HK1028051B

HK1028051B - Treatment of insulin resistance with growth hormone secretagogues

Info

Publication number: HK1028051B
Application number: HK00107485.7A
Authority: HK
Inventors: P‧A‧卡皮诺; C‧国锋‧赵; B‧A‧勒夫克; L‧C‧潘; J‧L‧特勒拉德瓦; M‧P‧萨威斯托斯基
Original assignee: 辉瑞产品公司
Priority date: 1997-06-25
Filing date: 1998-06-05
Publication date: 2004-11-12

Description

Treatment of insulin resistance with growth hormone secretagogues

Background

Growth Hormone (GH) secreted by the pituitary stimulates growth of all growth-competent tissues of the body, and is also known to have the following basic effects on metabolic processes of the body:

1. increasing the rate of protein synthesis in almost all body cells;

2. reducing the availability of carbohydrates in cells of the body;

3. promoting the metabolism of free fatty acids and the role of fatty acids in providing energy.

Deficiency of growth hormone results in a variety of diseases, which in children, cause dwarfism; in adults, the consequences of acquired GH deficiency include profound reduction in lean body mass and concomitant increase in fat throughout the body, particularly in the trunk area. The reduction in skeletal and cardiac muscle mass and the reduction in muscle strength results in a significant reduction in motor capacity; and bone density is also reduced. It has been shown that the administration of exogenous growth hormone can reverse a variety of metabolic changes, and that therapeutic benefits include lowering LDL cholesterol and promoting psychological well being.

In the case where an increase in growth hormone level is required, the above problems are generally solved by supplying exogenous growth hormone or administering an agent which stimulates the production and/or release of growth hormone. In both cases, the peptidic nature of the compound forces it to be administered by injection. The initial source of growth hormone was an extract from cadaveric pituitary, which resulted in expensive products and also risked that diseases associated with the pituitary source could be transmitted to recipients of growth hormone (e.g., Jacob-Crentzfeld disease). Recently, recombinant growth hormones have become available which no longer carry any risk of disease transmission, but are still very expensive products which have to be administered by injection or nasal spray.

Most GH deficiencies are caused by GH release defects, not by the initial defects of GH pituitary synthesis; thus, an alternative strategy to normalize serum GH levels is to stimulate GH release from growth hormone cells, with the goal of enhancing GH secretion by stimulating or inhibiting various neurotransmitter systems in the brain and hypothalamus; thus, research into synthetic growth hormone-releasing agents to stimulate pituitary GH secretion is ongoing and has several advantages over expensive and inconvenient GH replacement therapy. By acting along a physiologic regulatory pathway, most of the required agents will stimulate pulsatile GH secretion and by means of an intact negative feedback loop will be able to avoid excessive GH levels associated with the adverse side effects of feeding exogenous GH.

Physiological and pharmacological stimulators 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; these substances indirectly cause growth hormone to be released from the pituitary by acting in some manner in the hypothalamus, which may either decrease somatostatin secretion or enhance secretion of known GHRF or unknown endogenous growth hormone-releasing hormone or all of these.

Other compounds that stimulate endogenous growth hormone release have been developed such as analogues of peptide-based compounds related to GRF or peptide compounds described in US patent 4411890. The above peptide compounds, although much smaller than growth hormone, are still sensitive to various proteases. As with most peptides, their oral bioavailability is low. WO94/13696 relates to certain spiropiperidines and homologs thereof which promote secretion of growth hormone.

The compounds disclosed in WO94/11012 and WO94/13696 are reported to be effective in treating osteoporosis in combination with parathyroid hormone or a bisphosphonate.

In one aspect, the present invention relates to a method of treating insulin resistance conditions, such as non-insulin dependent diabetes mellitus (NIDDM) and the reduced glycemic control associated with obesity and aging in a mammal, in need thereof, which comprises administering to said mammal an effective amount of a compound of formula I as defined below or a pharmaceutically acceptable salt thereof.

The present invention relates to the use of growth hormone secretagogues, in particular Growth Hormone Releasing Peptide (GHRP) or GHRP mimetic (GHRP mimetic) of formula I as defined below for improving glycemic control. The above agents for increasing Growth Hormone (GH) levels are not expected to have such effects because GH causes diabetes in animals and humans, which is a long-established consensus. For acromegaly, glucose utilization and the ability to inhibit hepatic glucose production are attenuated (see Hansen, I., et al, Am J Physiol, 250: E269 (1986)). In the above-mentioned diseases of GH excess, impaired glucose handling and hyperinsulinemia can be reversed by pituitary surgery or chemotherapy which reduces GH levels (see Levin S.R., et al, Am J Med, 57: 526 (1974); Feek, C.M., et al, J Clin Endocrinol 22: 532 (1981)). Furthermore, it has been found in most studies that administration of GH to older subjects results in hyperglycemia, glucose intolerance and hyperinsulinemia (see Aloia, J.F., et al, J Clin Endocrinol Metab, 43: 992 (1976); Binnerts et al, J Clin Endocrinol Metab, 67: 1312 (1988); Marcus, R., et al, J Clin Endocrinol Metab, 70: 519 (1990)). Therefore, GH treatment is a contraindication for diabetics or individuals at risk of diabetes.

Obesity is a major risk factor for diabetes, and most NIDDM patients are obese; both conditions have the property that circulating insulin levels are elevated and GH levels are inhibited. GH treatment of adult GH deficiency (Jorgensen, J.O.L., et al, Lancet 1: 1221(1989)), obese women (Richelsen, B., et al, Am J Physiol, 266: E211(1994)), and older men (Rudman, D., et al, Horm Res 36(Suppl 1): 73(1991)) have been shown to increase liver and muscle mass in lean bodies while decreasing fat mass. Thus, GH treatment of diabetes would appear to be attractive without the diabetogenic effects of GH.

Alternative to exogenous GH administration, treatments that stimulate endogenous GH secretion have been shown to present large GH pituitary reserves in both patients with intact pituitary GH and older patients, and therefore, the reduction in serum GH levels is due to hyposecretion.

Several clinical manifestations of GH hyposecretion (obesity, aging, glucocorticoid suppression) are quite tolerant to GHRH stimulation (Gertz, b.j., et al, J Clin endocrinolometer, 79: 745 (1994); Arvat, e., et al, J Clin Endocrinol meter, 79: 1440 (1994); Maccario, m., et al, metablism, 44: 134 (1995)). In contrast, administration of GHRP or combined administration of GHRH and GHRP to these patients can cause a healthy GH response (Aloi, j.a., et al, J Clin Endocrinol Metab, 79: 943 (1994)). Single dose studies of GHRP demonstrated no acute effects on circulating insulin or glucose levels; insulin and glucose have not generally been monitored in chronic studies, except that no adverse changes have been described (Jacks, T., et al, J Endocrinol, 143: 399 (1993)).

Prior to the present invention, the use of GHRP or GHRP to improve glycemic control has not been specifically explored. A method of treating insulin resistance in a mammal comprises administering a compound of formula I to a patient having a functional hypothalamic-pituitary axis capable of producing GH-responsive secretion in response to GHRP and a diabetic patient (type I or type II), or an insulin resistant patient, or a patient exhibiting impaired tolerance to glucose.

In another aspect, the invention relates to a method of treating and preventing congestive heart failure, obesity and age-related debilitation in a mammal in need thereof, which comprises administering to said mammal a functional somatostatin antagonist (e.g., an alpha-2 adrenergic agonist such as clonidine, xylazine or medetomidine) in combination with a compound of formula I as defined below, simultaneously, in any order, or together. In a further aspect, the invention provides a method of accelerating fracture recovery and wound healing, attenuating the catabolic response to proteins following major surgery, and reducing cachexia and protein loss due to chronic disease in a mammal in need thereof, which comprises administering to said mammal an alpha-2 adrenergic agonist (such as clonidine, xylazine or medetomidine) simultaneously, in any order, or in combination with a compound of formula I as defined below. Clonidine is disclosed in US patent 3202660, hereby incorporated by reference; xylazine is disclosed in US patent 3235550, hereby incorporated by reference; and medetomidine is disclosed in US patent 4544664, which is hereby incorporated by reference. Alpha-2 adrenergic agonists have been shown to cause endogenous growth hormone release in humans and dogs (Cella et al, Life Sciences (1984), 34: 447-454; Hampshire J, AltsuzulerN. American Journal of mammalian Research (1981), 42: 6, 1073-1076; Valcalvi et al, Cl inorganic Endocrinology (1988), 29: 309-316; Morrison et al, American Journal of mammalian Research (1990), 51: 1, 65-70; and co-administration of alpha-2 adrenergic agonists and growth hormone releasing factors has been shown to restore unhealthy growth hormone secretion in older dogs (Areca et al, Brain Research (1990), 359: 362; Neoprene, 57: 432).

In yet another aspect, the invention provides a method for synthesizing a compound of formula Z

L-tartrate salt

Z

The process is described below

Furthermore, the present invention relates to a process for the preparation of certain intermediates (shown below) used in the synthesis of compounds of general formula Z.

The compounds of formula I and compounds of formula Z utilized in the present invention are disclosed and claimed in co-pending PCT application filed on 4.12.1996 under application number PCT/IB 96/01353, the same applicant as the present invention, in which it is disclosed that the above compounds have growth hormone secretagogue activity to elevate endogenous growth hormone levels.

Summary of The Invention

The compounds used in the method of the invention are compounds of general formula I,

Or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer, or a pharmaceutically acceptable salt or prodrug thereof,

wherein

e is 0 or 1;

n and w are each independently 0, 1 or 2;

provided that w and n are not both 0 at the same time;

y is oxygen or sulfur;

R¹is hydrogen, -CN, - (CH)₂)_qN(X⁶)C(O)X⁶，-(CH₂)_qN(X⁶)C(O)(CH₂)_t-A¹，

-(CH₂)_qN(X⁶)SO₂(CH₂)_t-A¹，-(CH₂)_qN(X⁶)SO₂X⁶，-(CH₂)_qN(X⁶)C(O)N(X⁶)(CH₂)_t-A¹，

-(CH₂)_qN(X⁶)C(O)N(X⁶)(X⁶)，-(CH₂)_qC(O)N(X⁶)(X⁶)，-(CH₂)_qC(O)N(X⁶)(CH₂)_t-A¹，

-(CH₂)_qC(O)OX⁶，-(CH₂)_qC(O)O(CH₂)_t-A¹，-(CH₂)_qOX⁶，-(CH₂)_qOC(O)X⁶，

-(CH₂)_qOC(O)(CH₂)_t-A¹，-(CH₂)_qOC(O)N(X⁶)(CH₂)_t-A¹，-(CH₂)_qOC(O)N(X⁶)(X⁶)，

-(CH₂)_qC(O)X⁶，-(CH₂)_qC(O)(CH₂)_t-A¹，-(CH₂)_qN(X⁶)C(O)O)X⁶，

-(CH₂)_qN(X⁶)SO₂N(X⁶)(X⁶)，-(CH₂)_qS(O)_mX⁶，-(CH₂)_qS(O)_m(CH₂)_t-A¹，-(C₁-C₁₀) Alkyl, - (CH)₂)_t-A¹，-(CH₂)_q-(C₃-C₇) Cycloalkyl, - (CH)₂)_q-Y¹-(C₁-C₆) Alkyl, - (CH)₂)_q-Y¹-(CH₂)_t-A¹Or- (CH)₂)_q-Y¹-(CH₂)_t-(C₃-C₇) A cycloalkyl group;

wherein R is¹Alkyl and cycloalkyl in the definitions are optionally substituted by (C)₁-C₄) Alkyl, hydroxy, (C)₁-C₄)

Alkoxy, carboxy, -CONH₂，-S(O)_m(C₁-C₆) Alkyl, -CO₂(C₁-C₄) An alkyl ester, a carboxylic acid,

1H-tetrazol-5-yl or 1, 2 or 3 fluoro;

Y¹is O, S (O)_m，-C(O)NX⁶-，-CH＝CH-，-C≡C-，-N(X⁶)C(O)-，

-C(O)NX⁶-，-C(O)O-，-OC(O)N(X⁶) -or-oc (o) -;

q is 0, 1, 2, 3 or 4;

t is 0, 1, 2 or 3;

said (CH)₂)_qAnd (CH)₂)_tMay each be optionally substituted by hydroxy, (C)₁-C₄) The alkoxy group, the carboxyl group,

-CONH₂，-S(O)_m(C₁-C₆) Alkyl, -CO₂(C₁-C₄) Alkyl esters, 1H-tetrazol-5-yl,

1.2 or 3 fluorine, or 1 or 2 (C)₁-C₄) Alkyl substitution; r²Is hydrogen, (C)₁-C₈) Alkyl, - (C)₀-C₃) Alkyl radical- (C)₃-C₈) Cycloalkyl, - (C)₁-C₄) alkyl-A¹Or A¹；

Wherein R is²Alkyl and cycloalkyl groups in the definition are optionally substituted by hydroxy,-C(O)OX⁶，

-C(O)N(X⁶)(X⁶)，-N(X⁵)(X⁶)，-S(O)_m(C₁-C₆) Alkyl, -C (O) A¹，

-C(O)(X⁶)，CF₃CN or 1, 2 or 3 halogen substitutions; r³Is A¹，(C₁-C₁₀) Alkyl, - (C)₁-C₆) alkyl-A¹，-(C₁-C₆) Alkyl radical- (C)₃-C₇) Cycloalkyl, - (C)₁-C₅) alkyl-X¹-(C₁-C₅) Alkyl, - (C)₁-C₅) alkyl-X¹-(C₀-C₅) alkyl-A¹Or- (C)₁-C₅) alkyl-X¹-(C₁-C₅) Alkyl radical- (C)₃-C₇) A cycloalkyl group;

wherein R is³Alkyl in the definitions is optionally substituted by-S (O)_m(C₁-C₆) Alkyl, -C (O) OX³，1、2、

3.4 or 5 halogens, or 1, 2 or 3 OX³Substitution;

X¹is O, S (O)_m，-N(X²)C(O)-，-C(O)N(X²)-，-OC(O)-，-C(O)O-，

-CX²＝CX²-，-N(X²)C(O)O-，-OC(O)N(X²) -or-C ≡ C-;

R⁴is hydrogen, (C)₁-C₆) Alkyl or (C)₃-C₇) A cycloalkyl group; or R⁴And R³Together with the carbon atom to which they are attached form (C)₅-C₇) Cycloalkyl group, (C)₅-C₇) Cycloalkenyl, partially saturated or fully saturated 4-to 8-membered rings having 1 to 4 heteroatoms independently selected from oxygen, sulfur and nitrogen, or bicyclic systems consisting of partially saturated or fully unsaturated 5-or 6-membered rings fused with partially saturated, fully unsaturated or fully saturated ringsA saturated 5-or 6-membered ring, and said ring optionally containing 1 to 4 heteroatoms independently selected from nitrogen, sulfur and oxygen; x⁴Is hydrogen or (C)₁-C₆) An alkyl group; or X⁴And R⁴And X⁴To the nitrogen atom and R⁴The attached carbon atoms together form a 5 to 7 membered ring;

R⁶is a key or

Wherein a and b are independently 0, 1, 2 or 3;

X⁵and X^5aEach independently selected from hydrogen, trifluoromethyl, A¹And optionally substituted (C)₁-C₆) An alkyl group;

wherein X⁵And X^5aOptionally substituted (C) in the definitions₁-C₆) Alkyl is optionally selected from A¹，OX²，-S(O)_m(C₁-C₆) Alkyl, -C (O) OX²，(C₃-C₇) Cycloalkyl, -N (X)²)(X²) and-C (O) N (X)²)(X²) Substituted with the substituent(s);

or with X⁵Or X^5aWith the carbon atom of (A) and with R⁷And R⁸Form 1 or 2 alkylene bridges, wherein each alkylene bridge contains 1 to 5 carbon atoms, with the proviso that when 1 alkylene bridge is formed, X⁵Or X^5aInstead of both being on carbon atoms, and R⁷Or R⁸But not both may be on a nitrogen atom; with the further proviso that when 2 alkylene bridges are formed, X⁵And X^5aNot on a carbon atom, and R⁷And R⁸Not on the nitrogen atom; or X⁵And X^5aTogether with the carbon atom to which they are attached form a partially or fully saturated 3-to 7-membered ring, or a partially or fully saturated ring containing 1 to 4 heteroatoms independently selected from oxygen, sulfur and nitrogenA 4-to 8-membered ring;

or X⁵And X^5aTogether with the carbon atoms to which they are attached form a bicyclic ring system consisting of a partially saturated or fully saturated 5-or 6-membered ring fused with a partially saturated, fully saturated or fully unsaturated 5-or 6-membered ring optionally containing 1 or 2 heteroatoms independently selected from nitrogen, sulfur and oxygen;

Z¹is a bond, O or N-X²Provided that Z is a value when a and b are both 0¹Is not N-X²Or^O；R⁷And R⁸Independently is hydrogen or optionally substituted (C)₁-C₆) An alkyl group;

wherein R is⁷And R⁸Optionally substituted in the definitions (C)₁-C₆) Alkyl is optionally independently substituted by A¹，

-C(O)O-(C₁-C₆) Alkyl, -S (O)_m(C₁-C₆) Alkyl, 1 to 5 halogens, 1 to 3

Hydroxy, 1 to 3-O-C (O) (C)₁-C₁₀) Alkyl or 1 to 3 (C)₁-C₆) Alkoxy substitution;

or R⁷And R⁸May be taken together to form- (CH)₂)r-L-(CH₂)r-；

Wherein L is C (X)²)(X²)，S(O)_mOr N (X)²) (ii) a In each case A¹Independently is (C)₅-C₇) Cycloalkenyl, phenyl, or a partially saturated, fully saturated or fully unsaturated 4-to 8-membered ring optionally containing 1 to 4 heteroatoms independently selected from oxygen, sulfur and nitrogen, a bicyclic ring system fused from a partially saturated, fully saturated or fully unsaturated 5-or 6-membered ring optionally containing 1 to 4 heteroatoms independently selected from nitrogen, sulfur and oxygen and a partially saturated, fully unsaturated or fully saturated 5-or 6-membered ring optionally containing 1 to 4 heteroatoms independently selected from nitrogen, sulfur and oxygenComposition is carried out;

in each case A¹Independently optionally substituted, said substitution occurring on 1 ring,

or at A¹In the case of bicyclic systems, optionally on both rings, the number of substituents

In an amount of up to 3 and each substituent is independently selected from F, Cl, Br, I, OCF₃，OCF₂H，

CF₃，CH₃，OCH₃，-OX⁶，-C(O)N(X⁶)(X⁶)，-C(O)OX⁶Oxo group (C)₁-C₆)

Alkyl, nitro, cyano, benzyl, -S (O)_m(C₁-C₆) Alkyl, 1H-tetrazol-5-yl, benzene

A group selected from phenoxy, phenylalkoxy, halophenyl, methylenedioxy, -N (X)⁶)(X⁶)，

-N(X⁶)C(O)(X⁶)，-SO₂N(X⁶)(X⁶)，-N(X₆)SO₂-phenyl, -N (X)⁶)SO₂X⁶，

-CONX¹¹X¹²，-SO₂NX¹¹X¹²，-NX⁶SO₂X¹²，-NX⁶CONX¹¹X¹²，-NX⁶SO₂NX¹¹X¹²，

-NX⁶C(O)X¹²Imidazolyl, thiazolyl and tetrazolyl, with the proviso that if A¹Is methylene dioxy

Substituted by a group, then it can only be substituted by 1 methylenedioxy group;

wherein X¹¹Is hydrogen or optionally substituted (C)₁-C₆) An alkyl group;

X¹¹optional in definitionSubstituted (C)₁-C₆) Alkyl is optionally substituted independently with phenyl, phenoxy,

(C₁-C₆) Alkoxycarbonyl, -S (O)_m(C₁-C₆) Alkyl, 1 to 5 halogens, 1 to 3

1 to 3 (C) hydroxyl groups₁-C₁₀) Alkanoyloxy or 1 to 3 (C)₁-C₆) An alkoxy group;

X¹²is hydrogen, (C)₁-C₆) Alkyl, phenyl, thiazolyl, imidazolyl, furyl or thia

A thienyl group, with the proviso that when X¹²When not hydrogen, X¹²Optionally 1 to 3 independently selected from

Cl，F，CH₃，OCH₃，OCF₃And CF₃Substituted with the substituent(s);

or X¹¹And X¹²Together form- (CH)₂)r-L¹-(CH₂)r-；

Wherein L is¹Is C (X)²)(X²)，O，S(O)_mOr N (X)²) (ii) a R in each occurrence is independently 1, 2 or 3; x in each case²Independently of hydrogen, optionally substituted (C)₁-C₆) Alkyl or optionally substituted (C)₃-C₇) A cycloalkyl group; wherein at X²Optionally substituted (C) in the definitions₁-C₆) Alkyl or optionally substituted (C)₃-C₇) Cycloalkyl is optionally independently substituted by-S (O)_m(C₁-C₆) Alkyl, -C (O) OX³1 to 5 halogens or 1 to 3 OX³Substitution; x in each case³Independently is hydrogen or (C)₁-C₆) An alkyl group; x⁶Independently of hydrogen, optionally substituted (C)₁-C₆) Alkyl radical (C)₂-C₆) Haloalkyl, optionally substituted (C)₃-C₇) Cycloalkyl group, (C)₃-C₇) Halocycloalkyl radicals in which X is⁶Optional substitution in the definitionIs (C)₁-C₆) Alkyl and optionally substituted (C)₃-C₇) Cycloalkyl is optionally independently substituted by 1 or 2 (C)₁-C₄) Alkyl, hydroxy, (C)₁-C₄) Alkoxy, carboxy, -CONH₂，-S(O)_m(C₁-C₆) Alkyl, formic acid (C)₁-C₄) Alkyl ester or 1H-tetrazol-5-yl; or when there are 2X's on 1 atom⁶And 2X⁶Are all independently (C)₁-C₆) When alkyl, said 2 (C)₁-C₆) Alkyl may optionally be taken together with the atom to which the 2X 6 are attached to form an optionally oxygen-, sulfur-or NX-containing radical⁷4-to 9-membered ring of (a);

X⁷is hydrogen or optionally substituted by hydroxy (C)₁-C₆) An alkyl group; and m in each occurrence is independently 0, 1 or 2; with the following conditions: in C (O) X⁶，C(O)X¹²，SO₂X⁶Or SO₂X¹²In (B) when X is⁶And X¹²Is with C (O) or SO₂When attached, they cannot be hydrogen; and when R is⁶When it is a bond, L is N (X)²) And is in- (CH)₂)r-L-(CH₂) Each r in the definition of r-is independently 2 or 3.

In one aspect, the present invention provides a method of treating insulin resistance in a mammal, said method comprising administering to said mammal an effective amount of a compound of formula I as defined above, or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof.

In preferred methods of the above methods, the disease associated with insulin resistance is type I diabetes, type II diabetes, hyperglycemia, glucose intolerance, or insulin resistance syndrome or state.

In another preferred method of the above method, the disease associated with insulin resistance is associated with obesity or aging.

In a preferred method of the above method, the compound of formula I is a compound of the formula

Or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof, wherein R is¹is-CH₂-phenyl, R²Is methyl, and R³Is- (CH)₂)₃-a phenyl group; r¹is-CH₂-phenyl, R²Is methyl, and R³Is 3-indolyl-CH₂-；R¹is-CH₂-phenyl, R²Is ethyl, and R³Is 3-indolyl-CH₂-；R¹is-CH₂-4-fluoro-phenyl, R²Is methyl, and R³Is 3-indolyl-CH₂-；R¹is-CH₂-phenyl, R²Is methyl, and R³is-CH₂-O-CH₂-a phenyl group; r¹is-CH₂-phenyl, R²Is ethyl, and R³is-CH₂-O-CH₂-a phenyl group; r¹is-CH₂-phenyl, R²is-CH₂CF₃And R³is-CH₂-O-CH₂-a phenyl group; r¹is-CH₂-4-fluoro-phenyl, R²Is methyl, and R³is-CH₂-O-CH₂-a phenyl group; r¹is-CH₂-phenyl, R²Is tert-butyl, and R³is-CH₂-O-CH₂-a phenyl group; or R¹is-CH₂-phenyl, R²Is methyl, and R³is-CH₂-O-CH₂-3, 4-difluoro-phenyl.

In another preferred method of the above method, the compound of formula I is a compound of the formula

Or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof, wherein R is²Is methyl, A¹Is 2-pyridyl, and R³is-CH₂-O-CH₂-a phenyl group; r²is-CH₂CF₃，A¹Is 2-pyridyl, and R³is-CH₂-O-CH₂-3-chloro-phenyl; r²is-CH₂CF₃，A¹Is 2-pyridyl, and R³is-CH₂-O-CH₂-4-chloro-phenyl; r²is-CH₂CF₃，A¹Is 2-pyridyl, and R³is-CH₂-O-CH₂-2, 4-dichloro-phenyl; r²is-CH₂CF₃，A¹Is 2-pyridyl, and R³is-CH₂-O-CH₂-3-chloro-thiophene; or R²is-CH₂CF₃，A¹Is 2-pyridyl, and R³is-CH₂-O-CH₂-2, 4-difluoro-phenyl.

In yet another preferred method of the above process, the compound of formula I or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof is a 3a (R, S), 1(R) diastereomeric mixture, 3a (R), 1(R) diastereomer or 3a (S), 1(R) diastereomer of a compound selected from the group consisting of: 2-amino-N- [1- (3 a-benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridine-5-carbonyl) -4-phenylbutyl ] isobutyramide; 2-amino-N- [2- (3 a-benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1- (1H-indol-3-ylmethyl) -2-oxo-ethyl ] isobutyramide; 2-amino-N- [2- (3 a-benzyl-2-ethyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1- (1H-indol-3-ylmethyl) -2-oxo-ethyl ] isobutyramide; 2-amino-N- [2- [3a- (4-fluoro-benzyl) -2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl ] -1- (1H-indol-3-ylmethyl) -2-oxo-ethyl ] isobutyramide; 2-amino-N- [2- (3 a-benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1-benzyloxymethyl-2-oxo-ethyl ] isobutyramide; 2-amino-N- [2- (3 a-benzyl-2-ethyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1-benzyloxymethyl-2-oxo-ethyl ] isobutyramide; 2-amino-N- {2- [3 a-benzyl-3-oxo-2- (2, 2, 2-trifluoro-ethyl) -2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl ] -1-benzyloxymethyl-2-oxo-ethyl } isobutyramide; 2-amino-N- { 1-benzyloxymethyl-2- [ (3a- (4-fluoro-benzyl) -2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -2-oxo-ethyl ] isobutyramide; 2-amino-N- [2- (3 a-benzyl-2-tert-butyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1-benzyloxymethyl-2-oxo-ethyl ] isobutyramide; 2-amino-N- [2- (3 a-benzyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyrid-5-yl) -1-benzyloxymethyl-2-oxo-ethyl ] isobutyramide.

In a preferred method of the method described immediately above, the compound of formula I is 2-amino-N- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl ] isobutyramide L-tartrate.

In a further preferred method of the above process, the compound of formula I or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof, is a 3a- (R, S), 1- (R) diastereomeric mixture, 3a- (R), 1- (R) enantiomer or 3a- (S), 1- (R) enantiomer of a compound selected from the group consisting of: 2-amino-N- [ 1-benzyloxymethyl-2- (2-methyl-3-oxo-3 a-pyridin-2-ylmethyl-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -2-oxo-ethyl ] -2-methyl-propionamide; 2-amino-N- {1- (3-chloro-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; 2-amino-N- {1- (4-chloro-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; 2-amino-N- {1- (2, 4-dichloro-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; 2-amino-N- {1- (4-chloro-thiophen-2-ylmethoxymethyl) -2-oxo-2- [ 3-oxo-3 a-pyridin-2-ylmethyl-2- (2, 2, 2-trifluoro-ethyl) -2, 3, 3a, 4, 5, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-6-yl ] ethyl } -2-methyl-propionamide; and 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.

In a more preferred method of the above method, there is also provided administering to a mammal in need thereof a growth hormone releasing hormone or a functional analogue thereof, prepared by methods known in the art and some examples of which are disclosed in European patent EP 511003.

In another aspect, the present invention provides a pharmaceutical composition for treating insulin resistance in a mammal, said composition comprising a pharmaceutically acceptable carrier and an effective amount of a compound of formula I as described above or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof.

In yet another aspect, the present invention provides a method of increasing endogenous growth hormone levels, said method comprising administering to a human or other mammal in need thereof an effective amount of a functional somatostatin antagonist and a compound of formula I as described above, or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched, enantiomerically pure isomer, or a pharmaceutically acceptable salt or prodrug thereof.

In a further aspect, the present invention provides a method of treating and preventing congestive heart failure, obesity or age-related frailty, which method comprises administering to a mammal in need thereof an effective amount of a functional somatostatin antagonist and a compound of formula I as described above, or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof. In the above method, the functional somatostatin antagonist is preferably an alpha-2 adrenergic agonist; whereas in the method just described, the alpha-2 adrenergic agonist is preferably selected from the group consisting of clonidine, xylazine, and medetomidine; furthermore, in the above process, the compound of formula I is preferably 2-amino-N- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl ] isobutyramide L-tartrate.

The invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an alpha-2 adrenergic agonist and a compound of formula I as defined above or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof.

The present invention further relates to a method of treating insulin resistance in a mammal, said method comprising administering to a mammal in need thereof an effective amount of a growth hormone releasing peptide or a ghrelin mimetic or a pharmaceutically acceptable salt thereof.

In one aspect, the invention relates to a method wherein ″ "represents a stereochemical center.

A process for the preparation of a compound of the general formula k,

said process comprising reacting a compound of formula g in the presence of an organic base, a peptide coupling agent and a reaction inert solvent at a temperature of about-78 ℃ to about-20 ℃

Free base

(g)

Reacting with a compound of a general formula j to generate a compound of a general formula k,

wherein Prt is an amino protecting group.

The above process is preferred wherein the peptide coupling agent is 1-propanephosphonic acid cyclic anhydride and the compound of formula g has the R configuration, the compound of formula j has the R configuration and the compound of formula k has the 3a- (R), 1- (R) configuration.

A process for the preparation of a compound of the general formula Z,

l-tartrate salt

Free base

(g)

With compounds of the formula j

To produce a compound of formula k;

deprotection of a compound of formula k to produce a compound of formula I;

then, the compound of the general formula I and L-tartaric acid are reacted in an alcohol solvent to generate the compound of the general formula Z.

Preferably, the above process wherein the peptide coupling agent is 1-propanephosphonic acid cyclic anhydride and the compound of formula g has the R configuration, the compound of formula j has the R configuration and the compounds of formulae k, I and Z all have the 3a- (R), 1- (R) configuration. A process for the preparation of a compound of the general formula g,

free base

(g)

Said process comprising reacting a compound of formula f in an inert solvent at a temperature of from about-50 ℃ to about-10 ℃

L-tartrate salt

(f)

Reaction with a base to produce a compound of formula g wherein the chirality of the benzyl group is maintained.

A process for the preparation of a compound of the general formula c,

the process comprises reacting a compound of the formula b in a reaction-inert solvent

Wherein Prt is an amino protecting group, with an inorganic or organic base and benzyl bromide to produce a compound of formula c.

A process for the preparation of a compound of the general formula f,

l-tartrate salt

(f)

The process comprises reacting a compound of the formula e in a reaction-inert organic solvent

Reacting with L-tartaric acid.

The invention also provides a mixture of the R, S-enantiomers, the R-enantiomer or the S-enantiomer of the compound shown in the formula,

wherein Prt is hydrogen or an amino protecting group.

Detailed Description

In general, the compounds of formula I utilized in the methods of the present invention, or their stereoisomeric mixtures, diastereomerically enriched, diastereomerically pure, enantiomerically enriched, enantiomerically pure isomers, or their pharmaceutically acceptable salts and prodrugs thereof, may be prepared by a variety of methods including those known in the chemical art.

In the above formulae and throughout the present application, the following terms have the meanings indicated below, unless otherwise indicated.

The alkyl group includes a straight chain or branched alkyl group of a specified length which may optionally contain a double bond or a triple bond, and examples thereof are methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, isohexyl, allyl, ethynyl, propenyl, butadienyl, hexenyl and the like.

When C appears in the definition₀Alkyl, represents a single covalent bond.

The alkoxy group includes a straight chain or branched alkoxy group of a specified length which may optionally contain a double bond or a triple bond, and examples thereof are methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, isopentyloxy, hexyloxy, isohexyloxy, allyloxy, 2-propynyloxy, isobutenyloxy, hexenyloxy and the like.

The term "halogen" or "halo" includes halogen atoms: fluorine, chlorine, bromine and iodine.

The term "haloalkyl" includes alkyl groups as defined above substituted with 1 or more halogen atoms as defined above.

The term "halocycloalkyl" includes cycloalkyl substituted with 1 or more halogen atoms as defined above.

The term "aryl" includes phenyl and naphthyl, as well as aromatic 5-and 6-membered rings with 1 to 4 heteroatoms, such as pyridine, thiophene, furan, benzothiophene, tetrazole, indole, N-methylindole, indoline, indazole, N-formylindole, benzimidazole, thiazole, pyrimidine and thiadiazole, or fused 5-or 6-membered bicyclic rings with 1 to 4 heteroatoms selected from nitrogen, sulfur or oxygen.

It is recognized by those of ordinary skill in the chemical arts that certain combinations of the heteroatom-containing substituents listed herein define compounds that are less stable under physiological conditions (e.g., under conditions containing an acetal or aminal bond), and thus, are less preferred.

The expression "prodrug" refers to a compound that is a precursor of a drug and, following administration, releases the drug in vivo via some chemical or physiological process (e.g., a prodrug is converted to the desired drug upon reaching physiological pH); typical prodrugs release the corresponding free acid upon cleavage, and the hydrolyzable ester-forming residues of the compounds of the present invention include, but are not limited to, carboxylic acid substituents (e.g., R)¹Is- (CH)₂)_qC(O)₂X⁶Wherein X is⁶Is hydrogen; or R²Or A¹Containing carboxylic acid) wherein the free hydrogen is substituted by (C)₁-C₄) Alkyl, (C)₂-C₁₂) Alkanoyloxymethyl group (C)₄-C₉)1- (alkanoyloxy) ethyl, 1-methyl-1- (alkanoyloxy) -ethyl having 5 to 10 carbon atoms, alkoxycarbonyl-oxymethyl having 3 to 6 carbon atoms, 1- (alkoxycarbonyloxy) ethyl having 4 to 7 carbon atoms, 1-methyl-1- (alkoxycarbonyloxy) ethyl having 5 to 8 carbon atoms, N- (alkoxycarbonyl) aminomethyl having 3 to 9 carbon atoms, 1- (N- (alkoxycarbonyl) amino) ethyl having 4 to 10 carbon atoms, 3-benzo [ c ] c]Furanone group, 4-crotonolactone group, gamma-butyrolactone-4-yl group, di-N, N- (C)₁-C₂) Alkylamino radical (C)₂-C₃) Alkyl (e.g.. beta. -dimethylaminoethyl), carbamoyl- (C)₁-C₂) Alkyl, N-di (C)₁-C₂) Alkylcarbamoyl- (C)₁-C₂) Alkyl and piperidino-, pyrrolidino-or morpholino (C)₂-C₃) Alkyl substitution.

Other exemplary prodrugs release alcohols of formula I wherein the hydroxy substituent (e.g., R)¹Containing hydroxy group) by free hydrogen (C)₁-C₆) Alkanoyloxymethyl, 1- ((C)₁-C₆) Alkanoyloxy) ethyl, 1-methylRadical-1- ((C)₁-C₆) Alkanoyloxy) ethyl group, (C)₁-C₆) Alkoxycarbonyloxymethyl, N- (C)₁-C₆) Alkoxycarbonylaminomethyl, succinyl, (C)₁-C₆) Alkanoyl, alpha-amino (C)₁-C₄) Alkanoyl, arylacetyl and alpha-aminoacyl or alpha-aminoacyl-alpha-aminoacyl [ wherein the alpha-aminoacyl moiety is independently an L-amino acid or P (O) (OH) of any naturally occurring protein₂、-P(O)(O(C₁-C₆) Alkyl radical)₂Or glycosyl (a group formed by removing a hydroxyl group from a hemiacetal of a carbohydrate).

Prodrugs of compounds of formula I wherein the carboxyl group of the carboxylic acid of formula I is replaced by an ester are prepared by reacting the carboxylic acid with a suitable alkyl halide in the presence of a base such as potassium carbonate in an inert solvent such as DMF at a temperature of from about 0 ℃ to about 100 ℃ for a period of from about 1 to about 24 hours. Alternatively, this is accomplished by reacting the carboxylic acid with a suitable alcohol as solvent in the presence of a catalytic amount of acid (e.g., concentrated sulfuric acid) at a temperature of about 20 ℃ to about 120 ℃, preferably under reflux, for a period of about 1 to about 24 hours. Yet another method is to react the acid in an inert solvent (such as THF) with the concomitant removal of water produced either by physical (such as dean stark trap) or chemical (such as molecular sieve).

Prodrugs of compounds of formula I wherein the alcohol function of formula I is derivatized to an ether are prepared by reacting the alcohol with a suitable alkyl bromide or iodide in the presence of a base (e.g., potassium carbonate) in an inert solvent (e.g., DMF) at a temperature of about 0 ℃ to about 100 ℃ for about 1 to about 24 hours. Alkanoylaminomethyl ethers can be prepared according to the method described in U.S. Pat. No. 4,984 by reacting an alcohol with bis- (alkanoylamino) methane in the presence of a catalytic amount of an acid in an inert solvent such as THF; alternatively, the above compounds can be prepared using the method described by Hoffman et al, j.org.chem.1994, 59, p.3530.

In the above formula, certain defined terms may occur more than once, and the definitions of each instance in which they occur are independent of each other.

Throughout the specification and claims, the following abbreviations are used with the following meanings:

BOC tert-butyloxycarbonyl radical

BOP benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate

CBZ benzyloxycarbonyl

CDI N, N' -carbonyldiimidazole

CH₂Cl dichloromethane

CHCl₃Chloroform

DCC dicyclohexylcarbodiimide

DMF dimethyl formamide

EDC 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride

EtOAc ethyl acetate

FMOC 9-fluorenylmethoxycarbonyl

h hours

Hex Hexane

HOAT 1-hydroxy-7-azabenzotriazole

HOBT hydroxybenzotriazole hydrate

HPLC high pressure liquid chromatography

MHz megahertz

MS Mass Spectrometry

NMR nuclear magnetic resonance

PTH parathyroid hormone

TFA trifluoroacetic acid

THF hydrofuran

TLC thin layer chromatography

TRH thyroid-stimulating hormone releasing hormone

TROC 2, 2, 2-trichloroethoxycarbonyl

As indicated by the asterisks in structural formula I above, the compounds utilized in the process of the present invention all have at least 1 asymmetric center. Other asymmetric centers may also be present in the molecule, determined by the nature of the various substituents in the molecule. Each such asymmetric center will give rise to 2 optical isomers and all such optical isomers are intended to be included within the scope of the present invention as isolated, purified or partially purified optical isomers, racemic mixtures or diastereomeric mixtures. In the case where the asymmetric center is indicated by an asterisk, it has been found that the more active, and thus preferred, absolute stereochemistry is represented by general formula IA. Likewise, the preferred absolute configurations described above also apply to formula I.

Wherein R is⁴The substituent is hydrogen, and the spatial configuration of the asymmetric center corresponds to D-amino acid; and, in most cases, it is designated as the R configuration, although dependent on the R used to determine the R-or S-stereochemical arrangement³And R⁴The above configuration may be changed.

The compounds utilized in the process of the present invention are generally isolated in the form of their pharmaceutically acceptable acid addition salts, e.g., salts formed using inorganic and organic acids. Examples of such acids are hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, formic acid, acetic acid, trifluoroacetic acid, propionic acid, maleic acid, succinic acid, D-tartaric acid, L-tartaric acid, malonic acid, methanesulfonic acid, and the like. In addition, certain compounds containing acidic functional groups, such as carboxyl groups, can be isolated in the form of their inorganic acid salts, where the counterion can be selected from sodium, potassium, lithium, calcium, magnesium, and the like, as well as from organic bases.

The pharmaceutically acceptable salts can be formed by taking about 1 equivalent of a compound of formula I and contacting it with about 1 equivalent of the appropriate acid corresponding to the desired salt. Methods for working up and isolating the salts formed are known to the person skilled in the art.

Within its scope, the present invention includes pharmaceutical compositions comprising as an active ingredient an insulin resistance treating amount of at least one compound of formula I, together with pharmaceutically acceptable excipients; furthermore, within its scope, the present invention also includes pharmaceutical compositions comprising as active ingredients at least one alpha-2 adrenergic agonist and at least one compound of formula I, together with pharmaceutically acceptable excipients; optionally, the pharmaceutical composition may further comprise an anabolic agent other than at least one compound of formula I, or other compounds exhibiting different activities, such as antibiotic growth-permitting agents (permetts), or other pharmaceutically active substances whose combination with the above substances increases efficacy and reduces side effects.

Test for stimulating GH Release from rat pituitary

Compounds having the ability to stimulate GH secretion from cultured rat pituitary cells were identified using the following protocol. The test is also used to compare to standards to determine dosage levels. Cells were isolated from the pituitary of 6-week male Wistar rats. After decapitation, the anterior pituitary is removed and placed in a cold sterile Hank's Balanced Salt Solution (HBSS) without calcium or magnesium. The tissue was minced and then treated with 10U/mL bacterial protein in HBSSThe enzyme (EC 3.4.24.4 Sigma P-6141, St.Louis, Missouri) performs two mechanical assisted enzymatic dispersion processes. The tissue-enzyme mixture was spun in a spinner at about 37 ℃ in 5% CO₂Stirring at 30rpm for about 30 minutes in a gas atmosphere; manual milling was performed after about 15 minutes and about 30 minutes using a 10ml pipette. The mixture was centrifuged at 200Xg for about 5 minutes. Horse serum (final concentration 35%) was added to the supernatant to neutralize excess protease. The pellet was resuspended in fresh protease (10U/mL), stirred under the conditions described above for approximately 30 more minutes, and manually ground to the point where it finally passed through a 23 gauge needle. Horse serum (final concentration 35%) was added again, and then the cells from the two digestions were pooled, pelleted (at 200Xg for about 15 minutes), resuspended in medium (Dulbecco's modified Eagle's medium (D-MEM) supplemented with 4.5g/L glucose, 10% horse serum, 2.5% fetal bovine serum, 1% non-essential amino acids, 100U/mL nystatin and 50mg/mL gentamicin sulfate, Gibco, Grand Island, New York) and counted. The cells are cultured at 6.0-6.5X 10⁴Cells/cm²Placing in a Costar with 48 holes^TM(Cambridge, Massachusetts) plates and cultured in medium for 3 to 4 days.

Before starting the GH secretion assay, the wells were rinsed twice with release medium and then equilibrated in release medium (25 mM Hepes buffered D-MEM, pH7.4 and containing 0.5% bovine serum albumin at 37 ℃) for approximately 30 minutes. Test compounds were dissolved in DMSO and then diluted into pre-warmed release medium. Experiments were performed in quadruplicate. The assay was started by adding 0.5mL of release medium (with vehicle or test compound) to each culture well. The culture was terminated by culturing at about 37 ℃ for about 15 minutes and then removing the release medium, and the medium was centrifuged at 2000Xg for about 15 minutes to remove the cell material. Rat growth hormone concentrations in the supernatants were determined by standard radioimmunoassay methods described below.

Determination of rat growth hormone

Rat growth hormone concentrations were determined by a double antibody radioimmunoassay procedure using a rat growth hormone control preparation (NIDDK-rGH-RP-2-) and rat growth hormone antiserum (NIDDK-anti-rGH-S-5) extracted from monkeys from Dr.A. Parlow (Harbor-UCLA medical center, Torrence, Calif.). Additional rat growth hormone (1.5U/mg, # G2414, Scripps Labs, San Diego, Calif.) was iodinated by chloramine T method to a specific activity of approximately 30 μ Ci/μ G for use as a tracer. Immune complexes were obtained by adding goat antiserum to monkey IgG (ICN/Cappel, Aurora, OH) plus polyethylene glycol, MW10000-20000, to a final concentration of 4.3%; and then the recovery is finished by centrifugation. On a basal level, the experimental operating range is 0.08-2.5 μ g rat growth hormone/tube.

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

Female Sprague-Dawley rats (Charles River Laboratory, Wilmington, MA) of 21 days of age were acclimatized to local zoo conditions (24 ℃, 12 hours light, 12 hours dark cycle) for about 1 week prior to compound testing. All rats were freely available with water and commercial pelleted feed (age Country Food, sympocuse NY). The test animals were kept and tested using NIH guidelines.

On the day of the assay, test compounds were dissolved in a vehicle salt solution containing 1% ethanol, 1mM acetic acid and 0.1% bovine serum albumin. Each experiment was performed with three rats. Rats were weighed and injected intraperitoneally with sodium pentobarbital (nembutal)^_50mg/kg body weight) was subjected to anesthesia. After 14 minutes of anesthesia, blood samples were obtained by inserting the tail tip and the blood was dropped into a microcentrifuge tube (baseline blood sample, approximately 100 μ l). After 15 minutes of anesthesia, the test compound was intravenously injected into the tail vein in an amount of 1mL/kg body weight in total injection volume. Another blood sample was taken from the tail 5, 10 and 15 minutes after compound administration. The blood samples were kept in ice until serum was separated by centrifugation (1430 Xg for 10 minutes at 10 ℃). The serum was stored at-80 ℃ until serum growth hormone was determined by the radioimmunoassay method described above.

Exogenously stimulated growth hormone release test in dogs following oral administration

On the day of administration, the appropriate dose of test compound is weighed out and dissolved in water. Each dosage regimen was administered to 2 to 4 dogs by gavage in a volume of 0.5-3mL/kg body weight. Blood samples (5mL) were collected from the jugular vein by direct venipuncture with 5mL vacutainer containing lithium heparin, before and at 0.17, 0.33, 0.5, 0.75, 1, 2, 4, 6, 8 and 24 hours after dosing. The plasma obtained was stored at-20 ℃ until analysis.

Determination of growth hormone in dogs

The concentration of dog growth hormone was determined by standard radioimmunoassay methods using dog growth hormone (antigen for iodination and control preparation AFP-1983B) and dog growth hormone antiserum extracted from monkeys (AFP-21452578) from dr.a. parlow (Harbor-UCLA medical center, Torrence, CA). The tracer was prepared by chloramine T-iodination to achieve a specific activity of 20 to 40. mu. Ci/. mu.g of dog growth hormone. Immune complexes were obtained by adding goat antiserum to monkey IgG (ICN/Cappel, Aurora, OH) plus polyethylene glycol, MW10000-20000, to a final concentration of 4.3%; and then the recovery is finished by centrifugation. The test has an operating range of 0.08-2.5 mug dog GH per tube.

Evaluation of dog growth hormone and insulin-like growth factor-1 levels following chronic oral dosing in dogs

Dogs were given test compounds daily for 7 or 14 days. On each day of administration, the appropriate dose of test compound is weighed out and dissolved in water. Each dosage regimen was administered to 5 dogs by gavage in a volume of 0.5-3mL/kg body weight. Blood samples were collected on days 0, 3, 7, 10, and 14. Blood samples (5mL) were collected by direct venipuncture from the jugular vein with 5mL vacutainer containing lithium heparin on days 0, 7 and 14 before and 0.17, 0.33, 0.5, 0.754, 1, 2, 3, 6, 8, 12 and 24 hours after dosing. In addition, blood was drawn before the administration on days 3 and 10 and at 8 hours. The plasma obtained was stored at-20 ℃ until analysis.

Female rat study

This study evaluated the effects of chronic treatment with GHRP on body weight, body composition and unphaged plasma concentrations of glucose, insulin, lactate and lipids in estrogen deficient and estrogen replete female rats. The ability of acute response to serum GH levels produced by intravenous administration of a GH-releasing agent was evaluated 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 assessed at the end of treatment.

Virgin Sprague-Dawley rats were obtained from Charles River Laboratories (Wilmington, MA) and were either bilateral ovariectomy (Ovx) or Sham (Sham) at approximately 12 weeks of age. Sham surgery involves removing the ovaries from the abdomen and replacing them with the abdominal cavity. After surgery, rats were housed individually in 20cm x 32cm x 20cm cages under standard zoo conditions (24 ℃, 12 hour light, 12 hour dark cycle). All rats were freely available with water and commercial pelleted feed (Agway prolob 3000, Agway CountryFood, inc., Syracuse NY). The test animals were kept and tested using NIH guidelines.

Approximately 7 months after surgery, Sham and Ovx rats were weighed and freely grouped. Rats were orally administered 1mL of vehicle (1% ethanol in distilled-deionized water), 0.5mg/kg or 5mg/kg growth hormone releasing agent by gavage daily for 90 days. Rats were weighed at weekly intervals throughout the study. 24 hours after the last oral administration, the acute response of plasma Growth Hormone (GH) to the test agent was evaluated by the following procedure. Rats were anesthetized with 50mg/kg sodium pentobarbital. The anesthetized rats were weighed and baseline blood samples (approximately 100 μ l) were collected from the tail vein. Then, the test agent (growth hormone releasing agent or excipient) was intravenously administered into the tail vein in an amount of 1mL volume. Approximately 10 minutes after injection, a second 100 μ l blood sample was taken from the tail. The blood was allowed to clot at about 4 ℃ and then centrifuged at 2000Xg for about 10 minutes. The serum was stored at about-70 ℃. Serum growth hormone concentrations were determined by radioimmunoassay methods as described above. Following this step, a whole body scan was performed on each anesthetized rat using a dual energy X-ray absorptiometer (DEXA, Hologic QDR 1000/W, Waltham MA). The final blood sample was collected into heparinized tubes by cardiac puncture. Plasma was centrifuged and stored frozen as described above.

Plasma insulin was measured by a radioimmunoassay using a kit from Binax Corp. (Portland, Maine). The coefficient of the difference of the interactive test is less than or equal to 10 percent. Respectively using AbbottVP^TMAnd VP Super System^_Automatic analyzer (Abbott Laboratories, Irving, Texas), A-Gent^TMTriglyceride, cholesterol and glucose test reagent systems and the lactate kit (Sigma) measure plasma triglyceride, total cholesterol, glucose and lactate levels. The activity of Growth Hormone Releasing Peptide (GHRP) or GHRP mimetics exemplified by the compounds of formula I to reduce plasma insulin, triglycerides, total cholesterol and lactate was determined by statistical analysis (unpaired t-test) against vehicle treated groups.

The compounds of formula I for use in the methods of the present invention may be administered orally, parenterally (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection or implant), intranasally, intravaginally, intrarectally, sublingually or transdermally; and may be formulated with pharmaceutically acceptable excipients into a dosage form suitable for each route of administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In the above solid dosage forms, the active compound is mixed with at least one pharmaceutically acceptable inert excipient, such as sucrose, lactose or starch. Typically, such dosage forms will include additional materials in addition to the inert diluents, such as lubricants, for example magnesium stearate. For capsules, tablets and pills, the dosage forms may also include buffering agents. Tablets and pills may also be formulated with 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 the inert diluents mentioned above, the compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, as well as sweetening, flavoring and perfuming agents.

Formulations for parenteral administration of the invention include sterile aqueous or nonaqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or excipients are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil and corn oil), gelatin, and injectable organic esters (such as ethyl oleate). The dosage form may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Sterilization may be performed, for example, by filtration through a filter that retains bacteria, by adding a bactericide to the composition, by irradiating the composition or by heating the composition, and the like. They may also be formulated as solid sterile compositions which are dissolved in sterile water or some other sterile injectable medium immediately prior to use.

Compositions for rectal or vaginal administration are preferably suppositories which comprise, in addition to the active substance, excipients such as cocoa butter or a suppository wax.

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

The dosage of the active ingredient in the compositions of the invention is variable; however, the amount of active ingredient must be such that a suitable dosage form is obtained. The choice of dosage depends on the desired therapeutic effect, the route of administration and the duration of the treatment. Generally, a dose of 0.0001 to 100mg/kg body weight/day is administered to a human or other animal, such as a mammal, to achieve effective release of growth hormone.

For humans, the preferred dosage range is 0.01 to 5.0mg/kg body weight/day and can be administered in a single dose or divided into multiple doses.

For animals other than humans, the preferred dosage range is 0.01 to 10.0mg/kg body weight/day, and can be administered in a single dose or divided into multiple doses. In the case of animals other than humans, the more preferable dosage range is 0.1 to 5.0mg/kg body weight/day, and can be administered in a single dose or divided into multiple doses.

The compounds of general formula I used in the process of the invention can be prepared by sequential or focused synthetic routes. A detailed synthetic procedure for the preparation of compounds of general formula I in a sequential manner is shown in the reaction scheme below.

Many protected amino acid derivatives are commercially available, wherein the protecting groups Prt, Z¹⁰⁰And Z²⁰⁰Is prepared from BOC, CBZ, benzyl, ethoxycarbonyl and CF₃C (O) -, FMOC, TROC, trityl or tosyl, etc. Other protected amino acid derivatives can be prepared by literature methods. Some 3-oxo-2-carboxypyrrolidines and 4-oxo-3-carboxypiperidines are commercially available, while many other related pyrrolidines and 4-substituted piperidines are known in the literature.

The inclusion of protecting groups Prt, Z is described in many of the schemes described below¹⁰⁰Or Z²⁰⁰The compound of (1). Of these, the benzyloxycarbonyl group can be removed by a number of methods including catalytic hydrogenation with hydrogen in the presence of palladium or platinum in a protic solvent such as methanol. Preferred catalysts are palladium hydroxide on carbon or palladium on carbon; hydrogen pressures of 1 to 1000psi, preferably 10 to 70psi, can be used. Alternatively, the benzyloxycarbonyl group can also be removed by transfer hydrogenation.

The BOC protecting group can be removed using a strong acid such as trifluoroacetic acid or hydrochloric acid in the presence or absence of a co-solvent such as dichloromethane, ethyl acetate, diethyl ether or methanol at about-30 to 70 c, preferably-5 to 35 c.

The benzyl ester of the amine can be removed by a number of methods including catalytic hydrogenation with hydrogen in a protic solvent such as methanol in the presence of a palladium catalyst. Hydrogen pressures of 1 to 1000psi, preferably 10 to 70psi, can be used. Greene has been discussed in organic synthetic protecting groups (John Wiley & Sons, New York, 1981) for the addition and removal of the above protecting groups or other protecting groups.

Scheme 1

Scheme 1: the above protected amino acid derivative 1 is commercially available in many cases, wherein the protecting group Prt is a group exemplified by BOC, FMOC or CBZ; other amino acids can be prepared by literature methods.

As shown in scheme 1, the coupling reaction of the amine of formula 2 with the protected amino acid of formula 1, wherein Prt is a suitable protecting group, is readily carried out in an inert solvent such as dichloromethane or DMF using a coupling agent such as EDC or DCC in the presence of HOBT or HOAT. In the case where the above-mentioned amine is present as the hydrochloride salt, it is preferred to add 1 or 2 equivalents of a suitable base to the reaction mixture; alternatively, the above coupling reaction is carried out in the presence of a coupling agent, such as BOP, in an inert solvent, such as methanol. The above-mentioned coupling reaction is carried out at a temperature of usually about-30 ℃ to about 80 ℃, preferably-10 ℃ to about 25 ℃. Other conditions for coupling peptides are discussed in Houben-Weyl, Vol.XV, part II, E.Wunsch, Ed., George Theime Verlag, 1974, Stuttgart. Isolation of unwanted side products and purification of intermediates are accomplished by silica gel chromatography using flash chromatography (w.c. still, m.kahn and a.mitra, journal of organic chemistry (j.org.chem.) 4329231978), by crystallization or by trituration.

The compound of formula 3 may be converted to an intermediate of formula 4 by removal of the protecting group Prt as described above. As described above, coupling of the intermediate of formula 4 to the amino acid of formula 5 can yield an intermediate of formula 6. Deprotection of the amine of formula 6 affords compounds of formula 7.

Scheme 2

And (2) a flow scheme: alternatively, the compounds of formula 7 may be prepared by the progressive route shown in scheme 2. The ester intermediates of formula 8 can be prepared by treating amino acid 1 (where Prt is a suitable protecting group) with a base (e.g., potassium carbonate) in a suitable solvent (e.g., DMF), followed by an alkyl halide (e.g., methyl iodide); deprotection of the amine can convert 8 to 9; alternatively, many of the amino acids of formula 9 are purchased directly as commercial products. Intermediate 10 is generated by coupling 9 to amino acid 5. The ester intermediate 10 may be converted to the acid intermediate 11 according to a number of methods known in the art, for example, the methyl and ethyl esters may be hydrolyzed with lithium hydroxide in a protic solvent (e.g., aqueous methanol or aqueous THF) at about-20 ℃ to 120 ℃, preferably about 0 ℃ to 50 ℃. In addition, benzyl groups can be removed by a number of reduction methods, including hydrogenation in a protic solvent (e.g., methanol) in the presence of a platinum or palladium catalyst. Acid 11 may then be coupled to amine 2 to produce an intermediate of formula 6. The conversion of 6 to 7 can be accomplished by removing the protecting group Z200.

Scheme 3

And (3) a flow path: the ester of formula 6 may be converted to the acid intermediate 13 according to a number of methods known in the art, for example, the methyl and ethyl esters may be hydrolyzed with lithium hydroxide in a protic solvent (e.g., aqueous methanol or aqueous THF) at about-20 deg.C to 120 deg.C, preferably about 0 deg.C to 50 deg.C. In addition, benzyl groups can be removed by a number of reduction methods, including hydrogenation in a protic solvent (e.g., methanol) in the presence of a platinum or palladium catalyst. Coupling of acid 13 to amine 16 produces an intermediate of formula 14. The conversion of 14 to 15 can be accomplished by removing the protecting group Z200.

Scheme 4

And (4) a flow chart: the esters of formula 17 can be prepared by treating the acids of formula 5 with hydroxysuccinimide in an inert solvent (e.g., dichloromethane) in the presence of a coupling agent (e.g., EDC), as described in scheme 4. Treatment of ester 17 with an amino acid of formula 1 in the presence of a base (e.g. diisopropylethylamine) in a solvent (e.g. dioxane, THF or DMF) may give 11.

Scheme 5

And (5) a flow chart: alkylation of the diphenyloxazinones of formula 18 with cinnamoyl bromide in the presence of sodium bis (trimethylsilyl) amide to 19 as described in scheme 5; then, by removing the protecting group (Prt) and in PdCl₂Hydrogenation over a catalyst converts 19 to the desired (D) -2-amino-5-phenylpentanoic acid 20.

Scheme 6

And (6) a flow path: as shown in scheme 6, treatment of the ester of formula 21 with a base such as sodium hydride in a solvent such as DMF followed by an alkyl halide 22 affords the compound of formula 23. Treating a compound of formula 23 with a hydrazine of formula 24 (e.g., hydrazine or methylhydrazine) in a solvent (e.g., refluxing ethanol), followed by concentration and heating the residue in toluene at or near reflux temperature to provide a compound of formula 25; alternatively, treatment of 23 with a hydrazine salt in the presence of sodium acetate in refluxing ethanol can also provide 25. Deprotection of the amine yields compounds of formula 28. Treatment of 25 with Lawesson's reagent in refluxing toluene or benzene forms thioamides of formula 26. Removal of the protecting group may complete the conversion from 26 to 27.

Scheme 7

Scheme 7: treating the compound of formula 21 with hydrazine of formula 24 in a solvent such as refluxing ethanol, followed by concentration and heating the residue in toluene at or near reflux temperature to provide the compound of formula 29; alternatively, treatment of 21 with a hydrazine salt in the presence of sodium acetate in refluxing ethanol can also give 29. Treatment of amides of formula 29 with a base (e.g., sodium hydride) in a solvent (e.g., DMF) followed by an alkyl halide affords 25. Deprotection of the amine yields compounds of formula 28.

Scheme 8

And (3) a process 8: the ketone ester of formula 30 is reacted with a chiral amine (e.g., α -methylbenzylamine) and a suitable aldehyde (e.g., formaldehyde), or the vinyl ketone ester of formula 31 is reacted with a chiral amine (e.g., α -methylbenzylamine) and a suitable aldehyde (e.g., formaldehyde) via a bis-Mannich reaction to produce the compound of formula 32. 32 with hydrazine to produce chiral compounds of formula 33. Deprotection of the nitrogen using hydrogen and a suitable catalyst such as palladium affords compounds of formula 34.

Scheme 9

And (3) a process 9: treatment of the compound of formula 81 with a reducing agent such as sodium borohydride and protection of the nitrogen affords the compound of formula 82. Protection of the alcohol gave 83. Saponification of the ester gives compounds of general formula 84. Reaction of 84 with thionyl chloride followed by treatment with diazomethane gives the acid identified by general formula 85. Esterification of 85 gives formula 86, and deprotection of the oxygen of 86 gives 87. Oxidation 87 affords ketones of the general formula 88. 88 with hydrazine, deprotection of the nitrogen affords compounds of formula 44.

Scheme 10

A process 10: treatment of a compound of formula 35 with a base such as sodium hydride in a solvent such as DMF followed by treatment with diethyl carbonate yields ethyl ester compound 36. Deprotection converts 36 to 37.

Scheme 11

Scheme 11: treating the malonate of formula 38 with a base (e.g., sodium hydride) in a solvent (e.g., DMF); the benzyl group is then hydrogenolysed in a suitable solvent such as methanol with hydrogen and a catalyst such as palladium to give the ester of formula 39. Deprotection of the amino group produces compounds of formula 40.

Scheme 12

And (3) a process 12: treatment of a ketone of formula 41 with a secondary amine such as piperidine in a suitable solvent such as benzene with removal of water affords an enamine of formula 42. In the presence of a suitable base (e.g. LDA or NaN (SiMe)₃)₂) By alkylation of an enamine with an alpha-haloester (e.g., ethyl bromoacetate) in a suitable solvent (e.g., benzene or THF) to provide a ketoester of formula 43. Then, reacting with hydrazine of formula 24 to obtain a compound of formula 44. Finally, deprotection of the nitrogen affords compounds of formula 45.

Scheme 13

Scheme 13: treatment of the ketoesters of general formula 37 with an iodonium salt (e.g., diphenyliodonium trifluoroacetate) in a suitable solvent (e.g., t-butanol) produces ketoesters of general formula 46. 46 with hydrazine to produce a compound of formula 47. The deprotection affords compounds of formula 48, for a detailed description see Synthesis, (9), 1984 p 709.

Scheme 14

The process 14: treatment of ketoesters of formula 37 with an olefin such as acrylonitrile produces ketoesters of formula 49. 49 with hydrazine to form a compound of formula 50. Deprotection of the nitrogen affords compounds of formula 51.

Scheme 15

Scheme 15: treatment of the ketoester of formula 37 with allyl bromide and a suitable base such as sodium hydride in a suitable solvent such as DMF affords the ketoester of formula 52. 52 with hydrazine to produce a compound of formula 53. Ozonolysis of 53 in a suitable solvent (e.g. dichloromethane); thereafter, treatment with a reducing agent such as dimethyl sulfide affords an aldehyde of the general formula 54. Oxidation of 54 affords carboxylic acids of formula 55. The 55 is subjected to a Curtius rearrangement followed by hydrolysis of the isocyanate intermediate to give the primary amine of formula 56. Treatment of compounds of formula 56 with isocyanates or carbamates affords ureas of formula 57. Removal of the protecting group on the nitrogen affords compounds of formula 58.

Scheme 16

And (3) a process 16: treatment of compounds of formula 54 with primary amines affords imines of formula 59. Reduction of the compound of formula 59 gives the compound of formula 60. Treating the compound of formula 60 with an acylating agent to provide a compound of formula 61. Removal of the protecting group on the nitrogen affords compounds of formula 62.

Scheme 17

Scheme 17: treatment of the compound of formula 54 with a reducing agent such as sodium borohydride affords a compound of formula 63. Reaction of 63 with an acylating agent such as an isocyanate or a carbamate affords compounds of formula 64. The protecting group on the nitrogen is removed to give the compound of formula 65.

Scheme 18

The process 18: treatment of a compound of formula 63 with a phosphine such as triphenylphosphine, an azo compound such as diethyl azocarboxylate and an oxindole affords a compound of formula 66. Removal of the protecting group on the nitrogen affords compounds of formula 67.

Scheme 19

Scheme 19: treatment of the ketoester of formula 37 with a chiral diol and an acid catalyst in a suitable solvent (e.g., benzene) with removal of water affords the chiral ketal of formula 68. Alkylation of 68 with an alkyl halide in the presence of a base such as LDA followed by acid-catalyzed hydrolysis of the ketal affords chiral ketoesters of formula 69. 69 with hydrazine to give chiral compounds of the formula 70. The protecting group on the nitrogen is removed to give the compound of formula 71.

Scheme 20

A process 20: treatment of the ketoesters of formula 37 with a chiral amino acid ester such as valine tert-butyl ester affords chiral enamines of formula 72. Alkylating 72 with an alkyl halide in the presence of a base such as LDA; the enamine is then subjected to acid-catalyzed hydrolysis to afford the chiral ketoester of formula 69. 69 with hydrazine to give chiral compounds of the formula 70. The protecting group on the nitrogen is removed to give the compound of formula 71.

Scheme 21

Scheme 21: the protecting group on the nitrogen of 25 is removed to give the compound of formula 28. Salt formation of 28 with a chiral acid gives a mixture of diastereomeric salts of formula 73. The diastereomeric salt is crystallized to give the acid salt of the chiral compound of formula 70. Salt 70 is decomposed with a base to release the chiral compound of formula 71.

Process 22

The process 22: alkylation of the compound of formula 25 with allyl acetate in the presence of a suitable catalyst such as tetrakis (triphenylphosphine) palladium affords the compound of formula 74. Deprotection of the nitrogen affords compounds of general formula 75, as discussed in more detail in Tetrahedron (50) p.515, 1994.

Scheme 23

Flow 23: alkylating the diester of formula 76 with an alkyl halide in the presence of a base such as sodium hydride; then, carrying out acid catalytic hydrolysis and decarboxylation; subsequently, esterification with methyl iodide and a suitable base affords compounds of general formula 77. The compound of formula 77 is reacted with an appropriate aldehyde, exemplified by formaldehyde, and benzylamine to provide the compound of formula 78. The compound of formula 78 is reacted with hydrazine to produce a chiral compound of formula 79. The protecting group on the nitrogen is removed to give the compound of formula 80.

Scheme 24

The process 24: treatment of the amine of formula 23 with the acid of formula 11 in an inert solvent such as dichloromethane or DMF in the presence of HOBT with a coupling agent such as EDC or DCC affords the compound of formula 89. The compound of formula 89 is reacted with hydrazine to produce a compound of formula 6. Removing the protecting group on the nitrogen to obtain the compound of the general formula 7.

Scheme 25

Flow 25: treatment of hydroxyacetoacetates of general formula 90 with an alkyl halide in the presence of a suitable base such as sodium hydride gives compounds of general formula 91. 91 with hydrazine to give a compound of formula 92. O-alkylating the carbonyl oxygen atom of 92 to give 93; and 93 is converted to halide 94. Substitution of the halogen X with a cyanide ion affords nitrile 95. Reduction 95 to give primary amine 96; deprotection and cyclization of 96 in the presence of formaldehyde gave 28.

Scheme 26

The process 26: treatment of a β -keto-protected aminovalerate such as 97 with an alkyl halide in the presence of a suitable base such as sodium hydride gives compounds of formula 98. The compound of formula 98 is reacted with hydrazine to produce a compound of formula 99. Deprotection of the compound of formula 99 affords a primary amine of formula 100. Cyclization of the compound of formula 100 is carried out in the presence of formaldehyde to give the compound of formula 28.

Scheme 27

The process 27: treatment of an amine of formula 23a with an acid such as 1 in the presence of EDC or HOAT in a suitable solvent produces a ketoester of formula 23 b. Treatment of ketoester 23b with a hydrazonium salt in refluxing ethanol in the presence of sodium acetate affords hydrazines of formula 23 c. Deprotection under suitable conditions affords the amine of formula 4. Coupling of the intermediate of formula 4 to the amino acid of formula 5 as described above gives the intermediate of formula 6. Deprotection of amine 6 affords compounds of formula 7.

Process 28

The process 28: prt represents an amino protecting group known to those of ordinary skill in the art. Although BOC is used as Prt in the present invention to illustrate the preferred protecting group, the scope of the present invention is not limited to the use of BOC alone. Furthermore, although the schemes illustrate synthetic methods for compounds of formula m using specific isomers, other isomers and/or isomer mixtures are also within the scope of the present invention.

Step A

First an inorganic or organic base (e.g. TEA, DMAP, hydroxide or carbonate, preferably TEA) and then an amino protecting group (preferably (Boc)₂O) to a solution of ethyl 4-oxo-piperidine-3-carboxylate hydrochloride in a reaction-inert organic solvent (e.g. IPE, THF, dichloromethane and EtOAc, preferably IPE and water) with or without water as a co-solvent. The mixture is stirred for about 1 to 24 hours, preferably overnight, and preferably under nitrogenThe process is carried out. The organic phase is separated off and worked up according to standard methods known to the person skilled in the art, and then concentrated to give the desired product in crystalline form.

Step B

To a solution of 1-tert-butyl 3-ethyl 4-oxo-piperidine-1, 3-dicarboxylate in an organic solvent (e.g. THF, IPE, alcohol, DNF or DMSO, preferably DMF) is added an inorganic or organic base (e.g. TEA, DMAP, hydroxide or carbonate, preferably lithium carbonate) followed by additional benzyl bromide. Heating the mixture to about 25 to 100 ℃, preferably 60 ℃; and stirred for about 1 to 24 hours, preferably 20 hours. The reaction mixture is then cooled to room temperature and extracted with an organic solvent (e.g. IPE, toluene, THF or EtOAc) and worked up according to standard methods well known to those skilled in the art to give the desired compound.

Step C

To a solution of 1-tert-butyl 3-ethyl 3-benzyl-4-oxo-piperidine-1, 3-dicarboxylate in an organic solvent such as an alcohol, THF or toluene at about 0 ℃ to room temperature is added methylhydrazine, followed by the addition of an acid such as sulfuric acid, hydrochloric acid, acetic acid or TsOH, preferably acetic acid. The reaction mixture is slowly heated to about 40 to 100 c, preferably about 65 c, and stirred for about 3 to 10 hours, preferably about 7.5 hours. After cooling to room temperature, the organic layer was washed with 10% sodium bicarbonate and worked up and concentrated according to standard methods well known to those skilled in the art to give the desired compound.

Step D

Mixing the concentrated solution from step C with an organic solvent such as IPE, cooling to about-10 to 10 deg.C (preferably 0 deg.C), and repeating the introduction of acid (e.g. MeSO)₃H. TFA or HCl, preferably HCl gas) and stirred at room temperature until hydrolysis is complete. Concentrating the mixture, adding an organic solvent (such as dichloromethane, IPE or THF), and adding a base (such as hydroxide, carbonate, preferably NH)₄OH). The mixture is then extracted with dichloromethane, IPE or THF and concentrated to give the desired compound。

Step E

L-tartaric acid is added to a solution of 3 a-benzyl-2-methyl-2, 3a, 4, 5, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-3-one in a mixture of acetone/water (1% to 11% water, preferably 5% water/acetone). The mixture is heated to 25 to 60 ℃, preferably about 50 ℃, and stirred, preferably overnight. The reaction mixture is cooled to preferably about 10 to 15 ℃, and the precipitate is filtered off, washed with cold acetone/water and dried to give the desired compound.

Step G

2-aminoisobutyric acid, a base (e.g. a hydroxide, preferably 1N NaOH), (Boc)₂O and an organic solvent (such as THF, IPE or dioxane) were mixed together and stirred at room temperature overnight. The reaction mixture is diluted with an organic solvent, such as ethyl acetate, and the pH is adjusted to about 3 to 7 by the addition of an aqueous solution of an acid, such as HCl. The organic phase is separated off and worked up according to standard methods known to the person skilled in the art to give the desired compound.

Step H

To a solution of 2-amino-3-benzyloxy-propionic acid in water and an inorganic or organic base, preferably TEA, is added a solution of 2-tert-butoxycarbonylamino-2-methyl-propionic acid 2, 5-dioxopyrrolidin-1-yl ester in an organic solvent, such as THF. The reaction mixture is stirred, preferably overnight, and preferably at room temperature under nitrogen. An aqueous acid solution, such as a 10% citric acid solution, is added to the mixture. The mixture is stirred for several minutes and then diluted with an organic solvent such as ethyl acetate. The organic phase is separated off and worked up according to standard methods known to the person skilled in the art, and then concentrated to give the desired compound in crystalline form.

Steps F and I

A base such as TEA is added to a solution of 3a- (R) -benzyl-2-methyl-2, 3a, 4, 5, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-3-one, L-tartrate in an organic solvent, such as ethyl acetate, at a temperature of about-78 to 20c, preferably about-66 c. The compound is stirred for 1 to 24 hours, preferably about 1.5 hours. After removal of the precipitated salt, 3-benzyloxy-2- (2-tert-butoxycarbonylamino-2-methylpropanamino) propionic acid and a base such as TEA are added at about-50 to 0 ℃, preferably about-35 ℃; then, a peptide coupling agent, preferably a 50% solution of 1-propanephosphonic acid cyclic anhydride (PPAA) in ethyl acetate is added. Stirring the reaction mixture at about-50 to 0 ℃, preferably about-20 ℃ to about-27 ℃, for about 1 to 6 hours, preferably about 2 hours; the temperature is then slowly raised to preferably about 0 ℃. The reaction mixture is poured into water and extracted with an organic solvent, for example IPE, and the organic phase is separated and worked up according to standard methods well known to those skilled in the art to give the desired compound.

Step J

TFA is added to a solution of {1- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethylcarbamoyl ] -1-methyl-ethyl } carbamic acid tert-butyl ester in an organic solvent, such as dichloromethane, at a temperature of about-10 to 10 ℃, preferably 0 to 5 ℃, and the temperature is preferably maintained below about 5 ℃. Then, the temperature was raised to room temperature. The mixture is stirred for about 1 to 6 hours, preferably about 3 hours. The dichloromethane is replaced by other organic solvents, such as ethyl acetate. The pH of the mixture is then adjusted to about 7 to 9, preferably 8, with an aqueous base, such as saturated sodium bicarbonate solution, followed by work-up according to standard methods well known to those skilled in the art to obtain the desired compound.

Step K

L- (+) -tartaric acid was added to a solution of 2-amino-N- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl ] -2-methyl-propionamide from step I in an alcohol (e.g. methanol) and the mixture was stirred overnight. The resulting solution was filtered and concentrated. An organic solvent, such as IPE or ethyl acetate, is added and the remaining alcohol is removed azeotropically. The separated solid was dissolved in ethyl acetate, and the solution was refluxed and then cooled to room temperature to obtain the desired product crystals.

The following examples are given for illustration only and are not intended to limit the scope of the invention.

Routine test procedure:

column chromatography was performed using 30 μ M Amicon silica gel with a 60-pore size. Melting points were measured on a Buchi 510 apparatus and uncorrected. Proton and carbon 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 low magnetic field from trisilane. Particle beam mass spectra were measured on a Hewlett-Packard 5989A spectrometer using ammonia as the chemical ionization source. The initial sample was dissolved with chloroform or methanol. Liquid phase secondary ion mass spectrometry (LSIMS) was measured on a Kratos concentrate-1S high resolution spectrometer using cesium ion bombardment with samples dissolved in a 1: 5 mixture of dithioerythritol and dithiothreitol or in a thioglycerol matrix. The initial sample was dissolved with chloroform or methanol. The data reported is the sum of 3 to 20 scans calibrated to cesium iodide. TLC analysis was done by visual inspection (after elution with the indicated solvent or solvents) on e.merck kieselgel 60F 254 silica gel plates stained with 15% ethanolic phosphomolybdic acid and heated on a hot plate.

Conventional procedure a (peptide coupling with EDC): a solution of 0.2-0.5M primary amine (1.0 equivalent) in methylene chloride (or primary amine hydrochloride and 1.0-1.3 equivalents triethylamine) is treated sequentially with 1.0-1.2 equivalents of the carboxylic acid coupled partner, 1.5-1.8 equivalents hydroxybenzotriazole Hydrate (HOBT) or HOAT, and 1.0-1.2 equivalents (stoichiometrically equivalent to the amount of carboxylic acid) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), and the mixture is stirred overnight in an ice bath (which can be heated, so that, typically, the reaction mixture is held at about 0 to 20 ℃ for about 4 to 6 hours, and at about 20 to 25 ℃ for the remainder). Dilute with ethyl acetate or other indicated solvent and wash the resulting mixture 2 times with 1N sodium hydroxide, 2 times with 1N hydrochloric acid (if the product is not basic), 1 time with brine, dry over sodium sulfate, and concentrate to give the crude product, which is purified in the indicated manner. The carboxylic acid component may be used as the dicyclohexylamine salt in the coupling with the primary amine or its hydrochloride, in which case triethylamine need not be used.

Example 1

2-amino-N- {1(R) -benzyloxymethyl-2- [3a- (R) - (4-fluoro-benzyl) -2-methyl-3-oxo Substituted-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3-c)]Pyridin-5-yl]-2-oxo-ethyl } iso Butylamide hydrochlorideAnd2-amino-N- {1(R) -benzyloxymethyl-2- [3a- (S) - (4-fluoro-benzyl) -2-methyl-3-oxo Substituted-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3-c)]Pyridin-5-yl]-2-oxo-ethyl } iso Butylamide hydrochloride

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

A mixture of 8.00g (38.5mmol) of ethyl 4-oxo-piperidine-3-carboxylate hydrochloride, 9.23g (42.4mmol) of di-tert-butyl dicarbonate and 3.89g (38.5mmol) of triethylamine in 150ml of THF is stirred at room temperature for approximately 72 hours. After concentrating the mixture, the residue was dissolved in ethyl acetate and washed three times with 10% aqueous hydrochloric acid, saturated sodium bicarbonate solution and brine, respectively, dried over magnesium sulfate and concentrated to give 10.0g of 1A white solid MS (Cl, NH)₃)272(MH⁺)。

B. 3- (R, S) - (4-fluoro-benzyl) -4-oxo-piperidine-1, 3-dicarboxylic acid 1-tert-butyl 3-ethyl ester

282mg (7.4mmol) of sodium hydride (60% oil dispersion) are added to a solution of 2.00g (7.4mmol) of 1A in 10mL of DMF, and the mixture is stirred at room temperature for about 15 minutes. To the stirred solution was added a solution of 1.39g (7.4mmol) of 4-fluorobenzyl bromide in 7mL of DMF and the mixture was cooled at room temperatureStirring was continued for about 72 hours. The mixture was diluted with ethyl acetate and washed once with water, four times with brine, dried over magnesium sulfate and concentrated to give 2.8g of 1BMS (Cl, NH)₃)380(MH⁺)。

C. 3a- (R, S) - (4-fluoro-benzyl) -2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazole And [4, 3-c ]]Pyridine-5-carboxylic acid tert-butyl ester

A solution of a mixture consisting of 2.54g (6.7mmol) of 1B and 309mg (6.7mmol) of methylhydrazine in 100mL of ethanol was heated at reflux for approximately 8 hours. The mixture was concentrated, and the residue was dissolved in 100mL of toluene and heated under reflux for about 17 hours. After concentration of the mixture, the residue was purified by chromatography on silica gel with an elution gradient (18: 82 v/v EtOAc: hexane) to (75: 25v/v EtOAc: hexane) to give 1.0g of 1C MS (Cl, NH, Cl) as a clear colorless oil₃)362(MH⁺)。

D. 3a- (RS) - (4-fluoro-benzyl) -2-methyl-2, 3a, 4, 5, 6, 7-hexahydro-pyrazolo [4, 3- c]Pyridin-3-one trifluoroacetate salt

At about 0 deg.C, 10mL of trifluoroacetic acid was added to 1.00g (2.8mmol) of 1C, and the mixture was stirred for about 1 hour. After addition of ethyl acetate, the mixture was concentrated to give 1.0g of 1DMS (Cl, NH)₃)263(MH⁺)。

E. (R) -3-benzyloxy-2- (2-tert-butoxycarbonylamino-2-methyl-propionylamino) propionic acid

1.02g (7.4mmol) of potassium carbonate are initially charged and 0.92g (6.5mmol) of methyl iodide are added to a solution of 1.83g (6.2mmol) of N-t-BOC-O-benzyl-D-serine in 35mL of DMF. The mixture was stirred at about 24 ℃ overnight under nitrogen atmosphere. The reaction mixture was diluted with 200mL of water and extracted three times with ethyl acetate. The combined organic phases were washed 5 times with water and once with brine, dried over magnesium sulfate and concentrated. The crude (R) -3-benzyloxy-2-tert-butoxycarbonylamino-propionic acid methyl ester was dissolved in 15mL of cold trifluoroacetic acid at about 0 deg.C and the mixture was stirred for about 2 hours. After concentrating the mixture, the residue was diluted with 1N sodium hydroxide and extracted three times with ethyl acetate. The combined organic extracts were washed with brine and dried over sodium sulfate to give 0.84g (4.02mmol) of (R) -2-amino-3-benzyloxy-propionic acid methyl ester; this was coupled to 0.81g (4.02mmol) N-t-BOC-. alpha. -methylalanine to give 1.80g of (R) -methyl 3-benzyloxy-2- (2-tert-butoxycarbonylamino-2-methylpropanamino) propionate. The above crude product was dissolved in 20mL of 4: 1 THF/water, and a solution of 335mg (7.98mmol) of lithium hydroxide hydrate in 1mL of water was added to the solution, and then the mixture was stirred at room temperature overnight. After concentration of the mixture, the residue was diluted with ethyl acetate, acidified with aqueous hydrochloric acid, and extracted three times with ethyl acetate. The organic extracts were combined and washed once with brine, dried over sodium sulfate and concentrated to give 1.60g of a 1E oil which solidified on standing.

¹H NMR(CDCl₃ 300 MHz)δ7.30(m，5H)，7.10(d，

1H)，5.07(bs，1H)，4.68(m，1H)，4.53(q，2H)4.09(m，1H)，3.68(m，1H)，1.3-1.5

(m，15H).

F. (1- {1(R) -benzyloxymethyl-2- [3a- (R, S) - (4-fluoro-benzyl) -2-methyl-3-oxo -2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c]Pyridin-5-yl]-2-oxoethylcarbamino Acyl } -1-methylethyl) carbamic acid tert-butyl ester

Following the general procedure outlined for general procedure A, 193mg (0.51mmol) of 1D and 196mg (0.51mmol) of 1E were coupled to give a mixture of diastereomers. The residue was purified by silica gel chromatography with an elution gradient (1: 1v/v ethyl acetate: hexane) to 100% ethyl acetate to give 60mg of the less polar 1F isomer 1 and 100mg of the more polar 1F isomer 2, MS (Cl, NH)₃)624(MH⁺) (the above-mentioned two isomers).

G. 2-amino group-N- {1(R) -benzyloxymethyl-2- [3a- (R) - (4-fluoro-benzyl) -2-methyl-3- Oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c)]Pyridin-5-yl]-2-oxoethyl } Isobutyramide hydrochloride

4mL of concentrated HCl was added to a solution of 60mg (0.10mmol) of 1F isomer 1 in 10mL of ethanol and the mixture was stirred at room temperature for about 2 hours. After concentrating the mixture, the residue was precipitated from ethanol/hexane to give 50mg of 1G isomer 1 as a white powder.

MS(Cl，NH₃)

524(MH⁺).¹HNMR(CD₃OD): (part) δ 7.32(m, 5H), 7.12(m, 2H), 6.91(m, 2H)

H)，5.15(m，1H)，4.54(s，2H)，3.78(m，2H)3.02(m，7H)，2.66(m，2H)，1.57(s，

6H).

H. 2-amino-N- {1(R) -benzyloxymethyl-2- [3a- (S) - (4-fluoro-benzyl) -2-methyl-3- Oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c)]Pyridin-5-yl]-2-oxoethyl } Isobutyramide hydrochloride

4mL of concentrated HCl was added to a solution of 100mg (0.16mmol) of 1F isomer 2 in 10mL of ethanol and the mixture was stirred at room temperature for about 2 hours. After concentrating the mixture, the residue was precipitated from ethanol/hexane to give 60mg of 1H isomer 2 as a white powder.

MS(Cl，NH₃)

524(MH⁺).¹HNMR(CD₃OD): (part) δ 7.32(m, 5H), 7.08(m, 2H), 6.95(m, 2H)

H)，6.80(m，2H)，5.30(m，1H)，4.61(m，3H)，3.80(m，2H)，2.58(m，3H)，1.58

(s，6H).

Example 2

2-amino-N- [2- [3a- (R, S) - (4-fluoro-benzyl) -2-methyl-3-oxo- 2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c)]Pyridin-5-yl]-1(R) - (1H-indole-3-) Ylmethyl) -2-oxo-ethyl } isobutyramide hydrochloride

A. (R) -2-amino-3- (1H-indol-3-yl) propionic acid methyl ester

2.46g (17.8mmol) of potassium carbonate are added, and 2.41g (17.0mmol) of methyl iodide are added to a solution of 4.92g (16.2mmol) of N-. alpha. -t-BOC-D-tryptophan in 100mL of DMF; and, the mixture was stirred at about 24 ℃ overnight under nitrogen atmosphere. The reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were washed 5 times with 500mL of water and once with brine, dried over magnesium sulfate and concentrated to give 4.67g of a white solid. 15mL of cold trifluoroacetic acid was added to the crude methyl (R) -2-tert-butoxycarbonylamino-3- (1H-indol-3-yl) propionate at about 0 deg.C and the mixture was stirred for about 2 hours. After concentrating the mixture, the residue was diluted with 1N sodium hydroxide and extracted three times with ethyl acetate. The combined organic extracts were washed with brine and dried over sodium sulfate to give (R) -2-amino-3- (1H-indol-3-yl) propionic acid methyl ester as an orange oil in quantitative yield.

B. (R) -2- (2-tert-Butoxycarbonylamino-2-methylpropionylamino) -3- (1H-indol-3-yl) Propionic acid methyl ester

1.55g (7.1mmol) of the crude product from 2A was coupled to 1.44g (7.1mmol) of N-t-BOC-. alpha. -methylalanine according to the procedure described in step A to give an oil; after the above oil was purified by silica gel chromatography and eluted with a gradient of 10%, 20%, 30%, 40% and 50% ethyl acetate/hexane, 1.32g of (R) -methyl 2- (2-tert-butoxycarbonylamino-2-methylpropanamino) -3- (1H-indol-3-yl) propionate was recovered.

C. (R) -2- (2-tert-Butoxycarbonylamino-2-methylpropionylamino) -3- (1H-indol-3-yl) Propionic acid

A solution of 381mg (9.1mmol) of lithium hydroxide in 2mL of water is added to a solution of 1.03g (2.64mmol) of 2B in 10mL of THF, and the mixture is stirred at room temperature overnight. Excess THF was removed by evaporation and the basic aqueous mixture was removed 3 times with ethyl acetate and then acidified to pH 4 with dilute acetic acid or hydrochloric acid. The product was extracted with ethyl acetate and the combined organic phases were washed with brine, dried over magnesium sulfate and evaporated to give 1.03g of a 2C orange foam.

MS

(Cl，NH₃)390(MH⁺).¹H NMR(CDCl₃ 300MHz)δ7.61(d，1H)，7.48(d，1H)，

7.27(t，1H)，7.10(t，1H)，4.81(bs，1H)，3.35(m，1H)，1.49(s，6H)，1.32(s，9H).

D. {1- [2- [3a- (R, S) - (4-fluoro-benzyl) -2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexa-position Hydro-pyrazolo [4, 3-c]Pyridin-5-yl]-1- (R) - (1H-indol-3-ylmethyl) -2-oxo- Ethylcarbamoyl radical]-1-methylethyl } carbamic acid tert-butyl ester

193mg (0.51mmol) of 1D and 200mg (0.51mmol) of 2C are coupled as outlined in general step A; and, the residue was purified by silica gel chromatography with an elution gradient (1: 1v/v ethyl acetate: hexane) to 100% ethyl acetate to give 230mg of 2DMS (Cl, NH)₃)633(MH⁺)。

E. 2-amino-N- [ -2- [3a- (R, S) - (4-fluoro-benzyl) -2-methyl-3-oxo- 2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c)]Pyridin-5-yl]-1(R) - (1H-indole-3-) Ylmethyl) -2-oxoethyl]Isobutyramide hydrochloride

4mL of concentrated HCl was added to a solution of 230mg (0.36mmol) of 2D in 10mL of ethanol, and the mixture was stirred at room temperature for about 2 hours. After concentrating the mixture, the residue was precipitated from ethanol/hexane to give 130mg of 2E white powder.

MS(Cl，NH₃)533

(MH⁺).¹HNMR(CD₃OD): (part) δ 7.79(d, 1H), 7.48(m, 1H), 7.33(m, 2H), 7.19-6.77(m, 7H), 6.54(m, 1H), 5.17(m, 1H), 4.02(m, 1H), 3.11-2.68(m, 6H), 2.47(m, 2H), 2.03(m, 2H), 1.59(m, 6H).

Example 3

2-amino-N- [2- (3a- (R, S) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro- Pyrazolo [4, 3-c]Pyridin-5-yl]-1(R) - (1H-indol-3-ylmethyl) -2-oxo-ethyl } Isobutyramide

A. 4-oxo-piperidine-1, 3-dicarboxylic acid 1-tert-butyl 3-methyl ester

To a mixture of 7.00g (36.2mmol) of methyl 4-oxo-piperidine-3-carboxylate and 8.82g (72.3mmol) of 4, 4-dimethylaminopyridine in 200mL of dichloromethane was added a solution of 7.88g (36.2mmol) of di-tert-butyl dicarbonate in 150mL of dichloromethane at about 0 ℃ over a period of about 30 minutes. The mixture was warmed to room temperature and then stirred for about 17 hours. After concentrating the mixture, the residue was diluted with chloroform and washed three times (each time with 10% aqueous hydrochloric acid, saturated aqueous sodium bicarbonate and brine), dried over magnesium sulfate and concentrated to give 9.18g of a clear yellow oil.

B. 3- (R, S) -benzyl-4-oxo-piperidine-1, 3-dicarboxylic acid 1-tert-butyl ester 3-methyl ester

745mg (7.4mmol) of sodium hydride (60% oil dispersion) was added to a solution of 5.00g (19.4mmol) of 3A in 10mL of DMF, and then the mixture was stirred at room temperature for about 15 minutes. To the stirring solution above was added a solution of 3.32g (19.4mmol) of benzyl bromide in 15mL of DMF through a cannula and the mixture was stirred at room temperature for about 42 hours. The mixture was diluted with ethyl acetate, washed once with water and four times with brine, dried over magnesium sulfate and concentrated to give 6.0g of 3B as a yellow oilA compound (I) is provided. MS (Cl, NH)₃)348(MH⁺)。C. 3a- (R, S) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3- c]Pyridine-5-carboxylic acid tert-butyl ester

A mixture of 4.00g (11.5mmol) of 3B and 530mg (11.5mmol) of methylhydrazine in 100mL of ethanol is heated under reflux for approximately 8 hours. The mixture was concentrated, and the residue was dissolved in 100mL of toluene and heated under reflux for about 17 hours. After concentration of the mixture, the residue was purified by chromatography on silica gel with an elution gradient (15: 85 v/v EtOAc: hexane) to (75: 25v/v EtOAc: hexane) to give 2.6g3C of MS (Cl, NH) as a clear colorless oil₃)344(MH⁺)。

D. 3a- (R, S) -benzyl-2-methyl-2, 3a, 4, 5, 6, 7-hexahydro-pyrazolo [4, 3-c)]Pyridine- 3-ketones

At about 0 deg.C, 20mL of trifluoroacetic acid was added to 2.60g (7.6mmol) of 3C, and the mixture was stirred for about 2.5 hours. After addition of ethyl acetate, the solution was washed with 6N sodium hydroxide, dried over magnesium sulfate and concentrated to give 1.8g of 3D. MS (Cl, NH)₃)244(MH⁺)。

E. {1- [2- (3a- (R, S) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazole And [4, 3-c ]]Pyridin-5-yl) -1R- (1H-indol-3-ylmethyl) -2-oxo-ethylcarbam-ine Acyl radical]-1-methylethyl } carbamic acid tert-butyl ester

125mg (4.6mmol) of 3C and 1.75g (0.51mmol) of 2C are coupled as outlined in general step A; and, the residue was purified by silica gel chromatography with an elution gradient (6: 4 v/v ethyl acetate: hexane) to 7% methanol/ethyl acetate to give 150mg of 3E.

F. 2-amino-N- [2- (3a- (R, S) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexa-amino Hydro-pyrazolo [4, 3-c]Pyridin-5-yl) -1R- (1H-indol-3-ylmethyl) -2-oxoethyl] Isobutyramide hydrochloric acidSalt (salt)

5mL of concentrated HCl was added to a solution of 150mg (0.24mmol) of 3E in 15mL of ethanol, and the mixture was stirred at room temperature for about 3 hours. After concentrating the mixture, the residue was crystallized from ethanol/hexane to give 100mg of 3F white powder.

MS(Cl，NH₃)515(MH⁺)¹HNMR

(CD₃OD)：δ7.20-6.91(m，9H)，6.56(m，1)，5.17(m，1H)，4.05(m，1H)，2.96(s，

3H)，2.62(m，1H)，2.38(m，1H)，2.06(m，2H)，1.61(m，8H).

Example 4

2-amino-N- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydropyrazine Azolo [4, 3-c ] s]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl]Isobutyramide Hydrochloride saltAnd2-amino-N- [2- (3a- (S) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydropyrazine Azolo [4, 3-c ] s]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl]Isobutyramide Hydrochloride salt

A. {1- [2- (3a- (R, S) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazole And [4, 3-c ]]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethylcarbamoyl Base of]-1-methylethyl } carbamic acid tert-butyl ester

Following the general procedure outlined in general procedure A, 1.12g (4.6mmol) of 3C and 1.75g (0.51mmol) of 1E were coupled to give a mixture of diastereomers; and the residue was purified by silica gel chromatography with an elution gradient (1: 1v/v ethyl acetate: hexanes) to 100% ethyl acetate to give 350mg of the less polar 4A isomer 1 and 250mg of the more polar 4A isomer 2, MS (Cl, NH)₃)606(MH⁺) (the above-mentioned two isomers).

B. 2-amino group-N- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro- Pyrazolo [4, 3-c]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxoethyl]Isobutyramide Hydrochloride salt

5mL of concentrated HCl was added to a solution of 250mg (0.41mmol) of 4A isomer 1 in 15mL of ethanol and the mixture was stirred at room temperature for about 5 hours. After concentration of the mixture, the residue was precipitated from ethanol/hexane and dried in vacuo to give 130mg of 4B isomer 1.

MS(Cl，

NH₃)506(MH⁺).¹HNMR(CD₃OD)：δ7.33(m，5H)，7.14(m，5H)，5.22(m，1H)，

4.57(m，3H)，3.80(m，2H)3.14(m，1H)，3.04(s，3H)，2.96(m，2H)，2.61(m，2

H)，1.63(m，7H).

C. 2-amino-N- [2- (3a- (S) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro- Pyrazolo [4, 3-c]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxoethyl]Isobutyramide Hydrochloride salt

5mL of concentrated HCl was added to a solution of 250mg (0.41mmol) of 4A isomer 2 in 15mL of ethanol and the mixture was stirred at room temperature for about 5 hours. After concentration of the mixture, the residue was precipitated from ethanol/hexane and dried in vacuo to give 120mg of 4C isomer 2.

MS(Cl，

NH₃)506(MH⁺).¹HNMR(CD₃OD)：δ7.31(m，5H)，7.13(m，5H)，6.78(m，1H)，

5.28(m，1H)，4.62(m，3H)，3.81(M，2H)，3.14(m，1H)，2.62(m，3H)，1.58(m，7

H).

D. 2-amino-N- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6,7-hexahydro Pyrazolo [4, 3-c]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl]Isobutyryl radical Amine methanesulfonate

Saturated aqueous sodium bicarbonate was added to 3.60g (6.6mmol) of 4B isomer 1 and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and concentrated. The residue was again dissolved in ethyl acetate, cooled to about 0 ℃ and then 0.43mL (6.6mmol) of methanesulfonic acid was added and the mixture was stirred for about 0.5 h. Hexane (200mL) was added to the solution and the mixture was stirred for about 1 hour and filtered to give 3.40g of a white solid. The solid was recrystallized from 3% ethyl acetate aqueous solution to give 2.55g of 4D isomer 1 as a white crystalline solid MS (Cl, NH)₃)506(MH⁺)。

Example 5

2-amino-N- [1- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydropyrazine Azolo [4, 3-c ] s]Pyridine-5-carbonyl) -4-phenyl- (R) -butyl]Isobutyramide hydrochlorideAnd2-amino-N- [1- (3a- (S) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydropyrazine Azolo [4, 3-c ] s]Pyridine-5-carbonyl) -4-phenyl- (R) -butyl]Isobutyramide hydrochloride

A. 2-oxo-5, 6-diphenyl-3- (3-phenyl-allyl) morpholine-4-carboxylic acid tert-butyl ester

To a solution of 13.8g (70.0mmol) of cinnamoyl bromide and 4.94g (14.0mmol) of tert-butyl (2S, 3R) - (+) -6-oxo-2, 3-diphenyl-4-morpholinecarboxylate in 350mL of anhydrous THF at about-78 deg.C was added 28mL (28mmol) of a 1M solution of sodium bistrimethylsilylamide in THF. The mixture was stirred at about-78 ℃ for about 1.5 hours and then poured into 750mL of ethyl acetate. The mixture was washed twice with brine, dried over magnesium sulfate and concentrated to give a yellow oil. The oil was stirred overnight in 150mL of hexane, and the precipitated solid was collected by filtration to give 3.2g of 5A as a white solid.

B. 5(S), 6(R) -diphenyl-3 (R) - (3-phenylallyl) morpholin-2-one

At about 0 deg.C, 20mL of trifluoroacetic acid was added to 2.97g (6.33mmol) of 5A, and the mixture was stirred for about 2 hours, followed by concentration. The residue was dissolved in water and basified with aqueous sodium hydroxide until the pH remained at 10. After extracting the mixture three times with ethyl acetate, the combined organic extracts were washed with brine, dried over magnesium sulfate and concentrated to give an orange oil; it was then purified by silica gel chromatography (10: 90 v/v ethyl acetate: ethane) to give 880mg of 5B as a white solid.

C. 2- (R) -amino-5-phenylpentanoic acid

A mixture consisting of 440mg (1.19mmol) of 5B and 120mg of palladium chloride in 20mL of ethanol and 10mL of THF was hydrogenated at 45psi for about 16 hours. The mixture was filtered through celite and concentrated, and the residue was triturated with ether to give 240mg of 5C white solid.

D. 2, 5-Dioxopyrrolidin-1-yl 2-Boc-amino-2-methylpropionate

To a slurry of 5.0g (24.6mmol) N-t-BOC-. alpha. -methylalanine in 13.5mL dichloromethane were added 3.40g (29.6mmol) N-hydroxysuccinimide and 5.65g (29.6mmol) EDC. The slurry was stirred at room temperature for about 17 hours. The mixture was diluted with ethyl acetate and washed twice with water, saturated sodium bicarbonate solution and brine each time; dried over magnesium sulfate and concentrated. The product was purified by silica gel chromatography (1: 1v/v ethyl acetate: hexane) to give 5.2g D parts of the title compound as a white solid.

E. (R) -2- (2-tert-Butoxycarbonylamino-2-methylpropionylamino) -5-phenylpentanoic acid

A mixture of 203mg (1.05mmol) of 5D, 378mg (1.26mmol) of 5C and 434mg (3.36mmol) of diisopropylethylamine in 2mL of DMF was stirred overnight. The mixture was diluted with ethyl acetate and extracted 2 times with 1N hydrochloric acid. The aqueous phase was extracted once more with ethyl acetate. The combined organic extracts were washed three times with water and once with brine. The resulting mixture was dried over magnesium sulfate and concentrated. The residue was purified by silica gel chromatography eluting with 80% chloroform/hexane followed by 100% chloroform and then 10% methanol/chloroform to give 127mg of 5E.

F. {1- [1- (3a- (R, S) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazole And [4, 3-c ]]Pyridine-5-carbonyl) -4-phenyl- (R) -butylcarbamoyl]-1-methylethyl } Carbamic acid tert-butyl ester

Following the general procedure outlined in general procedure A, 130mg (0.53mmol) of 3C and 200mg (0.53mmol) of 5E were coupled to give a mixture of diastereomers; and the residue was purified by silica gel chromatography with an elution gradient (1: 1v/v ethyl acetate: hexane) to 100% ethyl acetate to give 40mg of the less polar 5F isomer 1 and 40mg of the more polar 5F isomer 2, MS (Cl, NH)₃)604(MH⁺) (the above-mentioned two isomers).

G. 2-amino-N- [1- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro Pyrazolo [4, 3-c]Pyridine-5-carbonyl) -4-phenyl- (R) -butyl]Isobutyramide hydrochloride

4mL of concentrated HCl was added to a solution of 40mg (0.07mmol) of 5F isomer 1 in 10mL of ethanol and the mixture was stirred at room temperature for about 4 hours. After concentration of the mixture, the residue was precipitated from dichloromethane/hexane and dried in vacuo to give 30mg of 5G isomer 1.

MS(Cl，

NH₃)504(MH⁺)¹HNMR(CD₃OD): (part) δ 719(m, 10H), 4.37(m, 1H), 3.02

(m，6H)，2.67(m，4H)，1.83(m，4H)，1.62(s，6H)，1.28(m，1H).

H. 2-amino-N- [1- (3a- (S) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro Pyrazolo [4, 3-c]Pyridine-5-carbonyl) -4-phenyl- (R) -butyl]Isobutyramide hydrochloride

4mL of concentrated HCl was added to a solution of 40mg (0.07mmol) of 5F isomer 2 in 10mL of ethanol and the mixture was stirred at room temperature for about 4 hours. After concentration of the mixture, the residue was precipitated from dichloromethane/hexane and dried in vacuo to give 30mg of 5H isomer 2.

MS(Cl，

NH₃)504(MH⁺).¹HNMR(CD₃OD): (parts) 7.25(m, 9H), 6.88(m, 1H), 3.04(s,

3H)，2,71(m，4H)，2.48(m，2H)，1.75(m，4H)，1.62(m，6H)，1.28(m，1H).

example 6

2-amino-N- [2- (3a- (R, S) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro Pyrazolo [4, 3-c]Pyridin-5-yl) -1(R) -benzyloxymethyl-2-oxo-ethyl]Isobutyramide Hydrochloride salt

Following the general procedure outlined in general procedure A, 200mg (0.82mmol) of 3C and 320mg (0.82mmol) of 1E were coupled to give a mixture of diastereomers; and, the residue was purified by silica gel chromatography with an elution gradient (1: 1v/v ethyl acetate: hexane) to 10% methanol/ethyl acetate to give 170mg of 6A.

B. 2-amino-N- [2- (3a- (R, S) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexa-amino Hydro-pyrazolo [4, 3-c]Pyridin-5-yl) -1(R) -benzyloxymethyl-2-oxoethyl]Isobutyryl radical Amine hydrochloride

5mL of concentrated HCl was added to a solution of 170mg (0.28mmol) of 6A in 20mL of ethanol, and the mixture was stirred at room temperature for about 2.5 hours. After concentrating the mixture, the residue was precipitated from ethanol/hexane and dried in vacuo to give 70mg of 6B.

MS(Cl，NH₃)506(MH⁺).¹HNMR(CD₃OD)：

δ7.32(m，5H)；7.16(m，5H)，5.22(m，1H)，4.67(m，1H)，4.55(m，2H)，3.79m，2

H)，3.12(m，2H)，3.00(m，6H)，2.71(m，3H)，1.56(m，8H).

Example 7

2-amino-N- [2- (3 a-benzyl-2-ethyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4，3-c]Pyridin-5-yl]-1- (1H-indol-3-ylmethyl) -2-oxo-ethyl } isobutyramide Hydrochloride salt

A. 3a- (R, S) -benzyl-2-ethyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3- c]Pyridine-5-carboxylic acid tert-butyl ester

To 555mg (1.60mmol) of 3B in 27mL of ethanol was added 240mg (1.60mmol) of ethylhydrazine oxalate and the mixture was heated at reflux for about 4 hours. After concentration of the mixture, the residue was purified by silica gel chromatography with an elution gradient of (10: 1v/v hexane: ethyl acetate) to (3: 7 v/v hexane: ethyl acetate) to give 357mg of 7AMS (Cl, NH)₃)358(MH⁺)。

B. 3a- (R, S) -benzyl-2-ethyl-2, 3a, 4, 5, 6, 7-hexahydro-pyrazolo [4, 3-c)]Pyridine- 3-ketones

1.5mL of concentrated HCl was added to a solution of 350mg (0.98mmol) of 7A in 3mL of ethanol, and the mixture was stirred at room temperature for about 2 hours. The mixture was concentrated to give 257mg of 7BMS (Cl, NH)₃)258(MH⁺)。

C. {1- [2- (3a- (R, S) -benzyl-2-ethyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazole And [4, 3-c ]]Pyridin-5-yl) -1- (R) - (1H-indol-3-ylmethyl) -2-oxo-ethylamino Radical formyl radical]-1-methylethyl } carbamic acid tert-butyl ester

82mg (0.28mmol) of 7B and 100mg (0.26mmol) of 2C are coupled in accordance with the method outlined in general step A; and, the residue was purified by silica gel chromatography with an elution gradient of 100% dichloromethane to 2% methanol/dichloromethane to give 110mg of 7CMS (Cl, NH₃)629(MH⁺)。

D. 2-amino-N- [ -2- (3a- (R, S) -benzyl-2-ethyl-3-oxo-2, 3, 3a, 4, 6, 7-hexakis Hydro-pyrazolo [4, 3-c]Pyridin-5-yl) -1(R) - (1H-indol-3-ylmethyl) -2-oxoethyl Base of]Isobutyramide hydrochloride

1mL of concentrated HCl was added to a solution of 100mg (0.15mmol) of 7C in 2mL of ethanol, and the mixture was stirred at room temperature for about 2 hours. The mixture was concentrated to give 72mg of 7D colorless foam MS (Cl, NH)₃)529(MH⁺)。

Example 8

2-amino-N- [2- (3a- (R) -benzyl-2-ethyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydropyrazine Azolo [4, 3-c ] s]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl]Isobutyramide Hydrochloride saltAnd2-amino-N- [2- (3a- (S) -benzyl-2-ethyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydropyrazine Azolo [43-c ]]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl]Isobutyramide Hydrochloride salt

A. {1- [2- (3 a-benzyl-2-ethyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3- c]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethylcarbamoyl]-1-methyl group Ethyl } carbamic acid tert-butyl ester

Following the general procedure outlined in general procedure A, 85mg (0.29mmol) of 7B and 100mg (0.26mmol) of 1E were coupled to give a mixture of diastereomers; and the residue was purified by silica gel chromatography with an elution gradient from 100% dichloromethane to 2% methanol/dichloromethane to give 6mg of the less polar 8A isomer 1 and 11mg of the more polar 8A isomer 2, MS (Cl, NH)₃)620(MH⁺) (the above-mentioned two isomers).

B. 2-amino-N- [2- (3a- (R) -benzyl-2-ethyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro Pyrazolo [4, 3-c]Pyridin-5-yl) -1(R) -benzyloxymethyl-2-oxoethyl]Isobutyramide Hydrochloride salt

0.4mL of concentrated HCl was added to a solution of 5.7mg (0.009mmol) of 8A isomer 1 in 1mL of ethanol and the mixture was stirred at room temperature for about 3 hours. The mixture was concentrated to give 4.7mg of 8B isomer 1.

MS(Cl，NH₃)520

(MH⁺).¹HNMR(CD₃OD): (part) δ 7.41-7.05(m, 10H), 5.20(m, 1H), 4.61(m,

1H)，4.52(s，2H)，3.71(m，1H)，3.60(m，1H)，2.61(m，3H)，1.39(m，9H).

C. 2-amino-N- [2- (3a- (S) -benzyl-2-ethyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro- Pyrazolo [4, 3-c]Pyridin-5-yl) -1(R) -benzyloxymethyl-2-oxoethyl]Isobutyramide Hydrochloride salt

0.4mL of concentrated HCl was added to a solution of 10mg (0.016mmol) of 8A isomer 2 in 1mL of ethanol and the mixture was stirred at room temperature for about 3 hours. The mixture was concentrated to give 8mg of 8C isomer 2.

MS(Cl，NH₃)520

(MH⁺).¹HNMR(CD₃OD): (part) δ 7.43-7.00(m, 10H), 6.81(m, 1H), 5.32(m,

1H).4.63(m，2H)，4.53(m，1H)，3.72(m，1H)，1.37(m，9H).

example 9

2-amino-N- [2- (2-benzyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c)] Pyridin-5-yl]-1- (R) -benzyloxymethyl-2-oxo-ethyl } isobutyramide hydrochloride

A. 2-benzyl-3-hydroxy-2, 4, 6, 7-tetrahydro-pyrazolo [4, 3-c]Pyridine-, 5-carboxylic acid tert-butyl ester

A mixture of 800mg (3, 11mmol) of 3B and 495mg (3.11mmol) of benzylhydrazine dihydrochloride and 423(3.11mmol) of sodium acetate trihydrate in 15mL of ethanol was heated under reflux for about 17 hours. After concentrating the mixture, the residue was dissolved in 100mL of toluene and heated under reflux for about 48 hours. The mixture was diluted with ethyl acetate and washed with brine, dried over magnesium sulfate and concentrated; the residue was then purified by chromatography on silica gel eluting with 100% ethyl acetate followed by 5% methanol in dichloromethane to give 530mg of 9A as a light brown solid MS (Cl, NH)₃)330(MH⁺)。

B. 2-benzyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c]Pyridin-3-ols

10mL of concentrated HCl was added to a solution of 411mg (1.24mmol) of 3E in 30mL of ethanol, and the mixture was stirred at room temperature for about 30 minutes. After concentration of the mixture, the residue was crystallized from methanol/ethyl acetate to yield 353mg of 9B MS (Cl, NH)₃)230(MH⁺)。

C. {1- [2- (2-benzyl-3-hydroxy-2, 4, 6, 7-tetrahydro-pyrazolo [4, 3-c ]]Pyridine-5- 1- (R) -benzyloxymethyl-2-oxo-ethylcarbamoyl]-1-methylethyl) amino group (iv) Carboxylic acid tert-butyl ester

Following the general procedure outlined in general procedure A, 100mg (0.38mmol) of 9B and 145mg (0.38mmol) of 1E were coupled; and the residue was chromatographed on silica gel (95: 1)5v/v methanol: dichloromethane) to yield 42mg of 9C white solid MS (Cl, NH)₃)592(MH⁺)。

D. 2-amino-N- [2- (2-benzyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c)] Pyridin-5-yl) -1(R) -benzyloxymethyl-2-oxoethyl]Isobutyramide hydrochloride

6mL of concentrated HCl was added to a solution of 42mg (0.07mmol) of 9C in 20mL of ethanol, and the mixture was stirred at room temperature for about 30 minutes. The mixture was diluted with ethanol and concentrated, and the residue was precipitated from methanol/ethyl acetate to give 35mg of 9D white solid.

MS(Cl，NH₃)

492(MH⁺).¹HNMR(CD₃OD): (part) 7.41-7.16(m, 10H), 5.19(m, 3H), 4.48(m, 4H), 3.88(m, 1H), 3.74(m, 2H), 2.68(m, 2H), 1.58(m, 6).

Example 10

2-amino-N- {2- [3a- (R) -benzyl-3-oxo-2- (2, 2, 2-trifluoro-ethyl) - 2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3-c)]Pyridin-5-yl]-1(R) -benzyloxymethyl-2- Oxo-ethyl isobutyramide hydrochlorideAnd2-amino-N- {2- [3a- (S) -benzyl-3-oxo-2- (2, 2, 2-trifluoro-ethyl) - 2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3-c)]Pyridin-5-yl]-1(R) -benzyloxymethyl-2- Oxo-ethyl isobutyramide hydrochloride

A. 3a- (R, S) -benzyl-3-oxo-2- (2, 2, 2-trifluoro-ethyl) -2, 3, 3a, 4, 6, 7-hexa Hydro-pyrazolo [4, 3-c]Pyridine-5-carboxylic acid tert-butyl ester

A mixture of 840mg (2.42mmol) of 3B and 276mg (2.42mmol) of 2, 2, 2-trifluoroethylhydrazine (70% in water) in 20mL of ethanol is heated under reflux for about 5 hours and then concentrated. The residue was dissolved in 40mL of toluene and refluxedThe heating was carried out for about 17 hours. After concentrating the mixture, the residue was purified by silica gel chromatography (9: 1v/v hexane: ethyl acetate) to give 703mg of 10A as a yellow oil MS (Cl, NH)₃)412(MH⁺)。

B. 3a- (R, S) -benzyl-2- (2, 2, 2-trifluoro-ethyl) -2, 3a, 4, 5, 6, 7-hexahydro-pyrazole And [4, 3-c ]]Pyridin-3-ones

At about 0 deg.C, 3mL of cold trifluoroacetic acid was added to 600mg (1.46mmol) of 10A and the mixture was stirred for about 3 hours and the solution temperature was allowed to reach room temperature. After concentrating the mixture, the residue is dissolved in water and the solution is basified to pH 11 with 5N sodium hydroxide and then saturated with potassium carbonate. The solution was extracted three times with ethyl acetate and the combined organic extracts were washed with brine, dried over magnesium sulfate and concentrated to give 345mg of 10B opaque oil

MS(Cl，NH₃)312(MH⁺)。

C. (1- {2- [3a- (R, S) -benzyl-3-oxo-2- (2, 2, 2-trifluoro-ethyl) - 2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c)]Pyridin-5-yl]-1- (R) -benzyloxymethyl- 2-oxo-ethylcarbamoyl } -1-methylethyl) carbamic acid tert-butyl ester

Following the general procedure outlined in general procedure A, 137mg (0.44mmol) of 10B and 167mg (0.44mmol) of 1E were coupled to give a mixture of diastereomers; and the residue was purified by silica gel chromatography with an elution gradient from 100% dichloromethane to 5% methanol/dichloromethane to give 128mg of the less polar 10C isomer 1 and 63mg of the more polar 10C isomer 2, MS (Cl, NH)₃)674(MH⁺) (the above-mentioned two isomers).

D. 2-amino-N- {2- [3a- (R) -benzyl-3-oxo-2- (2, 2, 2-trifluoro-ethyl) - 2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c)]Pyridin-5-yl]-1(R) -benzyloxymethyl- 2-oxoethyl group]Isobutyramide hydrochloride

1.5mL of concentrated HCl was added to a solution of 120mg (0.18mmol) of 10C isomer 1 in 3.5mL of ethanol and the mixture was stirred at room temperature for about 2 hours. The mixture was concentrated to give 94mg of 10D isomer 1 as an off-white powder.

MS(Cl，NH₃)574(MH⁺).¹HNMR(CD₃OD): (part) δ 7.31(m, 5H),

7.18(m，5H)，5.21(m，1H)，4.57(m，3H)，4.26(m，1H)，4.08(m，1H)，3.79(m，2

H)，3.09(m，4H)，2.65(m，2H)，1.63(m，6H).

E. 2-amino-N- {2- [3a- (S) -benzyl-3-oxo-2- (2, 2, 2-trifluoro-ethyl) - 2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c)]Pyridin-5-yl]-1- (R) -benzyloxymethyl- 2-oxoethyl isobutyramide hydrochloride

1.5mL of concentrated HCl was added to a solution of 53mg (0.079mmol) of 10C isomer 2 in 3.5mL of ethanol and the mixture was stirred at room temperature for about 2 hours. The mixture was concentrated to give 41mg of 10E isomer 2 as a pale yellow solid.

MS(Cl，NH₃)574(MH⁺).¹HNMR(CD₃OD): (part) δ 7.33(m, 5H), 7.15(m, 4H), 6.81(m, 1H), 5.30(m, 1H), 4.67(m, 4H), 4.15(m, 2H), 3.77(m, 2H), 3.09(m, 3H), 2.64(m, 3H), 1.58(m, 6H).

Example 11

2-amino-N- [2- (3a- (R) -benzyl-2-tert-butyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro Pyrazolo [4, 3-c]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl]Isobutyryl radical Amine methanesulfonateAnd2-amino-N- [2- (3a- (S) -benzyl-2-tert-butyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro Pyrazolo [4, 3-c]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl]Isobutyryl radical Amine methanesulfonate

A. 3a- (R, S) -benzyl-2-tert-butyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4，3-c]Pyridine-5-carboxylic acid tert-butyl ester

To 2.07g (5.95mmol) of 14B in 40mL of ethanol was added 0.97g (7.7mmol) of t-butylhydrazine hydrochloride and 0.63g (7.7mmol) of sodium acetate and the mixture was heated at about 70 ℃ for about 17 hours. The mixture was cooled and the solution was decanted to remove precipitate and then concentrated. The residue was dissolved in 80mL of toluene and heated at reflux for about 6 hours. After concentrating the mixture, the residue was purified by silica gel chromatography (9: 1v/v hexane: ethyl acetate) to give 1.7g of 11A MS (Cl, NH)₃)386(MH⁺)。

B. 3a- (R, S) -benzyl-2-tert-butyl-2, 3a, 4, 5, 6, 7-hexahydro-pyrazolo [4, 3-c)]Pyridine (II) Pyridin-3-ones

Mu.l of methanesulfonic acid was added to a solution of 535mg (1.39mmol) of 11A in 20mL of dichloromethane and the mixture was stirred at room temperature for about 1.5 h. The mixture was diluted with ethyl acetate and washed twice with 1N sodium hydroxide, once with brine, dried over sodium sulfate and concentrated to give 246mg of 11B MS (Cl, NH)₃)286(MH⁺)。

C. {1- [2- (3a- (R, S) -benzyl-2-tert-butyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazine Azolo [4, 3-c ] s]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethylcarbamoyl Base of]-1-methylethyl } carbamic acid tert-butyl ester

Following the general procedure outlined in general procedure A, 246mg (0.86mmol) of 11B and 328mg of 14F were coupled to give a mixture of diastereomers; and the residue was purified by silica gel chromatography (6: 4 v/v hexane/ethyl acetate) to give 250mg of the less polar 11C isomer 1 and 90mg of the more polar 11C isomer 2, MS (Cl, NH)₃)648(MH⁺) (the above-mentioned two isomers).

D. 2-amino-N- [2- (3a- (R) -benzyl-2-tert-butyl-3-oxo-2, 3, 3a, 4, 6, 7-hexa-kis Hydro-pyrazolo [4, 3-c]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxoethyl]Chinese medicinal composition Amide methanesulfonate salt

Mu.l (0.44mmol) of methanesulfonic acid are added at approximately 0 ℃ to a solution of 210mg (0.32mmol) of 11C isomer 1 in 15mL of dichloromethane. After removal of the ice bath, the mixture was stirred for about 3 hours, diluted with 15mL of diethyl ether and the precipitate collected by filtration to give 100mg of 11D isomer 1.

MS(Cl，NH₃)548(MH⁺).¹H NMR(CD₃OD): (section) δ 7.33(m, 5H), 7.27-7.07(m, 5H), 5.21(m, 1H), 4.54(m, 3H), 3.86(m, 3H), 3.10(m, 4H), 2.61(s, 3H), 1.62(m, 6H), 1.18(s, 9H).

E. 2-amino-N- [2- (3a- (S) -benzyl-2-tert-butyl-3-oxo-2, 3, 3a, 4, 6, 7-hexa-kis Hydro-pyrazolo [4, 3-c]Pyridin-5-yl) -1(R) -benzyloxymethyl-2-oxoethyl]Isobutyryl radical Amine methanesulfonate

At about 0C, 21. mu.l (0.32mmol) of methanesulfonic acid are added to a solution of 85mg (0.13mmol) of the 11C isomer 2 in 10mL of dichloromethane. After removal of the ice bath, the mixture was stirred for about 3 hours, diluted with 20mL of diethyl ether and the precipitate collected by filtration to give 46mg of 11E isomer 2.

MS(Cl，NH₃)548(MH⁺).¹H NMR(CD₃OD): (part) δ 8.28(brd, 1H), 7.32(m, 5H), 7.18(m, 4H), 6.84(m, 1H), 5.31(m, 1H), 4.60(m, 3H), 3.70(m, 3H), 3.18-2.92(m, 3H), 2.68(s, 3H), 1.57(m, 6H), 1.13(s, 9H).

Example 12

2-amino-N- [1- (R) - (1H-indol-3-ylmethyl) -2- (2-methyl-3-oxo-3 a- (R, S) -pyridin-2-ylmethyl-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3-c)]Pyridine-5- 2-oxo-ethyl]Isobutyramide dihydrochloride

A. 4-oxo-3- (R, S) -pyridin-2-ylmethyl-piperidine-1, 3-dicarboxylic acid 1-tert-butyl 3-methyl Esters

468mg (11.7mmol) of sodium hydride (60% oil dispersion) are added to a solution of 2.00g (7.8mmol) of 3A in 32mL of THF at about 0 ℃ and the mixture is stirred for about 30 minutes. To the stirring solution above was added a solution of 762mg (6.0mmol) 2-pyridinecarboxylic acid chloride in 5mL THF over a period of 5 minutes; then, 432mg (2.6mmol) of potassium iodide was added. After removal of the ice bath, the mixture was heated at reflux for about 17 hours. The mixture was diluted with ethyl acetate and washed once with water, once with brine, dried over magnesium sulfate and concentrated. The residue was purified by chromatography on silica gel eluting first with (6: 4 v/v diethyl ether: hexane) and then with (6: 4 v/v ethyl acetate: hexane) to give 1.2g of 12AMS (Cl, NH)₃)349(MH⁺)。

B. 2-methyl-3-oxo-3 a- (R, S) -pyridin-2-ylmethyl-2, 3, 3a, 4, 6, 7-hexahydro-pir-ine Azolo [4, 3-c ] s]Pyridine-5-carboxylic acid tert-butyl ester

A mixture of 1.20g (3.45mmol) of 12A and 159mg (3.45mmol) of methylhydrazine in 20mL of ethanol was heated at reflux for about 6.5 hours. After concentrating the mixture, the residue was dissolved in 25mL of toluene and heated under reflux for about 17 hours. After concentrating the mixture, the residue was purified by silica gel chromatography (65: 35 v/v ethyl acetate: hexane) to give 450mg of 12B MS (Cl, NH)₃)345(MH⁺)。

C. 2-methyl-3 a- (R, S) -pyridin-2-ylmethyl-2, 3a, 4, 5, 6, 7-hexahydro-pyrazolo [4，3-c]Pyridin-3-one dihydrochloride

A mixture of 450mg (1.30mmol) of 12B in 2mL 4M HCl/dioxane was stirred at room temperature for about 4.5 hours. After concentration of the mixture, 450mg of 12C were obtained

MS(Cl，NH₃)245(MH⁺)。

D. {1-[1-(1-(R)-H-indol-3-ylmethyl) -2- (2-methyl-3-oxo-3 a- (R, S) - Pyridin-2-ylmethyl-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3-c)]Pyridin-5-yl) -2-oxo Substituted-ethylcarbamoyl radical]-1-methyl-ethyl } carbamic acid tert-butyl ester

108mg (0.31mmol) of 12C and 122mg (0.31mmol) of 2C are coupled as usual in step A; furthermore, the residue was purified by silica gel chromatography (95: 5v/v ethyl acetate: methanol) to give 118mg of 12D MS (Cl, NH)₃)616(MH⁺)。

E. 2-amino-N- [1- (R) - (1H-indol-3-ylmethyl) -2- (2-methyl-3-oxo-3 a- (R, S) -pyridin-2-ylmethyl-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3-c)]Pyridine-5- 2-oxo-ethyl]Isobutyramide dihydrochloride

A mixture of 110mg (0.18mmol) of 12D in 1mL of 4M HCl/dioxane was stirred at room temperature for about 17 hours. After concentration of the mixture, 51mg of 12E were obtained.

MS(Cl，NH₃)516(MH⁺).¹HNMR(CD₃OD): (part) δ 8.91-8.52(m, 2H), 8.04(m, 2H), 7.76-7.50(m, 3H), 6.82(m, 1H), 4.62(m, 1H), 3.36(s, 3H), 1.63(s, 6H).

Example 13

2-amino-N- [1- (R) -benzyloxymethyl-2- (2-methyl-3-oxo-3 a- (R, S) -pyridine- 2-ylmethyl-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3-c)]Pyridin-5-yl) -2-oxo-ethanes Base of]Isobutyramide dihydrochloride

A. {1- [1- (R) -benzyloxymethyl-2- (2-methyl-3-oxo-3 a- (R, S) -pyridin-2-yl Methyl-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3-c)]Pyridin-5-yl) -2-oxo-ethylamino Radical formyl radical]-1-methyl-ethyl } carbamic acid tert-butyl ester

86mg (0.27mmol) of 12C and 103mg (0.27mmol) of 1E are coupled as usual in step A; further, the residue was purified by silica gel chromatography (95: 5v/v ethyl acetate: hexane) to give 82mg of 13A.

B. 2-amino-N [1- (R) -benzyloxymethyl-2- (2-methyl-3-oxo-3 a- (R, S) -pyridine -2-ylmethyl-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3-c]Pyridin-5-yl) -2-oxo- Ethyl radical]Isobutyramide dihydrochloride

A mixture of 75mg (0.12mmol)13A in 1mL 4M HCl/dioxane was stirred at room temperature for about 17 hours. After concentration of the mixture, 80mg of 13B was obtained.

MS(Cl，NH₃)507(MH⁺).¹HNMR(CD₃OD): (part) Δ 8.78(m, 1H), 8.46

(m，1H)，8.13-7.82(m，2H)，7.32(m，5H)，4.57(m，3H)，3.96(m，1H)，3.82(m，

2H)，1.63(m，6H).

Example 14

2-amino-N- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pir-zine Azolo [4, 3-c ] s]Pyridin-5-yl) -1- (R) - (benzyloxymethyl) -2-oxo-ethyl]Isobutyryl radical Amines as pesticides

A. 4-oxo-piperidine-1, 3-dicarboxylic acid 1-tert-butyl 3-methyl ester

To a mixture of 100.0g (516.4mmol) of methyl 4-oxo-piperidine-3-carboxylate and 63g (516.4mmol) of 4, 4-dimethylaminopyridine in 1L of dichloromethane was added a solution of 113.0g (516.4mmol) of di-tert-butyl dicarbonate in 100mL of dichloromethane at about 0 ℃ over a period of 90 minutes. The mixture was slowly warmed to room temperature and then stirred for about 19 hours. The mixture was washed three more times (10% aqueous brine, saturated aqueous sodium bicarbonate and brine each time), dried over magnesium sulfate and concentrated to give 130.5g of 14A amorphous solid.

¹HNMR(CDCl₃)：δ4.03(br，2H)；3.74(s，3H)，3.56(t，2H)，2.36(t，2H)，1.42(s，9H).

B. 3- (R) -benzyl-4-oxo-piperidine-1, 3-dicarboxylic acid 1-tert-butyl 3-methyl ester

To a stirring suspension of 11.7g (293mmol) of sodium hydride (60% oil dispersion washed twice with 100mL of hexane) in 100mL of DMF at about 0 deg.C over a period of about 45 minutes was added a solution of 65.4g (254mmol) of 14A in 150mL of DMF. After removal of the ice bath, the mixture was stirred at room temperature for about 45 minutes. After the mixture was again cooled to about 0 ℃, a solution of 35.2mL (296mmol) of benzyl bromide in 200mL of DMF was added dropwise to the stirring solution, and the mixture was stirred at room temperature for about 23 hours. 550mL of water was carefully added to the solution and the mixture was stirred for approximately 30 minutes. After the mixture was extracted three times with ethyl acetate, the combined organic extracts were washed five times with water, once with brine, dried over magnesium sulfate and concentrated to give 98g of a yellow oil. The above oil was crystallized from hexane to give 71g of 14B as a white solid.

MS(Cl，NH₃)348(MH⁺).¹HNMR(CDCl₃): (part) δ 7.23(m, 3H), 7.13(m, 2H), 4.58(br m, 1H), 4.18(br, 1H), 3.63(s, 3H), 3.28-2.96(m, 4H), 2.72(m, 1H), 2.43(m, 1H), 1.44(s, 9H).

C. 3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c)] Pyridine-5-carboxylic acid tert-butyl ester

A mixture of 47.0g (135mmol) of 14B, 38.9g (270mmol) of methylhydrazine sulfate and 44.3g (540mmol) of sodium acetate in 900mL of ethanol is heated under reflux for about 17 hours under nitrogen. After concentrating the mixture, the residue was dissolved in ethyl acetate and washed three times with water and once with brine, dried over magnesium sulfate and concentrated to give a yellow oil. The oil was stirred in 750mL of hexanes for about 3 hours to give 41.17g of 14C white solid.

MS(Cl，NH₃)344(MH⁺) 1HNMR (CDCl 3): (part) δ 7.19(m, 3H), 7.05(m,

2H)，4.61(br m，2H)，3.24(m，1H)，3.09(s，3H)，3.01(m，1H)，2.62(m，4H)，

1.52(s，9H).

D. 3a- (R, S) -benzyl-2-methyl-2, 3a, 4, 5, 6, 7-hexahydro-pyrazolo [4, 3-c)]Pyridine- 3-keto hydrochloride

A solution of 24.55g (71.5mmol) of 14C in 800mL of diethyl ether was bubbled with anhydrous HCl at about 0 deg.C for about 12 minutes. The mixture was stirred for about 3 hours, during which time a white precipitate formed. The precipitated solid was collected by filtration to give 19.2g of 14D.

MS(Cl，NH₃)244(MH⁺).

¹HNMR(CD₃OD): (part) δ 7.25(m, 3H), 7.05(m, 2H), 3.77(m, 2H), 3.51(d, 1)

H)，3.25(m，1H)，3.17(m，3H)，3.03(s，3H)，2.81(m，1H).

E. 2-Boc-amino-2-methyl-propionic acid 2, 5-dioxopyrrolidin-1-yl ester

To a stirring solution of 100.0g (492mmol) Boc- α -methylalanine and 94.0g (492mmol) EDC in 2L dichloromethane at about 0 deg.C was added in portions 56.63g (492mmol) N-hydroxysuccinimide; then, the reaction was allowed to warm to room temperature. After the mixture was stirred for about 24 hours, washed twice (each with saturated aqueous sodium bicarbonate and brine, respectively), dried over sodium sulfate and concentrated to give 124.0g of 14E as a white solid.

¹HNMR(CDCl₃)：δ4.96(br，1H)，2.82(s，4H)，1.66(s，6H)，1.48(s，9H).

F. 3- (R) -benzyloxy-2- (2-tert-butoxycarbonylamino-2-methyl-propionylamino) propionic acid

A mixture of 50.5g (168mmol) of 14E, 33.5g (168mmol) of O-benzyl-D-serine and 51.05g (505mmol) of triethylamine in 400mL of dioxane was heated at about 45 ℃ for about 16 hours. The mixture was diluted with ethyl acetate and acidified with acetic acid to pH 2. After separation of the layers, the organic phase was washed with brine, dried over sodium sulfate and concentrated to give 650g of 14F as a white solid.

¹HNMR(CD₃OD): (part) δ 7.55(d, 1H), 7.29(m, 5H), 4.52(m, 1H), 4.48(s, 2H), 3.84(d of d, 1H), 3.69(dof d, 1H), 1.42(s, 6H), 1.38(s, 9H).

G. 3a- (R) -benzyl-2-methyl-2, 3a, 4, 5, 6, 7-hexahydro-pyrazolo [4, 3-c]Pyridine-3- Ketone L-tartrate salt

A mixture of 5.00g (20.6mmol) of 14D free base and 3.09g (20.6mmol) of L-tartaric acid in 80mL of acetone and 3.2mL of water was heated at about 70 deg.C for about 70 hours under nitrogen; during this time the reaction mixture became a thick suspension and 20mL of acetone was added. The reaction mixture was slowly cooled to room temperature and then filtered. The collected solid was washed with acetone and dried in vacuo to give 7.03G of 14G as a white solid.

H. 3a- (R) -benzyl-2-methyl-2, 3a, 4, 5, 6, 7-hexahydro-pyrazolo [4, 3-c]Pyridine-3- Ketones

1.72mL (25.4mmol) of ammonium hydroxide was added to a suspension of 5.00G (12.7mmol) of 14G in 80mL of dichloromethane at about 0 deg.C; and the mixture was stirred for about 15 minutes. The cold solution was filtered and used immediately in the next step.

I. {1- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4，3-c]Pyridin-5-yl) -1- (R) - (benzyloxymethyl) -2-oxo-ethylcarbamoyl Base of]-1-methyl-ethyl } carbamic acid tert-butyl ester

A mixture consisting of 4.83g (12.7mmol) of 14F, the solution from 14H, 2.60g (19.1mmol) of HOAT and 2.45g (12.8mmol) of EDC was stirred at about 0 ℃ for about 1 hour under nitrogen, warmed to room temperature and stirred for about 16 hours. After the mixture was filtered, the filtrate was washed with saturated aqueous sodium bicarbonate solution and water, dried over magnesium sulfate and concentrated to give 7.35g of 14I as a white solid.

J. 2-amino-N- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro- Pyrazolo [4, 3-c]Pyridin-5-yl) -1- (R) - (benzyloxymethyl) -2-oxo-ethyl]Chinese medicinal composition Amides of carboxylic acids

At about 0 deg.C, 3.5mL of cold trifluoroacetic acid is added to a solution of 755mg (1.25mmol) of 14I in 7mL of dichloromethane; and the mixture was stirred at about 0 ℃ for about 1 hour. The mixture was warmed to room temperature and stirred for about 2 hours. The mixture was concentrated and co-evaporated twice with toluene. The residue was dissolved in chloroform and washed twice with saturated aqueous sodium bicarbonate, once with water and brine. The mixture was dried over magnesium sulfate and concentrated to give 594mg of 14J as an oil.

Example 15

2-amino-N- [1- (R) -benzyloxymethyl-2- (2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexa-kis Hydro-pyrazolo [4, 3-c]Pyridin-5-yl) -2-oxo-ethyl]Isobutyramide hydrochloride

A. 2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c]Pyridine-5-carboxylic acid Tert-butyl ester

A mixture of 3.00g (11.66mmol) of 3A and 537mg (11.66mmol) of methylhydrazine in 100mL of ethanol was heated under reflux for about 17 hours. After concentrating the mixture, the residue was dissolved in 100mL of toluene and heated under reflux for about 17 hours. The mixture was diluted with ethyl acetate and washed twice with brine, dried over magnesium sulfate and concentrated. The residue was purified by silica gel chromatography eluting with a gradient of 100% ethyl acetate to 5% methanol in dichloromethane to give 2.28g of 15A as a white solid.

¹HNMR(CD₃OD)：δ4.20(s，2H)，3.67(t，2H)，3.43(s，3H)，2.58(t，2H)，1.48(s，9H).

B. 2-methyl-2, 3a, 4, 5, 6, 7-hexahydro-pyrazolo [4, 3-c)]Pyridin-3-one hydrochloride

10mL of concentrated HCl was added to a solution of 510mg (2.01mmol) of 15A in 30mL of ethanol, and the mixture was stirred at room temperature for about 35 minutes. After concentrating the mixture, the residue was crystallized from methanol/ethyl acetate to give 425mg 15B as a yellow solid.

¹HNMR(CD₃OD)：δ4.27(S，2H)，3.71(S，3H)，3.56(T，2H)，3.05(T，2H).

C. {1- [1(R) -benzyloxymethyl-2- (2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazine Azolo [4, 3-c ] s]Pyridin-5-yl) -2-oxo-ethylcarbamoyl]-1-methylethyl } amino group (iv) Carboxylic acid tert-butyl ester

Following the general procedure outlined in general procedure A, 100mg (0.53mmol) of 15B and 202mg (0.53mmol) of 1E were coupled; furthermore, the residue was purified by silica gel chromatography (95: 5v/v dichloromethane: methanol) to give 54mg of 15C white solid MS (Cl, NH)₃)516(MH⁺)。

D. 2-amino-N- [1- (R) -benzyloxymethyl-2- (2-methyl-3-oxo-2, 3, 3a, 4, 6, 7- Hexahydro-pyrazolo [4, 3-c]Pyridin-5-yl) -2-oxoethyl]Isobutyramide hydrochloride

10mL of concentrated HCl was added to a solution of 54mg (0.10mmol) of 15C in 30mL of ethanol, and the mixture was stirred at room temperature for about 40 minutes. After concentration of the mixture, the residue was precipitated from methanol/ethyl acetate to yield 50mg of 15D.

MS(Cl，NH₃)416(MH⁺).¹HNMR(CD₃OD): (part) δ 7.28(m, 5H), 5.18(m 1H), 4.69-4.38(m, 4H), 3.88(m, 1H), 3.73(m, 2H), 3.68(s, 2H), 3.61(m, 1H), 2.67(m, 1H), 1.57(s, 6H).

Example 16

2-amino-N- [2- (2-benzyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c)] Pyridin-5-yl]-1- (R) - (1H-indol-3-ylmethyl) -2-oxo-ethyl } isobutyramide salt Acid salts

A. 2-benzyl-3-hydroxy-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c]Pyridine-5-carboxylic acid Tert-butyl ester

A mixture of 800mg (3.11mmol) of 3A and 495mg (3.11mmol) of benzylhydrazine dihydrochloride in 15mL of ethanol was heated under reflux for about 17 hours. After concentrating the mixture, the residue was dissolved in 100mL of toluene and heated under reflux for about 48 hours. The mixture was diluted with ethyl acetate and washed twice with brine, dried over sodium sulfate and concentrated; the residue was then purified by silica gel chromatography eluting with a gradient of 100% ethyl acetate to 5% methanol in dichloromethane to give 530mg of 16A as a brown solid MS (Cl, NH)₃)330(MH⁺)。

B. 2-benzyl-2, 3a, 4, 5, 6, 7-hexahydro-pyrazolo [4, 3-c)]Pyridin-3-one hydrochloride

10mL of concentrated HCl was added to a solution of 411mg (1.24mmol) of 16A in 30mL of ethanol, and the mixture was stirred at room temperature for about 30 minutes. After concentrating the mixture, the residue was crystallized from methanol/ethyl acetate to yield 353mg of 16B as a yellow solid.

MS(Cl，NH₃)230

(MH⁺).¹ HNMR(CD₃OD)：δ7.26-7.40(m，5H)，5.22(s，2H)，4.12(s，2H)，3.53(t，

2H)，3.00(t，2H).

C. (R) -2- (2-tert-Butoxycarbonylamino-2-methyl-propionylamino) -3- (1H-indol-3-yl) Propionic acid

To a stirring solution of 30.6g (0.15mol) D-tryptophan, 30.4g (0.30mol) N-methylmorpholine in 450mL (4: 1) dioxane: water was added 45.0g (0.15mol)14E and the mixture was stirred for about 72 hours. Excess dioxane was removed by evaporation and ethyl acetate and water were added to the mixture. The pH of the solution was adjusted to 3 with concentrated hydrochloric acid and the layers were separated. The organic layer was washed with water and brine, dried over magnesium sulfate and concentrated; the residue was then crystallized from ethyl acetate/hexane to give 37.0g of an off-white solid.

D. {1- [2- (2-benzyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ]]Pyridine- 5-yl) -1- (R) - (1H-indol-3-ylmethyl) -2-oxo-ethylcarbamoyl]-1-A Ethyl carbamic acid tert-butyl ester

100mg (0.38mmol) of 16B and 202mg (0.53mmol) of 16C are coupled in accordance with the method outlined in general procedure A; furthermore, the residue was purified by silica gel chromatography (95: 5v/v dichloromethane: methanol) to give 45mg of 16D white solid MS (Cl, NH)₃)601(MH⁺)。

E. 2-amino-N- [2- (2-benzyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c)] Pyridin-5-yl) -1- (R) - (1H-indol-3-ylmethyl) -2-oxoethyl]Isobutyramide hydrochloric acid Salt (salt)

20mL of concentrated HCl was added to a solution of 45mg (0.07mmol) of 16D in 60mL of ethanol, and the mixture was stirred at room temperature for about 35 minutes. After concentrating the mixture, the residue was precipitated from methanol/ethyl acetate to give 30mg of 16E.

¹HNMR(CD₃OD): (part) delta 7.40(m, 4H),7.25(m，3H)，7.11(m，2H)，6.96(m，2H)，6.81(m，1H)，5.38-4.93(m，3H)，4.46(m，1H)，4.22(m，1H)，3.96(m，1H)，3.69(m，1H)，3.18(m，1H)，2.28(m，1H)，1.57(m，6H)，1.38(m，1H).

example 17

2-amino-N- [ 1-benzyloxymethyl-2- (2, 3 a-dimethyl-3-oxo-2, 3, 3a, 4, 6, 7- Hexahydropyrazolo [4, 3-c]Pyridin-5-yl) -2-oxo-ethyl]Isobutyramide hydrochloride

A. 3-methyl-4-oxo-piperidine-1, 3-dicarboxylic acid 1-tert-butyl 3- (R, S) -methyl ester

308mg (7.77mmol) of sodium hydride (60% oil dispersion) was added to a solution of 2.00g (7.77mmol) of 3A in 30mL of DMF, and the mixture was stirred at room temperature for about 25 minutes. To the stirring solution was added 0.50mL (7.77mmol) of methyl iodide, and the mixture was stirred at room temperature for about 17 hours. The mixture was then diluted with ethyl acetate and washed once with water, four times with brine, dried over magnesium sulfate and concentrated. The residue was purified by silica gel chromatography (7: 3 v/v hexane: ethyl acetate) to give 1.75g 17A as a clear oil MS (Cl, NH)₃)272(MH⁺)。

B. 2, 3a- (R, S) -dimethyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c)] Pyridine-5-carboxylic acid tert-butyl ester

A mixture of 1.62g (9.50mmol) of 17A and 435mg (9.50mmol) of methylhydrazine in 30mL of ethanol was heated at reflux for about 4 hours. After concentrating the mixture, the residue was dissolved in 50mL of toluene and heated under reflux for about 14 hours. The mixture was diluted with ethyl acetate, washed twice with brine, dried over sodium sulfate and concentrated. The residue was then purified by silica gel chromatography (7: 3 v/v hexane: ethyl acetate) to give 1.00g of 17B as a white solid MS (Cl, NH)₃)268(MH⁺)。

C. 2, 3a- (R, S) -dimethyl-2, 3a, 4, 5, 6, 7-hexahydro-pyrazolo [4, 3-c)]Pyridine-3- Ketohydrochlorides

8mL of concentrated HCl was added to a solution of 1.00g (3.74mmol) of 17B in 40mL of ethanol, and the mixture was stirred at room temperature for about 35 minutes. After concentrating the mixture, the residue was crystallized from methanol/ethyl acetate to give 850mg of 17C white solid

MS(Cl，NH₃)168(MH⁺)。

D. {1- [1- (R) -benzyloxymethyl-2- (2, 3a- (R, S) -dimethyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3-c)]Pyridin-5-yl) -2-oxo-ethylcarbamoyl]-1- Methyl-ethyl } carbamic acid tert-butyl ester

150mg (0.74mmol) of 17C and 514mg (1.35mmol) of 1E are coupled as outlined in general step A; further, the residue was purified by silica gel chromatography (85: 15 v/v hexane: ethyl acetate) to give 185mg of 17D white solid.

E. 2-amino-N- [1- (R) -benzyloxymethyl-2- (2, 3a- (R, S) -dimethyl-3-oxo-) 2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3-c)]Pyridin-5-yl) -2-oxo-ethyl]Isobutyryl radical Amine hydrochloride

15mL of concentrated HCl was added to a solution of 173mg (0.33mmol) of 17B in 40mL of ethanol, and the mixture was stirred at room temperature for about 1 hour. After concentrating the mixture, the residue was diluted with chloroform, washed with saturated aqueous sodium bicarbonate solution and brine, and dried over sodium sulfate; the residue was purified by silica gel chromatography eluting with a gradient from 100% ethyl acetate to 10% diethylamine/ethyl acetate. The residue obtained is then dissolved in ethanol and acidified with aqueous hydrochloric acid. After concentrating the mixture, the residue was crystallized from methanol/ethyl acetate to yield 65mg of 17E as a white solid.

MS(Cl，NH₃)502(MH⁺).¹HNMR(CD₃OD)：(part) δ 7.32(m, 5H), 5.14(m, 1H), 4.53(m, 3H), 3.71(m, 3H), 2.97(m, 1H), 2.83(m, 1H), 2.57(m, 1H), 1.98(m, 2H), 1.61(m, 6H), 1.38(s, 3H).

Example 18

2-amino-N- [2- (3a- (R) -benzyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4，3-c]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl]Isobutyramide hydrochloric acid Salt (salt)And2-amino-N- [2- (3a- (S) -benzyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4，3-c]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl]Isobutyramide hydrochloric acid Salt (salt)

A. 3-benzyl-4-oxo-piperidine-3-carboxylic acid methyl ester

To 200mg (0.58mmol) of 3B at about 0 ℃ was added 5mL of cold trifluoroacetic acid and the mixture was stirred for about 1 hour. After concentrating the mixture, the residue was co-evaporated together with ethyl acetate and hexane. The residue was made basic by adding 2N sodium hydroxide, and the mixture was extracted with chloroform. The combined organic extracts were dried over magnesium sulfate and concentrated to give 18A in quantitative yield.

B. 3- (R, S) -benzyl-1- [ 3-benzyloxy-2- (R) - (2-tert-butoxycarbonylamino-2-methyl-) Propionylamino) propionyl group]-4-oxo-piperidine-3-carboxylic acid methyl ester

Following the general procedure outlined in general procedure A, 1.77g (7.16mmol) of 18A and 3.04g (8.0mmol) of 14F were coupled to give a mixture of diastereomers; furthermore, the residue was purified by silica gel chromatography (7: 3 v/v hexane: ethyl acetate) to give 820mg of the less polar 18B isomer 1 and 1.14g of the more polar 18B isomer 2, MS (Cl, NH)₃)611(MH⁺) (the above-mentioned two isomers).

C. {1- [2- (3a- (R, S) -benzyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-) c]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethylaminoFormyl radical]-1-methyl group Ethyl } carbamic acid tert-butyl ester

342mg (2.63mmol) of hydrazine sulfate and 431mg (5.26mmol) of sodium acetate are added to a solution of 820mg (1.32mmol) of 18B isomer 1 in 13mL of ethanol and the mixture is heated under reflux for approximately 17 hours. After concentrating the mixture, the residue was diluted with ethyl acetate, washed with saturated sodium bicarbonate and brine, dried over magnesium sulfate and concentrated. The resulting residue was then purified by silica gel chromatography, eluting with a gradient of 75% ethyl acetate/hexane to 100% ethyl acetate, to give 550mg of 18C isomer 1.

485mg (3.73mmol) of hydrazine sulfate and 613mg (7.48mmol) of sodium acetate are added to a solution of 1.14g (1.86mmol) of the 18B isomer 2 in 20mL of ethanol and the mixture is heated under reflux for approximately 17 hours. After concentrating the mixture, the residue was diluted with ethyl acetate, washed with saturated sodium bicarbonate and brine, dried over magnesium sulfate and concentrated. The resulting residue was then purified by silica gel chromatography (75: 25v/v ethyl acetate: hexane) to give 710mg of 18C isomer 2.

D. 2-amino-N- [2- (3a- (R) -benzyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4，3-c]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl]Isobutyramide hydrochloric acid Salt (salt)

6mL of concentrated HCl was added to a solution of 200mg (0.34mmol) of 18C isomer 1 in 12mL of ethanol, and the mixture was stirred at room temperature for about 2.5 hours. The mixture was concentrated and co-evaporated three times with ethanol to give 20mg of 18D isomer 1.

MS(Cl，NH₃)492(MH⁺).¹HNMR(CD₃OD): (part) δ 8.42(br d, 1H), 7.35(m, 5H), 7.18(m, 5H), 5.23(m, 2H), 4.91(m, 1H), 4.54(m, 4H), 3.80(m, 2H), 3.63(m, 1H), 3.12(m, 1H), 3.07(m, 3H), 2.61(m, 3H), 1.62(m, 6H), 1.39(m, 1H).

E. 2-amino-N- [2- (3a- (S) -benzyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4，3-c]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl]Isobutyramide hydrochloric acid Salt (salt)

10mL of concentrated HCl was added to a solution of 200mg (0.34mmol) of 18C isomer 2 in 20mL of ethanol, and the mixture was stirred at room temperature for about 2.5 hours. The mixture was concentrated and co-evaporated with ethanol three times to give 30mg of 18E isomer 2.

MS(Cl，NH₃)492(MH⁺).¹HNMR(CD₃OD): (part) δ 8.29(br d, 1H), 7.30(m, 5H), 7.11(m, 4H), 6.88(m, 1H), 5.29(m, 1H), 4.92(m, 1H), 4.62(m, 3H), 3.91-3.70(m, 3H), 3.22-2.95(m, 3H), 2.66(m, 3H), 1.57(m, 6H), 1.30(m, 1H), 0.89(m, 1H).

Example 19

2-amino-N- [1- (R) -benzyloxymethyl-2- (2-methyl-3-oxo-3 a- (R, S) -thiazole- 4-ylmethyl-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3-c)]Pyridin-5-yl) -2-oxo-ethanes Base of]Isobutyramide dihydrochloride

A. 4-oxo-3- (R, S) -thiazol-4-ylmethyl-piperidine-1, 3-dicarboxylic acid 1-tert-butyl ester 3-ethane Esters

67mg (1.66mmol) of sodium hydride (60% oil dispersion) are added to a solution of 300mg (1.10mmol) of 1A in 5mL of THF at about 0 ℃ and the mixture is stirred for about 30 minutes. To the above cold solution was added a solution of 204mg (1.21mmol) 4-chloromethylthiazole (Hsiao, C.N; Synth. Comm. (synthetic communication) 20, p.3507(1990)) in 5mL THF; then, 87mg (0.53mmol) of potassium iodide was added, and the mixture was heated under reflux for about 17 hours. The mixture was diluted with water and extracted with ethyl acetate; after the combined organic extracts were dried over sodium sulfate and concentrated, the residue was purified by silica gel chromatography (7: 3 v/v hexane: ethyl acetate) to yield 90mg of the title compound MS (Cl, NH)₃)648(MH⁺)。

B. 2-methyl-3-oxo-3 a- (R, S) -thiazol-4-ylmethyl-2, 3, 3a, 4, 6, 7-hexahydro-pir-zine Azolo [4, 3-c ] s]Pyridine-5-carboxylic acid tert-butyl ester

11.2mg (0.24mmol) of methylhydrazine are added to a solution of 90mg (0.24mmol) of 19A in 2mL of ethanol, and the mixture is heated under reflux for approximately 17 hours. After a further 33.6mg (0.72mmol) of methylhydrazine are added, the mixture is heated at reflux for approximately 7 hours. After concentrating the mixture, the residue was dissolved in 3mL of toluene and heated under reflux for about 17 hours. After concentrating the mixture, the residue was purified by silica gel chromatography (6: 4 v/v hexane: ethyl acetate) to give 44mg of 19B MS (Cl, NH)₃)648(MH⁺)。

C. 2-methyl-3 a- (R, S) -thiazol-4-ylmethyl-2, 3a, 4, 5, 6, 7-hexahydro-pyrazolo [4，3-c]Pyridin-3-one dihydrochloride

A mixture of 44mg (0.10mmol)19B in 1mL 4M HCl/dioxane was stirred at room temperature for approximately 4 hours. The mixture was concentrated and co-evaporated with dichloromethane to give 40mg of 19C MS (Cl, NH)₃)251(MH⁺)。

D. {1- [1- (R) -benzyloxymethyl-2- (2-methyl-3-oxo-3 a- (R, S) -thiazol-4-yl Methyl-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3-c)]Pyridin-5-yl) -2-oxo-ethylamino Radical formyl radical]-1-methyl-ethyl } carbamic acid tert-butyl ester

40mg (0.12mmol) of 19C and 39mg (0.12mmol) of 14F are coupled as outlined in general step A; furthermore, the residue was purified by silica gel chromatography (9: 1v/v ethyl acetate: hexane) to give 40mg of 19D MS (Cl, NH)₃)613(MH⁺)。

E. 2-amino-N- [1- (R) -benzyloxymethyl-2- (2-methyl-3-oxo-3 a- (R, S) -thiazole -4-ylmethyl-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3-c]Pyridin-5-yl) -2-oxo- Ethyl radical]Isobutyramide dihydrochloride

A mixture of 40mg (0.06mmol)19D in 1mL 4M HCl/dioxane was stirred at room temperature for about 5 hours. The mixture was concentrated and co-evaporated with dichloromethane to give 40mg of 19E MS (Cl, NH)₃)513(MH⁺)。

Example 20

2-amino-N- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pir-zine Azolo [4, 3-c ] s]Pyridin-5-yl) -1- (R) - (benzyloxymethyl) -2-oxo-ethyl]Isobutyryl radical Amine L-tartrate salt

A solution of 1.36g L-tartaric acid in 20mL of methanol was added to a solution of 4.6g of the title compound of example 14 in 20mL of methanol at about 0 deg.C. The mixture was allowed to warm to room temperature, stirred for approximately 40 minutes, and concentrated under vacuum. The residue was diluted with 220mL of ethyl acetate and heated at reflux for about 1.5 hours, followed by stirring at about 72 ℃ for about 18 hours. The compound was cooled to room temperature and filtered to give 5.78g of the title compound as a colorless crystalline solid.

Example 21

3-benzyl-3-methoxycarbonylmethyl-4-oxo-piperidine-1-carboxylic acid tert-butyl ester

A. 3-benzyl-4-oxo-piperidine-1-carboxylic acid tert-butyl ester

A mixture consisting of beta-ketoester (4480mg, 12.9mmol) and LiCl (1100mg, 25.8mmol) was heated in DMF (2.0mL) at about 120 deg.C for about 17 hours. The reaction mixture was cooled to room temperature and extracted with ethyl acetate (3 × 100 mL). The combined organic extracts were dried and concentrated under vacuum. Chromatography of the crude product on silica, eluting with 20% ethyl acetate in hexanes, provided 1320mg of the desired product as a yellow oil.

¹H NMR(250MHz，CDCl₃)：d：7.4(m，5H)，

4.2(m，1H)，3.4(m，1H)，3.3(dd，1H)，3.05(dd，1H)，2.7(m，1H)，2.55(m，4H)，

1.5(s，9H)；MS(APCI)：190(M+1-BOC).

B. 3-benzyl-3-methoxycarbonylmethyl-4-oxo-piperidine-1-carboxylic acid tert-butyl ester

A solution of the product of step A, example 21 above (1320mg, 4.56mmol), pyrrolidine (972mg, 13mmol) and p-toluenesulfonic acid (33mg) in benzene (30mL) was refluxed over 3 molecular sieves for about 17 hours. The reaction mixture was cooled to room temperature and concentrated under vacuum. The residue was dissolved in benzene (10mL) and cooled to about 0 ℃. Methyl bromoacetate (1530mg, 10mmol) was added dropwise and the reaction mixture was slowly heated to room temperature, followed by heating at reflux for about 17 hours, at which point water (5mL) was added. After further refluxing for 2 hours, the reaction mixture was cooled to room temperature and extracted with ethyl acetate (3X 100 mL). The organic extracts were combined, dried and concentrated under vacuum. Chromatography of the crude residue on silica gel eluting with 15% ethyl acetate/hexanes provided 280mg of the product.

¹H NMR(250 MHz，

CDCl₃)：d 7.35(m，5 H)，4.5(m，1H)，3.8(s，3H)，3.4(dd，1H)，3.1(m，1H)，2.85

(m，4H)，2.6(m，1H)，2.4(m，1H)，1.5(s，9H)；MS(APCI)：362(M+1).

Example 22

6-oxo-1-phenyl-cyclohexane-1, 3-dicarboxylic acid 3-tert-butyl 1-methyl ester

Diphenylmercury (890mg, 2.5mmol) in CHCl was heated at about 40 deg.C under nitrogen₃(4mL) of the solution. Lead tetraacetate (1110mg, 2.5mmol) was added in small portions and the yellow-green solution was addedStirring at about 40 ℃ for 0.5 h then, beta-ketoester (520mg, 2.0mmol) was added; pyridine (0.2mL, 2.5mmol) was then added. After about 5 hours at about 40 ℃ for overdue, the reaction mixture was concentrated in vacuo and the residue was dissolved in ether (100mL) and filtered. The filtrate was washed with 3N sulfuric acid (3 ×), dried and concentrated to give 616mg of a yellow solid. Flash chromatography on silica gel eluting with 25% ethyl acetate/hexanes provided 368mg of the desired product.

¹H NMR(400

MHz，CDCl₃)：d 7.15(m，5H)，4.4(s，2H)，3.7(s，5H)，2.6(s，2H)，1.5(s，9H)；MS

(APCI)：334(M+1)

Example 23

(D) -2-amino-3- (2, 4-dichloro-benzyloxy) propionic acid hydrochloride

A. (D) -2-tert-butoxycarbonylamino-3- (2, 4-dichloro-benzyloxy) propionic acid

To a stirring solution of Boc-D-serine (8.2g, 40mmol) in DMF (75mL) at about 0 deg.C was added sodium hydride (60% dispersion, 3.2g, 80mmol) and the addition continued for about 10 minutes. The reaction mixture was stirred at about 0 ℃ for about 1.75 hours, then at room temperature for about 0.25 hours. After cooling to about 0 deg.C, a solution of 2, 4-dichlorotoluene (5.56mL, 40mmol) in DMF (5mL) was added dropwise. The reaction mixture was heated to about 23 ℃ and stirred for about 17 hours, then partitioned between diisopropyl ether and 10% hydrochloric acid. The resulting aqueous solution was extracted with diisopropyl ether (2 ×), and the combined organic extracts were washed with saturated brine, dried and concentrated to give 14.75g of crude product which was used without further purification.

¹H NMR(400MHz，

CDCl₃)：d7.6-7.2(m，3H)，5.4(d，1H)，4.6(s，2H)，4.0(d，1H)，3.8(dd，2H)，1.1

(s，9H)；MS(APCI)：264，266 (M+1，M+2).

B. (D) -2-amino-3- (2, 4-dichloro-benzyloxy) propionic acid hydrochloride

The product of example 23, step a, above (14.7g, 40mmol) was stirred in 4M HCl/dioxane (100mL) for about 17 hours. The reaction mixture was concentrated under vacuum to give 12g of a pale yellow solid (100%). Ms (apcl): 265(M + 1).

Example 24

The compound of example 24 having the general structure shown below was synthesized in analogy to the methods described in examples 3C to 3F, using the title mixture of example 21 as starting material:

wherein R is¹is-CH₂-phenyl and R²Is methyl. The R, R and S, R diastereomers (— represents the other stereoisomeric center at the C-3 carbon in the above structure) are separated. Mass spectrum (M +1) ═ 520; mass spectrometry is particle bombardment.

Examples 25 and 26

The compounds of examples 25 and 26 have the general structure shown below:

for examples 25 and 26, where R¹Is phenyl and R²Is methyl; example 25 is the R, R isomer, while example 26 is the S, R isomer. The compounds of examples 25 and 26 were synthesized in analogy to the procedure described for examples 3C to 3F, using the title compound of example 22 as starting material, followed byTwo separate isomers were separated by chromatography. Mass spectrum (M +1) ═ 493 for each sample; mass spectrometry is particle bombardment.

Examples 27 to 159

Examples 27 to 159, listed in the following table, were prepared according to the following scheme by coupling an appropriately substituted pyrazolone-piperidine of formula I (in the following scheme) with (D) -OBnSer derivative II (in the following scheme) in analogy to the procedures described in examples 3E and 3F.

Pyrazolone-piperidines of the general formula I can be prepared in analogy to the methods described in examples 3B and 3C, starting from suitable alkylating agents and alkylhydrazines; (D) -OBnSer derivative II can be prepared following three steps analogous to those described in the methods of example 23A, example 23B and example 5F.

Implementation of isomer R² R¹＝-CH₂-A¹ Ar MS MS

Example No. A¹Method of

27 d 1H 2-pyridylphenyl 493 PB

28 d 1H 4-thiazolyl phenyl 499 PB

29 d 2H 4-Thiazolylphenyl 499 PB

30 d 1H 5-Thiazolylphenyl 499 APCI

31 d1 Me phenyl 2, 4-di-Cl-Ph 574.5 APCI

32 d1 Me phenyl 2, 4-di-F-Ph 542 PB

33 d1 Me phenyl [2, 3-O-CH₂-O]Phenyl 550.2 PB

34 d1 Me phenyl 2-CF₃-Ph 575 PB

35 d1 Me phenyl 2-Me-Ph 520 PB

36 d1 Me phenyl 2-pyridyl 507 PB

37 d1 Me phenyl 3, 4-di-F-Ph 542 PB

38 d1, 2 Me phenyl 3, 5-di-CF₃Ph 642 PB

39 d1 Me phenyl 3, 5-di-Cl-Ph 576 APCI

40 d2 Me phenyl 3-CF₃-Ph 575 APCI

41 d1 Me phenyl 3-Cl-Ph 540 APCI

42 d1 Me phenyl 3-Cl-thiophene 546, 548 APCI

43 d1 Me phenyl 3-F-4-Cl-Ph 560 APCI

44 d1 Me phenyl 3-Me-Ph 520 PB

45 d1 Me phenyl 4-Cl-Ph 540 PB

46 d1 Me phenyl 4-pyridyl 507 PB

47 d1 Me phenyl 4-thiazolyl 513 PB

48 d1 Me phenyl 5-thiazolyl 513 APCI

49 d1, 2 Me Phenylbenzisoxazolyl 547 PB

50 d1 Me phenyl 4-pyrimidinyl 508 PB

51 d1, 2 Me 4-Ph-Ph 4-thiazolyl 589 APCI

52 d1, 2 Me 4-Ph-Ph 2-pyridinyl 583 APCI

53 d1 Me 4-F-Ph phenyl 524 PB

54 d2 Me 4-F-Ph phenyl 524 PB

55 d1 Me 4-F-Ph 3-Cl-Ph 558 PB

56 d2 Me 4-F-Ph 3-Cl-Ph 558 PB

57 d1 Me 4-F-Ph 3，4-di-F-Ph 560 APCI

58 d2 Me 4-F-Ph 3，4-di-F-Ph 560 APCI

59 d1, 2 Me 4-F-Ph 2-pyridinyl 525 APCI

60 d1，2 Me 4-F-Ph 2-CF₃-Ph 592 APCI

61 d1 Me 4-CF₃-Ph 4-Cl-Ph 609 APCI

62 d1，2 Me 4-CF₃-Ph 4-Cl-Ph 609 APCI

63 d1, 2 Me 3-pyridylphenyl 508 PB

64 d1 Me phenyl 3-pyridyl 508 PB

65 d1 Me 2-quinolylphenyl 594 PB

66 d2 Me 2-quinolylphenyl 594 PB

67 d1 Me 2-pyridylphenyl 506 PB

68 d2 Me 2-pyridylphenyl 506 PB

69 d1, 2 Me 2-pyridinyl 3-F-4-Cl-Ph 559, 561 APCI

70 d1 Me 2-pyridyl 3-Cl-thiophene 547, 549 APCI

71 d1 Me 2-pyridinyl 3-CF₃-Ph 575 PB

72 d1，2 Me 2，4-di-F-Ph 3，4-di-F-Ph 579 APCI

73 d1, 2 Me 2, 4-di-F-Ph 2-pyridinyl 544544 PB

74 d1 Me 4-Thiazolylphenyl 513 APCI

75 d2 Me 4-Thiazolylphenyl 513 PB

76 d1 Me 5-Thiazolylphenyl 513 PB

77 d1 Et 2-pyridylphenyl 521 PB

78 d1, 2 Et phenyl 4-thiazolyl 541 APCI

79 d1 Et phenyl 3, 5-di-CF₃-Ph 656 PB

80 d1, 2 Et phenyl 3, 4-di-F-Ph 556 PB

81 d1 Et 2，4-di-F-Ph 2，4-di-F-Ph 593 APCI

82 d2 Et 2，4-di-F-Ph 2，4-di-F-Ph 593 APCI

83 d1 Et 2，4-di-F-Ph 2-CF₃-Ph 625 APCI

84 d2 Et 2，4-di-F-Ph 2-CF₃-Ph 625 APCI

85 d1 Et 2，4-di-F-Ph 3，4-di-F-Ph 593 APCI

86 d2 Et 2，4-di-F-Ph 3，4-di-F-Ph 593 APCI

87 d1 Et 2-pyridinyl 3, 4-di-F-Ph 607 PB

88 d2 Et 2-pyridinyl 3, 4-di-F-Ph 607 PB

89 d1 Et 4-CF₃-Ph 2，4-di-F-Ph 625 APCI

90 d2 Et 4-CF₃-Ph 2，4-di-F-Ph 625 APCI

91 d1 Et 4-CF₃-Ph 3-Cl-Ph 623 APCI

92 d1 Et 4-CF₃-Ph 4-Cl-Ph 623 APCI

93 d2 Et 4-CF₃-Ph 4-Cl-Ph 623 APCI

94 d1 Et 4-CH₃-Ph 3-Cl-Ph 568 APCI

95 d2 Et 4-CH₃-Ph 3-Cl-Ph 568 APCI

96 d1 Et 4-Cl-Ph 3，4-di-F-Ph 590 PB

97 d2 Et 4-Cl-Ph 3，4-di-F-Ph 590 PB

98 d1 Et 4-Cl-Ph 3-5-di-Cl-Ph 622 PB

99 d2 Et 4-Cl-Ph 3-5-di-Cl-Ph 622 PB

100 d1 Et 4-Cl-Ph 3-Cl-Ph 589 PB

101 d2 Et 4-Cl-Ph 3-Cl-Ph 589 PB

102 d1 Et 4-F-Ph 3，4-di-F-Ph 574 PB

103 d2 Et 4-F-Ph 3，4-di-F-Ph 574 PB

104 d1 Et 4-F-Ph 3-Cl-Ph 572 APCI

105 d2 Et 4-F-Ph 3-Cl-Ph 572 APCI

106 d1，2 Et 4-Me-Ph 2-CF₃-Ph 602 APCI

107 d1，2 Et 4-Me-Ph 3，4-di-F-Ph 570 APCI

108 d1，2 CF₃CH₂Phenyl 4-thiazolyl 595 APCI

109 d1 CF₃CH₂Phenyl 3-CF₃-Ph 642.3 APCI

110 d1 CF₃CH₂Phenyl 3, 5-di-Cl-Ph 643 APCI

111 d2 CF₃CH₂Phenyl 3, 5-di-Cl-Ph 644 APCI

112 d1 CF₃CH₂Phenyl 3, 4-di-F-Ph 610.2 APCI

113 d2 CF₃CH₂Phenyl 3, 4-di-F-Ph 610.2 APCI

114 d1 CF₃CH₂Phenyl 3, 5-di-Cl-Ph 643 APCI

115 d2 CF₃CH₂Phenyl 3, 5-di-Cl-Ph 644 APCI

116 d1 CF₃CH₂Phenyl 3-CF₃-Ph 642.3 APCI

117 d1 CF₃CH₂Phenyl 3, 4-di-F-Ph 610.2 APCI

118 d2 CF₃CH₂Phenyl 3, 4-di-F-Ph 610.2 APCI

119 d1，2 CF₃CH₂Phenyl 4-thiazolyl 595 APCI

120 d1，2 CF₃CH₂2, 4-di-Cl-Ph 2-pyridinyl 643 APCI

121 d1，2 CF₃CH₂2, 4-di-Cl-Ph 4-thiazolyl 649 APCI

122 d1 CF₃CH₂ 2，4-F-Ph 2-CF₃-Ph 679 APCI

123 d2 CF₃CH₂ 2，4-F-Ph 2-CF₃-Ph 679 AFCI

124 d1 CF₃CH₂ 2，4-F-Ph 3，4-di-F-Ph 647 APCI

125 d2 CF₃CH₂ 2，4-F-Ph 3，4-di-F-Ph 647 APCI

126 d1，2 CF₃CH₂2, 4-F-Ph 4-thiazolyl 617 PB

127 d1 CF₃CH₂2-pyridinyl 2, 4-di-Cl-Ph 643 APCI

128 d2 CF₃CH₂2-pyridinyl 2, 4-di-Cl-Ph 643 APCI

129 d1 CF₃CH₂2-pyridyl 2, 4-di-F-Ph 611 PB

130 d2 CF₃CH₂2-pyridyl 2, 4-di-F-Ph 611 PB

131 d1 CF₃CH₂2-pyridyl-2-CF₃-4-F-Ph 661 APCI

132 d1 CF₃CH₂2-pyridyl-2-CF₃-Ph 643 PB

133 d2 CF₃CH₂2-pyridyl-2-CF₃-Ph 643 PB

134 d1 CF₃CH₂2-pyridinyl 3, 4-di-F-Ph 611 PB

135 d2 CF₃CH₂2-pyridinyl 3, 4-di-F-Ph 611 PB

136 d1 CF₃CH₂2-pyridinyl 3, 5-di-Cl-Ph 643 APCI

137 d1 CF₃CH₂2-pyridinyl 3-Cl-Ph 609 PB

138 d1 CF₃CH₂2-pyridinyl 3-Cl-thiophene 615, 617 APCI

139 d1，2 CF₃CH₂2-pyridinyl 3-F-4-Cl-Ph 627, 629 AFCI

140 d1 CF₃CH₂2-pyridyl-3-OCF₃-Ph 659 APCI

141 d1 CF₃CH₂2-pyridinyl 4-Cl-Ph 609 PB

142 d2 CF₃CH₂2-pyridinyl 4-Cl-Ph 609 PB

143 d1，2 CF₃CH₂3-pyridyl 2, 4-di-F-Ph 612 APCI

144 d1，2 CF₃CH₂3-pyridyl-2-CF₃-Ph 644 APCI

145 d1，2 CF₃CH₂3-pyridinyl 4-Cl-Ph 610 APCI

146 d1 CF₃CH₂ 4-CH₃-Ph 3-Cl-Ph 622 APCI

147 d2 CF₃CH₂ 4-CH₃-Ph 3-Cl-Ph 622 APCI

148 d1 CF₃CH₂ 4-Cl-Ph 3，4-di-F-Ph 644 PB

149 d2 CF₃CH₂ 4-Cl-Ph 3，4-di-F-Ph 644 PB

150 d1 CF₃CH₂ 4-Cl-Ph 3，5-di-Cl-Ph 675 PB

151 d2 CF₃CH₂ 4-Cl-Ph 3，5-di-Cl-Ph 675 PB

152 d2 CF₃CH₂ 4-Cl-Ph 3-Cl-Ph 642 PB

153 d1 CF₃CH₂ 4-Cl-Ph 3-Cl-Ph 642 PB

154 d1 CF₃CH₂ 4-F-Ph 3，4-di-F-Ph 628 PB

155 d2 CF₃CH₂ 4-F-Ph 3，4-di-F-Ph 628 PB

156 d1 CF₃CH₂ 4-F-Ph 3-Cl-Ph 626 PB

157 d2 CF₃CH₂ 4-F-Ph 3-Cl-Ph 626 PB

158 d1，2 CF₃CH₂ 4-Me-Ph 2-CF₃-Ph 656 APCI

159 d1，2 CF₃CH₂ 4-Me-Ph 3，4-di-F-Ph 624 APCI

Note that: in the above table, the definition of isomer refers to the stereochemistry at the C-3 position (indicated by an "-" in the structure) of the pyrazolone-piperidine group; d1 and d2 refer to chromatographically separated isomers; d1, 2 denotes the isomeric mixture, abbreviations used in the above tables: ph is phenyl; PB is particle bombardment; and APCI is atmospheric pressure chemical ionization. The following is the NMR data for the compounds shown in the table above.

Example 37:¹H NMR(400MHz，d4-MeOH)：d7.2(m，5H)，5.2(t，1H)，4.6(m，3H)，

3.8(d，2H)，3.1(d，1H)，3.0(s，3H)，2.6(dd，2H)，1.6(s，6H).

example 67&68：¹H NMR(300MHz，d4-MeOH)：d 8.85(s，1H)，8.6(t，1H)，8.1(d，

1H)，8.0(t，1H)，7.35(s，5H)，5.15(s，1H)，4.6(bs，3H)，3.85(m，2H)，3.65(m，2H)，

3.2(s，3H)，2.75(m，2H)，1.65(s，6H).

Example 128:¹H NMR(400MHz，d4-MeOH)：d 8.8(s，1H)，8.6(s，1H)，8.5(t，1H)，

7.96(t，1H)，7.9(d，1H)，7.45(d，1H)，7.33(d，1H)，5.2(s，1H)，4.6(s，3H)，4.4(m，

1H)，4.2(m，2H)，3.9(m，4H)，3.5(m)，3.2 (m，2H)，2.8(dd，2H)，1.6(s，6H).

example 129&130：¹H NMR(400MHz，d4-MeOH)：d 8.76(s，1H)，8.50(t，1H)，

7.92(dt，2H)，7.43(q，1H)，6.90(t，1H)，5.20(m，1H)，4.90(m)，4.30(m，1H)，4.20(m，

1H)，3.7-3.4(m)，3.30(s，2H)，3.20(m，1H)，2.80(dd，2H)，1.60(s，6H).

Example 137:¹H NMR(300MHz，d4-MeOH)：d8.7(1，1H)，8.45(t，1H)，7.9(t，2

H)，7.25(m，4H)，5.2(m，1H)，4.95(d，1H)，4.6(s，2H)，4.3(m，1H)，3.8(t，2H)，3.5

(dd，2H)，2.8(m，1H)，2.8(dd，2H)，1.6(s，6H).

example 138:¹H NMR(400MHz，d4-MeOH)：d8.8(dd，1H)，8.6(s，1H)，8.5(t，1H)，

7.95(t，1H)，7.9(s，1H)，7.3(s，1H)，7.0(s，1H)，5.2(s，1H)，4.85(s，3H)，4.4(m，

1H)，4.18(m，1H)，3.8(m，2H)，3.5(dd，2H)，3.2(d，2H)，2.8(dd，2H)，1.6(s，6H).

example 141&142：¹H NMR(300MHz，d4-MeOH)：d 8.75(m，1H)，8.5(m，1H)，

7.9(m，2H)，7.3(s，2H)，5.2(m，1H)，4.65(m，1H)，4.55(s，2H)，4.35(m，1H)，

4.20(m，1H)，3.8(t，1H)，3.5(dd，2H)，3.15(d，1H)，2.8(dd，2H)，1.6(s，2H).

Example 160-

Example 160-179, shown in the table below, was prepared by coupling the appropriate substituted pyrazolone-piperidine (I) (in scheme) with the (D) -Trp derivative (III) (see example 2C) in a manner analogous to that described in examples 3E and 3F, according to the scheme shown below.

Practice of

Example isomer R² R¹＝-CH₂-A¹MS MS method

160 d1 Me 4-CF₃-Ph 584 APCI

161 d1，2 Me 4-CF₃-Ph 584 APCI

162 d1 Me 4-F-Ph 533 PB

163 d2 Me 4-F-Ph 533 PB

164 d1 Me 4-Ph-Ph 591 APCI

165 d1，2 Et 2，4-di-Cl-Ph 597 APCI

166 d1，2 Et 2，4-F-Ph 566 APCI

167 d1 Et 4-CF₃-Ph 598 APCI

168 d1，2 Et 4-CF₃-Ph 598 APCI

169 d1 Et 4-Cl-Ph 563 PB

170 d2 Et 4-Cl-Ph 563 PB

171 d1，2 Et 4-F-Ph 547 APCI

172 d1，2 Et 4-Me-Ph 543 APCI

173 d1，2 CF₃CH₂ 2，4-di-Cl-Ph 651.5 APCI

174 d1，2 CF₃CH₂ 2，4-di-F-Ph 620 APCI

175 d1 CF₃CH₂ 4-Cl-Ph 617 PB

176 d2 CF₃CH₂ 4-Cl-Ph 617 PB

177 d1 CF₃CH₂ 4-F-Ph 601 AFCI

178 d2 CF₃CH₂ 4-F-Ph 601 APCI

179 d1，2 CF₃CH₂ 4-Me-Ph 597 APCI

Note that: in the above table, the definition of isomer refers to the stereochemistry at the C-3 position (indicated by an "+" in the structure) of the pyrazolone-piperidine group; d1 and d2 refer to chromatographically separated isomers; d1, 2 denotes the isomer mixture.

Example 180-

Prepared by coupling the appropriate substituted pyrazolone-piperidines I with the acid intermediate IV according to the scheme shown below, in analogy to the methods described in examples 3E and 3F

Example 180-183, shown in the table below.

Acid Intermediate (IV) was prepared by treating the amino acid with the product from example 5D using the established procedure described in example 5F.

EXAMPLE A¹

Example isomer R² (R¹＝-CH₂-A¹) Ar MS method

180 d1, 2 Me phenyl (CH)₂)₂Ph 504 PB

181 d1, 2 Me phenyl SCH₂Ph 559 PB

182 d1 Me phenyl 2-naphthyl 527 APCI

183 d1, 2 Me PhenylCH₂O-(4-F-Ph) 524 PB

Example 184

2-amino-N- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pir-zine Azolo [4, 3-c ] s]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl]-2-methyl Propionamide L-tartrate salt

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

To a solution of 4-oxo-piperidine-3-carboxylic acid ethyl ester hydrochloride (100g, 0.482mol) in IPE (725mL) and water (360mL) was added TEA (63.5g, 0.627mol) slowly followed by (Boc)₂O (115.7g, 0.53 mol). The mixture was stirred under nitrogen overnight. The organic phase was separated, washed with water and dried over sodium sulfate and concentrated in vacuo to give the desired product as a crystalline form (142.9g, 109% yield, containing a small amount of IPE).

B. 3-benzyl-4-oxo-piperidine-1, 3-dicarboxylic acid 1-tert-butyl 3-ethyl ester

To a solution of 4-oxo-piperidine-1, 3-dicarboxylic acid 1-tert-butyl ester 3-ethyl ester (73.36g, 0.27mol) in DMF (734mL) was added lithium carbonate (50g, 0.676mol), followed by benzyl bromide (55.44g, 0.324 mol). The mixture was heated to about 60 ℃ and stirred for about 20 hours. The reaction mixture was then cooled to room temperature and extracted with IPE, washed with water and dried over magnesium sulfate. The crude solid product was obtained after filtration and concentration in vacuo. The crude product was recrystallized from hexane to give a white solid (33.6g, yield 38.2%).

C. 3 a-benzyl-2-methyl-2, 3a, 4, 5, 6, 7-hexahydropyrazolo [4, 3-c)]Pyridin-3-ones

To a solution of 1-tert-butyl 3-ethyl 3-benzyl-4-oxo-piperidine-1, 3-dicarboxylate (1935.97g, 5.36mol) in toluene (9700mL) at about 8 deg.C was slowly added methyl hydrazine (299.2mL, 5.63mol) followed by acetic acid (325mL, 5.68 mol). The mixture was slowly heated to about 65 ℃ and stirred for about 7.5 hours. After cooling to room temperature, the organic layer was washed with 10% sodium bicarbonate, water and saturated sodium chloride solution and dried to a small volume in vacuo. The reaction was repeated twice on the same scale, and the concentrated product solutions from the three reactions were combined and combined with IPE (50L), cooled to about 0 ℃ again, and hydrogen chloride gas was repeatedly introduced and stirred at room temperature overnight until complete deprotection. The mixture was concentrated in vacuo to approximately half the initial volume, dichloromethane (24L) was added, followed by NH₄OH (22L). The mixture was then extracted with dichloromethane and concentrated to a small volume (6 to 7L). After addition of hexane (20L), the mixture was cooled to about 15 to 20 ℃. The free base product was collected as crystals and then dried in vacuo (2985 g total, 84.8% yield).

D. 3a- (R) -benzyl-2-methyl-2, 3a, 4, 5, 6, 7-hexahydropyrazolo [4, 3-c]Pyridine-3- Ketone L-tartrate salt

L-tartaric acid (67.55g, 0.45mol) was added to a solution of 3 a-benzyl-2-methyl-2, 3a, 4, 5, 6, 7-hexahydropyrazolo [4, 3-c ] pyridin-3-one (100g, 0.41mol) in a mixture of acetone/water (970mL/120mL), the mixture was heated to about 50 ℃ and stirred overnight, after which the reaction mixture was cooled to about 10 to 15 ℃, the resulting precipitate was filtered off, washed with cold acetone/water and dried under vacuum to give the product as a white solid (157.8g, yield 97.83%, 99% ee).

E. 2-Boc-amino-2-methyl-propionic acid

2-aminoisobutyric acid (140g, 1.36mol), 1N sodium hydroxide (1620mL, 1.63mol), (Boc)₂O (375mL, 1.63mol) and THF (420mL) were mixed together and stirred at room temperature overnight, the reaction mixture was diluted with ethyl acetate (700mL) and the pH was adjusted to 3.0 by the addition of 6N hydrochloric acid. The organic phase was separated, washed with saturated sodium chloride solution and concentrated to about the initial volume of 1/4, and after treatment with hexane, the product was isolated and collected as a white solid (125.8g, yield 45.44%). An additional 7.8g of product was recovered from the mother liquor,

F. 2, 5-dioxo-pyrrolidin-1-yl 2-Boc-amino-2-methylpropionate

EDC (100.09g, 0.522mol) was added with stirring to a solution of 2-tert-butoxycarbonylamino-2-methyl-propionic acid (100g, 0.492mol) and succinic anhydride (60.02g, 0.522mol) in dichloromethane (1000mL) under nitrogen, and then the reaction mixture was diluted with ethyl acetate (1L), washed with saturated sodium bicarbonate solution and water, then concentrated to a small volume under vacuum, white crystals precipitated from the solution, which were collected by filtration and dried under vacuum to give the product (104.9g +27.3g, yield 89.5%).

G. 3- (R) -benzyloxy-2- (2-tert-butoxycarbonylamino-2-methyl-propionylamino) propionic acid

A solution of 2, 5-dioxo-pyrrolidin-1-yl 2-tert-butoxycarbonylamino-2-methylpropionate (33.94g, 0.113mol) in THF (407mL) was added to a solution of 2-amino-3-benzyloxy-propionic acid (26.2g, 0.113mol) in water (101.8mL) and TEA (28.53g, 0.282mol)In the liquid. The mixture was stirred under nitrogen overnight at room temperature, then 10% citric acid solution (500mL) was added and after stirring the mixture for another 10 minutes, it was diluted with ethyl acetate (500 mL). The organic phase was separated from the mixture, washed with water and saturated sodium chloride solution, then concentrated in vacuo to give a thick oil, which was crude treated with IPE/hexane (50/50) and cooled to about 10 ℃ to give the product as a white solid (42.3g, 98.4% yield). H.{1- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4，3-c]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2 oxo-ethylcarbamoyl]- 1-methyl-ethyl } carbamic acid tert-butyl ester

TEA (8.43mL, 0.0605mol) was added to a solution of 3a- (R) -benzyl-2-methyl-2, 3a, 4, 5, 6, 7-hexahydropyrazolo [4, 3-c ] pyridin-3-one, L-tartrate (10.81g, 0.0275mol) in ethyl acetate (216.2mL) at about-66 ℃ and the mixture was stirred for about 1.5 hours. After removal of precipitated salts by filtration, 3-benzyloxy-2- (2-tert-butoxycarbonylamino-2-methyl-propionylamino) propionic acid (8.7g, 0.0229mol) and TEA (19.15mL, 0.1374mol) were added at about-35 ℃; then, a 50% solution of PPAA in ethyl acetate (27.5mL, 0.0458mol) was added dropwise. Stirring the mixture at a temperature of between about-20 and-27 ℃ for about 2 hours; then, stirring was continued for a further 1.5 hours, during which the temperature slowly rose back to about 0 ℃. The reaction mixture was poured into water and extracted with IPE, washed with 7% sodium chloride solution and concentrated under vacuum. The crude oil was crystallized by treatment with IPE/hexanes (50/50) to give the product as a white solid (10.3g, 74.3% yield).

I. 2-amino-N- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro- Pyrazolo [4, 3-c]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl]-2-methyl Cyclopropyl amides

Adding TFA (35mL) to a solution of tert-butyl {1- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethylcarbamoyl ] -1-methyl-ethyl } carbamate (10.3g, 0.017mol) in dichloromethane (68.6mL) at about 0 to 5 ℃ to maintain the temperature below about 5 ℃; then, the temperature was raised to room temperature. The mixture was stirred for about 3 hours, and dichloromethane was used as a solvent instead of ethyl acetate. The mixture was then adjusted to a pH of about 8 with saturated sodium bicarbonate solution, washed with saturated sodium chloride solution and concentrated to a small volume under vacuum. After treating the mixture with IPA and then hexane, the product was obtained as a white solid (7.4g, 86.1% yield). HPLC showed the product to contain 0.2% diastereomer.

J. 2-amino-N- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro- Pyrazolo [4, 3-c]Pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl]-2-methyl L-tartrate salt of propionamide

L- (+) -tartaric acid (114.5g, 0.761mol) was added to a solution of 2-amino-N- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl ] -2-methylpropanamide from step I (385g, 0.761mol) in methanol (4000mL) and the mixture was stirred overnight. The cloudy solution thus formed was filtered to give a clear solution, which was concentrated to remove most of the solvent. After addition of ethyl acetate (12L total), the remaining methanol was azeotropically removed between about 63 and 72 ℃. The solid separated was dissolved in ethyl acetate and the resulting solution was refluxed for about 16 hours and then cooled to room temperature overnight. The product was collected as a white solid (482.3g, yield 96.8%).

M.P.174-176℃。

Example 185

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

A. 4-oxo-3-pyridin-2-ylmethyl-piperidine-1, 3-dicarboxylic acid 1-tert-butyl 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 ℃. After stirring at about 0 ℃ for about 2 hours, the ice bath was removed and DABCO (973mg, 8.68mmol) was added. After stirring the reaction mixture for about 30 minutes, it was poured into a mixture of water and IPE. The organic layer was separated, washed with saturated aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution and dried over sodium sulfate, and concentrated in vacuo. The crude residue was crystallized from hexane to give a white solid (8.19g, yield 65%).

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(br s，1H)，7.17(d，1H)，7.54(m，1H)，8.40(s，1H).

B. 3-oxo-3 a-pyridin-2-ylmethyl-2- (2, 2, 2-trifluoro-ethyl) -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)₃CH₂NHNH₂(325mL, 1.986mol) (from Aldrich) in water. To a solution of the product of step A (600g, 1.655mol) in toluene (900mL) was added first the combined toluene extracts containing anhydrous 2, 2, 2-trifluoroethylhydrazine and then acetic acid (121.4g, 1.986 mol). After heating the reaction mixture at about 70 ℃ for about 2 hours, the remaining toluene extract (50g) of 70% aqueous 2, 2, 2-trifluoroethylhydrazine solution was added. After heating the reaction mixture at about 80 ℃ for about 3.5 hours, it was cooled to room temperature and diluted with saturated aqueous sodium bicarbonate (2L). The toluene layer was separated, washed with saturated aqueous sodium chloride, dried over sodium sulfate 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(br s，2H)，2.88(br s，1H)，3.31(m，2H)，3.97(m，1H)，4.19(m，1H)，4.46(brs，1H)，4.63(br s，1H)，7.06(m，2H)，7.51(m，1H)，8.34(m，1H).

C. 3 a-pyridin-2-ylmethyl-2- (2, 2, 2-trifluoroethyl) -2, 3a, 4, 5, 6, 7-hexahydropyrazole And [4, 3-c ]]Pyridin-3-ones

Methanesulfonic acid (11.6g, 120mmol) was added dropwise to a solution of the product of step B (10g, 24.2mmol) in dichloromethane (100mL) over approximately 30 minutes. The reaction mixture was stirred for about 1 hour, then cooled to about 0 ℃ and triethylamine (18.6mL, 133.1mmol) was added via addition funnel. The reaction mixture was allowed to warm to room temperature over about 1 hour, diluted with additional dichloromethane and washed with saturated aqueous sodium chloride, dried over sodium sulfate and concentrated in vacuo to afford the desired product as a white solid (7.2 g).

¹H-NMR (CDCl₃)δ：2.51-2.72(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).

D. 3 a-pyridin-2-ylmethyl-2- (2, 2, 2-trifluoroethyl) -2, 3a, 4, 5, 6, 7-hexahydropyrazole And [4, 3-c ]]Pyridin-3-one (D) -tartrate salt

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

E. 2-Boc-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 N-Boc- (D) -serine (452g, 2.2026mol) formed in a mixture of THF (7L) and DMF (3L) at about 0 deg.C. The reaction mixture was stirred at about 0 ℃ for about 30 minutes, then 2, 4-difluorobenzyl bromide (456.5g, 2.2051mol) was added. After the temperature was raised to room temperature, the reaction mixture was concentrated under vacuum to remove THF. The reaction mixture was partitioned between 4.5L of water and 4.5L of IPE. After the layer separation, the pH of the aqueous layer was adjusted to about 3 with 1N hydrochloric acid. The aqueous layer was extracted twice more with 4L IPE each time. The organic solution was then dried over sodium sulfate and concentrated in vacuo to give 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(br s，1H)，4.54(s，2H)，5.34(m，1H)，6.78(m，1H)，6.84(m，1H)，7.30(m，1H).

F. 2-amino-3- (2, 4-difluoro-benzyloxy) -propionic acid methanesulfonate salt

Methanesulfonic acid (1.72g, 17.95mmol) was added via syringe to a solution of the product of step E (1.19g, 3.59mmol) in dichloromethane/IPE (1: 1, 12mL) over about 10 minutes, and a solid precipitated out of solution immediately. After about 1h, the solid was filtered off and washed with a dichloromethane/IPE mixture (1: 1) to give 939mg of product (yield 80%).

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

2, 5-dioxo-pyrrolidin-1-yl 2-tert-butoxycarbonylamino-2-methylpropionate (438mg, 1.46mmol) and triethylamine (369mg, 3.65mmol) were added to a solution of the product of 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, the organic layer was separated, washed with saturated aqueous sodium chloride, dried over sodium sulfate and concentrated in vacuoTo obtain a foam. (534.1mg, yield 88%).¹H-NMR(CD₃OD)：δ1.38(br s，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)

H. (1- {1- (2, 4-difluoro-benzyloxymethyl) -2-oxo-2- [ 3-oxo-3 a-pyridin-2-yl Methyl-2- (2, 2, 2-trifluoro-ethyl) -2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c]Pyridine (II) Pyridin-5-yl]-Ethylcarbamoyl } -1-methyl-ethyl) carbamic acid tert-butyl ester

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

(b) The product of step G (425G, 1.02mol) was added to a solution of the product of step H (a), triethylamine (654mL, 4.69mol) and PPAA (50% in ethyl acetate, 916mL, 1.53mol) in ethyl acetate at about-30 ℃. The reaction mixture was stirred for about 1 hour, washed with water and saturated aqueous sodium chloride, dried over sodium sulfate and concentrated under vacuum to give the product as an oil (636g, 87.8% yield).

I. 2-amino-N- {1- (2, 4-difluoro-benzyloxymethyl) -2-oxo-2- [ 3-oxo-3 a-pyri-dine Pyridin-2-ylmethyl-2- (2, 2, 2-trifluoro-ethyl) -2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4，3-c]Pyridin-5-yl]-ethyl radical]-2-methylpropionamide

Methanesulfonic acid (258.3mL, 3.98mol) was added dropwise to a solution of the product of step H (566g, 0.796mol) in dichloromethane (11320mL) over about 55 minutes at about 15 deg.C in a 22L round bottom flask equipped with a mechanical stirrer, dried and purged with nitrogen. The mixture was stirred at about 20 ℃ for about 40 minutes, then saturated aqueous sodium bicarbonate (8490mL) was added until the pH was about 7.8. The organic layer was separated, washed with water and saturated aqueous sodium chloride, dried over sodium sulfate and concentrated under vacuum to give the product as an oil (388.8g, yield 80%).

J. 2-amino-N- {1- (2, 4-difluoro-benzyloxymethyl) -2-oxo-2- [ 3-oxo-3 a-pyri-dine 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-methylpropionamide L- (+) tartrate

In a dry and nitrogen purged 12L round bottom flask equipped with a mechanical stirrer, L- (+) -tartaric acid (90g, 0.6mol) was added to a solution of the product of step I (370g, 0.6mol) in methanol (4070 mL). The reaction mixture was stirred at about 22 ℃ for about 90 minutes, filtered and concentrated. The crude product was then diluted with ethyl acetate (4560mL), heated at about 70 ℃ and then slowly cooled to room temperature over about 17 hours. The resulting solid was filtered off and dried to give white crystals. mp188-189 deg.C (348.46g, 76% yield).

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).

Example A

The following is the result of the "female rat study" described hereinabove, in which the GH secretagogue compound 2-amino-N- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1- (R) - (benzyloxymethyl) -2-oxo-ethyl ] isobutyramide L-tartrate was administered to rats.

TABLE 1

Daily administration of GHRP, mean plasma insulin and metabolite levels after march

Non-fasting blood samples from the sacrificed rats were collected. Asterisks indicate distinct values from the corresponding excipient-treatment groups.

(p＜0.05)

	Dosage form	Insulin	Glucose	Lactic acid	Cholesterol	Triglyceride
	Dosage form	Insulin	Glucose	Lactic acid	Cholesterol	Triglyceride	Surgery	(mg/kg)	(uU/mL)	(mg/dl)	(mg/dl)	(mg/dl)	(mg/dl)
Sham	Excipient	118.8	181.7	4.6	97.9	254.8	Surgery	(mg/kg)	(uU/mL)	(mg/dl)	(mg/dl)	(mg/dl)	(mg/dl)
Sham	Excipient	118.8	181.7	4.6	97.9	254.8	Sham	0.5	94.9	^*142.7	3.7	95.4	219.6
Sham	5.0	95.7	^*139.9	^*3.2	^*80.6	227.2	Sham	0.5	94.9	^*142.7	3.7	95.4	219.6
Sham	5.0	95.7	^*139.9	^*3.2	^*80.6	227.2
Ovx	Excipient	112.8	194.0	3.9	106.8	182.7
Ovx	Excipient	112.8	194.0	3.9	106.8	182.7	0vx	0.5	^*78.7	179.7	3.6	92.5	181.9
Ovx	5.0	^*84.1	177.2	3.1	102.2	158.4	0vx	0.5	^*78.7	179.7	3.6	92.5	181.9

The data in table 1 show that the above treatments are associated with dose-dependent reductions in plasma glucose and/or insulin levels, consistent with improved glycemic control and insulin sensitivity by the above treatments; the above treatments are also associated with a tendency to decrease plasma lactate, cholesterol and triglycerides, which is also consistent with an improved effect on lipid structure and metabolic control as a result of the increased insulin sensitivity induced by the above treatments.

Claims

1. Use of an effective amount of a compound of formula I, or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer, or a pharmaceutically acceptable salt or prodrug thereof, for the preparation of a medicament for treating insulin resistance in a mammal:

wherein

e is 0;

n and w are each independently 1;

y is oxygen;

R¹is hydrogen, - (C)₁-C₁₀) Alkyl or- (CH)₂)_t-A¹，

Wherein t is 0, 1, 2 or 3;

R²is hydrogen or (C)₁-C₈) An alkyl group, a carboxyl group,

wherein R is²Alkyl in the definition is optionally substituted by CF₃Substitution;

R³is- (C)₁-C₆) alkyl-A¹Or- (C)₁-C₅) alkyl-X¹-(C₀-C₅) alkyl-A¹，

Wherein X¹Is O;

R⁴is hydrogen or (C)₁-C₆) An alkyl group;

X⁴is hydrogen or (C)₁-C₆) An alkyl group;

R⁶is that

Wherein a and b are independently 0;

X⁵and X^5aEach independently is (C)₁-C₆) An alkyl group;

Z¹is a bond;

R⁷and R⁸Independently is hydrogen or (C)₁-C₆) An alkyl group;

in each case A¹Independently phenyl, or a partially saturated, fully saturated or fully unsaturated 4-to 8-membered ring optionally containing 1 to 4 heteroatoms independently selected from oxygen, sulfur and nitrogen, a bicyclic ring system consisting of a partially saturated, fully saturated or fully unsaturated 5-or 6-membered ring optionally containing 1 to 4 heteroatoms independently selected from nitrogen, sulfur and oxygen;

in each case A¹Independently optionally substituted, the number of substituents being up to 3, an

Each substituent is independently selected from F, Cl, Br, I, CF₃，CH₃Or a phenyl group.

2. The use according to claim 1, wherein the compound of the general formula I is a compound of the formula or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof,

wherein

R²Is methyl, A¹Is 2-pyridyl, and R³is-CH₂-O-CH₂-a phenyl group;

R²is-CH₂CF₃，A¹Is 2-pyridyl, and R³is-CH₂-O-CH₂-3-chloro-phenyl;

R²is-CH₂CF₃，A¹Is 2-pyridyl, and R³is-CH₂-O-CH₂-4-chloro-phenyl;

R²is-CH₂CF₃，A¹Is 2-pyridyl, and R³is-CH₂-O-CH₂-2, 4-dichloro-phenyl;

R²is-CH₂CF₃，A¹Is 2-pyridyl, and R³is-CH₂-O-CH₂-3-chloro-thiophene; or

R²is-CH₂CF₃，A¹Is 2-pyridyl, and R³is-CH₂-O-CH₂-2, 4-difluoro-phenyl.

3. The use according to claim 1, wherein the compound of formula I, or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof, is a 3a (R, S), 1(R) diastereomeric mixture, 3a (R), 1(R) diastereomer or 3a (S), 1(R) diastereomer of a compound selected from the group consisting of:

2-amino-N- [1- (3 a-benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridine-5-carbonyl) -4-phenylbutyl ] isobutyramide;

2-amino-N- [2- (3 a-benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1- (1H-indol-3-ylmethyl) -2-oxo-ethyl ] isobutyramide;

2-amino-N- [2- (3 a-benzyl-2-ethyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1- (1H-indol-3-ylmethyl) -2-oxo-ethyl ] isobutyramide;

2-amino-N- [2- [3a- (4-fluoro-benzyl) -2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl ] -1- (1H-indol-3-ylmethyl) -2-oxo-ethyl ] isobutyramide;

2-amino-N- [2- (3 a-benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1-benzyloxymethyl-2-oxo-ethyl ] isobutyramide;

2-amino-N- [2- (3 a-benzyl-2-ethyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1-benzyloxymethyl-2-oxo-ethyl ] isobutyramide;

2-amino-N- {2- [3 a-benzyl-3-oxo-2- (2, 2, 2-trifluoro-ethyl) -2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl ] -1-benzyloxymethyl-2-oxo-ethyl } isobutyramide;

2-amino-N- { 1-benzyloxymethyl-2- [ (3a- (4-fluoro-benzyl) -2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -2-oxo-ethyl ] isobutyramide;

2-amino-N- [2- (3 a-benzyl-2-tert-butyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1-benzyloxymethyl-2-oxo-ethyl ] isobutyramide;

2-amino-N- [2- (3 a-benzyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1-benzyloxymethyl-2-oxo-ethyl ] isobutyramide.

4. Use according to claim 3, wherein the compound is 2-amino-N- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl ] isobutyramide L-tartrate.

5. The use according to claim 1, wherein the compound of formula I or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof, is a 3a- (R, S), 1- (R) diastereomeric mixture, 3a- (R), 1- (R) enantiomer or 3a- (S), 1- (R) enantiomer of a compound selected from the group consisting of:

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

2-amino-N- {1- (3-chloro-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;

2-amino-N- {1- (4-chloro-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;

2-amino-N- {1- (2, 4-dichloro-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;

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

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.

6. Use of an effective amount of a compound of formula I or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof, in combination with growth hormone releasing hormone or a functional analog thereof, according to claim 1, for the preparation of a medicament for the treatment of insulin resistance in a mammal.

7. A pharmaceutical composition for the treatment of insulin resistance in a mammal, said composition comprising a pharmaceutically acceptable carrier and an effective amount of a compound of formula I or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof,

wherein a and b are independently 0;

X⁵and X^5aEach independently is (C)₁-C₆) An alkyl group;

Z¹is a bond;

R⁷and R⁸Independently is hydrogen or (C)₁-C₆) An alkyl group;

in each case A¹Independently optionally substituted, the number of substituents being up to 3, and each substituent being independently selected from F, Cl, Br, I, CF₃，CH₃Or a phenyl group.

8. Use of an effective amount of a functional somatostatin antagonist and a compound of formula I, or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof, for the preparation of a medicament for increasing the endogenous growth hormone level in a human or other mammal;

wherein a and b are independently 0;

X⁵and X^5aEach independently is (C)₁-C₆) An alkyl group;

Z¹is a bond;

R⁷and R⁸Independently is hydrogen or (C)₁-C₆) An alkyl group;

9. Use of an effective amount of a functional somatostatin antagonist and a compound of formula I, or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof, for the preparation of a medicament for the treatment or prevention of congestive heart failure, obesity or age-related weakness in a mammal:

wherein a and b are independently 0;

X⁵and X^5aEach independently is (C)₁-C₆) An alkyl group;

Z¹is a bond;

R⁷and R⁸Independently is hydrogen or (C)₁-C₆) An alkyl group;

10. The use according to claim 9, wherein the functional somatostatin antagonist is an alpha-2 adrenergic agonist.

11. The use according to claim 10, wherein the alpha-2 adrenergic agonist is selected from the group consisting of clonidine, xylazine and medetomidine.

12. The use according to claim 11, wherein the compound of formula I is 2-amino-N- [2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl ] isobutyramide L-tartrate.

13. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an alpha-2 adrenergic agonist and a compound of formula I or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof,

wherein a and b are independently 0;

X⁵and X^5aEach independently is (C)₁-C₆) An alkyl group;

Z¹is a bond;

R⁷and R⁸Independently is hydrogen or (C)₁-C₆) An alkyl group;

14. The use according to claim 1, wherein the disease associated with insulin resistance is type I diabetes, type II diabetes, hyperglycemia, glucose intolerance or insulin resistance syndrome or state.

15. The use according to claim 1, wherein the disease associated with insulin resistance is associated with obesity or old age.

16. A process for the preparation of a compound of the general formula k,

Free base

(g)

wherein Prt is an amino protecting group.

17. The method of claim 16, wherein the peptide coupling agent is 1-propanephosphonic acid cyclic anhydride, and the compound of formula g has the R configuration, the compound of formula j has the R configuration, and the compound of formula k has the 3a- (R), 1- (R) configuration.

18. A process for the preparation of a compound of the general formula Z,

l-tartrate salt

Free base

(g)

With compounds of the formula j

To produce a compound of formula k;

deprotection of a compound of formula k to produce a compound of formula I;

19. The method of claim 18, wherein the peptide coupling agent is 1-propanephosphonic acid cyclic anhydride, and the compound of formula g has the R configuration, the compound of formula j has the R configuration, and the compounds of formulae k, I and Z all have the 3a- (R), 1- (R) configuration.

20. A process for the preparation of a compound of the general formula g,

free base

(g)

L-tartrate salt

(f)

Reaction with a base produces a compound of general formula g in which the habit of the benzyl group is retained.

21. A process for the preparation of a compound of the general formula c,

22. A process for the preparation of a compound of the general formula f,

l-tartrate salt

(f)

Reacting with L-tartaric acid.

23. A mixture of the R, S-enantiomers, the R-enantiomer or the S-enantiomer of a compound of the formula,

wherein Prt is hydrogen or an amino protecting group.

24. Use of an effective amount of a compound of formula I, or a stereoisomeric mixture, diastereomerically enriched, diastereomerically pure, enantiomerically enriched or enantiomerically pure isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof, for the preparation of a medicament for the treatment of sleep disorders in a mammal:

wherein a and b are independently 0;

X⁵and X^5aEach independently is (C)₁-C₆) An alkyl group;

Z¹is a bond;

R⁷and R⁸Independently is hydrogen or (C)₁-C₆) An alkyl group;

in each case A¹Independently phenyl, or a partially saturated, fully saturated or fully unsaturated 4-to 8-membered ring optionally containing 1 to 4 heteroatoms independently selected from oxygen, sulfur and nitrogen, a bicyclic ring system, a process for the preparation of a medicament, a medicament or a medicamentThe bicyclic ring system is composed of a partially saturated, fully saturated or fully unsaturated 5-or 6-membered ring optionally containing 1 to 4 heteroatoms independently selected from nitrogen, sulfur and oxygen;