FIELD OF INVENTION

The present invention relates to novel compounds, compositions containing them, and their use for treating medical disorders resulting from a deficiency in growth hormone.

BACKGROUND OF THE INVENTION

Growth hormone is a hormone which stimulates growth of all tissues capable of growing. In addition, growth hormone is known to have a number of effects on metabolic processes, e.g., stimulation of protein synthesis and free fatty acid mobilization and to cause a switch in energy metabolism from carbohydrate to fatty acid metabolism. Deficiency in growth hormone can result in a number of severe medical disorders, e.g., dwarfism.

Growth hormone is released from the pituitary. The release is under tight control of a number of hormones and neurotransmitters either directly or indirectly. Growth hormone release can be stimulated by growth hormone releasing hormone

(GHRH) and inhibited by so atostatin. In both cases the hormones are released from the hypothalamus but their action is mediated primarily via specific receptors located in the pituitary. Other compounds which stimulate the release of growth hormone from the pituitary have also been described. For example arginine, L-3,4-dihydroxyphenylalanine (L-Dopa) , glucagon, vasopressin, PACAP (pituitary adenylyl cyclase activating peptide) , muscarinic receptor agonists and a synthethic hexapeptide, GHRP (growth hormone releasing peptide) release endogenous growth hormone either by a direct effect on the pituitary or by affecting the release of GHRH and/or somatostatin from the hypothalamus.

In disorders or conditions where increased levels of grow hormone is desired, the protein nature of growth hormone mak anything but parenteral administration non-viable. Furthermor other directly acting natural secretagogues, e.g., GHRH a PACAP, are longer polypeptides for which reason or administration of them is not viable.

The use of certain compounds for increasing the levels growth hormone in mammals has previously been proposed, e.g.

EP 18072, EP 83 864, WO 89/07110, WO 89/01711, WO 89/10933, 88/9780, WO 83/02272, WO 91/18016, WO 92/01711, WO 93/04081 95/17422, WO 95/17423 and WO 95/14666.

The composition of growth hormone releasing compounds important for their growth hormone releasing potency as well their bioavailability. It is therefore the object of t present invention to provide compounds with growth hormo releasing properties which have improved properties relative known compounds of this type.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to a compound general formula I

wherein m is 0, 1 or 2,

R ¹ and R ² are independently hydrogen, aryl or C,_ ₆ -alkyl optionally substituted with halogen, amino, hydroxy or aryl, W is «S, =0, =NH or «N(CN), with the proviso that at least one of A, R ¹ or R ² is an aryl or branched or linear C,. ₆ -alkyl substituted with aryl;

A is hydrogen, -CONR ³ R ⁴ , -CONR ³ CHR ⁴ CONR ⁵ R ⁶ , -COOR ³ , -CH ₂ NR ³ R ⁴ or - CH _j OR ³ , wherein R ³ , R*, R ⁵ , and R ⁶ are independently hydrogen, aryl or C ₆ -alkyl optionally substituted with halogen, amino, hydroxy or aryl;

B is

optionally substituted with halogen, carboxamido, tetrazolyl, oxadiazolyl, thiadiazolyl, amino, hydroxy, C ₆ -alkyl or C.,_ ₆ - alkoxy, wherein R ⁷ is hydrogen or C,_ ₆ -alkyl;

D is

\

,N (CH ₂ ) _r (CR ¹⁰ R ¹¹ ) _S (CH ₂ ) _e

wherein R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ are independently hydrogen or C,. ₆ - alkyl optionally substituted with halogen, amino, hydroxy or aryl, R ^β and R ⁹ , R ¹⁰ and R ¹¹ , R ⁸ and R ¹⁰ or R ⁹ and R ¹¹ optionally forming -(CH ₂ )^-U-JCH _g ) _j -, wherein i and j are independently 1 or 2,

U is -0-, -S- or a valence bond, o and r are independently 0, 1, 2, 3 or 4, s is 0 or 1, and r + s is 1, 2, 3 or 4;

or a pharmaceutically acceptable salt thereof, and the compounds of formula I comprise any optical isomers thereof, in the form of separated, pure or patially purified optical isomers or racemic mixtures thereof.

It is believed that compounds of formula I exhibit an improved bioavailability because they contain no amide bonds susceptible to cleavage by proteolytic enzymes. The increased resistance to proteolytic degradation combined with the reduced size of the compounds of the invention in comparison with known growth hormone releasing compounds is expected to improve their bioavailability compared to that of the compounds suggested in the prior literature.

In the above structural formulas and throughout the present specification, the following terms have the indicated meanings:

The C ₆ -alkyl groups specified above are intended to include those alkyl groups of the designated length in either a linear or branched, or cyclic configuration. Examples of linear alkyl are methyl, ethyl, propyl, butyl, pentyl, and hexyl. Examples of branched alkyl are isopropyl, sec-butyl, tert-butyl, isopentyl, and isohexyl. Examples of cyclic alkyl are cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

Especially preferred C,_ ₆ -alkyl groups are the C.._ ₃ -alkyl groups. Preferred C-. _j -alkyl groups are methyl, ethyl, isopropyl, and cyclopropyl.

The C ₆ -alkoxy groups specified above are intended to include those alkoxy groups of the designated length in either a linear or branched or cyclic configuration. Examples of linear alkyloxy are methoxy, ethoxy, propoxy, butoxy, pentoxy, and hexoxy. Examples of branched alkoxy are isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, and isohexoxy. Examples of cyclic alkoxy are cyclopropyloxy, cyclobutyloxy, cyclopentyloxy and cyclohexyloxy.

Especially preferred C ₁ . ₆ -alkoxy groups are the C ₃ -alkoxy groups. Preferred C-. _j -alkoxy groups are methoxy, ethoxy, isopropoxy, and cyclopropyloxy.

The C ₁ . ₆ -alkylamino groups specified above are intended to include those alkylamino groups of the designated length in either a linear or branched or cyclic configuration. Examples of linear alkylamino are methylamino, ethylamino, propylamino, butylamino, pentylamino, and hexylamino. Examples of branched alkylamino are isopropylamino, sec-butylamino, tert-butylamino, isopentylamino, and isohexylamino. Examples of cyclic alkylamino are cyclopropylamino, cyclobutylamino, cyclopentylamino and cyclohexylamino.

Especially preferred C.,. ₆ -alkylamino groups are the ^c ι- alkylamino groups. Preferred C-.- _j -alkylamino groups a methylamino, ethylamino, isopropylamino, and cyclopropylamin

In the present context, the term "aryl" is intended to includ aromatic rings, such as carbocyclic and heterocyclic aromati rings selected from the group consisting of phenyl, naphthyl pyridyl, l-H-tetrazol-5-yl, thiazolyl, imidazolyl, indolyl pyrimidinyl, thiadiazolyl, pyrazolyl, oxazolyl, isoxazolyl thiopheneyl, quinolinyl, pyrazinyl, or isothiazolyl, optionall substituted by one or more C,_ ₆ -alkyl, C^-alkoxy, haloge amino or aryl. Aryl is preferably phenyl, thienyl, imidazolyl pyridyl, indolyl, guinoline or naphthyl optionally substitut with halogen, amino, hydroxy, C-. ₆ -alkyl or C*_ ₆ -alkoxy. The te "halogen" is intended to include Cl, F, Br and I.

The compounds of the present invention may have one or mo asymmetric centres and stereoisomers in the form of separate pure or partially purified stereoisomers or racemic mixtur thereof are intended to be included in the scope of t invention.

DETAILED DESCRIPTION OF THE INVENTION

Examples of specific compounds of the present invention are

l-Benzyl-3-(3-(morpholin-4-yl)propyl)-l- (naphth-2 yl) ethylthiourea

l-Benzyl-3- (3-dimethylaminopropyl) -1-phenylthiourea

2- [ 3- (3- (Morpholin-4-yl)propyl) -1- ( naphth-2-yl) methyl ■ thioureido]-3-phenylpropionamide

N-(4-Aminobutyl)-2-[3-( (3 -amino- 3 -methyl) butyl) -l-(naphth-2- yl) methylthioureido] -3-phenylpropionamide

N-(4-Aminobutyl)-2-(N-(naphth-2-yl)methyl-N'-(piperidin-3 yl)methyl-guanidino)-3-phenyl-propionamide

N-(4-Aminobutyl)-2-[l-methyl-3-(naphth-2-yl)methyl-3-(2 (piperidin-2-yl)ethyl)-thioureido]-3-(naphth-2-yl)propionami d

3-(3-(Morpholin-4-yl)propyl)-l-(naphth-2-yl)methyl-l-[2 ^, -(lH- tetrazol-5-yl)-biphenyl-4-ylmethyl]thiourea

N-( (l-Carbamoyl-2-phenyl)ethyl-N-methyl-2-[3-( (3-morpholin-4- yl)propyl)-thioureido]-3-(naphth-2-yl)propionamide

3- (3- (Dimethyl ami no) propyl )-l-(naphth-l-yl) methyl -1- phenylthiourea

l-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-phenylthiourea

l,l-Dibenzyl-3-( 3- (morpholin-4-yl) propyl )thiourea

l-Benzyl-3-( 3- (dimethyl amino) propyl )-l-((naphth-2 yl ) methyl ) thiourea

l-Benzyl-3-( 3- (morpholin-4-yl) propyl )-l-(pheneth-2-yl) thiourea

l-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(guinolin-3-yl)th iourea

l-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(pyridin-2-yl)thi oure

l-Benzyl-3- ( 3- (morpholin-4-yl) propyl) - 1 - ( 4 methoxyphenyl ) thiourea

l-Benzyl-3-(3-(morpholin-4-yl)propyl)-1-(4-([1 , 2 ,3]thiadiazol- 4-y1)benzyl)thiourea

3- (3-Dimethylaminopropyl) -1- ( (naphth-2-yl ) methyl) -1 phenylthiourea

Compounds of formula I may be prepared from natural or non natural amino acid residues as shown in one of the followin reaction schemes. The non-natural amino acid residues may b prepared according to methods known to those skilled in th art.

General Method A

Reaction Scheme I:

V~ NH _j Aldehyde, MeOH, 1 % HOAc _t "HN. ^{D i} °

B (CH ₂ ) _m NaCNBH _{3 B} (CH ₂ ) _m R ¹ ™ ^F '

1 2

Compounds of formula I may be prepared by the method shown in reaction scheme I starting with a primary amine 1, which may be either commercially available or prepared by methods known to those skilled in the art, e.g. peptide coupling methodologies described in the art (e.g. DCC coupling in DMF) , and an aldehyde under reductive conditions e.g. with sodium cyanoborohydride in methanol/acetic acid to give the compound 2. The compound 4 may be prepared from 2 and e.g. an isothiocyanate 3 in an appropriate solvent such as tetrahydrofuran to give the compound 4. The compound 4 may be deprotonated by a base such as sodium hydride in an appropriate solvent such as tetrahydrofuran and alkylated with an appropriate alkylhalogen such as benzylbromide to give the compound 5 which is a compound of the formula I. Functional groups in intermediates in reaction scheme I may be protected

and deprotected using a strategy known in the art a described by e.g. T.W. Greene (Protective Groups in Organ Synthesis, 2nd Ed., John Wiley and Sons 1991).

General Method B

Reaction Scheme II

10 11

Compounds of formula I may be prepared by the method shown reaction scheme II starting with a primary amine 6 which und basic conditions such as lithium diisopropylamide in appropriate solvent such as tetrahydrofuran may be alkylat with an alkyl halogenide 7 such as 2-bromo-3-phenyl-propano acid methyl ester to give the secondary amine 8. The compou

10 may be prepared from 8 and e.g. and isothiocyanate 9 in an appropriate solvent such as tetrahydrofuran to give the compound 10. The compound 10 may be deprotonated by a base such as sodium hydride in an appropriate solvent such as tetrahydrofuran and alkylated with an appropriate alkylhalogenide such as benzylbromide to give the compound 5 which is a compound of the formula I. In cases where the compounds 6,7,8,9 or/and 10 contains any primary or secondary amino functionalities an appropriate protection and deprotection strategy known in the art and described by e.g. T.W. Greene (Protective Groups in Organic Synthesis, 2nd Ed., John Wiley and Sons 1991) is used.

Pharmaceutically acceptable acid addition salts of compounds of formula I include those prepared by reacting the compound with an inorganic or organic acid such as hydrochloric, hydrobromic, sulfuric, acetic, phosphoric, lactic, maleic, phthalic, citric, glutaric, gluconic, methanesulfonic, salicylic, succinic, tartaric, toluenesulfonic, trifluoracetic, sulfamic or fumaric acid.

In another aspect, the present invention relates to a pharmaceutical composition comprising, as an active ingredient, a compound of the general formula I or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or diluent.

Pharmaceutical compositions containing a compound of the present invention may be prepared by conventional techniques, e.g. as described in Remington's Pharmaceutical Sciences. 1985. The compositions may appear in conventional forms, for example capsules, tablets, aerosols, solutions, suspensions or topical applications.

The pharmaceutical carrier or diluent employed may be a

conventional solid or liquid carrier. Examples of soli carriers are lactose, terra alba, sucrose, cyclodextrin, talc gelatin, agar, pectin, acacia, magnesium stearate, stearic aci or lower alkyl ethers of cellulose. Examples of liquid carrier are syrup, peanut oil, olive oil, phospholipids, fatty acids fatty acid amines, polyoxyethylene or water.

Similarly, the carrier or diluent may include any sustaine release material known in the art, such as glycery monostearate or glyceryl distearate, alone or mixed with a wax

If a solid carrier is used for oral administration, th preparation may be tabletted, placed in a hard gelatin capsul in powder or pellet form or it can be in the form of a troch or lozenge. The amount of solid carrier will vary widely bu will usually be from about 25 g to about 1 g. If a liqui carrier is used, the preparation may be in the form of a syrup emulsion, soft gelatin capsule or sterile injectable liqui such as an aqueous or non-aqueous liquid suspension o solution.

A typical tablet which may be prepared by conventiona tabletting techniques may contain:

Core:

Active compound (as free compound or salt thereof) 100 m Colloidal silicon dioxide (Aerosil) 1.5 m

Cellulose, microcryst. (Avicel) 70 m

Modified cellulose gum (Ac-Di-Sol) 7.5 m Magnesium stearate

Coating: HPMC approx. 9 m

♦Mywacett 9-40 T approx. 0.9 m

*Acylated monoglyceride used as plasticizer for film coating.

For nasal administration, the preparation may contain a compound of formula I dissolved or suspended in a liquid carrier, in particular an aqueous carrier, for aerosol application. The carrier may contain additives such as solubilizing agents, e.g. propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabenes.

Generally, the compounds of the present invention are dispensed in unit dosage form comprising 50-200 mg of active ingredient together with a pharmaceutically acceptable carrier per unit dosage.

The dosage of the compounds according to this invention is suitably 0.1-500 mg/day, e.g. from about 5 to about 50 mg, such as about 10 mg per dose, when administered to patients, e.g. humans, as a drug.

It has been demonstrated that compounds of the general formula I possess the ability to release endogenous growth hormone in vivo. The compounds may therefore be used in the treatment of conditions which require increased plasma growth hormone levels such as in growth hormone deficient humans or in elderly patients or livestock.

Thus, in a particular aspect, the present invention relates to a pharmaceutical composition for stimulating the release of growth hormone from the pituitary, the composition comprising, as an active ingredient, a compound of the general formula I or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or diluent.

In a further aspect, the present invention relates to a metho of stimulating the release of growth hormone from th pituitary, the method comprising administering to a subject i need thereof an effective amount of a compound of the genera formula I or a pharmaceutically acceptable salt thereof.

In a still further aspect, the present invention relates to th use of a compound of the general formula I or pharmaceutically acceptable salt thereof for the preparation o a medicament for stimulating the release of growth hormone fro the pituitary.

To those skilled in the art, it is well known that the curren and potential uses of growth hormone in humans are varied an multitudinous. Thus, compounds of formula I can be administere for purposes stimulating release of growth hormone from th pituitary and would then have similar effects or uses as growt hormone itself. The uses of growth hormone may be summarized a follows: stimulation of growth hormone release in the elderly; prevention of catabolic side effects of glucocorticoids, prevention and treatment of osteoporosis, stimulation of th immune system, acceleration of wound healing, accelerating bon fracture repair, treatment of growth retardation, treatin renal failure or insufficiency resulting from growt retardation, treatment of physiological short stature includin growth hormone deficient children and short stature associate with chronic illness, treatment of obesity and growt retardation associated with obesity, treating growt retardation associated with the Prader-Willi syndrome an Turner's syndrome; accelerating the recovery and reducin hospitalization of burn patients; treatment of intrauterin growth retardation, skeletal dysplasia, hypercortisolism an Cushing's syndrome; induction of pulsatile growth hormon release; replacement of growth hormone in stressed patients, treatment of osteochondrodysplasias, Noonan's syndrome,

schizophrenia, depressions, Alzheimer's disease, delayed wound healing and psychosocial deprivation, treatment of pulmonary dysfunction and ventilator dependency, attenuation of protein catabolic responses after major surgery, reducing cachexia and protein loss due to chronic illness such as cancer or AIDS; treatment of hyperinsulinemia including nesidioblastosis, adjuvant treatment for ovulation induction; to stimulate thymic development and prevent the age-related decline of thymic function, treatment of immunosuppressed patients, improvement in muscle strength, mobility, maintenance of skin thickness, metabolic homeostasis, renal homeostasis in the frail elderly, stimulation of osteoblasts, bone remodelling and cartilage growth, stimulation of the immune system in companion animals and treatment of disorder of aging in companion animals, growth promoter in livestock and stimulation of wool growth in sheep.

For the above indications the dosage will vary depending on the compound of formula I employed, on the mode of administration and on the therapy desired. However, generally dosage levels between 0.0001 and 100 g/kg body weight daily are administered to patients and animals to obtain effective release of endogenous growth hormone. Usually, dosage forms suitable for oral, nasal, pulmonal or transdermal administration comprise from about 0.0001 mg to about 100 mg, preferably from about 0.001 mg to about 50 mg of the compounds of formula I admixed with a pharmaceutically acceptable carrier or diluent.

The compounds of formula I may be administered in pharmaceutically acceptable acid addition salt form or, where appropriate, as a alkali metal or alkaline earth metal or lower alkylammonium salt. Such salt forms are believed to exhibit approximately the same order of activity as the free base forms.

Optionally, the pharmaceutical composition of the invention may

comprise a compound of formula I combined with one or mor compounds exhibiting a different activity, e.g., an antibioti or other pharmacologically active material. -

The route of administration may be any route which effectivel transports the active compound to the appropriate or desire site of action, such as oral, nasal, pulmonary, transdermal o parenteral, the oral route being preferred.

Apart from the pharmaceutical use of the compounds of formul I, they may be useful in vitro tools for investigating th regulation of growth hormone release.

Compounds of formula I may also be useful in vivo tools fo evaluating the growth hormone releasing capability of th pituitary. For example, serum samples taken before and afte administration of these compounds to humans can be assayed fo growth hormone. Comparison of the growth hormone in each seru sample would directly determine the ability of the patient pituitary to release growth hormone.

Compounds of formula I may be administered to commerciall important animals to increase their rate and extent of growth and to increase milk production.

A further use of growth hormone secretagogue compounds o formula I is in combination with other secretagogues such a GHRP (2 or 6) , GHRH and its analogues, growth hormone and it analogues or somatomedins including IGF-1 and IGF-2.

Pharmacological Methods

Compounds of formula I may be evaluated in vitro for thei efficacy and potency to release growth hormone in rat pituitar primary cultures.

The isolation of rat pituitary cells is a modification of O. Sartor et al., Endocrinology 116. 1985, pp. 952-957. Male albino Sprague-Dawley rats (250 +/- 25 grams) were purchased from Møllegaard, Lille Skensved, Denmark. The rats were housed 5 in group cages (four animals/cage) and placed in rooms with 12 hour light cycle. The room temperature varied from 19-24*C and the humidity from 30 - 60%.

The rats were decapitated and the pituitaries dissected. The neurointermediate lobes were removed and the remaining tissue

10 was immediately placed in icecold isolation buffer (Gey's medium (Gibco 041-04030) supplemented with 0.25% D-glucose, 2% non-essential amino acids (Gibco 043-01140) and 1% bovine serum albumine (BSA) (Sigma A-4503)). The tissue was cut into small pieces and transferred to isolation buffer supplemented with

153.8 mg/ml of trypsin (Worthington #3707 TRL-3) and 330 mg/ml of DNase (Sigma D-4527) . This mixture was incubated at 70 rotations/min for 35 min at 37*C in a 95/5% atmosphere of 0 ₂ /C0 ₂ . The tissue was then washed three times in the above buffer. Using a standard pasteur pipet, the tissue was then

20 aspirated into single cells. After dispersion, cells were filtered through a nylon filter (160 mm) to remove undigested tissue. The cell suspension was washed 3 times with isolation buffer supplemented with trypsin inhibitor (0.75 mg/ml, Worthington #2829) and finally resuspended in culture medium;

25 DMEM (Gibco 041-01965) supplemented with 25 M HEPES (Sigma H- 3375), 4 mM glutamine (Gibco 043-05030H) , 0.075% sodium bicarbonate (Sigma S-8875) , 0.1% non-essential amino acid, 2.5% fetal calf serum (FCS, Gibco 011-06290), 3% horse serum (Gibco 034-06050), 10% fresh rat serum, 1 nM T ₃ (Sigma T-2752) and 40

30 mg/L dexamethasone (Sigma D-4902) pH 7.3, to a density of 2 x 10 ⁵ cells/ml. The cells were seeded into microtiter plates (Nunc, Denmark), 200 ml/well, and cultured for 3 days at 37*C and 8% C0 ₂ .

Compound testing

After culturing, the cells were washed twice with stimulatio buffer (Hanks Balanced Salt Solution (Gibco 041-04020) supplemented with 1% BSA (Sigma A-4503) , 0.25% D-glucose (Sigm G-5250) and 25 mM HEPES (Sigma H-3375) pH 7.3) and preincubate for 1 hour at 37*C. The buffer was exchanged with 90 m stimulation buffer (37"C). Ten ml test compound solution wa added and the plates were incubated for 15 min at 37*c and 5 C0 ₂ . The medium was decanted and analyzed for GH content in a rGH SPA test system.

All compounds were tested in doses ranging from 10 pM to 10 mM. A dose-response relation was constructed using the Hil equation (Fig P, Biosoft) . The efficacy (maximal GH released, E _mM ) was expressed in % of the E^ of GHRP-6. The potency (EC ₅₀ ) was determined as the concentration inducing half maxima stimulation of the GH release.

Compounds of formula I may be evaluated for their metaboli stability.

Compounds were dissolved at a concentration of 1 mg/ml i water. 25 ml of this solution is added to 175 ml of th respective enzyme-solution (resulting in an enzyme:substrat ratio (w/w) of approximately 1:5). The solution is left at 37° overnight. 10 ml of the various degradation solutions i analyzed against a corresponding zero-sample using flo injection electrospray mass spectrometry (ESMS) with selecte ion monitoring of the molecular ion. If the signal ha decreased more than 20% compared to the zero-sample, th remainder of the solution is analyzed by HPLC and mas spectrometry in order to identify the extent and site(s) o degradation precisely.

Several standard peptides (ACTH 4-10, Angiotensin 1-14 and Glucagon) have been included in the stability tests in order to verify the. ability of the various solutions to degrade peptides.

Standard peptides (angiotensin 1-14, ACTH 4-10 and glucagon) were purchased from Sigma, MO, USA) .

Enzymes (trypsin, chymotrypsin, elastase aminopeptidase M and carboxypeptidase Y and B) were all purchased from Boehringer Mannheim GmbH (Mannheim, Germany) .

Pancreatic enzyme mix: trypsin, chymotrypsin and elastase in 100 mM ammoniumbicarbonate pH 8.0 (all concentrations 0.025 mg/ml) .

Carboxypeptidase mix: carboxypeptidase Y and B in 50 mM ammoniumacetate pH 4.5 (all concentrations 0.025 mg/ml).

Aminopeptidase M solution: aminopeptidase M (0.025 mg/ml) in 100 mM ammoniumbicarbonate pH 8.0.

Mass spectrometric analysis was performed using two different mass spectrometers. A Sciex API III triple quadrupole LC-MS instrument (Sciex instruments, Thornhill, Ontario) equipped with an electrospray ion-source and a Bio-Ion 20 time-of-flight Plasma Desorption instrument (Bio-Ion Nordic AB, Uppsala, Sweden) .

Quantification of the compounds (before and after degradation) was done on the API III instrument using single ion monitoring of the molecular ion in question with flow injection of the analyte. The liquid flow (MeOH:water 1:1) of 100 ml/min was controlled by an ABI 140B HPLC unit (Perkin-Elmer Applied

Biosystems Divisions, Foster City, CA) . The instrumen parameters were set to standard operation conditions, and SI monitoring was performed using the most intense molecular io (in most cases this corresponded to the doubly charge molecular ion) .

Identification of degradation products furthermore involved th use of plasma desorption mass spectrometry (PDMS) with sampl application on nitrocellulose coated targets and standar instrumental settings. The accuracy of the hereby determine masses is generally better than 0.1%.

Separation and isolation of degradation products was done usin a HY-TACH C-18 reverse phase 4.6x105 mm HPLC column (Hewlett Packard Company, Palo Alto, CA) with a standard acetonitril TFA separation gradient. The HPLC system used was HP1090 (Hewlett-Packard Company, Palo Alto, CA) .

+: Stable (less than 20% decrease in SIM signal after 24 fa in degradation solution) •: Unstable (mote than 20% decrease in SIM signal after 24 h in degradation solution)

Any novel feature or combination of features described herein is considered essential to this invention.

The invention is further illustrated in the following examples which are not in any way intended to limit the scope of the invention as claimed.

The present invention is further illustrated in the following examples which are not in any way intended to limit the scope of the invention as claimed.

EXAMPLES

Herinafter, TLC is thin layer chromatography and THF is tetrahydrofuran, CDC1 ₃ is deuterio chloroform, DMSO-d ₆ is hexadeuterio di ethylsulfoxide and CD ₃ OD is tetradeuterio methanol. The structure of the compounds are confirmed by either elemental analysis or NMR, where peaks assigned to characteristic protons in the title compounds are presented where appropriate. ¹ H NMR shift (d _H ) are given in parts per million (ppm) . M.p. is melting point and is given in *C and is not corrected. Column chromatography was carried out usin the technique described by W.C. Still et al., J. Org. Chem. (1978), 43, 2923-2925 on Merck silica gel 60 (Art. 9385). HPLC analysis was performed using a 5mm C18 4x250 mm column, eluting with 20-80 % gradient of 0.1 % trifluoroacetic acid/acetonitrile and 0.1 % trifluoroacetic acid/water over 30 minutes at 35 ^* C. All reactions were carried out under an atmosphere of nitrogen. THF was distilled over sodium and benzophenone before use. Compounds used as starting material are either known compounds or compounds which can readily be prepared by methods known per se.

Example 1

l-βeπzyl-3- ( ?- (porpfrol J-n-4-γl) propyl ) -I- ( (ne ht -?- vl ) methyl ) thJQMrea-hyflrpgh-), <?rifle

A solution of benzylamine (5.0 g, 47 mmol) and 2- naphthaldehyde (7.3 g, 47 mmol) in 200 ml of 99.9 % ethanol was refluxed overnight and cooled to room temperature. Sodium borohydride (1.8 g, 47 mmol) was added in small portion over a period of 15 minutes. After 30 minutes 400 ml of water was added and the solution was concentrated in vacuo to a minimum and extracted 4 times with 200 ml ethyl acetate and THF (1:1). The combined organic layer was dried over magnesium sulfate, filtered and concentrated in vacuo to give an oil which was dissolved in 100 ml of ethyl acetate and 50 ml of 3 M HC1 in ethyl acetate was added and a white solid precipitated. The precipitate was washed four times with 50 ml of ethyl acetate and dried overnight in vacuo to give 11.3 g (86 %) of N-benzyl-N-((naphth-2-yl)methyl)amine hydrochloride. The hydrochloride was dissolved in 600 ml of water and methanol (1:1) and 200 ml of saturated sodium bicarbonate was added. The solution was concentrated in vacuo to a minimum and extracted 4 times with 200 ml ethylacetate, dried over magnesium sulphate and concentrated in vacuo to give 9.9 g (86%) of N-benzyl-N-((naphth-2-yl)methyl)amine.

N-Benzyl-N-((naphth-2-yl)methyl)amine (1.0 g, 4.0 mmol) in 20 ml of THF was added to a solution of 3-(morpholin-4-yl)propyl isothiocyanate (750 mg, 4.0 mmol) in 30 ml of THF at -78*C over a period of 10 minutes. The mixture was stirred at room temperature overnight and the solvent was removed in vacuo and the obtained oil was chromatographed on 500 ml of silica gel in 10% methanol/methylene chloride to give an oil which was dissolved in 20 ml ethyl acetate to which was added 10 ml of 3 M HC1 in ethyl acetate. The solvent was removed in vacuo to give 1.4 g (73%) of l-benzyl-3-(3-(morpholin-4-yl)propyl)- l-( (naphth-2-yl)methyl)thiourea-hydrochloride as a white solid. M.p. 57*C (decomp.).

¹ H NMR (400 MHZ, CDC1 ₃ ) : d 1.70 (t, 2H) , 2.15 (d, 4H) , 2.25 (t, 2H) , 3.25 (d, 2H) , 3.80 (q, 2H) , 4.90 (s, 2H) , 5.10 (s, 2H) , 7.10-7.90 (m, 12H) .

Calculated for C ₂₆ H ₃₁ N ₃ OS , HC1 , H ₂ 0:C, 63.9 %; H, 6.4 %; N, 8.6 Found: C, 63.4 %; H, 7.2 %; N, 8.5

Reverse Phase HPLC: 28 min.

Exsrøφl-e

S-n-fDimethvlaminoϊpropvlϊ-l-fnaphth-l-v methvl-l- phenvlthiourea-hvdrochloride

A solution of aniline (7.0 g, 75 mmol) and 1-naphthaldehyde (11.7 g, 75 mmol) in 250 ml ethanol was stirred overnight at room temperature. Then small portions of sodium borohydride (2.8 g, 75 mmol) were added over a period of 20 minutes and the mixture was left to stir for 2 h. Then 250 ml of water was added and the mixture was concentrated in vacuo to a minimum and extracted 4 times with 200 ml of ethyl acetate, dried over magnesium sulfate and concentrated in vacuo to 15.9 (89%) of N-((naphth-1-yl)methyl)aniline as an oil.

N-((naphth-1-yl) )methylaniline (1.0 g, 4.3 mmol) was dissolved in 20 ml of THF at -78 ^β C and lithium diisopropylamide (2.4 ml of a 2.0 M solution in THF) was slowly added and a solution of (3-(diroethylamino)propyl) isothiocyanate in 15 ml of THF was added. The mixture was stirred overnight and concentrated in vacuo and the resulting oil was chromatographed on 250 ml of silica gel with 20% methanol/methylene chloride to give an oil which was dissolved in 20 ml ethyl acetate to which was added 10 ml of 3 M HCl in ethyl acetate. The solution was concentrated in vacuo to give 670 mg (38 %) of 3-(3-(dimethylamino)propyl)-l- (naphth-l-yl)methyl-l-phenyl-thiourea-hydrochloride as a hygroscopic foam which rapidly turned oily.

¹ H NMR (400 MHz, CDC1 ₃ , free base): d 2.00 (t, 2H) , 2.90 (s, 6H) , 3.12 (t, 2H) , 3.72 (t, 2H) , 5.90 (s, 2H) , 6.8-8.3 (m, 12H) .

Calculated for C ₂₃ H ₂₇ N ₃ S,HCl,3/2 H^O 62.6 %; H, 7.0 %; N, 9.5 % Found: C, 62.6 %; H, 7.3 %; N, 9.2 %

Reverse Phase HPLC: 23 min.

Example 3

l-Benzyl-3- ( 3- ( morpholin-4-yl ) propyl ) -1-phenyl thiourea

A solution of N,N-benzylphenylamine (0.5 g, 2.7 mmol) and 3- (morpholin-4-yl)propyl isothiocyanate (0.5 g, 2.7 mmol) in THF was heated at reflux for 20 minutes and cooled to room temperature. The solvent was removed in vacuo and the remaining oil was chromatographed on 300 ml of silicagel wit 20 % methanol/methylene chloride to give 170 mg (17%) of 1- benzyl-3-(3-(morpholin-4-yl)propyl)-l-phenylthiourea as an oil.

'H NMR (400 MHz, CDCl ₃ , free base): d 2.00 (t, 2H) , 3.07 (t, 2H) , 3.25 (m, 4H) , 3.70 (t, 2H) , 3.85 (m, 4H) , 5.45 (s, 2H) , 7.00-7.5 (m, 10H) .

Reverse Phase HPLC: 19 min.

Example 4

l-Benzvl-3-f3- (dimethvlamino) roovl )-1-Phenvlthiourea- h _Y flrpchlPride

To a solution of 3-(dimethylamino)propyl isothiocyanate (1.5 g, 17.8 mmol) in 25 ml of THF at -78*C was added N- benzylaniline (3.3 g, 17.8 mmol) in 20 ml of THF over a period of 15 minutes. The mixture was allowed to warm to roo temperature and stirred overnight. The solvent was removed i

vacuo and the resulting oil was chromatographed on 300 ml of silica gel with 20 % methanol/methylene chloride to give an oil which was dissolved in 30 ml of ethyl acetate to which was added 30 ml of 3M HCl in ethyl acetate. The solution was 5 concentrated in vacuo to give 2.5 g (38 %) of l-benzyl-3-(3- (dimethylamino)propyl)-l-phenylthiourea-hydrochloride as a white solid. M.p. 180-181 *C.

¹ H NMR (200 MHz, CDCl ₃ , free base): d 2.10 (t, 2H) , 2.55 (s, 6H) , 2.85 (t, 2H) , 3.75 (t, 2H) , 5.49 (s, 2H) , 6.3 (b, 1H) , 106.9-7.5 (m, 10H) .

Calculated for C ₁₉ H ₂₅ N ₃ S, HCl:

C, 62.6 %; H, 7.2 %; N, 11.5 %; Cl, 9.7 %

Found: C, 62.1 %; H, 7.3 %; N, 11.3 %; Cl, 9.3 %

Reverse Phase HPLC: 19 min.

Example 5

l.l-Dibenzvl-3-f3-(morpholin-4-vlιPropvHthiourea- hvdrochloride

To a solution of 3-( (morpholin-4-yl)propyl) isothiocyanate (1.0 g, 5.4 mmol) in 20 ml of THF at -78 *C was added N,N- dibenzylamine (1.1 g, 5.4 mmol) in 15 ml of THF over a perio of 15 min. The mixture was allowed to warm to room temperature and stirred overnight. The solvent was removed i vacuo and the residue was chromatographed on 300 ml of silicagel with 20 % methanol/methylene chloride to give an oil, which was dissolved in 30 ml of ethyl acetate to which was added 30 ml of 3M HCl in ethyl acetate. The solution was concentrated in vacuo to give 2.1 g (88 %) of l,l-dibenzyl-3 (3-(morpholin-4-yl)propyl)thiourea-hydrochloride as an amorphous powder.

¹ H NMR (200 MHz, CD ₃ 0D) : d 2.00 (t, 2H) , 3.00 (t, 4H) , 3.45 (t, 2H) , 3.7 (t, 4H) , 4.05 (t, 2H) , 5.00 (s, 4H) , 7.2-7.4 (m, 10H) .

Calculated for C ₂₂ H ₃₀ N ₃ OS , HCl :

C, 62.7 %; H, 7.4 %; N, 9.9 %; Cl, 8.4

Found : C , 62 . 6 % ; H , 7 . 4 % ; N , 9 . 9 % ; Cl , 8 . 4 %

Reverse Phase HPLC: 20 min. Example 6

l-Benzvl-3-(3-(dimethylamino)propyl)-1-( (naohth-2- vl )methvl )thiourea-hvdrochloride

To a solution of 3-(dimethylamino)propyl isothiocyanate (0.34 g, 4.0 mmol) in 20 ml of THF at -78 'C was added N-benzyl-N- ((naphth-2-yl)methyl)amine (1.0 g, 4.0 mmol, prepared as in example 1) in 10 ml of THF over a period of 10 min. The mixture was allowed to warm to room temperature and stirred overnight. The solvent was removed in vacuo and the residue was chromatographed on 300 ml of silica gel with 10 % methanol/methylene chloride to give an oil which was dissolved in 30 ml of ethyl acetate to which was added 30 ml of 3M HCl in ethyl acetate. The solution was concentrated in vacuo to give 0.56 g (33 %) of l-benzyl-3-(3-(morpholin-4- yl)propyl)-l-((naphth-2-yl)methyl)thiourea-hydrochloride as an amorphous powder.

¹ H NMR (200 MHz, CDC1 ₃ , free amine): d 1.65 (t, 2H) , 1.70 (s, 6H) , 2.30 (t, 2H) , 3.70 (t, 2H) , 4.90 (s, 2H) , 5.05 (s, 2H) , 7.2-7.9 (m, 12H) .

Calculated for C^H^S,HCl, 1/3 H ₂ 0: C, 66.3 %; H, 7.1 %; N, 9.7 %

Found: C, 66.1 %; H, 7.7 %; N, 9.5 %

Reverse Phase HPLC: 23 min.

Example 7

l-Benzyl-3- (3-(morpholin-4-yl) ropyl)-1- (pheneth-2- y1)thiourea-hydrochloride

To a solution of N-benzyl-N-(pheneth-2-yl)amine (3.0 g, 9.7 mmol) in 10 ml of THF at 0*C was slowly added 3-(morpholin-4 yl)propyl isothiocyanate in 10 ml of THF. After 15 minutes the solvent was removed and the brownish oil was chromatographed on 450 g of silica gel with 10 % methanol/methylene chloride to give a clear oil which was dissolved in 10 ml of ethyl acetate to which was added 10 ml

of 3 M HCl in ethyl acetate to give 1.8 g (40 %) of 1-benzyl- 3-(3-(morpholin-4-yl)propyl)-1-(pheneth-2-yl)thiourea- hydrochloride as a white solid. M.p. 159-160 *C.

^Λ H NMR (400 MHz, CDC1 ₃ , free amine): d 1.70 (t, 2H) , 2.30 (t, 4H) , 2.35 (t, 2H) , 3.00 (t, 2H) , 3.40 (t, 4H) , 3.70 (t, 2H) , 3.95 (t, 2H) , 4.70 (ε, 2H) , 6.80 (b, 1H) , 7.1-7.4 (m, 10H) .

Reverse Phase HPLC: 22 min.

Example 8

l-Benzvl-3- f 3- (morpholin-4-vl ■ roovl ) -1- f ouinolin-3- vl ) thiourea-dihvdrochlor ide

To a solution of 3-aminoquinoline (3.0 g, 21 mmol) in 100 ml of ethanol, which had been adjusted to pH=5 with HCl in ethanol, was added benzaldehyde (2.2 g, 21 mmol) in 50 ml of ethanol and stirred overnight. Then sodium borohydride (0.8 g, 21 mmol) was added in small portions at reflux and stirred for 1 hour. The mixture was cooled to room temperature, 300 ml of water was added and the solution was concentrated to a minimum in vacuo. The remaining aqueous layer was extracted 4

times with ethyl acetate and the combined extracts were drie over magnesium sulphate and concentrated in vacuo to an oil. This oil was chromatographed on 500 ml of silicagel with 10 ethyl acetate/heptane to give 3.73 g (78 %) of N-benzyl-N- (quinoline-3-yl)amine.

To a solution of N-benzyl-N-(quinoline-3-yl)amine (1.0 g, 4.3 mmol) in 30 ml of THF at -78 ^* C was added lithium diisopropylamide (2.4 ml of a 2.0 M solution in THF) and the mixture was allowed to stir for 10 minutes. Then 3- (morpholin-4-yl)propyl isothiocyanate (0.8 g, 4.3 mmol) in 1 ml of THF was added over a period of 10 minutes and stirred at room temperature overnight. The solution was concentrated in vacuo and the residue was chromatographed on 500 ml of silicagel with 5 % methanol/methylene chloride to an oil, which was dissolved in 15 ml of ethyl acetate to which was added 15 ml of 3M HCl in ethyl acetate. The solution was concentrated in vacuo to give 1.2 g (62 %) of l-benzyl-3-(3- (morpholin-4-yl)propyl)-l-(quinolin-3-yl)thiourea- dihydrochloride as an amorphous powder.

¹ H NMR (400 MHz, CDC1 ₃ , free amine): d 1.70 (t, 2H) , 2.15 (t, 4H) , 2.45 (t, 2H) , 3.15 (t, 4H) , 3.70 (m, 4H) , 5.60 (s, 2H) , 6.45 (S, 1H) , 7.2-7.8 (m, 9H) , 8.05 (d, 1H) , 8.60 (s, 1H) .

Calculated for C ₂₄ H ₂₈ N ₄ OS,2 HCl, H ₂ 0:

C, 56.5 %; H, 6.3 %; N, 11.0 %

Found: C, 56.4 %; H, 7.1 %; N, 11.1 %

Reverse Phase HPLC: 15 min.

Example 9

l-Benzyl-3-f3 morpholin-4-vl)propvl)-l-fpyridin-2-

yl)thiourea-hvdrochloride

To a solution of 2-(benzylamino)pyridine (1.0 g, 5.4 mmol) in 30 ml of THF at -78 *C was added lithium diisopropylamide (3.0 ml of a 2.0 M solution in THF) and the mixture was left to stir for 10 minutes. Then 3-(morpholin-4-yl)propyl isothiocyanate (1.0 g, 5.4 mmol) in 10 ml of THF was added over a period of 10 minutes and stirred at room temperature overnight. The solution was concentrated in vacuo. and the residue was chromatographed on 500 ml of silica gel with 10 % methanol/methylene chloride to give an oil which was dissolved in 15 ml of ethyl acetate to which was added 15 ml of 3M HCl in ethyl acetate. The solution was concentrated in vacuo to give 1.7 g (79 %) of l-benzyl-3-(3-(morpholin-4- yl)propyl)-l-(pyridin-2-yl)thiourea-hydrochloride as an amorphous powder.

'H NMR (400 MHz, CDC1 ₃ , free amine): d 1.95 (t, 2H) , 2.43 (t,

4H) , 2.46 (t, 2H) , 3.65 (t, 4H) , 3.85 (t, 2H) , 5.85 (s, 2H) ,

6.95-7.10 (m, 2H) , 7.20-7.35 (m, 5H) , 7.55 (t, 1H) , 8.25 (d, 1H) .

Calculated for C ₂₀ H ₂₆ N ₄ OS,HCl,2^H ₂ O:

C, 53.2 %; H, 7.1 %; N, 12.4 %;

Found: C, 53 . 5 % ; H, 7. 3 % ; N , 12 . 3

Reverse Phase HPLC: 15 min.

Example 10

l-Benzvl-3-t3-fmorpholin-4-vl)propvl)-l-U- methoxγphenyl)thiourea-hydrochloride

To a solution of N-benzyl-4-methoxyaniline (1.0 g, 4.7 mmol) in 30 ml of THF at -78 ^* C was added lithium diisopropylamide (2.6 ml of a 2.0 M solution in THF) and the mixture was left to stir for 10 minutes. Then 3-(morpholin-4-yl)propyl isothiocyanate (0.9 g, 4.7 mmol) in 20 ml of THF was added over a period of 10 minutes, and the mixture was stirred at room temperature overnight. The solution was concentrated in vacuo and the residue was chromatographed on 500 ml of silic gel with 10 % methanol/methylene chloride to an oil, which was dissolved in 15 ml of ethyl acetate to which was added 1 ml of 3M HCl in ethyl acetate. The solution was concentrated in vacuo to give 172 mg (8 %) of l-benzyl-l-(4- methoxypheny1)-3-(3-(morpholin-4-yl)propyl)thiourea- hydrochloride as an amorphous powder.

¹ H NMR (400 MHz, CDCl,, free amine): d 1.70 (t, 2H) , 2.25 (m,

6H), 2.45 (t, 2H), 3.50 (t, 2H) , 3.70 (t, 2H) , 3.80 (S, 3H) , 5.50 (S, 2H) , 5.80 (b, 1H) , 6.80-6.95 (m, 4H) , 7.20-7.35 (m, 5H) .

Calculated for C ₂₂ H ₂₉ N ₃ 0 ₂ S , HCl , 2H ₂ Ci 55.8 %; H, 7.2 %; N, 8.9 %; Found: C, 55.8 %; H, 7.2 %; N, 9.5 %;

Reverse Phase HPLC: 20 min.

Example U

l-Benzvl-3-f3-fmorpholin-4-vl)propyl)-1-(4-

(r1.2.31thiadiazol-4-vl)benzvl)thiourea-hvdrochloride

To a solution of benzylamine (1.7, 16 mmol) in 60 ml of THF was added 4-(4-bromomethylphenyl)-l,2,3-thiadiazole (1.0 g, 39 mmol) in 40 ml of THF over a period of 15 minutes. The mixture was stirred at room temperature overnight, 50 ml of saturated sodium hydrogencarbonate and 50 ml of water were added and the mixture was extracted 4 times with 50 ml ethyl

acetate. The combined extracts were dried over magnesium sulphate and concentrated in vacuo to an oil. The crude product was chromatographed on 600 ml of silicagel with 35 % ethyl acetate/heptane to give 1.05 g (95 %) of N-benzyl-N-(4 5 ([1,2,3]thiadiazol-4-yl)benzyl)amine.

To a solution of N-benzyl-N-(4-([l,2,3]thiadiazol-4- yl)benzyl)amine (0.95 g, 34 mmol) in 30 ml of THF at -78 *C was added 3-(morpholin-4-yl)propyl isothiocyanate in 30 ml o THF over a period of 10 minutes. The mixture was stirred at

10 room temperature for 2 hours and concentrated in vacuo to an oil. The crude mixture was chromatographed on 400 ml of silicagel with 10 % methanol/methylene chloride to give an oil which was dissolved in 20 ml of ethyl acetate to which was added 20 ml of 3 M of HCl in ethyl acetate and

15 concentrated in vacuo to give 1.37 g (81 %) of l-benzyl-3-(3 (morpholin-4-yl)propyl)-l-(4-([1,2,3]thiadiazol-4- yl)benzyl)thiourea-hydrochloride as an amorphous powder.

*H NMR (400 MHz, CDC1 ₃ , free amine): d 2.00 (t, 2H) , 3.00 (t,

4H) , 3.45 (d, 2H) , 3.75 (t, 4H) , 4.00 (d, 2H) , 5.05 (s, 2H) ,

205.10 (s, 2H) , 7.20-7.45 (m, 7H) , 8.05 (d, 2H) , 9.20 (s, IH) .

Calculated for C^H^OS _j ,HCl,H ₂ C; 55.2 %; H, 6.1 %; N, 13.4 % Found: C, 55.3 %; H, 6.4 %; N, 13.3 %

Reverse Phase HPLC: 23 min.

Exa le 2

253-f3-(Morpholin-4-vl)propvl)-l-ffnaphth-2-vl)methvl)-l- r2'- (lH-tetrazol-5-vl)-biPhenvl-4-vlmethvl1thiourea-hvdrochlorid

To a solution of 5-(4'-bromomethyl-biphenyl-2-yl)-l-trityl- lH-tetrazole (0.6 g, 1.1 mmol) prepared according to W.R. Schoen et al. (J. Med. Chem. (1994), 37, 897-906) in 40 ml of THF was added benzylamine (0.45 g, 4.3 mmol) in 30 ml of THF, and the mixture was stirred overnight. The solvent was removed in vacuo. and the remaining oil was chromatographed on 500 ml of silica gel with 70 % ethyl acetate/heptane to give 460 mg (73 %) of N-benzyl-N-[2'-(l-trityl-lH-tetrazol-5- yl)-(biphenyl-4-yl)methyl]amine.

To a solution of N-benzyl-N-[2'-(l-trityl-lH-tetrazol-5-yl)- (biphenyl-4-yl)methyl]amine (0.46 g, 0.79 mmol) in 20 ml of THF at -78 *C was added 3-(morpholin-4-yl)propyl isothiocyanate over a period of 10 minutes and the mixture was stirred for 2 hours at room temperature. The mixture was concentrated in vacuo to an oil which was dissolved in 20 ml of ethyl acetate, added 20 ml of 3M HCl in ethyl acetate and stirred for 90 minutes. The mixture was concentrated in vacuo and chromatographed on 300 ml of silica gel with 20 % methanol/79.5 % methylene chloride/0.5 % ammonia to give an oil which was dissolved in 20 ml of ethyl acetate to which was added 20 ml of 3 M HCl in ethyl acetate. This solution

was concentrated in vacuo to give 240 mg (54 %) of 3-(3- (morpholin-4-yl)propyl)-l-(naphth-2-yl)methyl-1-[2'-(1H- tetrazol-5-yl)-biphenyl-4-ylmethyl]thiourea- hydrochloride a an amorphous powder.

*H NMR (400 MHz, CDC1 ₃ , free amine): d 1.75 (t, 2H) , 2.65 (t, 2H) , 3.10 (t, 4H) , 3.60 (t, 4H) , 3.75 (t, 2H) , 4.65 (s, 2H) , 5.35 (S, 2H) , 7.10-7.60 (m, 13H) .

Reverse Phase HPLC: 21 min.

Example 13

3-(3-Dimethvlaminopropvl)-l-((naphth-2-vl)methvl)-l- phenylthiourea-hvdrochloride

A solution of 2-naphthaldehyde (5.0 g, 32 mmol) and aniline (3.0 g, 32 mmol) in 200 ml of ethanol was refluxed for 6 hours. Then small portions of sodium borohydride (1.2 g, 32 mmol) was added, and the mixture was stirred for 1 hour at room temperature. Then 200 ml of water was added and the solution was concentrated to a minimum and was extracted 4 times with 200 ml of ethyl acetate. The combined extracts

were dried over magnesium sulphate and concentrated in vacuo. The obtained oil was chromatographed on 700 ml of silicagel with 5 % ethyl acetate/heptane to give 5.2 g (70 %) of N- (naphth-2-yl)methyl-N-phenylamine.

To a solution of N-(naphth-2-yl)methyl-N-phenylamine (0.70 g, 3.0 mmol) in 30 ml of THF at -78 *c was added lithium diisopropylamine (1.7 ml of a 2 M solution in THF) and the mixture was stirred for 15 min. Then 3-(dimethylamino)propyl isothiocyanate (0.44 g, 3.0 mmol) in 20 ml of THF was added over a period of 10 minutes, and the mixture was left to stir overnight. The mixture was concentrated in vacuo and the residue was chromatographed on 500 ml of silicagel with 10 % methanol/methylene chloride to give 250 mg (20 %) of 3-(3- dimethylaminopropyl)-l-( aphth-2-yl)methyl-l-phenyl-thiourea as an oil which was dissolved in 20 ml of ethyl acetate to which was added 20 ml of 3M HCl in ethyl acetate to give 3- (3-dimethylaminopropyl)-1-(naph-2-yl)methyl-l-phenylthiourea - hydrochloride as an amorphous powder.

¹ H NMR (400 MHz, CDC1 ₃ , free amine): d 1.60 (t, 2H) , 1.70 (s, 6H) , 2.20 (t, 2H) , 3.75 (t, 2H) , 5.65 (s, 2H) , 6.9-7.9 (m, 12H) .

Calculated for C ₂₃ H ₂₉ N ₃ S,HCl: C, 66.7 %; H, 6.8 %; N, 10.1 %; Found: C, 62.6 %; H, 6.9 %; N, 9.5 %;

Reverse Phase HPLC: 25 min.