HK1036995A - Tartrate salt of a substituted dipeptide as growth hormone secretagogue - Google Patents

Description

Tartrate salt of substituted dipeptide as growth hormone secretagogue

Background

The present invention relates to the (L) - (+) -tartrate salt of 2-amino-N- {1- (R) - (2, 4-di-fluoro-benzyloxymethyl) -2-oxo-2- [ 3-oxo-3 a- (R) -pyridin-2-ylmethyl-2- (2,2, 2-trifluoro-ethyl) -2,3,3a,4,6, 7-hexahydro-pyrazolo [4,3-c ] pyridin-5-yl ] -ethyl } -2-methyl-propionamide, which is a growth hormone secretagogue.

Growth Hormone (GH) is secreted by the adenohypophysis and stimulates the growth of various viable tissues of the body. In addition, growth hormone is known to have the following basic effects on the metabolism of the body:

1. promoting the synthesis of proteins of virtually all cells of the organism;

2. inhibiting the utilization of carbohydrates in cells of the body; and

3. increase mobilization of free fatty acids and utilization of fatty acids.

Growth hormone deficiency leads to various diseases. Dwarfism can occur in children. Acquired GH deficiency in adults can cause a significant reduction in body non-fat mass with a concomitant increase in body total fat, particularly in the torso. The reduction in skeletal and cardiac muscle mass and muscle strength results in a significant reduction in motor capacity. Bone density also decreases. It has been observed that the use of exogenous growth hormone can reverse many metabolic changes. Other benefits of treatment include a decrease in LDL cholesterol and an improvement in mental health.

If elevated levels of growth hormone are desired, this problem is usually solved by providing exogenous growth hormone, or by applying drugs which stimulate the production and/or release of growth hormone. In both cases, the peptidyl nature of this compound determines that it needs to be administered by injection. The growth hormone was initially derived from an extraction of the pituitary gland from a cadaver gland. Such products are relatively expensive and are associated with a risk that diseases associated with the pituitary gland origin may be transmitted to the recipient of the growth hormone (e.g., Creutzfeldt-Jakob syndrome). Recently, recombinant growth hormone has become available, and although this is no longer accompanied by the risk of disease transmission, this product is still very expensive and it must be administered by injection or nasal aerosol.

Most GH deficiencies are due to a defect in GH secretion, not primarily pituitary GH synthesis. Thus, an alternative to normalizing serum GH is to stimulate its release from somatropic cells. Increased GH secretion can be achieved by stimulating or inhibiting various neurotransmitter systems in the brain and hypothalamus. Synthetic growth hormone releasing agents are being developed that stimulate pituitary GH secretion and have various advantages over expensive and inconsistent GH replacement therapies. Since this works through a physiologically regulated pathway, most desirable drugs stimulate pulsatile GH secretion and excessive GH levels associated with side effects of exogenous GH application can be avoided by virtue of an intact negative feedback loop.

Physiological and pharmacological stimulants of GH secretion include arginine, L-3, 4-dihydroxyphenylalanine (L-DOPA), glucagon, vasopressin, and insulin-induced hypoglycemia, as well as activities such as sleep and exercise, may indirectly cause growth hormone release from the pituitary by acting on the hypothalamus in a manner to decrease secretion of somatostatin or increase secretion of known GHRF or unknown endogenous GHRF, or all of them.

The present invention also relates to a method of treating insulin resistant states such as non-insulin dependent diabetes mellitus (NIDD) and the regulation of blood glucose lowering associated with obesity and aging in mammals. The method which is required comprises applying to the mammal in question an effective amount of the L- (+) -tartrate salt of the compound of formula I. The compounds of formula I are shown below.

Other compounds that stimulate endogenous growth hormone release have been developed, such as peptidyl-like compounds related to GRF or the peptides of us patent 4,411,890. These peptides are significantly smaller than growth hormone and are also sensitive to various proteases. For most peptides, their potential for oral bioavailability is relatively low.

WO 94/13696 mentions certain spiropiperidines and homologues which promote the release of growth hormone. The general structure of preferred compounds is as follows:

WO 94/11012 mentions certain dipeptides that promote the release of growth hormone. The general structure of these dipeptides is as follows:wherein L is

The compounds of WO 94/11012 and WO 94/13696 have been reported to be useful in combination with parathyroid hormone or a bisphosphonate for the treatment of osteoporosis.

A general description of the pharmaceutically acceptable salts of the compounds of formula I of the present application is disclosed and claimed as well as the free base of the compounds of formula I of the present invention in co-pending PCT application No. PCT/IB 96/01353 (International application date No. 12/4 1996, assigned to the assignee of the present invention).

It has been found that the L- (+) -tartaric acid salt of the compound of formula I below can be isolated in crystalline form, which has the following advantages: such as ease of formulation, higher solubility and good stability, and it is easier to purify than the non-crystalline form.

Summary of the invention

The present invention provides the L- (+) -tartrate salt of the compound of formula I.Wherein' means a standA body chemical center. The compound of formula I is preferably a stereochemical mixture or an isolated isomer having the configuration 3a- (S),1- (R); 3a- (S),1- (S); 3a- (R),1- (S) and/or 3a- (R),1- (R) isomers.

The present invention also provides a process for preparing a compound of formula (E) (D) -tartrate or (L) -tartrate,

the process comprises reacting a compound of formula (D) with (D) -tartaric acid or (L) -tartaric acid in a mixture of about 8: 1 to 9: 1 acetone: water at a temperature between 0 ℃ and room temperature.

The previous process is preferably carried out by reacting (D) -tartaric acid with a compound of formula (D), the compound of formula (E) having the R-configuration;

a process for the preparation of a compound of formula (J),

the process comprises reacting a compound of formula (E) with a compound of formula (X) in the presence of an organic base and a peptide coupling agent at a temperature of about-78 ℃ to-20 ℃,wherein Prt is an amine protecting group, X is OH, -O (C)₁-C₄) Alkyl or halogen. The method described immediately above is preferably: the peptide coupling agent is 1-propanephosphonic acid cyclic anhydride, the compound of formula (X) has the R-configuration, and the compound of formula (E) has the R-configuration. And more preferably Prt in the process just described is tert-butoxycarbonyl; and

a process for the preparation of the (L) - (+) -tartrate salt of the compound of formula I,

the process comprises reacting at a temperature of about-78 ℃ to-20 ℃ in the presence ofReacting a compound of formula (E) with a compound of formula (X) in the presence of an organic base and a peptide coupling agent to produce a compound of formula (J),wherein Prt is an amine protecting group, X is OH, -O (C)₁-C₄) An alkyl group or a halogen, in which,deprotecting the compound of formula (J) under suitable deprotection conditions to produce a compound of formula (K),(K)

reacting the compound of formula (K) with (L) - (+) -tartaric acid in a reaction-inert solvent to produce the (L) - (+) -tartrate salt of the compound of formula I. Prt is preferably tert-butoxycarbonyl in the immediately preceding method, and more preferably the peptide coupling agent is 1-propanephosphonic acid cyclic anhydride, and the compound of formula I has the absolute and relative configuration of 3a- (R),1- (R).

In another aspect, the invention also provides a method ofThe R, S-enantiomer mixture, R-enantiomer or S-enantiomer of the compounds, preferably here the (D) -tartrate or (L) -tartrate salt;

formula (II)

A 3a- (R, S), a 1- (R) diastereomer mixture, a 3a- (R), a 1- (R) diastereomer, or a 3a- (S),1- (R) diastereomer of the compound, where Prt is an amine protecting group selected from the group consisting of t-BOC, FMOC, and CBZ; and

formula (II)

A mixture of the R, S-enantiomers, the R-enantiomer or the S-enantiomer of the compound;

formula (II)

A mixture of R, S-enantiomers, R-enantiomer or S-enantiomer of a compound, wherein X is OH, -O (C)₁-C₄) Alkyl or halogen, Prt is an amine protecting group; and preferably X is OH, Prt is BOC, and the stereocenter is in the R-configuration.

In another aspect, the invention provides (wherein the compound of formula (I) is as shown above):

a method of increasing the endogenous growth hormone level in a human or other animal which comprises administering to said human or animal an effective amount of the (L) - (+) -tartrate salt of the compound of formula I;

a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an amount of the (L) - (+) -tartrate salt of the compound of formula I;

a pharmaceutical composition for increasing endogenous production or release of growth hormone in a human or other animal, which comprises a pharmaceutically acceptable carrier, an effective amount of the (L) - (+) -tartrate salt of the compound of formula I according to claim 1, and a growth hormone secretagogue. Wherein the growth hormone secretagogue is selected from the group consisting of GHRP-6, Hexarelin, GHRP-1, growth hormone releasing factor (GRF), IGF-1, IGF-2 and B-HT920 or an analog thereof;

a method for the treatment or prevention of osteoporosis which comprises administering to a human or other animal in need thereof an amount of a (L) - (+) -tartrate salt of a compound of formula I effective to treat or prevent osteoporosis;

a method of treatment or prophylaxis of a disease or condition which is treatable or preventable by growth hormone which comprises administering to a human or other animal in need of such treatment or prophylaxis an amount of a (L) - (+) -tartrate salt of a compound of formula I, which is effective to promote the release of endogenous growth hormone; the disease or condition in this method is preferably congestive heart failure, obesity or frailty associated with aging; the disease or condition in this method is also preferably congestive heart failure; the disease or condition in this method is further preferably frailty associated with aging;

methods for promoting fracture repair, reducing proteolytic reactions following major surgery, reducing cachexia and protein loss due to chronic disease, promoting wound repair, or promoting healing in burn patients or patients undergoing major surgery. The method comprises administering to a mammal in need of such treatment an amount of a (L) - (+) -tartrate salt of the compound of formula I effective to promote the release of endogenous growth hormone. This method is preferably used to promote healing in patients undergoing major surgery; this method is also preferably used to promote repair of bone fractures;

a method for improving muscle strength and mobility, the maintenance of skin thickness (thickness), metabolic homeostasis or renal homeostasis, the method comprising administering to a human or other animal in need of such treatment an amount of a (L) - (+) -tartrate salt of a compound of formula I effective to promote the release of endogenous growth hormone;

a method for the treatment or prevention of osteoporosis which comprises administering to a human or other animal suffering from osteoporosis an effective amount of a bisphosphonate and a (L) - (+) -tartrate salt of a compound of formula I; preferred bisphosphonate compounds in the method of treating osteoporosis are ibandronate; the preferred bisphosphonate compounds in the method of treating osteoporosis are alendronate:

a method of treating or preventing osteoporosis which comprises administering to a human or other animal suffering from osteoporosis an effective amount of estrogen or gestagen  and the (L) - (+) -tartrate salt of the compound of formula I, and optionally progesterone;

a method for the treatment of osteoporosis which comprises administering to a human or other animal suffering from osteoporosis an effective amount of a calcitonin in combination with the (L) - (+) -tartrate salt of the compound of formula I;

a method for increasing IGF-1 levels in a human or other animal deficient in IGF-1, which comprises administering to the human or other animal deficient in IGH-1 an effective amount of the (L) - (+) -tartrate salt of the compound of formula I;

a method of treating osteoporosis which comprises administering to a human or other animal suffering from osteoporosis an effective amount of an estrogen agonist or antagonist and the (L) - (+) -tartrate salt of the compound of formula I; preferred estrogen agonists or antagonists in this method are tamoxifen, droloxifene, raloxifene or idoxifene; preferred estrogen agonists or antagonists in this method are also cis-6- (4-fluoro-phenyl) -5- [4- (2-piperidin-1-yl-ethoxy) -phenyl ] -5,6,7, 8-tetrahydro-naphthalen-2-ol; (-) -cis-6-phenyl-5- [4- (2-pyrrolidin-1-yl-ethoxy) -phenyl ] -5,6,7, 8-tetrahydro-naphthalen-2-ol; cis-6-phenyl-5- [4- (2-pyrrolidin-1-yl-ethoxy) -phenyl ] -5,6,7, 8-tetrahydro-naphthalen-2-ol; cis-1- [6 '-pyrrolidino (pyrolodino) ethoxy-3' -pyridyl ] -2-phenyl-6-hydroxy-1, 2,3, 4-tetrahydro-naphthalene; 1- [ 4' -pyrrolidino (pyrrolidino) ethoxyphenyl ] -2- (4 "-fluorophenyl) -6-hydroxy-1, 2,3, 4-tetrahydroisoquinoline; cis-6- (4-hydroxyphenyl) -5- [4- (2-piperidin-1-yl-ethoxy) -phenyl ] -5,6,7, 8-tetrahydro-naphthalen-2-ol or 1- (4' -pyrrolidino (pyrrolidino) ethoxyphenyl) -2-phenyl-6-hydroxy-1, 2,3, 4-tetrahydroisoquinoline;

a method for increasing muscle mass which comprises administering to a human or other animal in need of such treatment an effective amount of a (L) - (+) -tartrate salt of a compound of formula I;

a method of promoting growth hormone deficiency in children, which method comprises administering to a growth hormone deficient child an effective amount of a (L) - (+) -tartrate salt of a compound of formula I;

a method of treating insulin resistance in a mammal which comprises administering to said mammal an effective amount of the (L) - (+) -tartrate salt of the compound of formula I; the condition associated with insulin resistance in the method is preferably type I diabetes, type II diabetes, hyperglycemia, impaired glucose tolerance, or insulin resistance syndrome; the condition associated with insulin resistance in this method is also preferably obesity or elderly;

a method for increasing endogenous growth hormone levels, which method comprises administering to a human or other animal in need thereof an effective amount of a functional somatostatin antagonist and the (L) - (+) -tartrate salt of the compound of formula I; preferably, the antagonist of a functional somatostatin in the method is an alpha-2 adrenergic agonist; and

a method of treating or preventing congestive heart failure, obesity or age-related frailty, which method comprises administering to a human or other animal in need thereof an effective amount of a functional somatostatin antagonist and the (L) - (+) -tartrate salt of the compound of formula I.

The compounds of formula I promote the release of growth hormone, are stable under a variety of physiological conditions, and can be administered parenterally, nasally, or orally.

Detailed description of the invention

The (L) - (+) -tartaric acid salt of the compound of formula I can be prepared by methods well known in the chemical arts for the production of the compound. Certain methods of producing the (L) - (+) -tartrate salt of the compound of formula I are further features of the invention and are illustrated by the reaction schemes shown below.

The absolute and relative configurations of the compounds of the invention are shown below:it is shown in the 3a- (R),1- (R) configuration. It can be prepared by the method described below.

The (L) - (+) -tartrate salt of the compound of formula I, which releases growth hormone, can be used as the only tool in vitro in elucidating how growth hormone secretion is regulated at the pituitary level. This includes use in evaluating many known or contemplated factors that affect growth hormone secretion, such as age, sex, nutritional factors, glucose, amino acids, fatty acids, and fasted and non-fasted states. In addition, the (L) - (+) -tartrate salt of the compound of formula I can be used to assess how other hormones alter the secretory activity of growth hormone. For example, somatostatin has been shown to inhibit growth hormone release.

The (L) - (+) -tartaric acid salt of the compound of formula I is useful in animals (including humans), which causes the release of growth hormone in vivo. The (L) - (+) -tartrate salt of the compound of formula I is useful for treating symptoms associated with GH deficiency; can stimulate the growth of animals providing meat products, or increase feeding efficiency to improve raw meat quality; the milk yield of the milk factory can be improved; can improve bone or wound healing; and can improve the function of important organs. The (L) - (+) -tartrate salt of the compound of formula I can alter body composition by inducing endogenous GH secretion, and can alter other GH-dependent metabolic, immunological or developmental processes. For example, the compounds of the invention may be used in poultry, turkeys, farm animals (such as sheep, pigs, horses, cattle, etc.), domestic animals (e.g. dogs), or in aquaculture to promote fish growth and improve the protein/fat ratio. Alternatively, the (L) - (+) -tartrate salt of the compound of formula I can be administered to humans as a diagnostic tool for directly determining whether the pituitary gland is capable of releasing growth hormone. For example, the (L) - (+) -tartrate salt of the compound of formula I can be applied to children. Serum samples can be assayed for growth hormone before and after administration. Comparing the amounts of growth hormone in these samples can be used as a direct measure of the ability of the pituitary to release growth hormone.

The invention accordingly also includes within its scope pharmaceutical compositions comprising the (L) - (+) -tartrate salt of the compound of formula I in association with a pharmaceutically acceptable carrier, wherein the (L) - (+) -tartrate salt of the compound of formula I is the active ingredient. In addition to the (L) - (+) -tartrate salt of the compound of formula I, the pharmaceutical composition may optionally contain an anabolic agent, or other compound having different activities, such as: an antibacterial growth permintant or a medicament for the treatment of osteoporosis, or in combination with other pharmaceutically active materials, which improves the therapeutic effect and reduces side effects.

Growth promoting and anabolic agents are well known in the art and include TRH, PTH, diethylstilbestrol, estrogens, β -agonists, theophylline, anabolic steroids, cerebrophenanthin, group E prostaglandins, compounds disclosed in U.S. patent 3,239,345 (the specification of which is incorporated herein by reference) (e.g., zenanol), compounds disclosed in U.S. patent 4,036,979 (the specification of which is incorporated herein by reference) (e.g., oxathiuron), and peptides disclosed in U.S. patent 4,411,890 (the specification of which is incorporated herein by reference).

(L) - (+) -tartrate salts of compounds of formula I in combination with other growth hormone secretagogues, such as the growth hormone releasing peptides GHRP-6 and GHRP-1 as described in US patent 4,411,890 (the specification of which is incorporated herein by reference), WO 89/07110, WO 89/07111, and B-HT920 and hexarelin and the newly discovered GHRP-2 or growth hormone releasing hormone (GHRH, also denoted GRF) as described in WO 93/04081 and analogues thereof, or growth hormones and analogues thereof or growth factors including IGF-1 and IGF-2 or adrenergic agonists, such as clonidine, xylazine, detomidine and medetomidine (clonidine, xylazine and medetomidine are disclosed in US patents 3,202,660, 3,235,550 and 4,544,664, respectively, the specifications of which are incorporated herein by reference) or 5-hydroxytryptamine 5HTID agonists such as sumitriptan, or agents inhibiting growth hormone release inhibitory factor or its release such as physostigmine and pyridostigmine can be used to increase endogenous GH levels in mammals. The (L) - (+) -tartrate salt of the compound of formula I in combination with GRF results in a synergistic increase in endogenous growth hormone.

It is well known to those skilled in the art that the known or potential uses of growth hormone are manifold [ see human growth hormone, Strobel and Thomas, for pharmacological reviews, pages 46,1-34 (1994); rosen et al, Horm Res, 1995; pages 43: 93-99; degerblad et al, J.Endocrinology Eur, 1995,133: 180-188; J.O. Jorgensen, J.E. Endocrinology, 1994,130: 224-; copeland et al, J.Clin Endocrinology and metabolism, Vol.78, No. 5, p.1040-1047; aloi et al, J.A. J.J.Clin Endocrinology & Metabolism, Vol.79, 4 th, 943-949; cordido et al, meta, clin. exp., (1995),44(6),745 pages 748; K.M.Fairhall et al, J.Endocrinal, (1995),145(3), p. 417-; friebose et al, neuroendocrinology, (1995),61(5), 584-. The (L) - (+) -tartrate salt of the compound of formula I thus stimulates the release of endogenous growth hormone, which has the same effect and action as growth hormone. The different effects of these growth hormones can be summarized as follows: stimulating the release of growth hormone in the elderly; treating adult growth hormone deficiency; prevention of side effects of glucocorticoid catabolism; treating osteoporosis; stimulating the immune system; promoting wound healing; promoting fracture repair; treating developmental retardation; an example of a method for determining the efficacy of growth hormone secretagogues in the treatment of congestive heart failure is disclosed in PCT publications WO 95/28173 and WO 95/28174 (r.yang et al, circulation, vol 92, p 2, 262, 1995); treating acute or chronic renal failure or insufficiency; treating physiologically-dwarfing, including growth hormone-deficient children; treating dwarfism associated with chronic disease; treating obesity; treating developmental delays associated with Prader-Willi syndrome and turner's syndrome; accelerating the recovery of burn patients or patients who undergo large-scale surgical operations such as digestive tract surgery and shortening the hospitalization period thereof; treating intrauterine growth retardation, skeletal dysplasia, adrenocortical hyperactivity and cushing's syndrome; replacement of growth hormone in stress patients; treatment of osteochondral dysplasia, Noonans syndrome, sleep disorders, alzheimer's disease, delayed wound healing and psychological sequestration; treating pulmonary dysfunction and ventilator dependence; reducing proteolytic reactions after major surgery; treating malabsorption syndrome; relieving cachexia and low protein caused by chronic diseases such as cancer or AIDS; promoting weight gain and protein gain in patients receiving TPN (total parenteral nutrition); treating hyperinsulinemia including islet cell proliferation; adjunctive therapy for ovulation induction and prevention and treatment of gastric and duodenal ulcers; stimulation of thymus development and prevention of age-related decline in thymus function; for adjunctive treatment of chronic hemodialysis patients; treating immunosuppressed patients and enhancing antibody responses following vaccine injection; improving muscle strength of the elderly with asthenia, increasing muscle mass and mobility, maintaining skin thickness, maintaining metabolic balance and stabilizing kidney function; stimulation of osteoblasts, bone remodeling and cartilage growth; treating neurological diseases such as peripheral neuropathy and drug-induced neuropathy, acute infectious polyneuritis, amyotrophic lateral sclerosis, multiple sclerosis, cerebrovascular accidents, and demyelinating diseases; stimulating the immune system of the domestic animal and treating the ageing disorder of the domestic animal; accelerating the growth of the livestock; and stimulating the growth of wool.

A number of compounds are now known to those skilled in the art for the treatment of the diseases or therapeutic indications listed above. The combined use of these therapeutic agents (some of which have been mentioned above) with growth promoters has the anabolic and desirable properties of these different therapeutic agents. In such combinations, the therapeutic agent and the (L) - (+) -tartrate salt of the compound of formula I may be administered separately and sequentially, or in combination, at a dosage level which is 1/100 to 1 times the dosage level at which the compound and secretagogue are effective when used alone. Combination therapy is provided by the combination of bisphosphonates and the (L) - (+) -tartrate salt of the compound of formula I, which combination therapy inhibits bone resorption, prevents osteoporosis, reduces bone fractures, improves healing of fractures, stimulates bone formation, and increases bone mineral density. See the publication of PCT WO 95/11029, which discusses the combination therapy of bisphosphonates with GH secretagogues. For example, Hamdy, N.A.T. in the role of bisphosphonates in metabolic bone disease Trend in Endocrinol.Metab.,1993, pages 4,19-25 have reviewed the use of bisphosphonates in these areas. Bisphosphonates having this utility include, but are not limited to, alendronate, tillutronate, dimethyl-APD, risedronate, etidronate, YM-175, clodronate, pamidronate, and BM-210995 (ibandronate). Based on their potency, patients are administered orally a daily dose of 0.1mg to 5g of a bisphosphonate and a daily dose of 0.01mg to 20mg per kg of body weight of a (L) - (+) -tartrate salt of a compound of formula I to achieve an effective treatment for osteoporosis.

The (L) - (+) -tartaric acid salt of the compound of formula I can be used in combination with mammalian estrogen agonists/antagonists. Any estrogen agonist/antagonist may be used as the second compound in this aspect of the invention. The term estrogen agonists/antagonists refers to compounds that bind to estrogen receptors, inhibit bone turnover, and prevent bone loss. In particular, an estrogen agonist is defined herein as a chemical compound that binds to estrogen receptors in mammalian tissues and mimics the action of estrogen in one or more tissues. An estrogen antagonist is defined herein as a chemical compound that binds to estrogen receptors in mammalian tissues and blocks the action of estrogen in one or more tissues. Such activity can be readily determined by one skilled in the art following standard assay methods. The assay methods for these standards include: estrogen receptor binding assays, standard bone tissue morphometry and densitometry (see Eriksen E.F. et al, bone tissue morphometry, Raven Press, New York, 1994, pages 1-74; Grier S.J. et al, application of the dual energy X-ray absorptiometry in animals, Inv.Radiol.,1996,31(1): 50-62; Wahner H.W. and Fogelman I., evaluation of osteoporosis: clinical dual energy X-ray absorptiometry, Martin Dunitz Ltd., London, 1994, pages 1-296). These various compounds are described below for reference. However, other estrogen agonists/antagonists will be well known to those skilled in the art. One preferred estrogen agonist/antagonist is droloxifene: (phenol, 3- [1- [4[2- (dimethylamino) ethoxy ] -phenyl ] -2-phenyl-1-butenyl ] -, (E) -) and related compounds, which are disclosed in U.S. Pat. No. 5,047,43, the specification of which is also incorporated herein by reference.

Another preferred estrogen agonist/antagonist is tamoxifen: (ethylamine, 2- [ -4- (1, 2-diphenyl-1-butenyl) phenoxy ] -N, N-dimethyl, (Z) -2-, 2-hydroxy-1, 2, 3-propanetricarboxylate (1: 1)) and related compounds, which are disclosed in U.S. Pat. No. 4,536,516, the specification of which is incorporated herein by reference. Another related compound is 4-hydroxyttamoxifen, which is disclosed in U.S. patent 4,623,660, the specification of which is incorporated herein by reference.

Another preferred estrogen agonist/antagonist is raloxifene: (methanones, [ 6-hydroxy-2- (4-hydroxyphenyl) benzo [ b ] thiophen-3-yl ] [4- [2- (1-piperidinyl) ethoxy ] phenyl ] -, hydrochloride) and related compounds, which are disclosed in U.S. Pat. No. 4,418,068, the specification of which is incorporated herein by reference.

Another preferred estrogen agonist/antagonist is idoxifene: pyrrolidine, 1- [ - [4- [ [1- (4-iodophenyl) -2-phenyl-1-butenyl ] phenoxy ] ethyl ] and related compounds, which are disclosed in U.S. patent 4,839,155, the specification of which is incorporated herein by reference.

Other preferred estrogen agonists/antagonists include the compounds described in commonly assigned U.S. patent 5,552,412, the specification of which is incorporated herein by reference. Particularly preferred compounds described herein are:

cis-6- (4-fluoro-phenyl) -5- [4- (2-piperidin-1-yl-ethoxy) -phenyl ] -5,6,7, 8-tetrahydro-naphthalen-2-ol;

(-) -cis-6-phenyl-5- [4- (2-pyrrolidin-1-yl-ethoxy) -phenyl ] -5,6,7, 8-tetrahydro-naphthalen-2-ol;

cis-6-phenyl-5- [4- (2-pyrrolidin-1-yl-ethoxy) -phenyl ] -5,6,7, 8-tetrahydro-naphthalen-2-ol;

cis-1- [6 '-pyrrolidino (pyrolodino) ethoxy-3' -pyridyl ] -2-phenyl-6-hydroxy-1, 2,3, 4-tetrahydronaphthalene;

1- (4' -pyrrolidino (pyrrolidino) ethoxyphenyl) -2- (4 "-fluorophenyl) -6-hydroxy-1, 2,3, 4-tetrahydroisoquinoline;

cis-6- (4-hydroxyphenyl) -5- [4- (2-piperidin-1-yl-ethoxy) -phenyl ] -5,6,7, 8-tetrahydro-naphthalen-2-ol; and

1- (4' -pyrrolidino (pyrrolidino) ethoxyphenyl) -2-phenyl-6-hydroxy-1, 2,3, 4-tetrahydroisoquinoline.

Other estrogen agonists/antagonists are described in U.S. Pat. No. 4,133,814 (the specification of which is incorporated herein by reference). U.S. Pat. No. 4,133,814 discloses derivatives of 2-phenyl-3-aroyl-benzothiophene and 2-phenyl-3-aroyl benzothiophene-1-oxide.

The following paragraphs provide preferred dosage ranges for the different anti-resorptive agents.

The amount of the anti-resorptive agent used is determined by its activity as a bone loss inhibitor. This activity is determined by the pharmacokinetics of the individual compounds and their minimum maximum effective dose to inhibit bone loss using a protocol such as those referenced above.

Generally for estrogen agonists/antagonists (when used in combination with the (L) - (+) -tartrate salt of the compound of formula I of the invention), for example in the treatment of osteoporosis, an effective dose of the activity of the invention is 0.01 to 200 mg/kg/day, preferably 0.5 to 100 mg/kg/day.

In particular, the effective dose of droloxifene is 0.1 to 40 mg/kg/day, preferably 0.1 to 5 mg/kg/day.

In particular, an effective dose of raloxifene is 0.1 to 100 mg/kg/day, preferably 0.1 to 10 mg/kg/day.

In particular an effective dose of tamoxifen is from 0.1 to 100 mg/kg/day, preferably from 0.1 to 5 mg/kg/day.

In particular cis-6- (4-fluoro-phenyl) -5- [4- (2-piperidin-1-yl-ethoxy) -phenyl ] -5,6,7, 8-tetrahydro-naphthalen-2-ol;

(-) -cis-6-phenyl-5- [4- (2-pyrrolidin-1-yl-ethoxy) -phenyl ] -5,6,7, 8-tetrahydro-naphthalen-2-ol;

cis-6-phenyl-5- [4- (2-pyrrolidin-1-yl-ethoxy) -phenyl ] -5,6,7, 8-tetrahydro-naphthalen-2-ol;

cis-1- [6 '-pyrrolidinoethoxy-3' -pyridyl ] -2-phenyl-6-hydroxy-1, 2,3, 4-tetrahydronaphthalene;

1- (4' -pyrrolidinoethoxyphenyl) -2- (4 "-fluorophenyl) -6-hydroxy-1, 2,3, 4-tetrahydroisoquinoline;

cis-6- (4-hydroxyphenyl) -5- [4- (2-piperidin-1-yl-ethoxy) -phenyl ] -5,6,7, 8-tetrahydro-naphthalen-2-ol; and

an effective dose of 1- (4' -pyrrolidinoethoxyphenyl) -2-phenyl-6-hydroxy-1, 2,3, 4-tetrahydroisoquinoline is 0.0001 to 100 mg/kg/day, preferably 0.001 to 10 mg/kg/day.

In particular, an effective dose of 4-hydroxytamoxifen is 0.0001 to 100 mg/kg/day, preferably 0.001 to 10 mg/kg/day.

Measurement of stimulation of GH Release from rat pituitary cells

The following protocol was applied to identify compounds capable of stimulating secretion of GH from cultured rat pituitary cells. This test is also used to compare with standards to determine dosage levels. Cells were isolated from the pituitary of 6-week-old male Wistar rats. After decapitation, the anterior pituitary is placed in a cold sterile Hank Balanced Salt Solution (HBSS) without calcium or magnesium. The tissue was finely minced and then subjected to two cycles of mechanolytic dispersion in HBSS using 10U/ml bacterial protease (EC 3.4.24.4, Sigma P-6141, St. Louis, Mo.). 5% CO at around 37 ℃₂The tissue-enzyme mixture was stirred in the spinner flask at 30rpm for about 30 minutes, approximately 15 minutes and after 30 minutes was manually triturated with a 10mL pipette under ambient conditions. This mixture was centrifuged at 200 Xg for about 5 minutes. Horse serum (35% final concentration) was added to the supernatant to neutralize excess protease. The pellets were resuspended in fresh protease (10U/mL), stirred under the previous conditions for more than about 30 minutes, and finally triturated manually through a 23-pin. Horse serum (35% final concentration) was added again, and then the cells of the two hydrolysates were combined, pelleted (200 Xg for about 15 minutes), resuspended in culture medium (Dulbecco's modified Eagle medium (D-MEM) supplemented with 4.5g/L glucose, 10% horse serum, 2.5% fetal bovine serum, 1% nonessential amino acids, 100U/mL nystatin and 50mg/mL gentamicin sulfate, Gibco, Grand Island, N.Y.), and counted. At 6.0-6.5X 10⁴/cm²Cells, placing the cells in 48-well Costar^TM(Cambridge, Massachusetts) plates and cultured in culture medium for 3-4 days.

Prior to GH secretion assays, the culture cells were washed twice with release medium and then equilibrated in release medium (25 mM Hepes buffered D-MEM at 37 ℃ C., pH 7.4, and containing 0.5% bovine serum albumin) for 30 minutes. Test compounds were dissolved in DMSO and then diluted in pre-warmed culture medium. The experiment was performed four times. The experiment was started by adding 0.5ml of release medium (containing excipients or test compounds) to each culture well. The incubation was carried out at about 37 ℃ for 15 minutes, and then the release medium was removed to terminate the incubation, and the release medium was centrifuged at 2000 Xg for about 15 minutes to remove the cell material. The concentration of rat growth hormone in the supernatant was determined by a standard radioimmunoassay protocol described below.

Determination of rat growth hormone

The concentration of rat growth hormone was determined by a double antibody radioimmunoassay using a rat growth hormone reference preparation (NIDDK-rGH-RP-2) obtained from dr.a. parlow (Harbor-UCLA medical center, Torrence, CA) and rat growth hormone antiserum raised in monkeys (NIDDK-anti-rGH-S-5). Additional rat growth hormone (1.5U/mg, # G2414, Scripps Labs, SanDiego, Calif.) was iodinated to have a specific activity of approximately 30. mu. Ci/μ G and used as a tracer by the chloramine T method. The immune complex was obtained by adding anti-monkey IgG goat antiserum (ICN/Cappel, Aurora, OH) plus polyethylene oxide with a molecular weight of 10,000-20,000 to a final concentration of 4.3%. Recovery was accomplished by centrifugation. The working range of this assay is 0.08-2.5 μ g rat growth hormone per tube on a basal level.

Determination of exogenously stimulated growth hormone Release in rats following intravenous administration of test Compounds

21-day-old Sprague-Dawley female rats (Charles river Laboratory, Wilmington, Mass.) were acclimatized to local breeding conditions (24 ℃,12 hours of sunlight, 12 hours of overnight circulation) for approximately 1 week prior to compound testing. All rats were allowed to eat water and a commercially available pellet diet (age Country Food, sympocuse NY). The experiment was guided by NIH guidelines for experimental animal feeding and use.

On the day of the experiment, the test compound was dissolved in vehicle containing 1% ethanol, 1mM acetic acid and saline containing 0.1% bovine serum albumin. Each experiment was performed in 3 rats. Rats were weighed and anesthetized by intraperitoneal injection of sodium pentobarbital (nembutal ,50mg/kg body weight). After 14 minutes of anesthesia administration, the rat tail was excised, and blood samples (baseline blood samples, approximately 100 μ l) were collected by dripping blood into a microcentrifuge tube. After 15 minutes of anesthetic administration, the test compound was injected into rats via tail vein in a total injection amount of 1ml/kg body weight. Additional blood samples were taken from the tail at 5, 10 and 15 minutes after compound application, respectively. The blood samples were placed on ice until the serum was separated by centrifugation (1430 Xg for 10 minutes at 10 ℃). The serum was stored at-80 ℃ until the serum growth hormone was assayed by the radioimmunoassay described above.

Assessment of exogenously stimulated growth hormone Release in dogs following oral administration

The day of administration the appropriate amount of test compound was weighed out and dissolved in water. 2-4 dogs were given gavage in an amount of 0.5-3mL/kg and each dosage regimen was accepted by the dogs. Blood samples (5ml) were collected from the jugular vein by direct venipuncture using a 5ml empty needle containing lithium heparin at 0.17, 0.33, 0.5, 0.75, 1,2, 4,6, 8 and 24 hours before and after administration. The plasma thus prepared was stored at-20 ℃ until analysis.

Determination of canine growth hormone

Canine growth hormone concentrations were determined by a standard radioimmunoassay protocol using canine growth hormone (iodinated antigen, and reference preparation AFP-1983B) obtained from dr.a. parlow (Harbor-UCLA medical center, Torrence, CA) and canine growth hormone antiserum (AFP-21452578) produced in monkeys. The tracer is formed by chloramine T-iodination of canine growth hormone, with a specific activity of 20-40. mu. Ci/. mu.g. The immune complex was obtained by adding anti-monkey IgG goat antiserum (ICN/Cappel, Aurora, OH) plus polyethylene oxide with a molecular weight of 10,000-20,000 to a final concentration of 4.3%. Recovery was accomplished by centrifugation. The working range for this assay is 0.08-2.5 μ g canine growth hormone per tube.

Assessment of canine growth hormone and insulin-like growth factor-1 levels in dogs following long-term oral dosing

Dogs were subjected to test compound daily for 7 to 14 days. In daily administration, the appropriate dose of test compound is weighed out and dissolved in water. The dose was administered to dogs receiving each dosage regimen by gavage in an amount of 0.5-3 mL/kg. Blood samples were collected on days 0, 3, 7, 10 and 14. Blood samples (5ml) were collected by direct jugular venipuncture using 5ml empty needles containing lithium heparin on days 0, 7 and 14 before administration, 0.17, 0.33, 0.5, 0.754, 1,2,3, 6, 8, 12 and 24 hours after administration. The plasma thus prepared was stored at-20 ℃ until analysis.

Female rat study

This study evaluated the effect of long-term treatment with GHRP analogs on body weight, body composition, and non-fasting plasma concentrations of glucose, insulin, lactate, and lipids in estrogen-deficient and estrogen-filled female rats. Acute responsiveness of serum GH levels to intravenous administration of a GH-releasing agent was assessed on the last day of administration. Body weight was monitored weekly throughout the treatment period; in addition, body composition and plasma levels of glucose, insulin, lactate, cholesterol and triglycerides were measured at the end of the treatment.

Unmated female Sprague-Dawley rats were obtained from Charles River laboratories (Wilmington, MA) and subjected to bilateral ovariectomy (Ovx) or Sham surgery (Sham) at approximately 12 weeks of age. For sham operators, their ovaries were exteriorized and returned to the abdominal cavity. The rats were individually housed in cages of 20cm x 32cm x 20cm under standard breeding conditions (about 24 ℃, about 12 hours sunlight/12 hours night cycle) post-operatively. All rats were fed water and commercially available pellets (age Prolab 3000, age Country Food, inc., sympulse, NY) ad libitum. The experiment was guided by NIH guidelines for experimental animal feeding and use.

After about 7 months post-surgery, Sham and Ovx rats were weighed and randomly grouped. 1ml of vehicle (distilled deionized water containing 1% ethanol), 0.5mg/kg or 5mg/kg of growth hormone releasing agent was administered to rats daily by oral gavage for 90 days. Rats were weighed weekly throughout the study. After 24 hours of the last administration, the acute response of serum Growth Hormone (GH) to the test agent was determined as follows. Rats were anesthetized with 50mg/kg sodium pentobarbital. Anesthetized rats were weighed and baseline blood samples (-100 μ Ι) were collected from their tail vein. 1ml of the test agent (growth hormone releasing agent or vehicle) was then administered intravenously via the tail vein. About 10 minutes after injection, a 2 nd 100. mu.l blood sample was collected from the tail. The blood was allowed to clot at around 4 ℃ and then centrifuged at 2000 Xg for around 10 minutes. Serum was stored at about-70 ℃. Serum growth hormone concentrations were determined according to the radioimmunoassay described above. Following this procedure, each anesthetized rat was scanned systemically by a dual energy X-ray absorptiometry (DEXA, Hologic QDR 1000/W, Waltham MA). The final blood sample was collected in heparinized tubes by cardiac puncture. Plasma was separated by centrifugation and stored frozen as described above.

Plasma insulin was measured by radioimmunoassay using the kit from Binax (Portland, Maine). The coefficient of variability between measurements is less than or equal to 10%. Plasma levels of triglyceride, total cholesterol, glucose and lactate were determined using an Abbott VPTM and VPSuperSystem  automated analyzers (Abbott laboratories, Irving, Texas), A-GentTM triglyceride, cholesterol and glucose reagent systems, respectively, and the lactate kit from Sigma. The plasma insulin, triglyceride, total cholesterol and lactic acid lowering activity of Growth Hormone Releasing Peptide (GHRP) or GHRP analogs (such as compounds of formula I) was determined by statistical analysis (unpaired T test) with vehicle treatment groups.

The (L) - (+) -tartaric acid salt of the compound of formula I can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual or topical routes of administration and can be prepared with pharmaceutically acceptable carriers to provide the appropriate form of administration for each route of administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In each solid administration form, the (L) - (+) -tartrate salt of the compound of formula I is mixed with at least one inert pharmaceutically acceptable carrier, such as sucrose, lactose or starch. As is customary, in addition to such inert diluents, the administration form may also comprise further substances, for example lubricants (such as magnesium stearate). In capsules, tablets and pills, the administration forms may also contain buffering agents. Tablets and pills may additionally comprise an enteric coating.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs containing inert diluents commonly used in the art, such as water. In addition to such inert diluents, the compositions may also include adjuvants such as wetting, emulsifying and suspending agents, as well as perfuming, flavoring and perfuming agents.

Formulations of the present invention for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or excipients are propylene glycol, polyoxyethylene, vegetable oils (such as olive oil and corn oil), gelatin, and injectable organic esters (such as ethyl oleate). Such administration forms may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. They can be rendered sterile, for example, by filtration through a bacteria-blocking filter, by mixing a bactericide into the composition, by irradiating the composition with radiation, or by heating the composition. They may also be prepared as sterile solid compositions which are dissolved in sterile water or some other sterile injection medium immediately prior to use.

Compositions for rectal or vaginal administration are preferably suppositories. In addition to the active substance, the suppository may contain excipients such as coca butter or suppository wax.

Compositions for nasal or sublingual administration may also be prepared using standard excipients well known in the art.

The dosage of the (L) - (+) -tartrate salt of the compound of formula I in this composition of the invention is variable and such variation in the amount thereof is required in order to obtain a suitable administration form. The dosage is selected according to the desired therapeutic effect, route of administration and course of treatment. Dosage levels typically used in humans and other animals (e.g., mammals) are from 0.0001 to 100mg/kg body weight per day to achieve effective release of growth hormone.

A preferred dosage range is 0.01 to 5.0mg/kg body weight per day, which can be administered once or in multiple divided doses.

The following figure illustrates the synthesis of the (L) - (+) -tartrate salt of the compound of formula I. The symbol "+" represents a stereochemical centre. In the scheme "Prt" refers to any suitable amine protecting group well known to those skilled in the art.

The reaction steps illustrated in the previous scheme are described below. In the following description, the amine protecting group Prt is exemplified by the preferred amine protecting group BOC.

Step a: the picolyl chloride hydrochloride, a carbonate salt (such as Li) is added at a temperature of about 0 deg.C to room temperature, preferably 0 deg.C₂CO₃、CsCO₃Or preferably potassium carbonate) and potassium iodide or tetrabutylammonium iodide are added to a solution of compound a in a reaction-inert polar aprotic solvent such as acetone, butanone or preferably DMF (dimethylformamide). Stirring at about-20 deg.C to 70 deg.C, preferably 0 deg.C for about 2 to 16 hours, preferably 2 hours, removing the ice bath, and adding DABCO (1, 4-diazabicyclo [2.2.2 ]]Octane). The reaction mixture is stirred for about 15 to 30 minutes and then poured into a mixture of water and a non-polar organic solvent such as toluene, diethyl ether or preferably IPE (isopropyl ether). The organic layer is separated and treated by standard methods well known in the art to yield compound B.

Step b: 70% CF₃CH₂NHNH₂Is used as an aqueous solution in ethanol, water or toluene, preferably the 70% CF is extracted with toluene₃CH₂NHNH₂An aqueous solution of (a). The toluene extract containing anhydrous 2,2, 2-trifluoroethylhydrazine is added to a solution of compound B in an organic solvent, such as ethanol or preferably toluene, followed by the addition of acetic acid. This reaction mixture is heated at about 60 ℃ to 110 ℃, preferably 70 ℃, for about 30 minutes to 12 hours, preferably 2 hours. The reaction mixture is cooled to room temperature and quenched with a solvent such as NaHCO₃Is neutralized with aqueous base. The organic layer is separated and treated by standard methods well known in the art to yield compound C.

Step c: adding an acid to a reaction-inert organic solvent (such as EtOH, IPE or preferably CH)₂Cl₂) In solution with compound C, the acid is for example HCl in IPE or ethanol, trifluoromethanesulfonic acid or an alkylsulfonic acid such as methanesulfonic acid. After stirring the mixture for about 1-2 hours, cooling to about 0 deg.C to room temperature, preferably 0 deg.C, and adding a solvent such as triethylamine or NH₄An amine base of OH is added to this mixture. This mixture is allowed to warm to room temperature, diluted with additional organic solvent, and then treated by standard methods well known in the art to yield compound D.

Step d: (D) -or (L) -tartaric acid, preferably (D) -tartaric acid, is added to compound D in acetone/water (about 8: 1 to 9: 1) at about room temperature. The mixture is stirred at room temperature for about 15 minutes to overnight, preferably overnight, the solid is filtered, collected and washed with cold acetone, thus yielding the compound of formula E, preferably compound E as a single enantiomeric (D) -tartrate salt.

Step e: the N-BuLi or potassium tert-butoxide solution is added to a solution of N-BOC-serine, preferably N-BOC- (D) -serine (compound F) in THF/DMF (about 1: 1 to 2: 1) at about 0 ℃. The reaction mixture is stirred at about 0 ℃ for about 10 to 30 minutes, preferably 20 minutes, and then 2, 4-difluorobenzyl bromide is added. Heating to room temperature and stirringAfter stirring for about 6-24 hours, the reaction mixture is concentrated in vacuo to remove THF, and an aqueous acid such as 1N HCl is added to adjust the pH to about 3. Then in water and an organic solvent (such as CH)₂Cl₂Or IPE) between the reaction mixtures. This organic solution is treated by standard methods well known in the art to give compound G, preferably in the R-configuration at the stereocenter, the so-called (D) -enantiomer.

Step f: adding an alkylsulfonic acid such as methanesulfonic acid to a solution of compound G in an organic solvent such as THF, CH₂Cl₂IPE or a mixture thereof, preferably CH₂Cl₂The ratio of/IPE (about 1: 1). Filtering the solid and using CH₂Cl₂the/IPE mixture (1: 1) is washed to give the compound H, preferably in the R-configuration at the stereocenter, the so-called (D) -enantiomer.

Step g: 2-tert-Butoxycarbonylamino-2-methyl-propionic acid-2, 5-dioxo-pyrrolidin-1-yl ester and an alkylamine such as triethylamine are added to a THF/water (ca. 4: 1) solution of compound H. The reaction mixture is stirred at room temperature for about 1-24 hours and quenched with an aqueous acid such as 10% aqueous citric acid. This mixture is partitioned with an organic solvent such as ethyl acetate, the organic layer is then separated and treated by standard methods well known in the art to yield compound X, preferably in the R-configuration at the stereocenter, the so-called (D) -enantiomer. The compound X may be an acid, alkyl ester or acid halide (X is OH, -O (C1-C4) alkyl or halide), preferably an acid.

Step h: (a) compound E, preferably the (D) -tartrate salt of the single enantiomer, is added to ethyl acetate at about-35 ℃ to 0 ℃, preferably-6 ℃. The solution is cooled to about-30 ℃ to about-50 ℃ and then an alkylamine, such as triethylamine, is added. The reaction mixture was stirred between about-78 deg.C and-20 deg.C for about 30-90 minutes and filtered to obtain a free base solution of Compound E.

(b) When X in compound X is OH, compound X, preferably compound X having R-configuration at the stereogenic center, is added to a reaction-inert organic solvent such as an ethyl acetate solution containing the free base of compound E obtained from step h (a), an alkylamine such as triethylamine, and PPAA (1-propanephosphonic acid cyclic anhydride) (50% in ethyl acetate) at about-78 deg.C to-20 deg.C, preferably-35 deg.C. The reaction mixture is stirred for about 1-24 hours and assayed by standard methods well known in the art to produce compound J, preferably having both absolute and relative 3a- (R),1- (R) configuration.

When X in compound X is Cl, compound X is added to a reaction-inert solvent such as a dichloromethane solution containing the free base of compound E and an alkylamine such as triethylamine at about-78 ℃. The reaction mixture is stirred at about 0-30 deg.C for about 1-24 hours and then assayed by standard methods well known in the art to produce compound J, preferably having both absolute and relative 3a- (R),1- (R) configuration.

When X in the compound X is-O (C)₁-C₄) Alkyl (here preferably methyl), compound X is added to a solution of the free or conjugated base of compound E in a reaction-inert solvent [ prepared by reacting the free amine base with the appropriate reagent (i.e. M = Li, butyllithium or LDA; m = Na, NaH or NaH (SiMe)₃)₂Or M = K, KH or KN (SiMe)₃)₂Or M = Mg, any alkyl grignard reagent, preferably diethylmagnesium bromide, or M = Al, any trialkylaluminum reagent, preferably trimethylaluminum) to produce the conjugate base of compound E (-NM, where M = Li, Na, K, Mg or Al, preferably aluminum)]Preferably aluminum, and then the reaction mixture is stirred at about-20 c to 110 c for about 1 to 24 hours and measured by standard methods well known in the art to produce compound J, preferably having both absolute and relative 3a- (R),1- (R) configuration.

Step i: adding an acid to the CH containing Compound J at about 0 deg.C to room temperature₂Cl₂IPE or THF, such as EtOH with HCl, methanesulfonic acid or CH with trifluoromethanesulfonic acid₂Cl₂. The mixture is cooled at room temperatureStirring for about 40 minutes to 4 hours, then adding for example NaHCO₃Until the solution is neutral in pH. The organic layer is separated and assayed by standard methods well known in the art to yield compound K, preferably having both absolute and relative 3a- (R),1- (R) configurations.

Step j: l- (+) tartaric acid is added to a solution of compound K in an alcohol, preferably methanol. The reaction mixture was stirred for about 1-12 hours, filtered and concentrated. The crude residue is diluted with an organic solvent such as ethyl acetate, heated and slowly cooled to room temperature. The solid is filtered and then dried to obtain the L- (+) tartrate salt of the compound of formula I as white crystals, preferably having absolute and relative 3a- (R),1- (R) configuration.

The following examples are intended to further illustrate the invention and are not intended to limit the invention.

Column chromatography was performed using silica gel. Melting points were determined on a Buchi 510 instrument and were not corrected. Proton NMR spectra were recorded at 25 ℃ on Varian XL-300, Bruker AC-300, Varian Unity 400 or Bruker AC-250. Chemical shifts are expressed in parts per million by low magnetic field of trisilane.

Example 1

2-amino-N- {1- (2, 4-difluoro-benzyloxymethyl) -2-oxo-2- [ 3-oxo-3 a-pyridin-2-ylmethyl-2- (2,2, 2-trifluoro-ethyl) -2,3,3a,4,6, 7-hexahydro-pyrazolo [4,3-c ] pyridin-5-yl ] -ethyl } -2-methyl-propionamide L- (+) tartrate

A.4-oxo-3-pyridin-2-ylmethyl-piperidine-1, 3-dicarboxylic acid 1-tert-butyl ester 3-ethyl ester

Picolyl chloride hydrochloride (5.7g,34.7mmol), potassium carbonate (14.4g,104.1mmol) and potassium iodide (5.76g,34.7mmol) were added to a solution of 4-oxo-piperidine-1, 3-dicarboxylic acid 1-tert-butyl ester 3-ethyl ester (10.34g,38.2mmol) in DMF (40ml) at about 0 ℃. Stirring at about 0 deg.CAfter stirring for about 2 hours, the ice bath was removed and DABCO (973mg,8.68mmol) was added. The reaction mixture was stirred for about 30 minutes and then poured into a mixture of water and IPE. The organic layer was separated and saturated aqueous NaHCO was used₃And a saturated aqueous NaCl rinse over Na₂SO₄Dried and concentrated in vacuo. The crude residue was crystallized from hexane to give a white solid (8.19g, 65% yield).¹H-NMR(CDCl₃)δ1.17(t,3H),1.48(s,9H),1.55(s,2H),2.61(m,1H),2.71(m,1H),3.31-3.50(m,3H),4.11(d,2H),4.49(d,1H),7.06(brs,1H),7.17(d,1H),7.54(m,1H),8.40(s,1H)。

3-oxo-3 a-pyridin-2-ylmethyl-2- (2,2, 2-trifluoroethyl) -2,3,3a,4,6, 7-hexahydro-pyrazolo [4,3-c ] pyridine-5-carboxylic acid tert-butyl ester

Extraction of 70% CF with toluene (3X 1200ml)₃CH₂NHNH₂Aqueous solution (325ml,1.986mol) (obtained from Aldrich). The combined toluene extract containing anhydrous 2,2, 2-trifluoroethylhydrazine was added to a solution of the product from step A (600g,1.655mol) in toluene (900ml) followed by acetic acid (121.4g,1.986 mol). The reaction mixture was heated at about 70 ℃ for about 2 hours, and then another toluene extract containing 70% aqueous 2,2, 2-trifluoroethylhydrazine (50g) was added. The reaction mixture was heated at about 80 ℃ for about 3.5 hours, then cooled to room temperature and quenched with saturated aqueous NaHCO₃And (2L) diluting. The toluene layer was separated, washed with saturated aqueous NaCl and passed over Na₂SO₄Dried and concentrated in vacuo to give an oil (754.8 g). Crystallization from methanol/water afforded the desired product as a white solid (609.5 g).¹H-NMR(CDCl₃)δ1.50(s,9H),2.53(d,1H),2.70(brs,2H),2.88(brs,1H),3.31(m,2H),3.97(m,1H),4.19(m,1H),4.46(brs,1H),4.63(brs,1H),7.06(m,2H),7.51(m,1H),8.34(m,1H)。

3 a-pyridin-2-ylmethyl-2- (2,2, 2-trifluoroethyl) -2,3a,4,5,6, 7-hexahydro-pyrazolo [4,3-c ] pyridin-3-one

Methanesulfonic acid (11.6g,121mmol) was added dropwise over a period of more than 30 minutes to the solution containing step BProduct (10g,24.2mmol) in CH₂Cl₂(100ml) in solution. After stirring the mixture for about 1 hour, it was cooled to about 0 ℃ and triethylamine (18.6ml,133.1mmol) was added to the mixture via an addition funnel. The mixture was allowed to warm to room temperature over 1 hour, with additional CH₂Cl₂Diluted and then washed with saturated aqueous NaCl over Na₂SO₄Dried, filtered and concentrated in vacuo to afford a white solid (7.2 g).¹H-NMR(CDCl₃)δ2.51-2.72(m,4H),3.35(m,2H),3.49(m,2H),4.03(m,1H),4.25(m,1H),7.08(d,2H),7.51(t,1H),8.37(d,1H)。

3 a-pyridin-2-ylmethyl-2- (2,2, 2-trifluoroethyl) -2,3a,4,5,6, 7-hexahydro-pyrazolo [4,3-c ] pyridin-3-one (D) -tartrate

(D) -tartaric acid (129g,0.86mol) was added to the compound obtained in step C (243g,0.78mol) in acetone/water (ca. 9: 1,2430ml) in a dry 5L round-bottom flask equipped with a mechanical stirrer and purged with nitrogen at around 17 ℃. The mixture was stirred at room temperature overnight, the solid was filtered, collected and washed with cold acetone and dried under vacuum. The product was obtained as a yellow solid (284g, yield 78.8%).

E.2-tert-Butoxycarbonylamino-3- (2, 4-difluoro-benzyloxy) -propionic acid

A solution of potassium tert-butoxide (515.8g,4.5963mol) was added to a solution of a mixture of THF (7L) and DMF (3L) containing N-BOC- (D) -serine (452g,2.2026mol) at about 0 ℃. The reaction mixture was stirred at about 0 ℃ for about 30 minutes, then 2, 4-difluorobenzyl bromide (456.5g,2.2051mol) was added. After warming to room temperature, the reaction mixture was concentrated in vacuo to remove THF. The reaction mixture was then partitioned between 4.5L of water and 4.5 LIPE. The layers were separated and the pH of the aqueous layer was adjusted to around 3 with 1N HCl. The aqueous layer was extracted twice with 4L of IPE each. Through Na₂SO₄The organic solution was dried and concentrated in vacuo to afford a yellow waxy solid (518.0g, 70.9% yield).¹H-NMR(CDCl₃)δ1.44(s,9H),3.73(m,1H),3.94(d,1H),4.44(brs,1H)，4.54(s,2H),5.34(m,1H),6.78(m,1H),6.84(m,1H),7.30(m,1H)。

2-amino-3- (2, 4-difluoro-benzyloxy) -propionic acid, methanesulfonic acid salt

Methanesulfonic acid (1.72g,17.95mmol) was added to the CH containing the product from step E (1.19g,3.59mmol) via a syringe over a period of about 10 minutes₂Cl₂In a solution of/IPE (1: 1,12 ml). A solid precipitated out of solution immediately. After about 1 hour, the solid is filtered and washed with CH₂Cl₂the/IPE mixture (1: 1) was washed to obtain 939mg of product (80% yield).

G.2- (2-tert-Butoxycarbonylamino-2-methyl-propionylamino) -3- (2, 4-difluoro-benzyloxy) -propionic acid

2-tert-Butoxycarbonylamino-2-methyl-propionic acid-2, 5-dioxo-pyrrolidin-1-yl ester (438mg,1.46mmol) and triethylamine (369mg,3.65mmol) were added to a solution of the product obtained in step F (520mg,1.46mmol) in THF/water (4: 1,10 ml). The reaction mixture was stirred at room temperature for about 1 hour and quenched with 10% aqueous citric acid (10 ml). After about 15 minutes, ethyl acetate (50ml) was added, then the organic layer was separated and washed with saturated aqueous NaCl over Na₂SO₄Dried and concentrated in vacuo to give a foam (534.1mg, 88% yield).¹H-NMR(CD₃OD)δ1.38(brs,15H),3.77(d,1H),3.92(d,1H),4.52(m,3H),6.92(m,1H),7.41(m,1H),7.58(d,1H)。

(1- {1- (2, 4-difluoro-benzyloxymethyl) -2-oxo-2- [ 3-oxo-3 a-pyridin-2-ylmethyl-2- (2,2, 2-trifluoro-ethyl) -2,3,3a,4,6, 7-hexahydro-pyrazolo [4,3-c ] pyridin-5-yl ] -ethylcarbamoyl } -1-methyl-ethyl) -carbamic acid tert-butyl ester

(1) The compound obtained in step D (517g,1.12mol) was added to ethyl acetate (5170ml) at about-6 ℃ in a dry 12L round bottom flask equipped with a mechanical stirrer and purged with nitrogen. The solution was cooled to around-40 ℃ and triethylamine (398ml,2.86mol) was added over about 45 minutes. The reaction mixture was stirred between about-50 ℃ and-40 ℃ for about 90 minutes, filtered into a 22L nitrogen purged round bottom flask and rinsed with ethyl acetate (2068ml, pre-cooled to-50 ℃) to afford the free base as a white solid.

(2) The compound obtained in step G (425G,1.02mol) was added to a solution of the compound obtained in step H (1), triethylamine (654ml,4.69mol) and PPAA (1-propanephosphonic cyclic anhydride) (50% in ethyl acetate, 916ml,1.53mol) in ethyl acetate at around-30 ℃. The reaction mixture was stirred for about 1 hour, washed with water and saturated aqueous NaCl, and passed over Na₂SO₄Dried and concentrated in vacuo to give the product as an oil (636g, 87.8% yield).

2-amino-N- {1- (2, 4-difluoro-benzyloxymethyl) -2-oxo-2- [ 3-oxo-3 a-pyridin-2-ylmethyl-2- (2,2, 2-trifluoro-ethyl) -2,3,3a,4,6, 7-hexahydro-pyrazolo [4,3-c ] pyridin-5-yl ] -ethyl } -2-methyl-propionamide

Methanesulfonic acid (258.3ml,3.98mol) was added dropwise to CH containing the product obtained in step H (566g,0.796mol) in a dry, nitrogen purged, 22L round bottom flask equipped with a mechanical stirrer at about 15 ℃ over about 55 minutes₂Cl₂(11,320 ml). The mixture was stirred at about 20 ℃ for about 40 minutes, then saturated aqueous NaHCO was added₃(8,490ml) until the pH of the solution was 7.8. The organic layer was separated, washed with water and saturated aqueous NaCl and passed over Na₂SO₄Dried and concentrated in vacuo to afford the product as an oil (388.8g, 80% yield).

2-amino-N- {1- (2, 4-difluoro-benzyloxymethyl) -2-oxo-2- [ 3-oxo-3 a-pyridin-2-ylmethyl-2- (2,2, 2-trifluoro-ethyl) -2,3,3a,4,6, 7-hexahydro-pyrazolo [4,3-c ] pyridin-5-yl ] -ethyl } -2-methyl-propionamide L- (+) tartrate

In a 12L round-bottomed flask equipped with a mechanical stirrer, L- (+) tartaric acid (90g,0.6mol) was added to the solution containing the product obtained in step I (370g,0.6mol)Methanol (4,070 ml). The reaction mixture was stirred for about 90 minutes at about 22 ℃, filtered and concentrated. The crude residue was diluted with ethyl acetate (4,560ml), heated at about 70 ℃ and allowed to cool slowly to room temperature over 17 hours. The solid was filtered and then dried to obtain white crystals with melting point 188-.¹H-NMR(MeOH,d4)δ:8.28(d,1H),7.59(t,1H),7.41-7.39(m,1H)7.18-7.13(m,1H),6.92(t,1H),5.2(t,1H),4.56(bs,3H),4.36(s,2H),4.31-4.25(m,1H),4.13-4.06(m,1H),3.78(d,2H),3.21(t,1H),3.18-2.96(m,2H),2.65-2.55(m,2H),1.57(d,6H)。MS:MH+611。[a]⁵⁸⁹+22.03(c=11.9,MeOH)。

Claims (49)

1. (L) - (+) -tartaric acid salt of a compound of formula I

2. The (L) - (+) -tartaric acid salt of the compound of formula I according to claim 1, wherein the stereochemical configuration is 3a- (S),1- (R).

3. The (L) - (+) -tartrate salt of the compound of formula I according to claim 1, wherein the stereochemical configuration is 3a- (S),1- (S).

4. The (L) - (+) -tartaric acid salt of the compound of formula I according to claim 1, wherein the stereochemical configuration is 3a- (R),1- (S).

5. The (L) - (+) -tartaric acid salt of the compound of formula I according to claim 1, wherein the stereochemical configuration is 3a- (R),1- (R).

6. A process for the preparation of a compound of formula (E) (D) -tartrate or (L) -tartrate,it comprises reacting a compound of formula (D) with (D) -tartaric acid or (L) -tartaric acid in a mixture of about 8: 1 to 9: 1 acetone: water at a temperature between 0 ℃ and room temperature.

7. A process according to claim 6, wherein (D) -tartaric acid is reacted with a compound of formula (D), formula (E) having the R-configuration.

8. A process for the preparation of a compound of formula (J),which comprises reacting a compound of formula (E) with a compound of formula (X) in the presence of an organic base and a peptide coupling agent at a temperature of about-78 ℃ to-20 ℃,wherein Prt is an amine protecting group, X is OH, -O (C)₁-C₄) Alkyl or halogen.

9. A process according to claim 8 wherein the peptide coupling agent is 1-propanephosphonic acid cyclic anhydride, the compound of formula (X) having the R-configuration and the compound of formula (E) having the R-configuration.

10. A process according to claim 9, wherein Prt is tert-butoxycarbonyl.

11. A process for the preparation of the (L) - (+) -tartrate salt of the compound of formula I,which comprises reacting a compound of formula (E) with a compound of formula (X) in the presence of an organic base and a peptide coupling agent at a temperature of about-78 ℃ to-20 ℃ to produce a compound of formula (J),wherein Prt is an amine protecting group, X is OH, -O (C)₁-C₄) An alkyl group or a halogen, in which,deprotecting the compound of formula (J) under suitable deprotection conditions to produce a compound of formula (K),

reacting the compound of formula (K) with (L) - (+) -tartaric acid in a reaction-inert solvent to produce the (L) - (+) -tartrate salt of the compound of formula I.

12. A process according to claim 11, wherein Prt is tert-butoxycarbonyl.

13. A process according to claim 12 wherein the peptide coupling reagent is 1-propanephosphonic acid cyclic anhydride and the compound of formula I has the absolute and relative configuration of 3a- (R),1- (R).

14. A method of increasing the endogenous growth hormone level in a human or other animal which comprises administering to said human or animal an effective amount of the (L) - (+) -tartrate salt of the compound of formula I of claim 1.

15. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an amount of the (L) - (+) -tartrate salt of the compound of formula I of claim 1.

16. A pharmaceutical composition for increasing endogenous production or release of growth hormone in a human or other animal comprising a pharmaceutically acceptable carrier, an effective amount of the (L) - (+) -tartrate salt of the compound of formula I of claim 1, a growth hormone secretagogue selected from the group consisting of GHRP-6, Hexarelin, GHRP-1, growth hormone releasing factor (GRF), IGF-1, IGF-2 and B-HT920, or an analog thereof.

17. A method for the treatment or prevention of osteoporosis which comprises administering to a human or other animal in need thereof an amount of a compound of formula I (L) - (+) -tartrate salt of claim 1 effective in the treatment or prevention of osteoporosis.

18. A method of treatment or prophylaxis of diseases or conditions which are treatable or preventable by growth hormone which comprises administering to a human or other animal in need of such treatment or prophylaxis an amount of a (L) - (+) -tartrate salt of a compound of formula I as claimed in claim 1, which is effective in promoting the release of endogenous growth hormone.

19. A method according to claim 18 wherein the disease or condition is congestive heart failure, obesity or frailty associated with aging.

20. A method according to claim 19 wherein the disease or condition is congestive heart failure.

21. A method according to claim 19 wherein the disease or condition is frailty associated with aging.

22. A method for promoting bone fracture repair, reducing proteolytic responses following major surgery, reducing cachexia and protein loss due to chronic disease, promoting wound repair, or promoting healing in a patient suffering from a burn or undergoing major surgery comprising administering to a mammal in need thereof an amount of the (L) - (+) -tartrate salt of the compound of formula I of claim 1 effective to promote the release of endogenous growth hormone.

23. A method according to claim 22, wherein the method is used to promote healing in patients undergoing major surgery.

24. A method according to claim 22, wherein the method is used to promote repair of a bone fracture.

25. A method for increasing muscle strength and mobility, maintaining skin thickness, maintaining metabolic homeostasis or renal homeostasis which comprises administering to a human or other animal in need of such treatment an amount of (L) - (+) -tartrate salt of the compound of formula I of claim 1 effective to promote the release of endogenous growth hormone.

26. A method for the treatment or prevention of osteoporosis which comprises administering to a human or other animal suffering from osteoporosis an effective amount of a bisphosphonate compound and the (L) - (+) -tartrate salt of a compound of formula I of claim 1.

27. A method for treating osteoporosis of claim 26 wherein the bisphosphonate is ibandronate.

28. A method for treating osteoporosis according to claim 26 wherein the bisphosphonate is alendronate.

29. A method for the treatment or prophylaxis of osteoporosis which comprises administering to a human or other animal suffering from osteoporosis an effective amount of estrogen or gestagen  and the (L) - (+) -tartrate salt of the compound of formula I of claim 1, and optionally progesterone.

30. A method of treating osteoporosis which comprises administering to a human or other animal suffering from osteoporosis an effective amount of a calcitonin and a (L) - (+) -tartrate salt of a compound of formula I as claimed in claim 1.

31. A method for increasing IGF-1 levels in a human or other animal deficient in IGF-1, which comprises administering to the human or other animal deficient in IGH-1 an effective amount of the (L) - (+) -tartrate salt of the compound of formula I of claim 1.

32. A method of treating osteoporosis which comprises administering to a human or other animal suffering from osteoporosis an effective amount of an estrogen agonist or antagonist and the (L) - (+) -tartrate salt of the compound of formula I of claim 1.

33. A method according to claim 32 wherein the estrogen agonist or antagonist is tamoxifen, droloxifene, raloxifene or idoxifene.

34. A method according to claim 32 wherein the estrogen agonist or antagonist is cis-6- (4-fluoro-phenyl) -5- [4- (2-piperidin-1-yl-ethoxy) -phenyl ] -5,6,7, 8-tetrahydro-naphthalen-2-ol; (-) -cis-6-phenyl-5- [4- (2-pyrrolidin-1-yl-ethoxy) -phenyl ] -5,6,7, 8-tetrahydro-naphthalen-2-ol; cis-6-phenyl-5- [4- (2-pyrrolidin-1-yl-ethoxy) -phenyl ] -5,6,7, 8-tetrahydro-naphthalen-2-ol; cis-1- [6 '-pyrrolidinoethoxy-3' -pyridyl ] -2-phenyl-6-hydroxy-1, 2,3, 4-tetrahydro-naphthalene; 1- [ 4' -pyrrolidinoethoxyphenyl ] -2- (4 "-fluorophenyl) -6-hydroxy-1, 2,3, 4-tetrahydroisoquinoline; cis-6- (4-hydroxyphenyl) -5- [4- (2-piperidin-1-yl-ethoxy) -phenyl ] -5,6,7, 8-tetrahydro-naphthalen-2-ol or 1- (4' -pyrrolidinoethoxyphenyl) -2-phenyl-6-hydroxy-1, 2,3, 4-tetrahydroisoquinoline.

35. A method of increasing muscle mass which comprises administering to a human or other animal in need of such treatment an effective amount of the (L) - (+) -tartrate salt of the compound of formula I of claim 1.

36. A method of promoting growth hormone deficiency in children, which comprises administering to a growth hormone deficient child an effective amount of the (L) - (+) -tartrate salt of the compound of formula I of claim 1.

37. A method of treating insulin resistance in a mammal which comprises administering to said mammal an effective amount of the (L) - (+) -tartrate salt of the compound of formula I of claim 1.

38. A method according to claim 37, wherein the condition associated with insulin resistance is type i diabetes, type ii diabetes, hyperglycemia, impaired glucose tolerance, or insulin resistance syndrome.

39. A method according to claim 37 wherein the state associated with insulin resistance is associated with obesity or elderly.

40. A method for increasing endogenous growth hormone levels which comprises administering to a human or other animal in need thereof an effective amount of a functional somatostatin antagonist and the (L) - (+) -tartrate salt of the compound of formula I of claim 1.

41. A method according to claim 40 wherein the functional somatostatin antagonist is an alpha-2 adrenergic agonist.

42. A method of treating or preventing congestive heart failure, obesity or age-related frailty comprising administering to a human or other animal in need thereof an effective amount of a functional somatostatin antagonist and the (L) - (+) -tartrate salt of the compound of formula I of claim 1.

43. Formula (II)A mixture of the R, S-enantiomers, the R-enantiomer or the S-enantiomer of the compound.

44. The (D) -tartrate or (L) -tartrate salt of the compound of claim 43.

45. Formula (II)A 3a- (R, S), a 1- (R) diastereomer mixture, a 3a- (R), a 1- (R) diastereomer, or a 3a- (S),1- (R) diastereomer of a compound, wherein Prt is an amine protecting group selected from the group consisting of t-BOC, FMOC, and CBZ.

46. Formula (II)A mixture of the R, S-enantiomers, the R-enantiomer or the S-enantiomer of the compound.

47. Formula (II)A mixture of R, S-enantiomers, R-enantiomer or S-enantiomer of a compound, wherein X is OH, -O (C)₁-C₄) Alkyl or halogen, Prt is an amine protecting group.

48. A compound according to claim 47, wherein X is OH, Prt is BOC, and the stereocenter is in the R-configuration.

49. A method of treating a sleep disorder in a mammal suffering from a sleep disorder which comprises administering to said mammal an effective amount of the (L) - (+) -tartrate salt of the compound of formula I of claim 1.