WO2002056873A2

WO2002056873A2 - 2-substituted dibenzo[a,e]1,2,3-triazolo[4,5-c][7]annulen-8-ones as growth hormone secretagogues

Info

Publication number: WO2002056873A2
Application number: PCT/US2001/048258
Authority: WO
Inventors: Brian R. Dixon; Cedo M. Bagi; William J. Scott; Gerald Ranges
Original assignee: Bayer Pharmaceuticals Corporation
Priority date: 2000-12-22
Filing date: 2001-12-14
Publication date: 2002-07-25
Also published as: WO2002056873A3

Abstract

The invention relates to methods of treating or preventing diseases or conditions caused by deficiencies in growth hormone by administration of 2-substituted dibenzo[a,e]1,2,3-triazolo[4,5-c][7]annulen-8-ones. Such diseases or conditions include osteoporosis, osteopenia and obesity. Furthermore, the invention relates to methods of stimulating the release of growth hormone in a mammal by administration of 2-substituted dibenzo[a,e]1,2,3-triazolo[4,5-c][7]annulen-8-ones. Finally, the invention further relates to promoting bone growth in a mammal, particularly endocortical, periosteal, or cancellous bone growth, by administration of 2-substituted dibenzo[a,e]1,2,3-triazolo[4,5-c][7]annulen-8-ones.

Description

2-SUBSTITUTED DBENZO[A,E] 1 ,2,3-TRIAZOLO[4,5-C] [7] ANNULETS-ONES AS GROWTH HORMONE SECRETAGOGUES

Field of the Invention

The invention relates to the use of 2-substituted dibenzo[a,e]l,2,3-tria2;olo[4,5- c][7]annulen-8-ones as growth hormone secretagogues. More particularly, the invention relates to methods of treating or preventing diseases or conditions caused by deficiencies in growth hormone by administering to a mammal a 2-substituted dibenzo[a,e] 1 ,2,3- triazolo[4,5-c][7]annulen-8-one growth hormone secretagogue.

Background

Growth hormone, a hormone that is both produced by and secreted from the pituitary gland, stimulates growth of all tissues in the body that are capable of growing. Additionally, growth hormone regulates metabolic processes, such as the rate of protein synthesis, the rate of carbohydrate metabolism, and the mobilization of free fatty acids and their use for energy.

Deficiencies in growth hormone can result in various diseases and conditions, such as osteoporosis, obesity, growth retardation, skeletal displasia, etc. In children, it causes growth retardation or dwarfism. In adults, the consequences of growth hormone deficiency can include reduction of lean body mass and concomitant increase in total body fat. The consequences of growth hormone deficiency in adults can further include decreased skeletal and muscle mass and decreased bone density. Administration of exogenous growth hormone has been shown to reduce or reverse the effects of such metabolic disturbances.

To increase levels of growth hormone, exogenous growth hormone has historically been administered. The peptidyl nature of the compound, however, required that it be administered by injection. Furthermore, early sources of growth hormone included the pituitary glands of cadavers. This resulted in an expensive product that carried with it the risk of disease transmission (e.g., Creutzfel- Jacob disease).

Most deficiencies of growth hormone result from defects in release of growth hormone from the pituitary gland, however, and not from defects in the synthesis of growth hormone. The release of growth hormone is controlled by a number of hormones and neurotransmitter systems in the brain, particularly the hypothalamus. The release of growth hormone can be stimulated by growth hormone releasing factor (GHRF) and inhibited by somatostatin. In both the cases, the hormones are released from the hypothalamus, but the release of growth hormone itself is mediated primarily by specific receptors in the pituitary gland.

Methods of treating or preventing diseases or conditions caused by growth hormone deficiencies therefore involve stimulating release of growth hormone, such as by administration of a growth hormone secretagogue. Compounds that stimulate the release of growth hormone include arginine, L,-3,4-dihydroxyphenylalanine (L-Dopa), glucagon, vasopressin, pituitary adenylyl cyclase activating peptide (PACAP), and growth hormone releasing peptide (GHRP). Such compounds stimulate release of growth hormone by influencing the hypothalamus to either increase GHRF production or decrease somatostatin production.

Some known growth hormone secretagogues are analogs of these naturally occurring peptides. The peptidyl nature of these compounds, however, requires that they be administered by injection.

Many known growth hormone secretagogues are therefore proteins, which require parenteral administration and suffer from low bioavailability. Other types of compounds that increase levels of endogenous growth hormone are known. The present invention further provides methods of increasing endogenous levels of growth hormone by administration of non-protein compounds.

Summary of the Invention

The present invention provides methods of increasing endogenous levels of growth hormone in a mammal, and therefore treating or preventing diseases or conditions caused by deficiencies in growth hormone, by the administration of a non-protein growth hormone secretagogue. Such compounds follow the general formula I:

wherein

n is a number from 2 to 4;

R¹ and R² are independently selected from hydrogen and alkyl of 1-3 carbon atoms; or

R¹ and R² join to form a 3-7 membered ring, wherein the R¹ and R² that form the ring can be on the same carbon atom, adjacent carbon atoms, or carbon atoms that are 2-3 carbon atoms away from one another;

R³ is selected from hydrogen, alkyl of 1-6 carbon atoms, and C(=0)-R⁵-NH2; or

R³ and R¹ join together with the nitrogen atom to which they are attached when n is 2 or 3 to form a 5-6 membered ring;

R⁴ is selected from hydrogen and alkyl of 1-6 carbon atoms; or

R³ and R⁴ join together with the nitrogen atom to which they are attached to form a 5-8 membered ring, which may contain an additional heteroatom selected from O and NR⁶;

R⁵ is alkyl of 1-6 carbon atoms;

R⁶ is selected from hydrogen and alkyl of 1-3 carbon atoms;

R⁷, R⁸, R⁹ and R¹⁰ are independently selected from hydrogen, halogen, alkyl of 1- 3 carbon atoms, alkoxy of 1-3 carbon atoms, NO2, CN and CF3; and

pharmaceutically acceptable salts thereof. Methods of the invention relate to administration of a growth hormone secretagogue according to formula I for the treatment of any disease or condition that is treatable by increasing endogenous levels of growth hormone in a mammal. Methods of the invention relate to the administration of a growth hormone secretagogue of formula I for (1) stimulating the release of growth hormone in a mammal; (2) treating or preventing a disease or condition resulting from a deficiency of growth hormone in a mammal; (3) promoting/ enhancing bone growth, such as periosteal, endocortical or cancellous bone growth, in a mammal; and (4) promoting/ enhancing bone fracture healing in a mammal.

Diseases and conditions resulting from deficiencies in growth hormone include osteoporosis and osteopenia, bone fracture, delayed wound healing, skin burns, decreased muscle mass, congestive heart failure, growth retardation, obesity, immune deficiencies, physiological short stature, in rauterine growth retardation and hyperinsulinemia.

Methods of the invention for the treatment or prevention of osteoporosis or osteopenia include the administration of bone anti-resorptive agents or bone anabolic agents in addition to a growth hormone secretagogue of formula I. Finally, methods of the invention involve the co-administration of a second growth hormone secretagogue with a growth hormone secretagogue of formula I.

The present invention therefore provides methods for the treatment or prevention of diseases and conditions caused by growth hormone deficiencies. These and other aspects of the invention will be more apparent from the following drawings, description and claims.

Brief Description of the Drawings

Figure 1 shows the release of growth hormone from rat pituitary cells upon exposure to a compound of formula I at 0.1 μM, 1 μM, 10 μM and 100 μM or to GHRP-6 at 100 nM for 15 minutes. Figure 2 shows the change in bone mineral area of the femoral midshaft of ovariectomized rats treated with a compound of formula I versus controls.

Figure 3 shows the change in bone formation rate of the periosteal bone envelope of the femoral midshaft of ovariectomized rats treated with a compound of formula I versus controls.

Figure 4A shows the growth plate chondrocy tes of the distal femor from sham operated rats.

Figure 4B shows the growth plate chondrocytes of the distal femor from ovariectomized rats.

Figure 4C shows the growth plate chondrocytes of the distal femor from ovariectormized rats treated with a compound of formula I.

Detailed Description of the Invention

Methods of the invention provide for increasing endogenous levels of growth hormone in a mammal, and therefore treating or preventing diseases or conditions caused by deficiencies in growth hormone, by the administration of a growth hormone secretagogue. A growth hormone secretagogue for use in methods of the invention includes compounds of the general formula I:

wherein

n is a number from 2 to 4; R¹ and R² are independently selected from hydrogen and alkyl of 1-3 carbon atoms; or

R⁴ is selected from hydrogen and alklyl of 1-6 carbon atoms; or

R⁵ is alkyl of 1-6 carbon atoms;

R⁶ is selected from hydrogen and alkyl of 1-3 carbon atoms;

R⁷, R⁸, R⁹ and R¹⁰ are independently selected from hydrogen, halogen, alkyl of 1-

3 carbon atoms, alkoxy of 1-3 carbon atoms, N0₂, CN and CF₃; and

pharmaceutically acceptable salts thereof.

In preferred embodiments of the invention, R¹ and R² are selected from hydrogen and alkyl of 1-3 carbon atoms, and R³ is selected from C(=0)-R⁵-NH2 or joins with R⁴ and the nitrogen atom to which it is attached to form a 5-6 membered ring.

As used herein the term "alkyl" includes straight-chain or branched alkyls of between 1 and 6 carbon atoms. An "alkyl" in compounds for use in the invention may be a terminal alkyl or it may be a linker between two other parts of the compound. Growth hormone secretagogues useful in methods of the invention include, but are not limited to, the following:

Compounds of formula I are useful in the treatment or prevention of diseases or conditions resulting from deficiencies in endogenous growth hormone levels. A growth hormone secretagogue of formula I may be employed for a wide variety of indications. The known and potential uses of growth hormone are varied. See, e.g., Strobl, et al., Pharm. Rev.. 46, 1-34 (1994); Rosen, etal, Horm. Res.. 43, 93-99 (1995); Degerbald, et al, Eu. J. Endocrinol.. 133, 180-188 (1995); Jorgensen, Eu. J. Endocrinol.. 130, 224-228

(1994); Aloi, et al, J. Clin. Endocrinol. and Metab.. 79(4), 943-949 (1994); Cordido, et al, Metab. Clin. Exp.. 44(6), 745-748 (1995); Frieboes, et al, Neuroendocrinol. 61(5), 584-589 (1995); and Llovera, et al, Inst. J. Cancer. 61(1), 138-141 (1995).

Administration of compounds of formula I stimulate the release of growth hormone and can therefore have the same effect as administration of exogenous growth hormone. Methods of the invention therefore relate to the administration of growth hormone secretagogues of formula I for the treatment of any known disease or condition that is treatable by increasing the levels of growth hormone in a mammal including, but not limited to:

(1) diseases or conditions characterized by low bone mass (See, Shimon, et al, Growth Hormone Secretagogues 315 (1999) and Degerbald, supra), including osteoporosis, osteopenia, osteotomy, childhood idiopathic bone loss, bone loss associated with periodontitis and bone metastases;

(2) bone fractures;

(3) delayed wound healing;

(4) skin burns;

(5) decreased muscle mass (See, Llovera, supra);

(6) cardiovascular disease, including congestive heart failure (See, Nass, et al, Growth Hormone Secretago ues 263 (1999), Berti, etal, Growth Hormone Secretagogues 301 (1999), and Rosen, supra);

(I) growth retardation (See, Strobl, supra), which may result from any number of causes, including renal failure or renal insufficiency, chronic illness, obesity, Prader-Willi syndrome, Turner's syndrome, Down's syndrome, or Noonan's syndrome;

(8) obesity (See, Nass, supra and Nam, Horm. Res.. 53 (Suppl. 1), 87 (2000));

(9) immune deficiencies (See, Foster, et al, Clin. Immunol.. 96, 140 (2000));

(10) physiological short stature (See, Strobl, supra);

(I I) intrauterine growth retardation (See, Strobl, supra); and (12) hyperinsulinemia (See Saenger, Horm. Res. 53 (Suppl. l):60-69 (2000), Nam, Strobl and Rosen, supra).

Compounds of formula I are preferably used in the treatment or prevention of osteopenia and osteoporosis, and in the promotion of bone growth and bone fracture healing.

The present invention also includes use of pharmaceutically acceptable salts of the compounds of formula I. Suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of inorganic and organic acids, such as hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, trifluoromethanesulfonic acid, sulphonic acid, acetic acid, trifluoroacetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid, and mandelic acid. In addition, pharmaceutically acceptable salts include acid salts of inorganic bases, such as salts containing alkaline cations (e.g., Li⁺ Na⁺ or K⁺), alkaline earth cations (e.g., Mg⁺², Ca⁺² or Ba⁺²), the ammonium cation, as well as acid salts of organic bases, including aliphatic and aromatic substituted ammonium, and quaternary ammonium cations such as those arising from protonation or peralkylation of triethylamine, NN-diethylamine, N,N- dicyclohexylamine, pyridine, NN-dimethylaminopyridine (DMAP), 1,4- diazabicyclo[2.2.2]octane (DABCO), l,5-diazabicyclo[4.3.0]non-5-ene (DBΝ) and 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU).

A number of the compounds of formula I possess asymmetric carbons and can therefore exist in racemic and optically active forms. Methods of separation of enantiomeric and diastereomeric mixtures are well known to those skilled in the art. The present invention encompasses the use of any racemic or optically active forms of the compounds described in formula I for the treatment or prevention of diseases or conditions resulting from deficiencies in growth hormone.

The therapeutic agents of the invention may be employed alone or concurrently with other therapies. Particularly, a growth hormone secretagogue of formula I may be administered in combination, either concurrently or sequentially, with other known growth hormone secretagogues, anabolic agents or bone antiresorptive agents. The compounds may be administered in dose ranges from one one-hundredth to one times the dose levels which are effective when these compounds are used alone. Other known growth hormone secretagogues include, but are not limited to, the growth hormone releasing peptides (GHRP-1, GHRP-2, and GHRP-6), growth hormone releasing factor (growth honnone releasing honnone) and its analogs, B-HT920, hexarelin, somatomedins (IGF-1 and IGF-2), μ-adrenergic agonists (clonidine), serotonin 5HTlDa agonists (sumitriptan), and agents that inhibit somatostatin or its release

(physostigmine and pyridostigmine). Additionally, growth hormone secretagogues disclosed, for example, in U.S. Pat. Nos. 5,536,716; 5,723,616; 5,773,441; 6,110,932; and 6,127,391, the disclosures of which are hereby incorporated by reference, may be useful in combination with the growth hormone secretagogues of formula I. Additionally, compounds disclosed, for example, in GB 2324726, EP 0 761 220, EP 0

761 219, EP 1 031 575, EP 1 002 802, WO 00/49037, WO 98/18815, WO 97/15573, WO 95/03289, WO 94/07483, WO 98/58949, and WO 98/58947 may be useful in methods of the invention in combination with a growth hormone secretagogue of fonnula I. Any growth hormone secretagogue may be useful in combination with a growth hormone secretagogue of formula I in methods of the invention.

Anabolic agents that may be useful in combination with compounds of formula I include, but are not limited to, bone morphogenic proteins or active fragments thereof, thyrotropin releasing hormone or an active fragment thereof, diethylstilbeserol, estrogens, β-agonists, parathyroid hormone or an active fragment thereof, theophylline, anabolic steroids, enkephalins, prostaglandins and their agonists/antagonists, and sodium fluoride. Additionally, anabolic agents disclosed, for example, in U.S. Pat. Nos. 3,239,345; 4,036,979; 4,411,890, the disclosures of which are hereby incorporated by reference, may be useful in combination with the growth hormone secretagogues of formula I. Any anabolic agent may be useful in combination with a growth hormone secretagogue of formula I in methods of the invention.

When employed as a treatment for osteoporosis or osteopenia, the compounds of formula I may be used in combination with a calcium source, vitamin D or analogues of vitamin D, and/or bone antiresorptive therapies, such as estrogen replacement therapy, treatment with a fluoride source, or treatment with calcitonin or a calcitonin analogue. Additionally, combined therapy to inhibit bone resorption, prevent osteoporosis and/or osteopenia, reduce skeletal fracture, enhance bone fracture healing, stimulate bone formation and increase bone mineral density can be effectuated by combinations of the compounds of formula I and a bisphosphonate. See, e.g., Hamdy, Trends in Endocrinol. Metab.. 4, 19-25 (1993). Bisphosphonates with these utilities include, but are not limited to, alendrorirate, tiludronate, dimethyl- APD, risedronate, etidronate, YM-175, clodronate, pamidronate, BM-210995 (ibandronate), tiludronic acid, zoledronic acid, alendronic acid, ibandronic acid, risedronic acid, etidronic acid, clodronic acid and pamidronic acid. Any bone antiresorptive agent may be useful in combination with a growth hormone secretagogue of formula I in methods of the invention.

Bone antiresorptive agents that may also be used in combination with a growth hormone secretagogue of formula I include estrogen agonist/antagonist. The phrase "estrogen agonist/antagonist" refers to compounds that bind the estrogen receptor, inhibit bone turnover and prevent bone loss. Examples of estrogen agonist/antagonist include, but are not limited to, droloxifene, raloxifene, tamoxifen, 4-hydroxy tamoxifen, toremifene, clometherone, delmadinone, centchroman, levormeloxifene, nafoxidine, nitromifene, ormeloxifene, trioxifene, and idoxifene. Estrogen agonist/antagoinst are disclosed, for example, in U.S. Pat. Nos. 3,274,213; 4,133,814; 4,230,862; 4,323,707; 4,380,635; 4,400,543; 4,418,068; 4,536,516; 5,254,594, the disclosures of which are hereby incorporated by reference. Other estrogen agonist/antagonist are disclosed in, for example, EP 0 062 503, EP 0 054 168, EP 0 260 066, EP 0 470 310, EP 0 651 998, and EP 0 652 005. Any estrogen receptor agonist or antagonist may be useful in combination with a growth hormone secretagogue of formula I in methods of the invention.

The methods of the invention are intended to be employed for treatment of diseases or conditions resulting from deficiencies in growth hormone in both humans and other mammals. For example, the compounds of foπnula I may also be useful in stimulating the immune system in companion animals, treating disorders of aging in companion animals, promoting growth in livestock, or stimulating wool growth in sheep.

In methods of the invention, the growth hormone secretagogues of formula I, and/or any co-administered compounds, may be administered orally, dermally, parenterally, by injection, by inhalation or spray, or sublingually, rectally or vaginally in dosage unit formulations. The term "administered by injection" includes intravenous, intraarticular, intramuscular, subcutaneous and parenteral injections, as well as use of infusion techniques. Dermal administration may include topical application or transdermal administration. One or more compounds may be present in association with one or more non-toxic phannaceutically acceptable carriers and, if desired, other active ingredients.

Compositions intended for oral use may be prepared according to any suitable method known to the art for the manufacture of pharmaceutical compositions. Such compositions may contain one or more agents selected from the group consisting of diluents, sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide palatable preparations.

Tablets contain the growth hormone secretagogue of formula I in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; and binding agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. These compounds may also be prepared in solid, rapidly released form.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions containing a growth hormone secretagogue of formula I in admixture with excipients suitable for the manufacture of aqueous suspensions may also be used. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or «-propyl,

one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavoring and coloring agents, may also be present.

The growth honnone secretagogue of formula I may also be in the form of non- aqueous liquid formulations, e.g., oily suspensions which may be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or peanut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Methods of the invention may also include administration of pharmaceutical compositions containing a growth hormone secretagogue of formula I in the form of oil- in-water emulsions. The oil phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents. A growth hormone secretagogue of formula I may also be administered in the form of suppositories for rectal or vaginal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal or vaginal temperature and will therefore melt in the rectum or vagina to release the drug. Such materials include cocoa butter and polyethylene glycols.

Methods of the invention may also include transdermal administration of a growth hormone secretagogue of formula I using methods known to those skilled in the art (see, e.g., Chien; "Transdermal Controlled Systemic Medications"; Marcel Dekker, Inc.; 1987. Lipp et al. WO 94/04157 3Mar94). For example, a solution or suspension of a compound of formula I in a suitable volatile solvent optionally containing penetration enhancing agents can be combined with additional additives known to those skilled in the art, such as matrix materials and bacteriocides. After sterilization, the resulting mixture can be formulated following known procedures into dosage forms. In addition, on treatment with emulsifying agents and water, a solution or suspension of a compound of formula I may be formulated into a lotion or salve.

Suitable solvents for processing transdermal delivery systems are known to those skilled in the art, and include lower alcohols such as ethanol or isopropyl alcohol, lower ketones such as acetone, lower carboxylic acid esters such as ethyl acetate, polar ethers such as tetrahydrofuran, lower hydrocarbons such as hexane, cyclohexane or benzene, or halogenated hydrocarbons such as dichloromethane, chloroform, trichlorotrifluoroethane, or trichlorofluoroethane. Suitable solvents may also include mixtures of one or more materials selected from lower alcohols, lower ketones, lower carboxylic acid esters, polar ethers, lower hydrocarbons, halogenated hydrocarbons.

Suitable penetration enhancing materials for transdermal delivery systems are known to those skilled in the art, and include, for example, monohydroxy or polyhydroxy alcohols such as ethanol, propylene glycol or benzyl alcohol, saturated or unsaturated C₈- Ci₈ fatty alcohols such as lauryl alcohol or cetyl alcohol, saturated or unsaturated C₈-Cι₈ fatty acids such as stearic acid, saturated or unsaturated fatty esters with up to 24 carbons such as methyl, ethyl, propyl, isopropyl, ra-butyl, sec-butyl isobutyl tert-butyl or monoglycerin esters of acetic acid, capronic acid, lauric acid, myristinic acid, stearic acid, or palmitic acid, or diesters of saturated or unsaturated dicarboxylic acids with a total of up to 24 carbons such as diisopropyl adipate, diisobutyl adipate, diisopropyl sebacate, diisopropyl maleate, or diisopropyl fumarate. Additional penetration enhancing materials include phosphatidyl derivatives such as lecithin or cephalin, terpenes, amides, ketones, ureas and their derivatives, and ethers such as dimethyl isosorbid and diethyleneglycol monoethyl ether. Suitable penetration enhancing formulations may also include mixtures one or more materials selected from monohydroxy or polyhydroxy alcohols, saturated or unsaturated C₈-Cι₈ fatty alcohols, saturated or unsaturated C₈-Cι₈ fatty acids, saturated or unsaturated fatty esters with up to 24 carbons, diesters of saturated or unsaturated dicarboxylic acids with a total of up to 24 carbons, phosphatidyl derivatives, terpenes, amides, ketones, ureas and their derivatives, and ethers.

Suitable binding materials for transdermal delivery systems are known to those skilled in the art and include polyacrylates, silicones, polyurethanes, block polymers, styrene-butadiene coploymers, and natural and synthetic rubbers. Cellulose ethers, derivatized polyethylenes, and silicates may also be used as matrix components.

Additional additives, such as viscous resins or oils may be added to increase the viscosity of the matrix.

For all methods of administering a growth hormone secretagogue of formula I disclosed herein, the daily oral dosage regimen will preferably be from 0.01 to 200 mg/Kg of total body weight. The daily dosage for administration by injection, including intravenous, intramuscular, subcutaneous and parenteral injections, and use of infusion techniques will preferably be from 0.01 to 200 mg/Kg of total body weight. The daily rectal dosage regimen will preferably be from 0.01 to 200 mg/Kg of total body weight. The daily vaginal dosage regimen will preferably be from 0.01 to 200 mg/Kg of total body weight. The daily topical dosage regimen will preferably be from 0.1 to 200 mg administered between one to four times daily. The transdermal concentration will preferably be that required to maintain a daily dose of from 0.01 to 200 mg/Kg. The daily inhalation dosage regimen will preferably be from 0.01 to 10 mg/Kg of total body weight.

It will be appreciated by those skilled in the art that the particular method of administration will depend on a variety of factors, all of which are considered routinely when administering therapeutics. It will also be understood, however, that the specific dose level for any given patient will depend upon a variety of factors, including, but not limited to the activity of the specific compound employed, the age of the patient, the body weight of the patient, the general health of the patient, the gender of the patient, the diet of the patient, time of administration, route of administration, rate of excretion, drug combinations, and the severity of the condition undergoing therapy. It will be further appreciated by one skilled in the art that the optimal course of treatment, i.e., the mode of treatment and the daily number of doses of a compound of formula I or a pharmaceutically acceptable salt thereof given for a defined number of days, can be ascertained by those skilled in the art using conventional treatment tests.

The growth hormone secretagogues of formula I may be prepared by use of known chemical reactions and procedures, from known compounds (or from starting materials which, in turn, are producible from known compounds) through the preparative methods described below, as well as by other reactions and procedures known to those skilled in the art. Such reactions and procedures include, but are not limited to, esterification, hydrolysis, alkylation, acylation neutralization, coupling, oxidation, reduction, condensation, elimination and substitution reactions. Nevertheless, the following general preparative methods are presented to aid practitioners in synthesizing the compounds of the invention, with more detailed particular examples being presented in the experimental section. The examples are for illustrative purposes only and are not intended, nor should they be construed, to limit the invention in any way.

Within the scope of each method, optional substituents may appear on reagents or intermediates which may act as protecting groups or other non-participating groups. Utilizing methods well known to those skilled in the art, such groups are introduced and/or removed during the course of the synthetic schemes to provide the compounds of the present invention. All variable groups not defined below are as described hereinabove.

General Preparative Methods

The compounds of formula I may be prepared by the use of known chemical reactions and procedures, some from starting materials that are commercially available. Some of the compounds of formula I are known in the art and methods of preparing them have been described. (See, Tochtermann, DE 1,938,583 and DE 1,938,584.) Nevertheless, general preparative methods are provided below to aid one skilled in the art in synthesizing these compounds.

Aromatic synthesis and functional group interconversions may be accomplished using standard methods (March, Advanced Organic Chemistry, 3^rd Ed.; John Wiley: New York (1985); Larock, Comprehensive Organic Transformations; VCH Publishers: New York (1989)). 5H- Dibenzo[a,d]cyclohepten-5-one and substituted 5H-dibenzo[a,d]cyclo-hepten-5- ones (ketone 1, Scheme 1) may be prepared using known methods (Cope, T. Am. Chem. Soc. 1951, 73, 1673; Klinkhammer, Chem. Ber. 1951, 84, 671; Campbell, Helv. Chim. Acta 1953, 36, 1489). Treatment of ketone 1 with bromine affords dibromoketone 2. Subsequent dehydrobromination using a base, such as KOH, delivers the vinyl bromide 3 (Tochtermann, Justus Liebigs Ann. Chem. 1967, 705, 169). Further potassium tert-butoxide-mediated dehydrobromination of vinyl bromide 3 in the presence of NaN₃ then affords triazole 4 (Tochtermann, Chem. Ber. 1964, 97, 1318).

Scheme 1

Formation of the anion of triazole 4, either on treatment with a base, such as NaOH (Tochtermann, Chem. Ber. 1964, 97, 1318), or abase such as NaOEt (Tochtennann DE 1,938,583 (1971)), followed by treatment with electrophile 5, in which L stands for a leaving group, such as a halogen (CI, Br or I) or a sulf onate ester, affords amine 6 (Scheme 2). In some cases, use of a base is not required for the reaction of triazole 4 with electrophile 5 to generate amine 6 (Tochtermann DE 1,938,583 (1971)).

Scheme 2

Compounds of formula I stimulate the release of growth hormone in a mammal and promote the formation of bone in a mammal.

Biological Example 1 - Release of Growth Hormone

A primary culture of rat anterior pituitary cells was prepared and the Ca ,2+ . flux response and growth hormone release to a GHRP-6 challenge was tested. Thirty Sprague Dawley male rats (Harlan Sprague Dawley, Inc., Indianapolis, IN) at ages of 50-55 days were used in this experiment. The rats were sacrificed using CO₂. The rats were dipped in a 70% aqueous ethanol solution, and the pituitary was removed using instruments that were pre-sterilized in a 70% aqueous ethanol solution. Three sets of instruments were used - one to peel the skin, cut the head off, and break the bone between eyes; a second to cut the head bone layer and take the brain tissue away; and a third to remove the pituitary gland. The pituitary glands were placed in Hanks' balanced salt solution modified to include a Penicillin Streptomycin solution (HBSS+P/S; 1% Penicillin Streptomycin solution (Gibco 15140-122) in HBSS (Gibco 14025-092)) in a 50 mL conical tube cooled to 0 °C over an ice bath. The glands were taken to a class II cell culture hood in which all subsequent operations were performed.

The pituitary glands were washed three times with HBSS + P/S (ten-fold volume, 30 mL) and transferred to a petri dish. The glands were minced with sterile disposable scalpels #21 into fine pieces. The minced tissue was then transferred to a 50 mL disposable tube by suspending the tissue fragments in three successive 10 mL aliquots of digestion mixture (0.2% Collagenase Type I (Gibco 1700-017) and 0.2% hyaluronidase (Sigma H3506) in HBSS). (This technique is a modification of the method of Cheng, et al, Endocrinology 1989, 124, 2791.) Cell dispersion was carried out in a 37 °C water bath for 30 min with vortexing every 10-15 min. The tube was then allowed to sit for about 3 min so that the undigested pituitary fragments settled. The supernatant was then transferred to a new tube. Fresh digestion mixture (30 mL) was added to the undigested pituitary fragments. Digestion was allowed to occur for another 30 min at 37 °C. The first batch supernatant was spun at 800 x g for 10 min and the cell pellet resuspended into 25 mL of culture medium. The cells were kept on ice. These steps are then repeated for the second batch of supernatant. The two cell suspensions were then combined and centrifuged again at 800 x g for 10 min. The cell pellet was resuspended in 50 mL of culture media and strained through a cell strainer with 100 υM. The cells were seeded at lxl 0⁵ cells/1 mL/well in 24-well poly(lysine) coated plates.

The cells were placed in a humidified incubator at 37 °C in a culture medium under a 5% CO₂ atmosphere for 3 days. The culture medium consisted of (1) DMEM (Gibco 11995-065); (2) 10% heat inactivated horse serum (Gibco 26050-070); (3) 2.5% heat inactivated Fetal Bovine Serum (FBS) (Gibco 10082-139); (4) 1% nonessential amino acids (Gibco 11140-050 lot 1025957); (5) 1% MEM Amino Acids (Gibco 11130- 051); (6) 1% L-Glutamine (Gibco 25030-081, 200mM stock); (7) 1% Nystatin (Gibco

15340-052); (8) 0.1% Gentamicin (Gibco 15750-060); and (9) 0.37% NaHCO₃.

After 3 days in culture, the cell media was replaced with 1 mL fresh culture medium plus 20 nM somatostatin. The cells were then incubated at 37 °C overnight. The next day, the cells are washed twice with challenge medium (culture medium with 25 mM HEPES, pH 7.4), then the cells were challenged with a compound of formula I or

GHRP-6 for 15 min. The supernatant was removed, spun down and frozen in aliquots until analyzed. The media was analyzed using a BIOTRAK Rat Growth Honnone Elisa kit (Amersham RPN2561).

The compounds of the invention stimulated the release of growth hormone from the rat pituitary cells. Figure 1 shows the amount of released growth hormone (GH) from the rat pituitary cells by a compound of formula I compared to the amount of released growth hormone from rat pituitary cells by GHRP-6.

Biological Example 2 - Promotion of Bone Growth

Laboratory animals

OVX rat model is a small animal model for estrogen deficient bone loss in humans (postmenopausal osteoporosis) (Wronski, et al., 1985, Calcif Tissue Int

37, 324-328; Frost and Jee, 1991, Bone Mineral 18, 227-236; Kalu, Bone Mineral, 1991, 15, 175-192; Miller, et al, 1991, Bone 12, 439-446 ).

Fifty female Sprague-Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, IN) were obtained at 90 days of age. Thirty rats had been ovariectomized and twenty had been sham operated at Harlan Sprague Dawley using a dorsal approach. Rats were allowed to acclimate in-house for 10 weeks. At week ten, rats were divided into groups based on body weight with OVX rats in three groups of ten and Sham rats in two groups of seven and one group of six. Fourteen previously Sham-operated rats (two groups of seven) were ovariectomized using a dorsal approach (Waynforth HB, 1987, Academic Press,

London). Throughout the 5 weeks of the experiment, the Sham-operated and OVX rats had free access to standard laboratory diet (PMI Feeds, Inc., Richmond, IN) and water. Fluorochrome bone markers for histomorphometric measurements were administered to all rats in this study as intraperitoneal injections before treatment started; time 0 (30 mg/Kg demeclocycline HC1 salt,

Sigma D6140), 10 days (15 mg/Kg calcein, Sigma C0875), and 3 days before euthanasia (Demeclocycline HC1, 30 mg/Kg Sigma D6140). Experimental protocol

All rats in this study were dosed daily with an oral dose of vehicle, progesterone (20 mg/Kg) or a growth hormone secretagogue of formula I (20 mg/Kg) during the 5 week period. Rats were dosed according to average group weight.

Vehicle, Compound of Formula I, and Progesterone

All solutions were made up once a week and stored at 4°C. Prior to dosing, solutions were sonicated at room temperature for 10 min.

Vehicle was distilled water from a pico-pure hydro distilling system (HYDRO, West Haven, CT). All animals in the vehicle groups were dosed at 10 mL/Kg using the average body weight of the group.

A 2 mg/mL solution of the compound of formula I was made by dissolving 500 mg of the compound in 0.5 mL of 100% ethanol and diluting to 250 mL with vehicle. Animals were dosed at 10 mL/Kg based on the average group body weight.

Body and Organ Weights

Body weights were recorded weekly throughout the experiment. At necropsy, liver, kidney, adrenal, and uterus were collected, and percent of body weight was determined.

Dual Energy X-Ray Absorptiometry (DEXA)

At day zero and day 34, the following were measured in all rats using

Dual Energy X-ray Absorptiometry with a Hologic QDR 1000+ (Hologic,

Waltham, MA): total bone area, total bone mineral content, and total bone mineral density (Ammann, et al, T Bone Min Res, 1992, 7, 311-315). The heads and tails of the rats were not included in any of these determinations.

Additionally, the total bone area, bone mineral content, and bone mineral density were determined in the whole left femurs and at the femoral neck sites after necropsy. For that purpose, the machine was adapted for ultra-high resolution with line spacing set at 0.0254 cm, resolution 0.0127 cm, and a collimator of 0.9 cm diameter. The Bone Mineral Area (BMA) was expressed in cm .

Histology and Histomorphometry

Following DEXA measurements, the left femurs were cut into halves. Following fixation with NBF for 48 hours, the distal halves were dehydrated with a series of graded ethanol and embedded in poly (methyl methacrylate). Sections were cut with a Jung Polycut E microtome (Reinchart Jung, Germany) into longitudinal sections 4 μm thick. Those sections were used unstained for dynamic histomorphometric analyses of the cancellous bone. Right femurs were fixed in NBF for 48 hours and dehydrated through a graded ethanol series, undecalcified femurs were cut in halves. Proximal halves were used for cortical bone measurements at the mid-diaphyses. Three consecutive sections, each 60 μm thick, were cut with a precision bone saw (Isomet 1000, Buehler, Lake Bluff, IL), and ground to 10 μm in thickness (Pethro-thin, Buehler, Lake Bluff, IL) before being used for microradiography (Faxitron, Faxitron X-Ray Corp., Buffalo

Grove IL). Microradiographs were taken using 1 A ultra microradiography plates (IMTEC, Sunnyvale, CA). The distal halves were used for static bone histomorphometry of the cancellous bone (metaphyses and epiphyses) and assessment of the longitudinal bone growth at distal femoral growth plate.

Dynamic histomorphometric measurements were performed on unstained, undecalcified sections (3 sections per animal) at 10 and/ or 20 X magnification using a fluorescence microscope (Nikon, Japan) interfaced with a digitizer. The data were collected using a software package specifically written for bone histomorphometry ("Stereology", KSS Computer Engineers, Magna, UT). The histomorphometric parameters were calculated according to Parfitt, et ah, T Bone Min Res, 1987, 2:595-610. Measurements were made on the entire periosteal surface, on the endocortical surfaces, which were free of endocortico- trabecular intersections, and on the entire metaphyseal spongiosa 0.5 mm below the growth plate. The thickness of the growth plate, and size and number of the growth plate chondrocytes was assessed at the growth plate at the distal femur.

Statistical analyses

Differences among groups were tested for significance in a one-way analysis of variance. When the analysis of variance indicated significant differences among means, the differences were evaluated using Fisher's protected least significant difference (PLSD) method for multiple comparisons. Statistical significance was considered at P<0.05, and results are expressed as the mean + standard error (SE).

Results

Changes in the bone mineral area at femoral midshaft in ovariectomized rats treated with a compound of formula I were observed, as shown in Figure 2. The P value indicates significance of the change in rats treated with a compound of formula I versus the Ovx controls.

Changes in the bone fonriation rate measured at the periosteal bone envelope at the femoral midshaft in ovariectomized rats treated with a compound of formula I were observed, as shown in Figure 3. The P value indicates significance of the change in rats treated with a compound of formula I versus the Ovx controls.

Figures 4A, 4B and 4C show the growth plate chondrocytes at the distal femur from Sham (4A), Ovx (4B) and Ovx rats treated with a compound of formula I (4C). Note the difference in chondrocyte size between groups indicated by arrows. E = epiphysis; M = metaphysis; Magnification = x20.

Conclusions of the In Vivo Pharmacology

Based on the histologic and histomorphometric measurements, and DEXA evaluation, as described above and as shown in Figures 2, 3, A, 4B and 4C, compounds of formula I promote bone growth and bone formation. Oral treatment with a compound of formula I promoted cortical bone formation at the periosteal cortical envelope, as judged by bone formation rate, and longitudinal bone growth, as judged by bone mineral area, growth plate width and number and size of the growth plate chondrocytes.

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing examples are included by way of illustration only. Accordingly, the scope of the invention is limited only by the scope of the appended claims.

Claims

ClaimsWhat is claimed is:

1. A method of stimulating the release of growth hormone in a mammal, comprising administering to the mammal a growth hormone secretagogue of formula I:

wherein

n is a number from 2 to 4;

R³ is selected from hydrogen, alkyl of 1-6 carbon atoms, and C(=0)-R⁵- NH₂; or

R⁴ is selected from hydrogen and alkyl of 1-6 carbon atoms; or

R³ and R⁴ join together with the nitrogen atom to which they are attached to form a 5-8 membered ring, which may contain an additional heteroatom selected from O and NR⁶; R⁵ is alkyl of 1-6 carbon atoms;

R⁶ is selected from hydrogen and alkyl of 1-3 carbon atoms;

R⁷, R⁸, R⁹ and R¹⁰ are independently selected from hydrogen, halogen, alkyl of 1-3 carbon atoms, alkoxy of 1-3 carbon atoms, N0₂, CN and CF₃; and

pharmaceutically acceptable salts thereof.

2. The method of claim 1, wherein

R¹ and R² are selected from hydrogen and alkyl of 1-3 carbon atoms;

R³ is selected from C(=0)-R⁵-NH2 or joins with R⁴ and the nitrogen atom to which it is attached to form a 5-6 membered ring; and

pharmaceutically acceptable salts thereof.

3. The method of claim 1, wherein said growth hormone secretagogue is selected from:

and pharmaceutically acceptable salts thereof.

4. A method of treating or preventing a disease or condition resulting from a deficiency of growth hormone in a mammal, comprising administering a growth hormone secretagogue of formula I according to claim 1.

5. The method of claim 4, wherein said disease or condition is selected from:

a) delayed wound healing; b) skin burns; c) decreased muscle mass; d) congestive heart failure; e) growth retardation; f) obesity; g) immune deficiencies; h) physiological short stature; i) intrauterine growth retardation; and j) hyperinsulinemia.

6. The method of claim 5, wherein said growth retardation results from

a) renal failure or renal insufficiency; b) chronic illness; c) obesity; d) Prader-Willi syndrome; e) Turner's syndrome; f) Down's syndrome; and g) Noonan's syndrome.

7. A method of treating or preventing a disease or condition that is treatable by increasing levels of growth hormone in a mammal, comprising administering a growth hormone secretagogue of formula I according to claim 1.

8. A method of treating or preventing a disease or condition characterized by low bone mass, comprising administering a growth hormone secretagogue of formula I according to claim 1.

9. The method of claim 8, wherein the disease or condition is selected from osteoporosis, osteopenia, osteotomy, childhood idiopathic bone loss, bone loss associated with periodontitis and bone metastases.

10. The method of claim 8, further comprising administering a bone antiresorptive agent in combination with a growth hormone secretagogue of formula I.

11. The method of claim 10, wherein said bone anti-resorptive agent is selected from droloxifene, raloxifene, tamoxifen, 4-hydroxy-tamoxifen, toremifene, centchroman, clometherone, delmadinone, levormeloxifene, idoxifene, nafoxidine, nitromifene, onneloxifene, trioxifene, tiludronic acid, zoledronic acid, alendronic acid, ibandronic acid, risedronic acid, etidronic acid, clodronic acid, pamidronic acid, dimethyl-APD, YM-175 and BM-210995.

12. The method of claim 8, further comprising administering a bone anabolic agent in combination with a growth hormone secretagogue of formula I.

13. The method of claim 12, wherein said bone anabolic agent is selected from bone morphogenic proteins or active fragments thereof, thyrotropin releasing hormone or an active fragment thereof, diethylstilbeserol, estrogens, β-agonists, parathyroid hormone or an active fragment thereof, theophylline, anabolic steroids, enkephalins, prostaglandins and their agonists/ antagonists, and sodium fluoride.

14. A method of promoting or enhancing bone growth in a mammal, comprising administering a growth hormone secretagogue of formula I according to claim 1.

15. The method of claim 14, wherein said bone growth is selected from periosteal bone growth, endocortical bone growth and cancellous bone growth.

16. A method of promoting or enhancing healing of a bone fracture in a mammal, comprising administering a growth hormone secretagogue of formula I according to claim 1.

17. The method of claim 1, 4, 7, 8, 14 or 16, further comprising the administration of a second growth hormone secretagogue in combination with a growth hormone secretagogue of formula I.

18. The method of claim 17, wherein said second growth hormone secretagogue is selected from hexarelin, GHRP-6, GHRP-1, GHRP-2, GHRF or an analog thereof, IGF-1, IGF-2, B-HT920, clondine, sumitriptan, physostigmine and pyridostigmine.