacol. Exp. Ther. 219:192-198 (1981); 
Zukin, R. S., et al., Mol. Pharm. 20:246-254 (1981); and Tam, S. W., Eur. 
J. Pharmacol. 109:33-41 (1985). [.sup.3 H]DTG binds specifically and with 
high affinity to a single class of binding sites in guinea pig brain 
membranes. The binding characteristics and the drug specificity profile of 
these sites are concordant with those proposed for the sigma receptor, 
including 1) naloxone insensitivity and stereo-selectivity for 
dextrorotatory isomers of benzomorphan opiates such as (+)SKF 10,047, 
(+)cyclazocine and (+)pentazocine; 2) high affinity for haloperidol and 
certain phenothiazine antipsychotic drugs; 3) stereo-selectivity for 
(-)butaclamol; and 4) insensitivity to dopamine and apomorphine. [.sup.3 
H]--DTG is one of only two known tritiated compounds that are selective 
for the sigma site. The other, (+)[.sup.3 H]3-PPP, originally proposed to 
be a dopamine autoreceptor agonist, has recently been shown to be 
selective for sigma sites in rat brain membrane binding assays. Largent et 
al. (1984), supra. Our experiments confirm these findings in the guinea 
pig and show that [.sup.3 H]DTG and (+)[.sup.3 H]3-PPP have virtually 
identical receptor binding characteristics and drug selectivity profiles. 
Previous studies have shown that sigma sites can also be labeled with 
(+)[.sup.3 H]SKF 10,047, (+)[.sup.3 H]-ethylketazocine and with 
(.+-.)[.sup.3 H]SKF 10,047. However, these ligands are not selective for 
the sigma site and require the presence of appropriate drugs in the 
binding assays to mask cross-reacting non-sigma binding sites. 
[.sup.3 H]DTG has a number of advantages as a sigma ligand. It is highly 
selective for the sigma site (unlike [.sup.3 H]SKF 10,047 and (+)[.sup.3 
H]ethylketazocine), it has a high degree of specific binding (90-97% of 
total binding) and it has a relatively simple chemical structure that is 
not chiral (unlike (+)[.sup.3 H]3-PPP and the benzomorphan opiates). These 
characteristics make it a good starting compound for the synthesis of 
analogs for structure-activity studies and for the design of irreversible 
(after photolysis) sigma receptor ligands, e.g., compounds of Formulae I 
and II wherein at least one of R and R' is azido-substituted carbocyclic 
aryl. 
The sigma site labeled with [.sup.3 H]DTG is clearly not related to 
conventional (mu, delta, kappa) opioid receptors as it is naloxone 
insensitive and shows stereoselectivity for dextrorotatory isomers of 
benzomorphan drugs. This is a reversed stereoselectivity compared to 
naloxone-sensitive opioid receptors which are selective for levorotatory 
isomers of opiates. Sigma receptors should therefore not be referred to as 
sigma "opioid" receptors. The drug selectivity of sigma sites for 
dextrorotatory isomers of psychotomimetic opiates does, however, correlate 
well with the pharmacological profile of dextrorotatory versus 
levorotatory opiates in animal tests designed to differentiate between 
conventional opioid receptor activity and sigma (behavioral) activity of 
benzomorphan drugs. Cowan, A., Life Sci. 28:1559-1570 (1981); Brady, K. 
T., et al., Science 215:178-180 (1982); and Khazan, N., et al., 
Neuropharmacol. 23:983-987 (1984). 
Autoradiography studies using [.sup.3 H]DTG visualize the sigma site in 
slide-mounted rodent brain sections and confirm that sigma sites are 
different from mu, delta, and kappa opioid receptors as the distribution 
of [.sup.3 H]DTG binding is rather distinct from the distribution of mu, 
delta, kappa opioid receptors. The anatomical distribution of [.sup.3 
H]DTG binding sites is, however, very similar if not identical to the 
distribution of [.sup.3 H]3-PPP binding sites, further confirming that the 
two radioligands label identical binding sites. The high affinity of the 
[.sup.3 H]DTG binding site for haloperidol and for certain phenothiazine 
antipsychotics (TABLE I) which are also dopamine D.sub.2 receptor 
antagonists raises the question as to the relation of sigma receptors to 
dopamine D.sub.2 receptors. The results presented show that the [.sup.3 
H]DTG site is clearly distinct from dopamine D.sub.2 receptors, because 
the autoradiographic distribution of dopamine receptor is dissimilar and 
because dopamine, apomorphine and many other dopamine receptor ligands do 
not interact with the [.sup.3 H]DTG binding site. 
Furthermore the sigma site labeled with [.sup.3 H]DTG is stereoselective 
for (-)butaclamol which is a reversed stereoselectivity compared to the 
dopamine D.sub.2 receptors which are stereoselective for (+)butaclamol. 
The haloperidol-sensitive sigma site labeled with [.sup.3 H]DTG was found 
to have a moderate affinity for the potent hallucinogen PCP in competition 
experiments. This is in agreement with findings by others who use 
(+)[.sup.3 H]SKF 10,047, (.+-.)[.sup.3 H]SKF 10,047 or (.+-.)-[.sup.3 
H]3-PPP to label sigma sites. In PCP receptor binding assays, however, 
[.sup.3 H]-PCP labeled predominantly (but not exclusively) a 
haloperidol-insensitive PCP binding site, termed PCP/sigma opiate receptor 
by Zukin and colleagues, Zukin et al. (1981, 1986), supra, which is 
separate from the haloperidol-sensitive sigma site labeled with [.sup.3 
H]DTG or (+)[.sup.3 H]3-PPP. In contrast, [.sup.3 H]DTG appears to label 
exclusively the haloperidol sensitive sigma site, since all specific 
binding is displaceable by haloperidol and the anatomical distribution of 
[.sup.3 H]DTG binding is distinct from the distribution of PCP receptors. 
Furthermore, unlabeled DTG is virtually inactive in a [.sup.3 H]-PCP 
binding assay (S. William Tam, E. l. DuPont De Nemours & Co., Wilmington, 
Del., personal communication). There is some controversy as to which of 
the two binding sites is responsible for causing the behavioral effects of 
PCP and psychotomimetic benzomorphan opiates and would therefore 
correspond to the sigma receptor postulated by Martin et al. (1976), 
supra. Zukin and his collaborators have argued that the behavioral effects 
of both PCP and psychotomimetic benzomorphan opiates are mediated by the 
haloperidol-insensitive PCP site, to which benzomorphan opiates bind with 
moderate affinity. Largent et al. (1986), supra, cited circumstantial 
evidence suggesting that it is equally likely that the behavioral effects 
of both PCP and psychotomimetic opiates are mediated through the 
haloperidol sensitive sigma site. As [.sup.3 H]DTG exclusively labels the 
haloperidol sensitive sigma site and does not interact significantly with 
the haloperidol-insensitive PCP site, behavioral studies using DTG or 
other substituted guanidines of this invention as prototypical sigma 
ligands, taking into account of whether they are agonists or antagonists 
(see below), should resolve this issue. 
Perhaps the most important aspect of the findings on the drug specificity 
of sigma sites that have emerged from this and other studies is that they 
interact with certain very potent antipsychotic drugs (haloperidol, 
phenothiazines) that are used clinically to treat schizophrenia. This 
intriguing drug selectivity profile facilitates studies aimed at 
investigating the role of sigma receptors in antipsychotic drug action and 
abnormal brain function. The availability of DTG and like 
N,N'-disubstituted guanidines as a selective sigma ligand should serve to 
facilitate such studies. 
The compounds of this invention have highly selective affinity for the 
sigma receptor. Consequently, they may have some of the activities of the 
benzomorphans, i.e., those produced by binding to the 
haloperidol-sensitive sigma receptor but not those produced by the binding 
of benzomorphans to other non-sigma receptors. For instance, benzomorphans 
may act at sigma receptors to cause mydriasis and tachycardia and 
pronounced psychotomimetic effects. DTG is therefore an effective tool to 
demonstrate the physiological effects mediated by the sigma receptor 
which, to date, have been obscured by cross-reactivity of benzomorphans 
with non-sigma receptors. 
The compounds of this invention are particularly valuable in the treatment 
of humans afflicted with a psychotic disease, e.g., schizophrenia, or with 
chronic hypertension. In this regard, they can be employed in 
substantially the same manner as known antipsychotic agents and 
anti-hypertensive agents, respectively. 
Without further elaboration, it is believed that one skilled in the art 
can, using the preceding description, utilize the present invention to its 
fullest extent. The following preferred specific embodiments are, 
therefore, to be construed as merely illustrative, and not limitative of 
the remainder of the disclosure in any way whatsoever. The entire text of 
all applications, patents and publications, if any, cited above and below 
are hereby incorporated by reference. 
EXAMPLES 
In the foregoing and in the following examples, all temperatures are set 
forth uncorrected in degrees Celsius and unless otherwise indicated, all 
parts and percentages are by weight. 
Preparations 
General Procedures. Melting points are uncorrected. N M R spectra were 
recorded on a General Electric QE-300 spectrometer, operating at 300 MHz. 
Chemical shifts (.delta.) are given in ppm using the residual proton 
signal of the deuterated solvent as reference (CHD.sub.2 OD .delta.3.300, 
CHCl.sub.3 .delta.7.260, HDO .delta.4.80), or the .sup.13 C signal of the 
solvent (CD.sub.3 OD .delta.49.00, CDCl.sub.3 .delta.77.00). All solvents 
were reagent grade quality. Where dry solvents were needed, these were 
distilled from CaH.sub.2 or Na before use. 
Adamantan-1-ylcyanamide was prepared from the amine with cyanogen bromide 
in Et.sub.2 O, as previously described by Geluk, N. W., et. al., J. Med. 
Chem. 12:712-716 (1969). (2-Methylphenyl)cyanamide was prepared similarly. 
N,N'-Di-(adamantan-1-yl)guanidine hydrochloride was prepared according to 
the procedure of Geluk et al., supra. 
Example 1 
N,N'-Di-(4-bromo-2-methylphenyl)guanidine (4-Br--DTG) 
To a stirred solution of cyanogen bromide (846 mg, 8.06 mmol) in distilled 
water (70 ml) was added in small portions 2.997 g (16.11 mmol) of 
4-bromo-2-methylaniline (Aldrich, recrystallized from etherpentane). A 
white precipitate formed during the addition. The mixture was stirred at 
80.degree. C. for 4 h. Upon cooling at 0.degree. C. for 12 h, a sticky 
yellow oil separated out and was discarded. The clear aqueous phase was 
concentrated to about 30 ml. The white precipitate which formed was 
redissolved by heating the mixture. This was then set aside at 4.degree. 
C. for 12 h. Filtration gave 430 mg of white solid. A 200-mg portion was 
dissolved in 10 ml of hot water and treated with 5 ml of 10 KOH solution. 
The mixture was extracted with CHCl.sub.3 and the extract was washed with 
brine and then dried (MgSO.sub.4). Evaporation of the solvent gave 171 mg 
of a brown solid which was crystallized from CHCl.sub.3, giving 120 mg 
(8%) 4-Br--DTG as small white needles: mp 209.degree.-210.degree.; NMR 
(300 MHz, CD.sub.3 OD, TMS) .delta.2.24 (s, 3), 7.12-7.25 (AB, 2, J=8 Hz), 
7.35 (s, 1 ); IR (KBr) 3460, 3340, 1630 cm.sup.-1. Analysis calculated for 
C.sub.15 H.sub.15 N.sub.3 Br.sub.2 : C, 45.37; H, 3.81; N, 10.58. Found: 
C, 45.34; H, 3.56; N, 10.50. 
Example 2 
[.sup.3 H]-N,N'-di-ortho-tolyl-guanidine ([.sup.3 H]DTG) 
Twenty-five mg (0.1 mmol) of the thus-produced 4-Br--DTG were submitted to 
Amersham Corporation (Arlington Heights, Ill.) for catalytic reduction in 
the presence of 20 Ci of [.sup.3 H]-gas. Two mCi portions of the crude, 
radioactive product in 0.2 ml/each of 25% ethanol were purified by reverse 
phase high performance liquid chromatography (RP-HPu) on a Vydac TP218 
octadecasilica column using a CH.sub.3 OH gradient (0-35% in 60 minutes) 
in 0.1% trifluoroacetic acid for elution. How rate was 1 ml/min. One 
minute fractions were collected. Aliquots of the fractions were diluted 
100 fold and 10 ul aliquots of the diluted fractions corresponding to 0.1 
ul of the original fractions were dissolved in 10 ml scintillation fluid 
and counted in a scintillation spectrometer. The equipment consisted of 2 
Waters HPLC pumps, and automated electronic gradient controller and a 
Kratos variable wave length spectrophotometer. The radioactivity eluted as 
a major symmetrical peak coinciding with a major, symmetrical UV (220 nm) 
absorbing peak at 41 minutes. This is the same elution time at which 
authentic, unlabeled DTG emerges from the column in this RP HPLC system. 
The specific activity of [.sup.3 H]DTG was found to be 52 Ci/mmol based on 
the amount of DTG under the major UV absorbing peak as determined by 
quantitative UV-spectrophotometry and the amount of radioactivity 
associated with this peak as determined by quantitative liquid 
scintillation spectrometry. 
Following this procedure, or one of another conventional labelling 
synthetic techniques well known in the art, the tritium labeled versions 
of other N,N'-disubstituted guanidines of this invention, e.g., those of 
Table I, can be produced. If only one of the N- and N'-groups employed as 
starting material bears a functional group convertible to an [.sup.3 H] 
bearing group, the resulting N,N'-disubstituted guanidine will be singly 
tagged. If both bear such a functional group, the resulting 
N,N'-disubstituted guanidine will be polytagged. 
Example 3 
N-(2-Methyl-4-isothiocyanatophenyl-N'-(2-methylphenyl)-guanidine 
[Di-tolyl-Guanidine-Isothiocyanate (DIGIT)] 
a. N-(2-Methyl-4-nitrophenyl)-N'-(2-methyl-phenyl)guanidine Hydrochloride 
and the corresponding free base 
A vigorously stirred mixture of 2-methylphenyl-cyanamide [1.107 g, 0.380 
mmol, prepared from o-toluidine by the method of Safter et al. (1948)] and 
2-methyl-4-nitroaniline hydrochloride in chlorobenzene (45 ml) was heated 
at 90.degree. for 3 h and then allowed to cool to 25.degree.. The 
resulting pale yellow precipitate was collected, washed with CH.sub.2 
Cl.sub.2, and dried, giving 2.284 g (91%) of the hydrochloride of the 
desired product (pure by NMR). A 681 mg sample was recrystallized twice 
from absolute EtOH to give 600 mg (88%) of the desired product as pale 
yellow microcrystals suitable for the next reaction: mp 196-200; (CD.sub.3 
OD, 300 MHz) .delta.2.37 (s, 3), 2.47 (s, 3), 7.33-7.40 (m, 4), 7.59 (d, 
1), 8.18 (dd, 1), 8.27 (d, 1). A 473 mg sample of the hydrochloride was 
dissolved in 50 ml of hot water, filtered, cooled to 25.degree., and 
treated with 5 ml of 5N NaOH. The resulting bright yellow precipitate of 
the title compound (free base) was dried (403 mg, 92%) and then 
recrystallized twice from 95% EtOH to give the analytical sample as yellow 
platelets: mp 177-179; (DC.sub.3 OD, 300 MHz) .delta.2.31 (s, 6), 
7.01-7.35 (m, 5), 7.98 (dd, 1), 8.06 (d, 1). Analysis calculated for 
C.sub.15 H.sub.16 N.sub.4 O.sub.2 ; C, 63.37; H, 5.67; N, 19.71. Found: C, 
63.41; H, 5.42; N, 19.90. 
b. N-(2-Methyl-4-aminophenyl)-N'-(2-methylphenyl)-guanidine hydrochloride 
A Parr hydrogenation flask was charged with a solution of 
N-(2-methyl-4-nitrophenyl)-N'-2-(methylphenyl)-guanidine hydrochloride 
(478 mg) in 30 ml of absolute EtOH. 30% Pd on charcoal (71 mg) was added 
and then the mixture was hydrogenated at 60 psi at 25.degree. C. for 12 h. 
All further operations were carried out under an atmosphere of Ar. The 
mixture was centrifuged, filtered through Celite and the filtrate was 
concentrated in vacuo to 2 ml. Ether (21 ml) was added and after 4 h at 
20.degree., the white precipitate was collected and dried, giving 406 mg 
(78%) of the title compound (amine hydrochloride) suitable for the next 
reaction: mp 232.5.degree.-234.5.degree., (CD.sub.3 OD, 300 MHz) 
.delta.2.22 (s, 3), 2.34 (s, 3), 6.60 (dd, 1), 6.66 (d, 1), 6.98 (d, 1), 
7.26-7.38 (m, 4). 
c. N-(2-Methyl-4-isothiocyanatophenyl)-N'-(2-methylphenyl)-guanidine 
Hydrochloride (DIGIT) 
NaOAc (1.767 g, 21.5 mmol) and HOAc (0.839 g, 14.0 mmol) were dissolved in 
dry MeOH such that the final volume of the solution was 100 ml. A 12.7 ml 
aliquot of this solution was added to 199 mg (0.686 mmol) of 
N-(2-methyl-4-aminophenyl)-N'-(2-methylphenyl)guanidine hydrochloride 
under an Ar atmosphere. Thiophosgene (54.7 ul, 86.7 mg, 0.754 mmol, 
freshly distilled) was then injected into the stirred reaction mixture. 
The reaction was complete upon mixing as judged by silica gel TLC 
(500:100:1, CHCl.sub.3 --MeOH--HOAc). Water (20 ml) was added and the 
mixture was concentrated in vacuo to 19 ml. 
The next steps were done in rapid succession in order to minimize the 
possible reaction of the isothiocyanate group with the guanidine free base 
grouping of another molecule. The above 19 ml concentrate was cooled to 
0.degree. and treated with ice-cold saturated NaHCO.sub.3 (15 ml). The 
resulting mixture containing a white precipitate was extracted with 
CHCl.sub.3 (4.times.15 ml). The combined extracts were washed with ice 
cold brine, dried (MgSO.sub.4), filtered through Celite, and cooled to 
0.degree.. Next, excess HCl gas was bubbled through the colorless solution 
and then the solution was concentrated in vacuo to 25 ml and hexane (20 
ml) was added. Four additional times the mixture was concentrated again 
and more hexane was added. Evaporation of the final mixture to dryness 
gave crude hydrochloride as a gummy white solid (263 mg, 115%). This was 
taken up in absolute ethanol (2 ml), and diluted with ether (80 ml) to 
give a cloudy white suspension from which small clusters of white needles 
formed on standing. The crystals were centrifuged, washed with ether 
(3.times.5 ml), and dried, giving 173 mg (76%) of the title compound: m.p. 
195.degree.-197.degree. C. (preheated bath); (CD.sub.3 OD), 300 MHz) 
.delta.2.347 (s, 3), 2.351 (s, 3), 7.23 (d, 1), 7.31 (s, 1), 7.34 (d, 1), 
7.26-7.40 (m, 4); IR (KBr) 3466, 3138, 2927, 2152, 2119, 1646, 1631 
cm.sup.-1. Analysis calculated for C.sub.16 H.sub.17 ClN.sub.4 S: C, 
57.74; H, 5.15; N, 16.83. Found: C, 57.60; H, 5.20; N, 16.64. 
A tri-tritiated version of DIGIT is prepared starting with tritiated 
N-(2-methyl-4-aminophenyl)-N'-(2-methylphenyl)-guanidine, which was 
prepared by catalytic tritiation (Amersham) of 
N-(2-methyl-4-nitro-6-bromophenyl)-N'-(2-methyl-4,6-dibromophenyl)-guanidi 
ne. The tri-tritiated amino compound was also used as the immediate 
precursor for the preparation of the tritritiated version of the 4-azido 
compound the preparation of which is described hereinafter and which was 
used in the solubilization of sigma receptors. 
Example 4 
N, Adamantan-1-yl-N'-cyclohexylguanidine HCl 
Adamantan-1-ylcyanamide (514 mg, 2.92 mmol), and cyclohexylamine 
hydrochloride (398 mg, 2.93 mmol) were finely ground together, and heated 
at 200.degree. C. for 10 min. The resultant glassy solid was pulverized, 
and extracted twice with 50 ml boiling 5% HCl. The insoluble material was 
filtered, and dried. 346 mg, mp 264.degree.-270.degree. (p.h.b. 
240.degree. C.). On cooling the combined aqueous extracts to 25.degree., a 
white ppt. formed, and was filtered off, 56 mg. Combined crude yield: 44%. 
Crystallization of a 50 mg sample of the crude product from EtOH/Et.sub.2 
O afforded white needles (25 mg, mp 269.degree.-271.degree. C. (p.h.b. 
240.degree.), lit. 268.degree.-269.degree. C.). .sup.1 H (CD.sub.3 OD) 
.delta.1.208-1.474 (m, 5H), 1.744 (s, 9H), 1.967 (s, 8H), 2.119 (5, 3H), 
3.434 (m, 1H), .sup.13 C (CD.sub.3 OD) (broad band decoupled) 25.55, 
26.26, 30.96, 33.74, 36.74, 42.73. 
Example 5 
N-Cyclohexyl-N'-(2-methylphenyl)guanidine 
A suspension of cyclohexylamine hydrochloride (310 mg, 2.28 mmol) and 
(2-methylphenyl)cyanamide (365 mg, 2.76 mmol) in dry chlorobenzene was 
heated at 125.degree.-135.degree. for 4 hrs. The solvent was evaporated in 
vacuo with heating, and the residue partitioned between CH.sub.2 Cl.sub.2 
and 20.times.8 ml 10% HCl. The aqueous extracts were made alkaline to pH 
9-9.5, and the white precipitate collected after standing at 4.degree. C. 
overnight. Two recrystallizations from EtOH/H.sub.2 O gave 140 mg (22%) of 
white needles, mp 145.degree.-146.degree., 1H (CD.sub.3 OD) 
.delta.1.149-2.054 (m, 10H), 2.164 (s, 3H), 3.50 (m, 1H), 6.875 (d, 1H, 
J=7.5 Hz), 6.968 (t, 1H, J=7.5 Hz), 7.118 (t, 1H, J=7.5 Hz), 7.177 (d, 1H, 
J=7.5 Hz). Anal. Calcd for C.sub.14 H.sub.21 N.sub.3 : C, 72.69; H, 9.15; 
N, 18.16. Found: C, 72.72; H, 9.24; N, 18.18. 
Example 6 
N-(Adamantan-1-yl)-N'-(2-methylphenyl)guanidine 
A suspension of adamantan-1-ylcyanamide (151 mg, 0.857 mmol) and 
o-toluidine hydrochloride (186 mg, 0.857 mmol) in dry chlorobenzene was 
heated between 100.degree.-130.degree. C. for 4.5 hrs. The chlorobenzene 
was evaporated in vacuo with heating and the residue (285 mg) taken up in 
18 ml of water. A gummy, insoluble material was discarded. On adjusting 
the aqueous extract to pH 9-9.5, a precipitate formed (194 mg, 71%). Two 
recrystallizations from EtOH/H.sub.2 O gave the analytical sample; mp 
160.degree.-161.degree., .sup.1 H (CD.sub.3 OD) .delta.1.742 (s, 6H), 
2.050 (d, 6H, J=2.4 Hz), 2.029 (s, 3H), 6.956 (j, 1H, J=8.1 Hz), 7.056 (t, 
1H, J=8.1 Hz), 7.168 (t, 1H, J=7.5 Hz), 7.213 (d, 1H, J=7.5 Hz). Anal. 
Calcd for C.sub.18 H.sub.25 N.sub.3 : C, 76.28; H, 8.89; N, 14.83. Found: 
C, 76.25; H, 8.86; N, 14.60. 
Example 7 
N-(Adamantan-1-yl)-N'-(2-iodophenyl)guanidine HCl 
Adamantan-1-ylcyanamide (299 mg, 1.70 mmol) and o-iodoaniline hydrochloride 
(433 mg 1.70 mmol) were finely ground together and heated at 200.degree. 
for 10 min. The resultant black glassy solid was pulverized, and 
recrystallized 5 times from EtOH/Et.sub.2 O. The resultant faintly blue 
needles (80 mg) were dissolved in 4 ml EtOH, and passed through a short 
(&lt;1 cm) column packed from bottom to top with Celite, charcoal, activated 
alumina, and sand. The colorless eluate was then diluted with 5 ml 
Et.sub.2 O, and allowed to stand in an Et.sub.2 O diffusion chamber 
overnight. The white needles were collected by suction filtration, 40 mg, 
mp 274.degree.-275.degree. C. Addition of another 5 ml Et.sub.2 O yielded 
a second crop (31 mg) of identical material. Combined yield: 21%. .sup.1 H 
(CD.sub.3 OD) .delta.1.779 (s, 6H), 2.090 (s, 6H), 2.164 (s, 3H), 7.168 
(t, 1H, J=8.1 Hz), 7.388 (d, 1H, J=9.1 Hz), 7.503 (t, 1H, J=7.8 Hz), 8.004 
(d, 1H, J=7.8 Hz). 
Example 8 
N-(2-Methyl-4-azido-phenyl)-N'-(2-methylphenyl)guanidine 
N-(2-Methylphenyl)-N'-(2-methyl-4-aminophenyl)guanidine dihydrochloride 
(112 mg, 0.341 mmol) were dissolved in 2 ml H.sub.2 O and 100 ul cone. HCl 
(1.2 mmol). The solution was cooled in an ice bath, and a solution of 
NaNO.sub.2 (42 mg, 0.61 mmol) in 450 ul H.sub.2 O were added. The reaction 
mixture turned yellow, and was stirred for 45 min before solid NaN.sub.3 
(43 mg, 0.661 mmol) was added in a single portion. After N.sub.2 evolution 
was caused, a foamy solid (11 mg) was removed and discarded. Solid NaOH 
(96 mg, 2.41 mmol) was added, and the bright yellow precipitate was 
extracted with Et.sub.2 O (3.times.5 ml). Evaporation of the combined 
Et.sub.2 O layers gave a yellow solid which was crystallized from 
EtOH/H.sub.2 O: NMR (CD.sub.3 OD) .delta.2.279 (s, 3H), 2.284 (s, 3H), 
6.864 (dd, 1H, J=2.4 Hz, 8.4 Hz), 6.914 (d, 1H, J=2.1 Hz), 7.055 (td, 1H, 
J=1.8 Hz, 7.2 Hz), 7.136-7.229 (m, 4H). 
Example 9 
N-(2-Methyl-4-nitro-6-bromophenyl)-N'-(2-methyl-4,6-dibromophenyl)guanidine 
N-(2-Methyl-4-nitrophenyl)-N'-(2-methylphenyl)guanidine, as the free base 
(281 mg, 0.987 mmol) was dissolved in 4 ml MeOH, and cooled in an 
ice-bath. N-bromosuccinimide (freshly recrystallized from H.sub.2 O) (531 
mg, 2.98 mmol) was added in two portions over 15 min. After 1.5 hrs the 
brown sludgy reaction mixture was diluted with 4 ml MeOH, and allowed to 
warm to 25.degree. C. A brown solid was filtered off (266 mg), and 
crystallized from acetone/H.sub.2 O, to afford brown needles (2.6 mg, 42%, 
mp 193.degree.-195.degree. C.). Sublimation of a 56 mg sample of these 
crystals at 0.01 mm Hg and 170.degree. afforded the analytical sample as a 
bright yellow amorphous solid (38 mg, mp 210.degree.-213.degree. C.). NMR 
(CD.sub.3 OD) .delta.2.357 (s, 3H), 2.488 (s, 3H), 7.444 (d, 1H, J=1.5 
Hz), 7.669 (d, 1H, J=1.8 Hz), 8.033 (d, 1H, 2.1 Hz), 8.267 (d, 1H, 2.4 
Hz). Anal. Calcd for C.sub.15 N.sub.13 Br.sub.3 N.sub.4 O.sub.2 : C, 
34.58; N, 2.52; N, 10.75. Found: C, 34.64; N, 2.41; N, 10.65. 
Example 10 
N,N'-Bis(2-iodophenyl)guanidine 
A solution of cyanogen bromide (4.4042 g, 38.2 mmol) and 2-iodoaniline 
(4.138 g, 18.9 mmol) in H.sub.2 O (70 ml) was heated at 
70.degree.-80.degree. C. for 5 h. The reaction mixture was decanted from 
an off-white solid (1.90 g) which was discarded, and the supernatant was 
heated at the same temperature an additional 16 h. On cooling to 
25.degree. C., the title compound precipitated from solution as its 
hydrobromide salt and was centrifuged off, and dried (500 mg, 10%). This 
white powder was dissolved in boiling H.sub.2 O (20 ml), and 5N NaOH (2 
ml) was added to the clear solution. The resulting white precipitate (290 
mg) was washed with H.sub.2 O (3.times.4 ml), and crystallized from 95% 
EtOH, to give the title compound (119 mg, 39% from the hydrobromide salt) 
as long white needles: mp 161.degree.-162.degree. C. One further 
crystallization provided the analytical sample: mp 161.degree.-162.degree. 
C. Anal. Calcd for C.sub.13 N.sub.11 N.sub.3 I.sub.2 : C, 33.72; N, 2.39; 
N, 9.07. Found: C, 33.80; N, 2.26; N, 8.78. .sup.1 N NMR: .delta.6.790 (t, 
J=7.8 Hz, 2H), 7.304 (t, 3=7.8 Hz, 2H), 7.506 (d, 3=7.8 Hz, 2H), 7.817 (d, 
3=7.8 Hz, 2H). IR: 729, 753, 1467, 1502, 1572, 1613, 1647, 3056, 3387 
cm.sup.-1. 
Example 11 
N,N'-Bis(3-methylphenyl)guanidine 
Cyanogen bromide (788 mg, 7.44 mmol) was placed in a 25 ml round bottom 
flask, and m-toluidine (1.89 g, 17.6 mmol) was added dropwise. After the 
exothermic reaction had subsided, the residue was taken up in CH.sub.2 
Cl.sub.2 (20 ml), and was extracted with 5% HCl (5.times.10 ml). The 
aqueous extracts were adjusted to pH 10 with 6N NaOH. The resulting 
precipitate (674 mg, 38%) was filtered off and crystallized from 
EtOH/H.sub.2 O to give the title compound (240 mg, 14%) as white needles: 
mp 105.degree.-106.degree. C. .sup.1 H NMR: .delta.2.289 (s, 6H), 6.814 
(d, 2H, J=7.5 Hz), 6.939 (d, 2H, J=7.5 Hz), 6.981 (s, 2H), 7.141 (t, 2H, 
J=7.5 Hz). Anal. Calcd for C.sub.15 H.sub.17 N.sub.3 : C, 75.28; H, 7.16; 
N, 17.56. Found: C, 75.42; H, 7.11; N, 17.43. 
References 
.sup.1 Geluk, H. W., et al., J. Med. Chem. 12:712 (1969). 
.sup.2 Kazarinova, N. F., et al., Zn. Anal. Khim. 28:1853 (1973); Chem. 
Abstr. 80:97021 (1973). 
Example 12 
Synthesis of N,N'-Di(o-tolyl)-2-imino-imidazolidine 
a. Synthesis of N,N' Ditolyl oxalodiamide 
Oxalyl chloride (32 mmol) in methylene chloride (16 mL) was added dropwise 
to a solution of o-toluidine (67 mmol) in methylene chloride (4 mL) over a 
period of 10 min at 4.degree. C. After the exotherm subsided, the solution 
was removed from the ice bath and stirred at ambient temperature for 2 h. 
A white precipitate had formed. The precipitate was filtered off, dried, 
and found to weigh 724.6 mg (90%). The amide and the hydrooxalate salts 
were partially dissolved in methylene chloride (20 mL) and extracted with 
1N HCl (5.times., 15 mL). The resultant white suspension was filtered to 
provide a white solid (61%, mp 103.degree.-104.degree. C.). .sup.1 H NMR 
(CD.sub.3 CN/DMSO): .delta.2.259 (6H, s), 7.128-7605 (8H, m). IR (KBr): 
1298.7, 1642.9 (amide). 
b. Synthesis of N,N' Bistolyl ethylene diamine 
The following procedure was adapted from H. C. Brown, J. Org. Chem. 38:912 
(1978). Diborane (5 mmol) in THF was added dropwise to a THF solution of 
N,N' Ditolyl oxalodiamide (443.0 mg, 1.65 mmol) over 10 min at 0.degree. 
C. After 30 min, the reaction mixture was allowed to stir at ambient 
temperature for 6 h. Then the solution was refluxed for 3 h. The resulting 
yellowish solution was allowed to cool to 25.degree. C. The solution was 
then acidified dropwise with 15% HCl (15 mL) via an additional funnel over 
20 min. Gas evolution was noted and a white precipitate had formed. The 
THF was then distilled off from the water through rotoevaporation at 
25.degree. C. The water was then made basic with an excess of NaOH and 
then extracted with ether. The ether layer was washed with brine, then 
dried over anhydrous potassium carbonate. The ether layer was then 
concentrated to dryness to provide a tannish brown liquid. The liquid was 
immediately taken up again in dry ether. Following this procedure, the 
solution was made acidic by adding ethereal HCl (10 mL) dropwise. A white 
solid had formed. This solid was immediately filtered off and dissolved in 
ethanol (5 mL) and placed into an ether diffusion chamber. After 2 days, 
white prisms were found (158.1 mg, 30.6%), mp 268.degree.-270.degree. C. 
IR (KBr): in comparison with IR of diamide the bands at 1643.8 and 1298.7 
cm.sup.-1 had disappeared. 
c. Synthesis of N,N' Bistolyl-2-imino-imidazolidine 
N,N' Bistolyl ethylene diamine (105.8 mg, 0.44 mmol) was taken up in EtOH 
(3 mL) to provide a light purple solution. This solution was then placed 
in a one-necked round-bottom flask (25 mL) equipped with magnetic stirbar 
and reflux condenser. To this solution, cyanogen bromide (50 mg, 0.47 
mmol) in ethanol (2 mL) was added in a single portion. The resultant 
reaction mixture was stirred for 1 h. It was noted that the solution 
turned clear. The solution was then brought to reflux and maintained at 
that temperature for 16 h. The reflux condenser was then removed, allowing 
the solvent to evaporate. The reaction mixture was then fused at 
150.degree. C. for 30 min to provide a brown solid. This solid was 
immediately taken up in ethanol (4 mL) and placed into a centrifuge tube. 
To this solution 1N NaOH was added (8 mL) to provide a "whispy" tan 
precipitate. After several failed attempts to pellet the precipitate, the 
solution was simply extracted with chloroform (20 mL). The chloroform was 
concentrated to dryness to provide a clear oil (108.1 mg, 92.6%). 
Example 13 
Synthesis of N-Cyclohexyl-N'-(2-methyl-4-bromophenyl)guanidine 
A solution of bromine (0.336 g, 2.1 mmol) in glacial acetic acid (1 ml) was 
added dropwise to a stirred solution of 
N-cyclohexyl-N'-(2-methylphenyl)guanidine (0.231 g, 1 mmol) in glacial 
acetic acid (2 ml) at room temperature. After the addition, the addition 
funnel was replaced by a reflux condenser and the red reaction solution 
was heated on a warm water bath at 70.degree. C. for about an hour. The 
reaction mixture was allowed to cool to room temperature and then poured 
in cold ice water (30 ml) containing a 200 mg of sodium bisulfite. The 
mixture was then extracted with dichloromethane (3.times.15 ml) and the 
combined organic layers were dried over sodium sulfate, filtered and 
concentrated to give a colorless liquid (0.475 g). This liquid was 
purified on a flash silica gel column to give a bright foamy product (0.25 
g), mp 89.degree.-91.degree. C. 
.sup.1 H NMR (CDCl.sub.3): .delta.7.4 (s, 1H, Ar-3-H); 7.32 (dd, 1H, J=8.29 
and 1.52 Hz, Ar-5-H); 6.99 (d, 1H, J=8.34 Hz, Ar-6-H); 2.22 (s, 
3H--Ar--CH3) and 1.95-1.11 (m, 11H, cyclohexyl). 
Mass (CI): 310, 312 (M.sup.+), 232 (M.sup.+ --Br). 
Example 14 
Synthesis of 
N-(Adamantan-1-yl)-N'-(2-trifluoromethyl-4-fluorophenyl)guanidine 
1-Adamantylcyanamide--A 2 liter glass three neck round bottom flask 
equipped with a mechanical stirrer, thermometer and 250 ml pressure 
equalizing addition funnel was charged with 1-adamantanamine (72.25 g, 
0.48 mol), anhydrous ethyl ether (1L) and the solution cooled in an ice 
bath (0.degree.-5.degree. C.). The addition funnel was charged with a 
solution of cyanogen bromide (327.0 g, 0.31 mol) in anhydrous ethyl ether 
(200 ml). The reaction apparatus was purged with N.sub.2 and a N.sub.2 
atmosphere maintained throughout the reaction. The cyanogen bromide was 
added dropwise with stirring so as to maintain the temperature of the 
reaction mixture below 5.degree. C. The addition required 35 minutes and 
upon completion the ice bath was removed and the reaction mixture allowed 
to reach room temperature. The reaction mixture was allowed to stir 
overnight. The reaction mixture was filtered (sintered glass funnel) to 
remove the adamantylamine hydrobromide and the filtrate concentrated in 
vacuo (Buchi Rotovap RE 111) at 10 torr (aspirator vacuum) to afford the 
crude product. The crude product was dissolved in hot ethanol (100 ml) and 
distilled water (200 ml, room temperature) was added or until the point 
where turbidity was observed. The flask and contents were placed in the 
refrigerator over night. The product crystals were collected by filtration 
and air dried briefly (15 min). The product was then dried in a large 
capacity Abderhalden drying pistol at room temperature at 0.1 mm for 48 h 
to afford 46.01 g of 1-adamantylcyanamide. 
N-(Adamantan-1-yl)-N-(2-trifluoromethyl-4-fluorophenyl)guanidine 
Hydrochloride. A single neck 50 ml round bottom flask was charged with 
chlorobenzene (14 ml), 2-trifluoromethyl-4-fluoroaniline hydrochloride (L. 
M. Weinstock et al., Tet. Lett. 1419 (1978)) (1.0 g, 4.64 mmol) and 
1-adamantylcyanamide (0.82 g, 4.64 mmol). The reaction apparatus was 
purged with N.sub.2, and the reaction heated at reflux under N.sub.2, 
whereupon a clear colorless solution resulted. Within minutes, a thick 
white precipitate had formed. After 4.5 hr, the heating mantle was removed 
and after cooling to room temperature diethyl ether (30 ml) was added and 
the white solid (1.3 g) was collected by filtration. A portion of this 
white solid (0.6 g) was crystallized from iso-propanol and the resulting 
white crystals were filtered and dried in vacuo at 100.degree. C. to give 
the title product (0.35 g), mp 260.degree.-61.degree. C. 
Anal. Calcd for C.sub.18 H.sub.22 N.sub.3 ClF.sub.4 (391.83): C, 55.17; H, 
5.66; N, 10.72. Found: C, 55.32; H, 5.58; N, 10.88. 
Example 15 
Synthesis of N-(Adamantan-1-yl)-N'-(2-trifluoromethylphenyl)guanidine) 
A single neck glass 100 ml round bottom flask was charged with 
chlorobenzene (25 ml), o-(trifluoromethyl)-aniline hydrochloride (prepared 
from the free base, Aldrich, by dissolving in 5% HCl in MeOH and 
concentrating, 5.93 g, 30.0 mmol) and 1-adamantylcyanamide (5.29 g, 30.0 
mmol). 
The reaction apparatus was purged with N.sub.2 and the reaction heated at 
reflux under N.sub.2, whereupon a clear colorless solution resulted. 
Within minutes a thick white precipitate had formed and an additional 15 
ml of chlorobenzene was added to facilitate more efficient stirring. After 
3 h, the heating mantle was removed and after cooling to room temperature 
the white solid was collected by filtration. This material was dissolved 
in boiling EtOH, filtered to remove insolubles, and ether added to the 
filtrate until turbid. The white solid which crystallized on standing was 
collected by filtration and dried in vacuo at room temperature to give the 
title product mp 259.degree.-260.degree. C. 
Anal. Calcd for C.sub.18 H.sub.23 N.sub.3 ClF.sub.3 (373.83): C, 57.82; H, 
6.20; N, 11.24. Found: C, 57.67; H, 6.20; N, 11.46. 
Example 16 
Synthesis of N-(Adamantan-1-yl)-N'-(2,4-difluorophenyl)guanidine 
A single neck glass 100 ml round bottom flask was charged with 
chlorobenzene (20 ml), 2,4-difluoroaniline hydrochloride (prepared from 
the free base, Aldrich, by dissolving in 5% HCl in MeOH and concentrating, 
2.0 g, 12 mmol) and 1-adamantylcyanamide (2.13 g, 12 mmol). 
The reaction apparatus was purged with N.sub.2, and the reaction heated at 
reflux under N.sub.2, whereupon a clear colorless solution resulted. 
Within minutes a thick white precipitate had formed. After 2 h, the 
heating mantle was removed and after cooling to room temperature the white 
solid was collected by filtration. This material was recrystallized two 
times from ethanol and the resultant white solid dried in vacuo at room 
temperature to give the title compound mp 258.degree.-260.degree. C. 
Anal. Calcd for C.sub.17 H.sub.22 N.sub.3 ClF.sub.2 (341.82): C, 59.73; H, 
6.49; N, 12.29. Found: C, 59.62; H, 6.44; N, 12.23. 
Example 17 
Synthesis of N,N'-Bis (3-ethylphenyl)-2-imino-imidazolidine 
N,N'-di-(3-ethylphenyl)oxalodiamide Oxalyl chloride (19 mmol) in methylene 
chloride (16 ml) was added dropwise to a solution of 3-ethylaniline (40 
mmol) in methylene chloride (4 ml) over a period of 10 minutes at 
4.degree. C. After the exotherm subsided, the solution was removed from 
the ice bath and stirred at 25.degree. C. for 2 hours. The reaction 
mixture was diluted with another 40 ml of CH.sub.2 Cl.sub.2. The methylene 
chloride solution was washed by aqueous HCl (1N, 40 ml.times.2), aqueous 
NaOH (3%, 30 ml.times.1), and brine (20 ml), dried, and concentrated. The 
crude product was washed with CH.sub.2 Cl.sub.2 (10 ml) as well as aqueous 
HCl (1N, 5-8 ml), the the precipitate was collected by filtration and 
dried under vacuum to provide a white solid 
N,N'-di(3-ethylphenyl)-oxalodiamide in 55% yield. Mp 131.degree. C.; 
.sup.1 H NMR (60 MHz, CDCl.sub.3): .delta.1.2 (t, 6H), 2.6 (q, 4H), 
6.8-7.6 (m, 8H). IR (CH.sub.2 Cl.sub.2)=1690, 1520 cm.sup.-1. 
N,N'-Bis-(3-ethylphenyl)ethylene diamine Diborane (27.4 mmol, 27.4 ml, 1M) 
in THF was added dropwise to a THF solution of 
N,N'-di-(3-ethylphenyl)oxalodiamide (6.76 mmol, 2 g) over 10 minutes at 
0.degree. C. After 30 minutes, the reaction mixture was refluxed for 16 
hours. The resulting yellowish solution was allowed to cool to 25.degree. 
C. The solution was then acidified by adding aqueous HCl (15%, 15 ml) over 
20 minutes; gas evolution was noted and a white precipitate had formed. 
The THF was then distilled off from the water through rotoevaporation at 
25.degree. C. The water was then made basic with excess of NaOH and then 
extracted with CH.sub.2 Cl.sub.2. The CH.sub.2 Cl.sub.2 layer was washed 
with brine, then dried over anhydrous potassium carbonate. The CH.sub.2 
Cl.sub.2 layer was then concentrated to dryness to provide an oil in 66% 
yield. .sup.1 H NMR (60 MHz,, CDCl.sub.3): .delta.1.2 (t, 6H), 2.5 (q, 
4H), 3.2 (s, 4H), 6.3-7.2 (m, 8H). IR (CH.sub.2 Cl.sub.2)=1420, 1600 
cm.sup.-1. 
N,N'-Bis (3-ethylphenyl)-2-imino-imidazolidine 
N,N'-Bis(3-ethylphenyl)-ethylene diamine (0.4 g, 1.49 mmol) in EtOH (2 ml) 
was placed in a one necked round bottom flask equipped with a magnetic 
stir bar and reflux condenser. To this solution, cyanogen bromide (174 mg, 
1.64 mmol) was added in a single portion. The solution was then brought to 
reflux for 15 hours. After reaction, the mixture was concentrated and 
recrystallized in MeOH/ether to yield a white crystal in 54% yield. MP 
155.degree. C.; .sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.1.25 (t, J=7.6 
Hz, 6H), 2.72 (q, J=7.6 Hz, 4H), 4.36 (s, 4H), 7.21-7.39 (m, 8H). .sup.13 
C NMR (300 MHz, CDCl.sub.3): 15.08, 28.47, 49.45, 122.40, 124.48, 128.62, 
130.31, 134.91, 147.00, 154.39. IR (CH.sub.2 Cl.sub.2)=1470, 1550 
cm.sup.-1. Elemental analysis. Calcd: C, 58.76; H, 6.49; N, 10.82. Found: 
C, 59.14; H, 6.83; N, 10.85. 
Example 18 
Characteristics of [.sup.3 H]DTG binding to guinea pig brain membranes 
Synthesis [.sup.3 H]DTG resulted in a pure homogenous product of high 
specific radioactivity (52 Ci/mmol). [.sup.3 H]DTG bound specifically, 
saturably, reversibly, and with high affinity to guinea pig brain 
membrane. In a typical experiment with 0.9 nM [.sup.3 H]DTG (30,000 cpm, 
50% counting efficiency) the total binding was 2700 cpm while the 
nonspecific binding in the presence of 10 uM DTG or 10 uM haloperidol was 
50-150 cpm. Routinely, a specific binding to 90-97% of total binding was 
observed. At room temperature the binding of [.sup.3 H]DTG reached 
equilibrium after 60-90 minutes and it was fully reversible after addition 
of 10 uM unlabeled DTG. Specific binding was linear with tissue 
concentration between 2-40 mg tissue (original wet brain weight per assay 
tube). Binding of radioactivity to the glass fiber filters in the absence 
of membranes was 10-20 cpm. Boiling of membranes at 100.degree. C. for 10 
minutes prior to assay almost completely (90%) abolished specific [.sup.3 
H]DTG binding as did treatment of the membranes with trypsin and pronase 
(0.01 mg/ml for 30 min at room temperature), indicating that protein 
components are important for the receptors binding ability. 
To determine the equilibrium saturation binding of [.sup.3 H]DTG to guinea 
pig brain membranes, membranes prepared as described herein were incubated 
with [.sup.3 H]DTG at various concentrations from 0.3 nM to 90 nM in 1 ml 
50 nM Tris/HCl buffer, pH 7.4, for 120 minutes at room temperature. Values 
obtained were the mean of quadruplicate determinations. 
A Scatchard analysis of the saturation data shows a linear Scatchard plot 
with an apparent K.sub.D of 28 nM and a maximum number of binding sites 
(Bmax) of 85 pmol/g brain tissue (original wet weight). Analysis of the 
binding data with the curve fitting program LIGAND, Munson, P. J., et al., 
Anal. Biochem. 107:220-239 showed high compatibility with a one site 
binding model. 
Example 19 
Radioligand Binding Assays 
Frozen guinea pig brains (Pel-Freeze, Rogers, Ariz.) were homogenized in 10 
volumes (w/v) of 0.32M sucrose using a Polytron homogenizer. The 
homogenate was spun at 900.times.g for 10 minutes at 4.degree. C. The 
supernatant was collected, and spun at 22,000.times.g for 20 minutes at 
4.degree. C. The pellet was resuspended in 10 volumes of 50 mM Tris/HCl 
buffer, pH 7.4, incubated at 37.degree. C. for 30 minutes and spun again 
at 22,000.times.g for 20 minutes at 4.degree. C. The pellet was then 
resuspended in 10 volumes of 50 mM Tris/HCl buffer, pH 7.4 and 10 ml 
aliquots of this membrane suspension were stored frozen at -70.degree. C. 
until used in the binding assay. No effects of prolonged storage (&gt;3 
months) of the membranes at -70.degree. C. on sigma receptor number or 
affinity for [.sup.3 H]DTG binding were observed. 
For radioreceptor assays aliquots of the frozen membrane suspension were 
thawed and diluted tenfold with 50 nM Tris/HCl buffer, pH 7.4. To 
12.times.75 mm polystyrene or glass test tubes were added 0.8 ml of 
membrane suspension, 0.1 ml [.sup.3 H]DTG or (+)[.sup.3 H]3-PPP for a 
final concentration of 0.9 nM, and 0.1 ml of unlabeled drugs or buffer. 
The protein concentration in the 1 ml final incubation volume was 800 ug, 
corresponding to 32 mg of brain tissue (original wet weight). Nonspecific 
binding was defined as that remaining in the presence of either 10 uM DTG 
or haloperidol for both the [.sup.3 H]DTG and the (+)[.sup.3 H]3-PPP 
binding. After incubation for 90 minutes at room temperature the membrane 
suspension was rapidly filtered under vacuum through Whatman GF/B glass 
fiber filters using a Brandel 48 well cell harvester (Brandel, 
Gaithersburg, Md.). The filters were washed with 3.times.5 ml ice-cold 50 
nM Tris buffer (pH 7.4 at room temperature). The filters were dissolved in 
10 ml each of Cytoscint (Westchem Products, San Diego, Calif.) and 
radioactivity was measured by liquid scintillation spectrometry at a 
counting efficiency of 35-50%. Saturation data were evaluated by Scatchard 
analysis using both the EBDA NcPherson, G. A., Computer Programs Biomed. 
17:107-114 (1983), and LIGAND Munson, P. J., et al., Anal. Biochem. 
107:220-239 (1980), data analysis programs on an IBM Personal Computer-AT. 
IC.sub.50 values were determined by plotting displacement curves onto 
semilogarithmic graph paper followed by interpolation or by computerized 
non-linear least squares curve fitting (Fischer, J. B. et al., J. Biol. 
Chem. 263:2808-2816 (1988). 
Utilizing the radioligand binding assay described above, the sigma receptor 
binding activity based on [.sup.3 H]DTG displacement activity (IC.sub.50 
value) for the sixteen (16) N,N'-disubstituted guanidine compounds listed 
in Table I below was determined. The IC.sub.50 value for each compound is 
reported in Table I. 
TABLE I 
______________________________________ 
IC.sub.50 vs 
Compound [.sup.3 H]DTG (nM) 
______________________________________ 
N,N'-di-o-tolyl-guanidine 
32 .+-. 1 
N,N'-di-n-butyl-guanidine 
750 .+-. 33 
N,N'-diphenyl-guanidine 397 .+-. 21 
N,N'-diadamantyl-guanidine 
17 .+-. 3 
N-adamantyl-N'-2-methylphenyl-guanidine 
3 .+-. 1 
N,N'-di(2-methyl-4-bromophenyl)guanidine 
37 .+-. 3 
N-(2-iodophenyl)-N'-(2-methylphenyl)- 
21 .+-. 1 
guanidine 
N-(2-methyl-4-nitrophenyl)-N'-(2-methyl- 
37 .+-. 5 
phenyl)-guanidine 
N,N'-di-(2,6-dimethylphenyl)guanidine 
90 .+-. 18 
N-(2,6-dimethylphenyl)-N'-(2-methylphenyl)- 
70 .+-. 3 
guanidine 
N-(adamantyl)-N'-(cyclohexyl)guanidine 
13 .+-. 2 
N,N'-di(cyclohexyl)guanidine 
71 .+-. 7 
N-(2-iodophenyl)-N'-(adamantyl)guanidine 
5 .+-. 1 
N-(2-methylphenyl)-N'-(cyclohexyl)guanidine 
17 .+-. 5 
N-(2-methyl-4-azidophenyl)-N'-(2-methyl- 
20 .+-. 1 
phenyl)-guanidine 
N-(2-methylphenyl)-N'-(4-amino-2-methyl- 
280 .+-. 14 
phenyl)-guanidine 
______________________________________ 
Fourteen of these compounds were found to be potent ligands of sigma 
receptors as determined by their ability to displace [.sup.3 H]DTG from 
sigma receptors in guinea-pig brain homogenates, the most potent being 
N-(2-methylphenyl)-N'-(adamantan-1-yl)guanidine with an IC.sub.50 of 
2.6.+-.0.6 nM (n=6). 
In a second series of experiments, selected compounds were tested against 
[.sup.3 H]MK-801 and [.sup.3 H]DTG. The results depicted in Table II are 
as follows (nM): 
TABLE II 
______________________________________ 
IC.sub.50 vs* 
Chemical name [.sup.3 H]DTG 
[.sup.3 H]MK-801 
______________________________________ 
N-(1-Naphthyl)-N'- 
40.1 .+-. 
7.3 (4) 209 .+-. 
50 (4) 
(2-iodophenyl)-guanidine 
N-(Cyclohexyl)-N'-(4- 
4.67 .+-. 
1.04 (4) 
23860 .+-. 
8540 (5) 
bromo-2-methylphenyl)- 
guanidine 
N,N'-Di-(4-indanyl)- 
28.5 .+-. 
7.5 (3) 506 .+-. 
99 (2) 
guanidine 
N-(Adamantan-1-yl)-N'-(2- 
7.44 .+-. 
0.50 (3) 
23100 .+-. 
13100 (2) 
trifluoromethyl-phenyl)- 
guanidine 
N-(Adamantan-1-yl)-N'-(2- 
22.6 .+-. 
1.9 (4) not done 
methylphenyl)-N'-methyl- 
guanidine 
N-(Adamantan-1-yl)-N'-(6- 
21.9 .+-. 
0.7 (4) &gt;10,000 (2) 
coumarinyl)-guanidine 
N-(Adamantan-1-yl)-N'-(8- 
43.9 .+-. 
2.0 (4) 6,700 (1) 
coumarinyl)-guanidine 
N-(Adamantan-1-yl)-N'- 
8.92 .+-. 
0.48 (4) 
&gt;10,000 (1) 
(2,4-difluorophenyl)- 
guanidine 
N-(Adamantan-1-yl)-N'-(2- 
4.27 .+-. 
0.45 (4) 
31,900 (1) 
trifluoromethyl-4-fluoro- 
phenyl)-guanidine 
______________________________________ 
*[s.e.m. (n) 
Example 20 
Drug Specificity of [.sup.3 H]DTG Binding 
Displacement experiments were performed with drugs that are considered 
typical sigma ligands, as well as with drugs considered to be prototypical 
ligands for other neurotransmitter, neuromodulator, and drug receptors. 
The IC.sub.50 vs [.sup.3 H]DTG and vs. [.sup.3 H]3-PPP results are given 
in Table III below. These experiments showed that the [.sup.3 H]DTG 
binding site is stereoselective for dextrorotatory benzomorphan opiates 
and for (-)butaclamol; does not significantly interact with drugs that 
have high affinities for acetylcholine, benzodiazepine, GABA, nor with mu, 
delta, or kappa opioid receptors; has a high affinity for haloperidol and 
several drugs belonging to the phenothiazine class of antipsychotics 
(haloperidol had the highest displacement potency of all drugs tested); 
and has a moderate affinity for several other classes of psychoactive 
drugs, which included several tricyclic antidepressants, PCP, and the 
kappa opioid receptor ligand U50, 488H. 
TABLE III 
______________________________________ 
IC.sub.50 vs. 
IC.sub.50 vs. 
[.sup.3 H]DTG (nM) 
(+)[.sup.3 H]3-PPP (nM) 
Drug (.+-. SEM) (.+-. SEM) 
______________________________________ 
Haloperidol 5 .+-. 0.3 17 .+-. 1 
DTG 28 .+-. 1 53 .+-. 9 
Perphenazine 42 .+-. 10 21 .+-. 3 
(+)Pentazocine 
43 .+-. 2 8 .+-. 3 
(-)Pentazocine 
135 .+-. 3 81 .+-. 1 
(.+-.)Pentazocine 
69 .+-. 1 ND 
(+)3-PPP 76 .+-. 4 33 .+-. 12 
(-)3-PPP 280 .+-. 21 235 .+-. 
60 
(+)Cyclazocine 
365 .+-. 25 47 .+-. 12 
(-)Cyclazocine 
2,600 .+-. 
210 1,000 .+-. 
0 
Spiperone 690 .+-. 21 ND 
(-)Butaclamol 
530 .+-. 49 183 .+-. 
5 
(+)Butaclamol 
2,150 .+-. 
250 2,100 .+-. 
71 
(+)SKF 10,047 
625 .+-. 88 93 .+-. 5 
(-)SKF 10,047 
4,000 .+-. 
566 2,850 .+-. 
390 
PCP 1,050 .+-. 
106 1,000 .+-. 
71 
U50,488H 1,350 .+-. 
106 ND 
Trifluoperazine 
345 .+-. 4 ND 
Trifluopromazine 
605 .+-. 67 ND 
Chlorpromazine 
1,475 .+-. 
265 ND 
Amitriptyline 
300 .+-. 7 ND 
Imipramine 520 .+-. 14 ND 
Desipramine 4,000 .+-. 
566 ND 
Nortriptyline 
2,000 .+-. 
640 ND 
Guanabenz 4,600 .+-. 
283 ND 
Clonidine &gt;10,000 ND 
Cocaine &gt;10,000 ND 
______________________________________ 
*IC.sub.50 is the molar concentration of the drug needed to produce 
halfmaximal displacement of [.sup.3 H]DTG from sigma receptors. This is a 
direct measure of the sigma receptor binding potency of the drug. ND = no 
determined. 
The above IC.sub.50 s represent the average from 2-4 separate experiments 
(in triplicate). The following compounds caused no significant 
displacement at a 10 uM concentration: scopolamine, 5-OH-tryptamine, 
diazepam, bicuculline, picrotoxin, hexamethonium, dopamine, apomorphine, 
GABA, gamma-guanidino butyric acid, morphine, DAGO, metorphamide, 
dynorphin A, [leu.sup.5 ] enkephalin, beta-endorphin, naloxone, guanidino 
acetic acid, creatine, creatinine, 1,1-dimethyl-4-guanidine, 
methyl-guanidine, beta-guanidino propionic acid and cimetidine. 
Example 21 
Drug specificity of [.sup.3 H]DTG binding compared to (+)-[.sup.3 H]3-PPP 
binding 
Comparing the drug specificity of [.sup.3 H]--DTG binding with that of 
(+)[.sup.3 H]3-PPP in the guinea pig, it was found that (+)[.sup.3 H]3-PPP 
bound specifically, saturably (linear Scatchard plot), reversely and with 
high affinity to guinea pig brain membranes (K.sub.D =30 nm, Bmax=80 
pmol/g fresh brain weight). The drug specificity profile of the (+)[.sup.3 
H]3-PPP binding in the guinea pig (Table II) was found to be very similar 
to that reported in the rat. Largent et al. (1984), supra. Moreover, the 
drug specificity profiles of typical sigma receptor active drugs in the 
(+)[.sup.3 H]3-PPP and [.sup.3 H]--DTG binding assays were highly 
correlated (r=0.95; p.ltoreq.0.00001) which is consistent with the two 
compounds labeling the same sites. 
Example 22 
Autoradiography Studies 
Male Sprague Dawley rats (200-250 g) and NIH guinea pigs (300-350 g) were 
sacrificed, their brains rapidly removed and processed for receptor 
autoradiography according to the method of Herkenham et al. J. 
Neuroscience 2:1129-1149 (1982). 
Fifteen um thick slide-mounted brain sections were incubated for 45 minutes 
in 50 nM Tris-HCl (pH 8.0, 22.degree. C.) containing 1 mg/ml bovine serum 
albumin (BSA) and 2 nM [.sup.3 H]DTG. Adjacent sections were incubated 
with 10 uM haloperidol or 10 uM DTG to measure nonspecific binding. 
Incubations were terminated by 4.times.2 minute washes in 10 nM Tris-HCl 
(pH 7.4, 4.degree. C.) with 1 mg/ml BSA, rapidly dried under a stream of 
cool air and placed in x-ray cassettes with .sup.3 H-sensitive film 
(.sup.3 H-ultrofilm, LKB). Films were developed 6-8 weeks later (D-19, 
Kodak). 
Example 23 
Autoradiographic visualization of [.sup.8 H]DTG binding 
Receptor autoradiography studies on guinea pig and rat brain sections using 
[.sup.3 H]DTG showed a low density of specific binding diffusely 
distributed throughout the gray matter of the rat and guinea pig brain. 
Superimposed on this homogeneous binding patterns was a heterogeneous 
distribution of enriched binding in limbic and sensorimotor structures. 
The pattern of binding was more distinct in the guinea pig than rat. 
Similar observations for (+)[.sup.3 H]3-PPP autoradiography have been 
reported. Largent et al. (1986), supra. Thus, description of [.sup.3 H]DTG 
binding was drawn primarily from the guinea pig. In the forebrain, limbic 
structures moderately to densely labeled by [.sup.3 H]DTG were the 
diagonal band of Broca, septum, hypothalamus (especially the 
paraventricular nucleus), anterodorsal thalamic nucleus and zona incerta. 
Sensorimotor thalamic nuclei moderately to densely labeled included the 
thalamic taste relay and reticular nuclei. Other thalamic nuclei labeled 
were the paraventricular and habenular nuclei. Very dense binding was seen 
in the choroid plexus. In the cortex dense [.sup.3 H]DTG labeling occupied 
layer III/IV of retrospenial piriform, and entorhinal cortices. The rest 
of the cortex contained a low level of homogeneous binding. The 
hippocampal formation exhibited discrete binding in the pyramidal granular 
cell layers. Sensorimotor areas of the midbrain were selectively labeled 
by [.sup.3 H]DTG. The oculomotor nucleus, and more caudally, the trochlear 
nucleus were very densely labeled, and the superior colliculus and red 
nucleus had moderate levels of binding. Other midbrain nuclei labeled were 
the dorsal raphe, interpeduncular nucleus, central gray, and the 
substantia nigra, para compacta. The selective labeling of the para 
compacta in the guinea pig contrasted with the low to moderate density of 
labeling present throughout the substantia nigra of the rat. In addition, 
very dense binding was found in the subcommissural organ. In the hindbrain 
the locus coeruleus was the most densely labeled nucleus. Sensorimotor 
nuclei enriched in [.sup.3 H]DTG binding sites were the trigeminal motor 
nucleus, nucleus of the facial nerve, nucleus of the solitary tract, 
dorsal motor nucleus of the vagus, and the hypoglossal nucleus. Moderate 
to dense binding was also found throughout the gray matter of the 
cerebellum, and in the pontine reticular nuclei. 
Example 24 
Drug Specificity of [.sup.3 H]AZ--DTG binding 
The haloperidol-sensitive sigma receptor binds [.sup.3 
H](+)3-[3-hydroxyphenyl]-N-(1-propyl)piperidine([.sup.3 H](+)-3-PPP) and 
[.sup.3 H]1,3-di-o-tolylguanidine ([.sup.3 H]DTG), with high affinity. In 
order to elucidate its structure, photoaffinity labeling of the sigma 
receptor from guinea pig brain was accomplished using a novel radioactive 
photolabile derivative of DTG, [.sup.3 H]-m-azido-1,3-di-o-tolylguanidine 
([H]AZ--DTG). In the dark, [.sup.3 H]AZ--DTG binds reversibly to sigma 
sites in brain membranes with high affinity (kd=28 nM). The drug 
specificity profile of [.sup.3 H]AZ--DTG binding to brain membranes is 
identical to that of the prototypical sigma ligands [.sup.3 H]DTG and 
[.sup.3 H](+)-3-PPP. For photoaffinity labeling, membrane suspensions 
containing protease inhibitors were preincubated in the dark with [.sup.3 
H]AZ--DTG, then filtered and washed over Whatman GF/B glass fiber filters. 
The filters were then irradiated with long-wavelength UV light for a 15 
minute period. Filter-bound proteins were solubilized with 50 mM Tris pH 
7.4, 0.1% sodium dodecyl sulfate. Solubilized proteins were subjected to 
SDS-polyacrylamide gel electrophoresis. Fluorography of the SDS-PAGE gels 
revealed that [.sup.3 H]AZ--DTG was selectively incorporated into a 29 kD 
polypeptide. Labeling of this polypeptide was completely blocked by the 
sigma ligands DTG, (+)-3-PPP, (+)pentazocine, and haloperidol at a 
concentration of 10 uM, while labeling was unaffected by morphine, 
serotonin, dopamine, scopolamine, or GABA at the same concentration. These 
results represent the first estimate of the size of the binding subunit of 
the haloperidol-sensitive sigma receptor. 
Example 25 
Additional sigma receptor binding assays 
Sigma receptor binding assays using guinea pig brain membrane homogenates 
and the radioligands [.sup.3 H]DTG and (+)[.sup.3 H]3-PPP were done as 
previously described (Weber et al., P.N.A.S. (USA) 83:8784-8788 (1986)). 
Briefly, frozen whole guinea-pig brains (Biotrol, Indianapolis, Ind.) were 
homogenized in 10 volumes (w/v) of ice-cold 320 mM sucrose using a 
Brinkman polytron. The homogenate was centrifuged at 1,000.times.g for 20 
minutes at 4.degree. C. The supernatant was centrifuged at 20,000.times.g 
for 20 minutes at 4.degree. C. The resulting pellet was resuspended in 10 
initial volumes of 50 mM Tris/HCl buffer at pH 7.4 and centrifuged at 
20,000.times.g for 20 minutes at 4.degree. C. The resulting pellet was 
resuspended in 5 initial volumes ice-cold 50 mM Tris/Hcl (pH 0.4), and the 
final volume was adjusted to yield a protein concentration of 3 mg/ml, as 
determined by dye-binding protein assay (Biorad) using BSA as the 
standard. Aliquots of 20-ml were stored at -70.degree. C. until used, with 
no detectable loss of binding. 
For [.sup.3 H]DTG binding assays, 20-ml aliquots of the frozen membrane 
suspension were thawed and diluted 1:3 in 50 mM Tris/HCl (pH 7.4). To 
12.times.75 mm polystyrene test tubes were added 0.8 ml of diluted 
membrane suspension, 0.1 ml of [.sup.3 H]DTG (46 Ci/mmol; see Weber et 
al., P.N.A.S (USA) 83:8784-8788 (1986) or (+)[.sup.3 H]3-PPP (NEN, 98 
Ci/mmol) to yield a final concentration of 1.4 nM, and 0.1 ml of 
unlabelled drugs or buffer. The protein concentration in the 1-ml final 
incubation volume was 800 ug/ml, corresponding to 32 mg of brain tissue 
(original wet weight) and to a tissue concentration within the linear 
range for specific binding. Non-specific binding was defined as that 
remaining in the presence of 10 uM haloperidol. Specific binding 
constituted &gt;90% of total [.sup.3 H]DTG binding. Incubations were 
terminated after 90 minutes at room temperature by addition of 4 ml of 
ice-cold 50 mM Tris/HCl (pH 7.4) and rapid filtration of the membrane 
suspension through Whatman GF/B glass-fiber filters under vacuum, using a 
48-well cell harvester (Brandel, Gaithersburg, Md.). The filters were 
washed 2 times with 4 ml of 50 mM Tris/HCl (pH 7.4). Total filtration and 
washing time was less than 20 seconds. Each filter was dissolved in 10 ml 
Cytoscint (Westchem, San Diego, Calif.), and radioactivity was measured by 
liquid scintillation spectrometry at a counting efficiency of 
approximately 50%. IC.sub.50 values were determined by interpolation from 
displacement-curve plots on semilogarithmic graph paper. 
The IC.sub.50 binding values (nM) are as follows: N,N-di(o-tolyl)guanidine 
(DTG, 32); N-(2-iodophenyl)-N'-(adamant-1-yl)guanidine (AdIpG, 
6.2.+-.0.7); N-(o-tolyl)-N'-(adamant-1-yl)guanidine (AdTG, 7.6.+-.0.3); 
N,N'-di(adamant-1-yl)guanidine (DAG, 11.8.+-.3.4); 
N-(cyclohexyl)-N'-(adamant-1-yl)guanidine (AdChG, 12.5.+-.2.2); 
N-(o-tolyl)-N'-(cyclohexyl)guanidine (13.0.+-.1.0); 
N,N'-di-(2,6-dimethylphenyl)guanidine (DXG, 90.+-.18); 
N-(o-tolyl)-N'-(4-amino-2-methylphenyl)guanidine (NH.sub.2 --DTG, 
280.+-.20); N,N'-di(phenyl)guanidine (DPG, 397.+-.21); 
N-(o-tolyl)-N'-(o-iodophenyl)guanidine (IC.sub.50 =21); 
N,N'-di-(p-bromo-o-methylphenyl)guanidine (IC.sub.50 =37); 
N,N'-di-(m-n-propylphenyl)guanidine (IC.sub.50 =36); 
N-(o-tolyl)-N'-(p-nitro-otolyl)guanidine (IC.sub.50 =37); 
N,N'-di-(1-tetralinyl)guanidine (IC.sub.50 =58); 
N-(o-tolyl)-N'-(o-xylyl)guanidine (IC.sub.50 =70); 
N,N'-di-(cyclohexyl)guanidine (IC.sub.50 =71); 
N-(3,5-dimethyl-1-adamantanyl)-N'-(o-tolyl)guanidine (IC.sub.50 =15); 
N-(3,5-dimethyl-1-adamantanyl)-N'-(oiodophenyl)guanidine (IC.sub.50 =16); 
N-(1-adamantyl)-N'-(o-nitrophenyl)guanidine (IC.sub.50 =30); 
N,N'-di-(endo-2-norbornyl)guanidine (IC.sub.50 =16); 
N-(exo-2-isobornyl)-N'-(o-iodophenyl)guanidine (IC.sub.50 =18); 
N,N'-di-(exo-2-norbornyl)guanidine (IC.sub.50 =22); 
N-(exo-2-isobornyl)-N'-(o-tolyl)guanidine (IC.sub.50 =25); 
N-(o-iodophenyl)-N'-(t-butyl)guanidine (IC.sub.50 =20); 
N,N'-dibenzylguanidine (IC.sub.50 =90); 
N-(adamant-1-yl)-N'-(o-isopropylphenyl)guanidine (IC.sub.50 =24); 
N-(adamant-2-yl)-N'-(p-iodophenyl)guanidine (IC.sub.50 =2.7); 
N-(cyclohexyl)-N'-(p-bromo-o-tolyl)guanidine (IC.sub.50 =5.5); 
N-(adamantan-2-yl)-N'-(o-iodophenyl)guanidine (IC.sub.50 =5.2); 
2-imino-1,3H-dibenzo[d,f]-[1,3]-diazepine (Bridge-DPG, &gt;10,000); 
N,N'-di(methyl)guanidine (DMG, &gt;10,000); (+)-3-PPP (76.+-.4); (-)-3-PPP 
(280.+-.21); (+)-pentazocine (43.+-.2); (-)-pentazocine (135.+-.3); 
(-)-cyclazocine (2600.+-.210); (-)-SKF10047 (4000.+-.566); haloperidol 
(5.+-.0.3); BMY 14802 (120.+-.15); rimcazole (1400.+-.100); tiospirone 
(233.+-.52); perphenazine (42.+-.10); chlorpromazine (1475.+-.265); 
sulpiride (&gt;10,000); TCP (1100.+-.110); PCP (1050.+-.106); and MK-801 
(&gt;10,000). 
Example 26 
Effect of Diazepam and N,N'-Disubstituted Guanidines on Light/Dark 
Exploration of Mice 
The inventors wish to thank Brenda Costall, University of Bradford, 
Bradford, BD7 1DP, England, for screening the sigma receptor ligands 
according to the procedure disclosed by her and others in Jones et al., 
Br. J. Pharmacol. 93:985-993 (1988). According to this procedure, male 
albino BKW mice, 25-30 g, were housed 10 to a cage and allowed free access 
to food and water. They were kept on reversed light cycle with the lights 
on between 22 h 00 min and 10 h 00 min. 
The apparatus was an open-topped box, 45 cm long, 27 cm wide and 27 cm 
high, divided into a small (2/5) area and a large (3/5) area by a 
partition that extended 20 cm above the walls. There was a 7.5.times.7.5 
opening in the partition at floor level. The small compartment was painted 
black and the large compartment white. The floor of each of the 
compartments was marked into 9 cm squares. The white compartment was 
illuminated by a 100 W tungsten bulb 17 cm above the box and the black 
compartment by a similarly placed 60 W red bulb. The laboratory was 
illuminated with red light. 
All tests were performed between 13 h 00 min and 18 h 00 min. Each mouse 
was tested by placing it in the center of the white area and allowing it 
to explore the novel environment for 5 min. Its behavior was recorded on 
videotape and the behavioral analysis was performed subsequently from the 
recording. Five parameters were measured: the latency to entry into the 
dark compartment, the time spent in each area, the number of transitions 
between compartments, the number of lines crossed in each compartment and 
the number of rears in each compartment. 
The N,N'-disubstituted guanidines, dissolved in distilled water, were 
administered subcutaneously 40 min before testing over the dosage range of 
0.01 ug to 0.1 mg/kg (5 mice per dosage level). Diazepam (0.063-10 mg/kg) 
was dissolved in the minimum quantity of polyethylene glycol, diluted to 
the appropriate volume with distilled water and administered 
intraperitoneally to five mice for each dosage level. The results are 
shown in FIGS. 1-6 (The cross hatched area=light area; solid columns=dark 
area). 
As shown in FIG. 1, diazepam dose-dependently increased the proportion of 
time the mice spent in the larger, lighted area of the test chamber. The 
numbers of line crossings and rears in the light compartment increased at 
the expense of those in the dark compartment. At the highest dose of 
diazepam (10 mg/kg), the numbers of rears and line crossings decreased 
significantly showing that the drug was markedly sedative. The latency to 
entering the dark compartment increased with a peak effect at 0.25 mg/kg 
(latency=30 sec) while controls showed a latency of 9 sec (S.E.M.s&lt;12.1%, 
P&lt;0.001). 
As shown in FIG. 2, N,N'-di-(adamantan-1-yl)guanidine tended to increase 
the proportion of time the mice spent in the larger, lighted area of the 
test chamber, with a peak at 0.01 ug/kg. The numbers of line crossings and 
rears in the light compartment increased at the expense of those in the 
dark compartment. At the highest dose of N,N'-di-(adamantan-1-yl) 
guanidine (0.1 mg/kg), the numbers of rears and line crossings did not 
decrease significantly showing that the drug was not sedative at this 
level. The latency to entering the dark compartment increased with a peak 
effect at 0.01 ug/kg (latency=20 sec) while controls showed a latency of 
12 sec (P&lt;0.05&lt;0.001). 
As shown in FIG. 3, N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine dose 
dependently increased the proportion of time the mice spent in the larger, 
lighted area of the test chamber. The numbers of line crossings and rears 
in the light compartment increased at the expense of those in the dark 
compartment. At the highest dose of 
N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine (0.1 mg/kg), the numbers 
of rears and line crossings did not decrease significantly showing that 
the drug was not sedative at this level. The latency to entering the dark 
compartment also increased in a dose-dependent manner. 
As shown in FIG. 5, N-(adamantan-1-yl)-N'-o-iodophenyl-guanidine also 
increased, in a dose-dependent manner, the proportion of time the mice 
spent in the larger, lighted area of the test chamber. The numbers of line 
crossings and rears in the light compartment increased at the expense of 
those in the dark compartment. At the highest dose of 
N-(adamantan-1-yl)-N'-o-iodophenyl guanidine (0.1 mg/kg), the numbers of 
rears and line crossings did not decrease significantly showing that the 
drug was not sedative at this level. The latency to entering the dark 
compartment also increased in a dose-dependent manner. 
For comparison, a compound with a relatively low sigma receptor affinity 
was tested for anxiolytic activity. As shown in FIG. 6, 
N-(3,5-dimethyladamantan-1-yl)-N'-{[(E)-2-phenylethenyl]phenyl}guanidine 
(IC.sub.50 =1,000 nM) generally did not increase the proportion of time 
the mice spent in the larger, lighted area of the test chamber. The 
numbers of line crossings and rears in the light compartment tended to 
remain the same except at the highest dosage. At the highest dose of 
N-(3,5-dimethyladamantan-1-yl)-N'-{[(E)-2-phenylethenyl]phenyl}guanidine 
(0.1 mg/kg), the numbers of rears and line crossings did not decrease 
significantly showing that the drug was not sedative at this level. 
As shown in FIG. 7, N,N'-di-(2-methylphenyl)guanidine did not affect 
significantly the number of rears, crossings, or the % time in the black 
compartment at the concentrations tested. This result is surprising in 
light of the high sigma receptor binding of this compound (IC.sub.50 
=32.0.+-.1). Although the inventors do not wish to be bound by any 
particular theory, it would appear that DTG does not exhibit anxiolytic 
activity in the in vivo assay since it is quickly metabolized and thereby 
deactivated by the animal. 
Next, N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine, 
N,N'-di-(adamantan-1-yl)guanidine and 
N-(adamantan-1-yl)-N'-(o-iodophenyl)guanidine were orally administered to 
the mice at a dose of 1 mg/kg. As shown in FIG. 4, orally administered 
N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine increased significantly 
the proportion of time the mice spent in the larger, lighted area of the 
box. Moreover, the numbers of line crossings and rears in the light 
compartment increased at the expense of those in the dark compartment. 
As shown in FIG. 8, N,N'-di-(adamantan-1-yl)guanidine and 
N-(adamantan-1-yl)-N'-(o-iodophenyl)guanidine caused an increase in the 
numbers of line crossings and rears in the light compartment at the 
expense of those in the dark compartment. Compared with controls, the 
numbers of rears and line crossings did not decrease significantly showing 
that the two drugs were not sedative at the dosage level administered. The 
two drugs also caused an increase in the latency to entering the dark 
compartment in comparison to controls. These experiments confirm that 
N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine, 
N,N'-di-(adamantan-1-yl)guanidine and 
N-(adamantan-1-yl)-N'-(o-iodophenyl)guanidine have anxiolytic activity 
when orally administered. 
As shown in FIG. 9, N-cyclohexyl-N'-(2-methylphenyl)guanidine increases 
significantly the proportion of time the mice spent in the larger, lighted 
area of the box. Moreover, the numbers of line crossings and rears in the 
light compartment increased in a dose-dependent manner at the expense of 
those in the dark compartment. Compared with controls, the total number of 
rears and line crossings did not decrease significantly, showing that the 
drug was not sedative at the dosage levels administered. The drug also 
caused an increase in the latency to entering the dark compartment 
compared to controls. 
As shown in FIG. 10, N-((.+-.)-endo-2-norbornyl)-N'-(2-iodophenyl)guanidine 
caused a significant dose-dependent increase in the number of rears in the 
light compartment at the expense of those in the dark compartment. 
Compared with controls, the total number of rears and line crossings did 
not decrease significantly, showing that the drug was not sedative at the 
dosage levels administered. The drug also caused a dose-dependent increase 
in the latency to entering the dark compartment compared to controls. 
As shown in FIG. 11, N-(exo-2-norbornyl)-N'-(2-methylphenyl)guanidine 
caused an increase in the number of rears in the light compartment at the 
expense of those in the dark compartment. Compared with controls, the 
total number of rears and line crossings did not decrease significantly, 
showing that the drug was not sedative at the dosage level administered. 
The drug also caused an increase in the latency to entering the dark 
compartment when administered at 0.1 mg/kg in comparison to controls. 
As shown in FIG. 12, N-(adamantan-1-yl)-N'-cyclohexylguanidine caused an 
increase in the number of rears in the light compartment at the expense of 
those in the dark compartment. Compared with controls, the total number of 
rears and line crossings did not change significantly, showing that the 
drug was not sedative at the dosage levels administered. The drug also 
caused a dose-dependent increase in the latency to entering the dark 
compartment in comparison to controls. 
As shown in FIG. 13, N-(cyclohexyl)-N'-(2-methylphenyl) guanidine, when 
administered orally, caused an increase in the total numbers of rears in 
the light compartment at the expense of those in the dark compartment. 
Compared with controls, the number of rears and line crossings did not 
change significantly, showing that the compound was not sedative at the 
dosage level administered. The compound also caused a slight increase in 
the latency to entering the dark compartment in comparison to controls. 
However, a significant decrease in the percentage of time in the black 
compartment was observed. 
As shown in FIG. 14, N-((.+-.)-endo-2-norbornyl)-N'-2-iodophenyl)guanidine, 
administered orally, caused an increase in the number of rears in the 
light compartment at the expense of those in the dark compartment. 
Compared with controls, the total number of rears and line crossings did 
not decrease significantly, showing that the drug was not sedative at the 
dosage level administered. The drug also caused an increase in the latency 
to entering the dark compartment in comparison to controls and a decrease 
in the percentage time spent in the black compartment. 
As shown in FIG. 15, N-(exo-2-norbornyl)-N'-(2-methylphenyl)guanidine, when 
administered orally, caused an increase in the number of rears in the 
light compartment at the expense of those in the dark compartment. 
Compared with controls, the total number of rears and line crossings did 
not decrease significantly, showing that the drug was not sedative at the 
dosage levels administered. The drug also caused an increase in the 
latency to entering the dark compartment in comparison with controls, and 
a decrease in the percentage time spent in the black compartment. 
As shown in FIG. 16, N-(2-styrylphenyl)-N'-(2-iodophenyl)guanidine, a 
non-active control compound, did not affect the numbers of line crossings 
or rears in the light compartment. Moreover, the drug actually decreased 
the latency to entering the dark compartment in comparison with controls. 
No significant change in the percentage time in the black compartment was 
observed. 
Example 27 
The Anxiolytic Potential of Diazepam and 
N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine in a Rat Social 
Interaction Test 
Male Hooded Lister rats (Glaxo bred, 200-250 g), were housed 5 to a cage 
and were kept in the laboratory environment for at least a week before 
testing. Rats paired in the test were taken from separate cages. 
The compounds were screened for anxiolytic activity by Brenda Costall 
according to the disclosure of Jones, B. J. et al., Br. J. Pharmacol. 
93:985-993 (1988) and File, S. E. et al., Br. J. Pharmacol. 62:19-24 
(1978). The test arena consisted of an open-topped box, 
62.times.62.times.33 cm with a 7.times.7 matrix of infra-red photocell 
beams in the walls, 2.5 cm from the floor. Diazepam and 
N-(adamantan-1-yl)-N'-(2-methylphenyl)-guanidine were tested by treating 
both members of a pair of rats with the same treatment 40 min. before 
testing (Rats were placed singly in small cages immediately after dosing 
until they were tested). 
Testing involved placing each member of a pair of rats in opposite comers 
of the arena and then leaving them undisturbed for 10 min. while recording 
their behavior remotely on videotape. The behavioral assessments were made 
subsequently from the recordings. The time spent in social interaction was 
measured and expressed as a cumulative total for the 10 min session. The 
behaviors that comprised social interaction were: following with contact, 
sniffing (but not sniffing of the hindquarters), crawling over and under, 
tumbling, boxing and grooming. 
Intraperitoneally administered diazepam was tested over the dose range of 
0.125-1 mg/kg. N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine was tested 
over the range of 0.001-0.1 mg/kg. The results appear in FIG. 17 (n=6, 
P&lt;0.001). 
As shown in FIG. 17, both diazepam and 
N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine significantly increased 
social interactions. However, 
N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine produced about the same 
result at one-tenth the dose of diazepam (1 mg/kg for diazepam and 0.1 
mg/kg for N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine). These results 
provide strong indication that 
N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine will have anxiolytic 
activity in man, especially since the social interaction test in the rat 
is one of the most extensively validated tests (See File et al., supra). 
The preceding examples can be repeated with similar success by substituting 
the generically or specifically described reactants and/or operating 
conditions of this invention for those used in the preceding examples. 
From the foregoing description, one skilled in the art can easily ascertain 
the essential characteristics of this invention, and without departing 
from the spirit and scope thereof, can make various changes and 
modifications of the invention to adapt it to various usages and 
conditions.