Indian Patents
Title of Invention

"A BRANCHED CHAIN AMINO ACID-DEPENDENT AMINOTRANSFERASE INHIBITORS COMPOUND"
Abstract A COMPOUND OF FORMULA IV. R1-R4 are hydrogen or alkyl; X is NR5 or O: R5 is hydrogen or alkyl, R6 is hydrogen, alkyl. benzyl. alkanoyl. alkoxyalkanoyl. aryJalkyl. alkoxy. cyclonikyl, allyl, alkylcycloalkyl, alkoxy, cycloalkyl, alkylcycloalkyl. trisubstituted halogenalkyl. and wherein R1-R4 are each hydrogen the R6 is not hydrogen or methyl; or a pharmaceutically acceptable salt, ester, prodrug. or amide.thereof.,.
Full Text BACKGROUND OF THE INVENTION
More than 14 million peope.in the United States have diabetes. All people with diabetes are at risk of retinal complications. However, people with type 1, i.e.. insulin-dependent diabetes, face a greater risk of severe vision loss than people with type II. i.e.. non-insulin dependent diabetes.
Retinopathy is any non-inflammatory disease of the retina. Diabetic retinopathy is any retinopathy associated with any form of diabetes mellitus.
Initially, the high blood glucose level in diabetic people causes an increase in growth factors in their eyes. This condition is known as the "pre-diabctic retinopathy stage" and can lead to full diabetic retinopathy. if not prophylactically treated.
Retinopathy will affect the majority of diabetic people to some extent during their lifetimes. It is the leading cause of blindness in Americans of age 20 to 74 today, and is expected to impair vision in approximately one-third of diabetic people in the United States. Each year in the United States, as many as 40.000 new cases of blindness occur among diabetic people (CDC. unpublished data. 1993). Diabetic people arc 25 times more I'kely than the general population to become blind due to retinopathy.
Diabetic retinopathy has two stages-a nonproliferative stage, which typically occurs first, and a proliferativc stage. The nonproliferative stage, which is also referred to as "background diabetic retinopathy." is characterized by thickening of the basement membrane, loss of retinal pericytes, microvascular abnormalities, intrarctinal microaneurysms. retinal hemorrhages (also known as "dot blot" hemorrhages), retinal edema, in particular diabetic macular edema, capillary closure associated with retinal ischemia or poor retinal perfusion (i.e., poor vessel development) and soft and hard exudates. The proliferative stage, which affects an estimated 700.000 Americans (Chen et aL J. Miss. State Med. Assoc. 36(7): 201-208 (1995)). is characterized by neovascularization and fibrovascular growth (i.e., scarring involving glial and fibrous elements) from the retina or optic nerve over the inner surface of the retina or disc or into the vitreous cavity.
The proliferative sthge can lead to rubeotic or neovascular glaucoma. Macular edema can occur in either stage and it, along with complications from retinal neovascularization, are the two major retinal problems that cause the diabetes-related vision loss.
While the pathological stages of diabetic retinopathy are well-described, the molecular events underlying diabetic retinopathy are poorly understood. This is due, in part, to the fact that the disease progresses over ten to thirty years, depending on a given individual.
Tight control of glycemia and hypertension and ophthalmic screening of diabetics appears beneficial in preventing the disease. Current treatment consists of regular observation by an ophthalmologist, laser photocoagulation and vitrectomy.
Macular edema threatening or involving the macular center is treated with local macular photocoagulation. Small (50 mu in diameter), mild-intensity laser burns arc targeted at areas of leakage in the macula (Murphy, Amer. Family Physician 51(4): 785-796 (1995)). If the macular edema persists, retreatment may be necessary.
Patients with severe to very severe nonprolifcrative retinopathy and patients, who are at high risk for proliferative retinopathy or who already have early or advanced proliferative rctinopathy. arc treated with scatter or panretinal photocoagulation. Panretinal photocoagulation involves 1.500-2.000 laser bums, which are 500 mu in diameter, in the midperiphcral and peripheral portion of the retina (Murphy (1995). supra).
The best-documented biochemical mechanism for the development of microvascular complications of diabetes is the sorbitol pathway. In the sorbitol pathway. the enzyme aldose reductase catalyzes the conversion of glucose to sorbitol. and of" galactose to galactitol. Aldose reductase has a low substrate affinity for glucose. Accordingly, when glucose concentrations arc normal, the pathway is inactive. During hyperglycemia, the sorbitol pathway becomes active. Activation of the sorbitoi pathway is important for retinal pericytes, for example, which do not require insulin for glucose penetration. Similarly, retinal capillary celte'appear to contain substantial amounts of aldose reductase (Ferris, Hospital Practice: 79-89 (May 15, 1993).
Given the prevalence of diabetic retinopathy, there remains a need for an effective prophylactic and therapeutic treatment of the disease. Accordingly, it is a principal object of the present invention to provide a method of prophylactically and therapeuiically
treating diabetic retinopathy, including treatment at the pre-diabetic retinopathy stage, the nonproliferative diabetic retinopathy stage, and the proliferative diabetic retinopathy stage. This and other objects of the present invention will become apparent from the detailed description provided herein.
SUMMARY OF THE INVENTION The present invention is directed to a method for the prophylactic and therapeutic treatment of diabetic retinopathy, including treatment at the pre-diabetic relinopathy stage, the nonproliferative diabetic retinopathy stage, and the proliferative diabetic retinopathy stage. The method comprises the administration of an inhibitor of the branched chain amino acid-dependent aminotransferase pathway. Preferably, the inhibitor of the branched chain amino acid-dependent aminotransferase pathway (BCAT) is a compound of Formulas 1, II, and/or III:
(Formula Removed)
wherein:
R9 is H: alkyl; cycloalkyl; substituted alkyl containing halogen, amine. alkoxy. cycloalkyl, or hydroxy; allyl; alkynyl; alkanoyl: alkoxyalkanoyl: sullbnyl; phenyl benzyl; orarylalkyl; m and n are independently an integer of 1-3;
R1 - R8 and R10 - R14 are independently H. alkyl. or substituted alkyl; and
X = NR14, O, or S; or a pharmaceutically acceptable salt, ester, prodrug. or amide thereof.
The compounds can have one or more asymmetric carbon atoms and each asymmetric center can be racemic (R & S) or chiral (R or S).
Preferred compounds are those of Formula I, II. or HI wherein m and n are 1; X is NR14; R9 is H; R4 is methyl; R4 and R5 are methyl; R8 is methyl; R10 is methyl; R7 and R8 are methyl; R4 and R8 are methyl; R1-R8 and R10-R13 are H; R9 is alkyl; R9 is benzyl; RM is alkyl; R9 is arylalkyl; R9 is cycloalkyl; R1,R8 are H; R1,R8 and R10-R11 are H: R1, R2 and R7-R8 are H; or R2 is methyl.
More preferred compounds are those of Formula II wherein R3 is alkyl. R1-R.2 and R4-R11 and R14 are hydrogen, and m and n are 1. and X is NR11 : R3 and R11 are alkyl. R1-R2 and R4-R10 and R14 are hydrogen, m and n are 1. and X is NR14; R3 and R11, are alkyl, R1-R2 and R4-R10 and RM are hydrogen, m and n are 1. R9 is alkyl. and X is NR14: and R1-R11 and R14 are hydrogen, m and n are 1. and X is O.
Especially preferred compounds are selected from: (1 -Allylaminomethyl-cyclohexyl)-acetic acid; (1-Prop-2-ynylaminomethyI-cyclohexyl)-acetic acid; (1-[(2.2.2-Trifluoro-ethylamino)-methyl]-cyclohexyl}-acciic acid; (1-[(3,3.3-Trifluoro-propylamino)-methyl]-cyclohexyl}-acetic acid; 1α,3ß.5ß- (l-Ally!aminomethyl-3.5-dimcthyl-cyclohexyl)-aceiic acid: 1α.3ß.5ß-(3.5-Dimcthyl-l-prop-2-ynylaminomethyl-cyclohexyl(-acetic acid: 1α,3ß,5ß-{3.5-Dimethyl-l-[(2,2.2-trifluoro-cihvlamino)-mcthyl]-cyclohcxyl|-acetic acid;
1α,3ß,5ß-{3.5-Dimethyl-l-[(3.3.3-trinuoro-propylamino)-methyl]-cyclohcxyl |-aceiic acid;
trans-((lR_3R)-l-Allylaminomethyl-3-methyl-cyclohexyl)-acetic acid; trans-((lR.3R)-3-Methyl-l-prop-2-ynylaminomethyl-cyclohexyl)-acetic acid; trans-{(lR.3R)-3-Mcthyl-l-[(2,2.2-trifluoro-ethylamino)-methylj-cyclohexy!}-accuc acid;
trans-{(lR.3R)-3-Methyl-l-[(3.3,3-trifluoro-propylamino)-metliyl]-cyclohcxylj-acetic acid; trans-{(lR,3R)-3-Methyl-l-[(4,4.4-triflubro-butylamino)-melhyl]-cyclohexyl} -acetic acid;
1α,3ß,5ß-{3,5-Dimelhyl-l-[(4,4,4-trifluoro-butylamino)-methyl]-cyclohexyl}-acetic acid;
1α,3ß,5ß-(1-[(Cycloprop>lmethyl-amino>raethyl]-3!5-dimethyl-cyclohexyl}-aceticacid;
trans- {(!R,3R)-l-[(Cyclopropylmethyl-amino)-melhyI]-3-methyl-cyclohexyl} -acetic
acid;
trans-(( 1 R,3R)-3-MethyI-1 -methylaminomethyl-cyclohexyl)-acetic acid;
trans-(( 1 R,3R)-1 -Ethylaminomethyl-3-methyl-cyclohexyl)-acetic acid;
trans-(( 1 R,3R)-3-Methyl-1 -propylaminomethyl-cyclohexyl)-acetic acid;
trans-{(lR,3R)-l-Butylaminomethyl-3-jnethy]-cyclohexyl)-aceticacid:
trans-((lR,3R)-] -Hydroxymethyl-3-methyl-cyclohexyl)-acetic acid;
1α,3ß,5ß-(1-[(Hydroxymethyl-amino)-methyI]-3,5-dimethyl-cyclohexyl}-acetic acid;
1α,3ß,5ß-(l-Aminomethyl-3,5-diethyl-cyclohexyl)-aceticacid. hydrochloride;
trans-(lR.3R)(l-Aminomethyl-3-methyl-cycIohexyI)-acetic acid, hydrochloride;
(l-Aminomethyl-2-methyI-cyclohexyl)-acetic acid, hydrochioridc;
(l-Aminomethyl-3.3-dimethyI-cyclohexyl)-acetic acid, hydrochloride;
(±)-(trans)-(l -AminomethyI-3,4-dimethyl-cyclopentyl)-acetic acid, hydrochloride;
(cis/transH3R) (l-Aminomethyl-3-methyl-cyclopentyl)-acetic acid, hydrochloride;
(+)-(trans)-( l-Arninomethyl-3.4-dimethyl-cyclopentyn-acetic acid, hydrochloride:
(+)-(trans)-( 1 -Aminomethyl-3.4-dimethyl-cyclopcntyl)-acetic acid, hydrochioridc:
1α.3ß.5ß-n-Aminorncthyl-3.5-dimethyl-cyclohcxyl)-acetic acid, hydrochloride;
1α,3ß.5ß-(3.5-Dimclhyl-)-propylaminomcthy]-cyclohexyl)-acciic aL'id. hydrochioridc
salt;
1α,3ß.5ß-(l-Ethylaminomethyl-3.5-dimcthyl-cyc]ohcxyl)-acciic acid, hydrochloride salt:
1 a,3ß,5ß-(l-Bcnzylamino-methyl)-3.5-dimethyl-cyclohexyl]-acctic acid, hydrochloride
salt;
1 cc,3ß,5ß-( 1 -Dimethylaminomethyl-3,5-dimethyl-cyclohexyl)-acctic acid, hydrochloride
salt;
1α,3ß,5ß-(l-Butylaminomethyl-3,5-dimethyi-cyclohexyl)-acctic acid, hydrochioridc salt;
1α,3ß,5ß-(1-[(Benzyl-methyl-amino)-methyl]-3.5-dimethy!-cyclohexyl}-acciic acid.
hydrochloride salt;
1α,3ß.5ß-(3,5-Dimethyl-l-methylaminomethyl-cyclohexyl)-acetic acid, hydrochioridc
salt;
1α,3ß,5ß-[l-(Acetylamino-methyl)-3,5-dimethyl-cyclohcxyl]-acetic acid;
1α,3ß,5ß-(l-(Isobutylamino-methyl)-3,5-dimethyl-cyclohexyI]-accticacid, hydrochloride salt;
1α,3ß,5ß-[3,5-Dimethyl-l-(pheneuhylamino-methyl>cyclohexyl]-aceticacid, hydrochloride salt;
1α,3ß,5ß-{3,5-DimethyI-l-[(3-phenyi-propylamino)-methyl]-cyclohexyl}-acetic acid. hydrochloride salt;
(1-[(Cyclobutylmethyl-amino)-methyr]-tycloriexyl}-acetic acid, hydrochloride salt; 1α,3ß.5ß-(l-(lsopropylamino-methyl)-3,5-dimethyl-cyclohexyl]-acetic acid. hydrochloride salt;
1 -Aminomethyl-1 -cyclohexane-acetic acid; 1-Aminomethyl-l-cyclopentane-acetic acid; 1-Aminomenthyl-l-cyclopentane-acetic acid, sodium salt; ]-(hydroxymethyl)cyclohexane-acetic acid, sodium salt; (1-[(2-Methyl-butylamino)-methyl]-cyclohcxy!}-acetic acid, hydrochloride salt: (1-[(4,4.4-Trifluoro-butylamino)-methyl]-cyclohexyi}-acetic acid, hydrochloride salt; (l-Ethylaminomethyl-cyclohexyl)-acetic acid, hydrochloride salt; (1-[(Cyclopropylmethyl-amino)-methyl]-cyclohexy]}-acetic acid, hydrochloride salt: (1-[(2-Hydroxy-l-methyl-ethylamino)-methyl]-cyclohexyi;-acetic acid, hydrochloride salt;
[l-(Isobutylamino-methy])-cyclohexy!]-acetic acid, hydrochloride salt: (l-Propylaminomethyl-cyciohexyl)-acetic acid, nydrochloride salt: [l-(lsopropylamino-mcthyl)-cyclohexyi]-acetic acid, hydrochloride salt; (l-Cyclohexylaminomethyl-cyclohexyl)-acetic acid, hydrochloride salt; [l-(Benzylamino-methyl)-cyclohexyl]-acetic acid, hydrochloride salt; ((lR,3R)-3-Methyl-l-propylaminomethyl-cyclohexyl)-acetic acid, hydrochloride salt; (1-[Cyclopentylmethyl-amino)-methyI]-cyclohexyl |-acetic acid, hydrochloric sail; (1-[(Cyc]ohexylmethyI-amino)-methyl]-cyclohexyl}-acetic acid, hydrochloric salt; [1 -(tert-Bmoxycarbony)amino-meihyl)-cyclohexyl)-acetic acid; [1 -(Acetylamino-methyl)-cyclohexyl]-aceti6 acid:
((3R, 5S)-l-Cyclobutylaminomethyl-3.5-dimethyl-cyclohexyl)-acetic acid, hydrochloride salt;
{(3R,5S)-3,5-Dimethyl-l-[(2-methyl-butylamino)-methyl]-cyclohexyl}-aceticacid,
hydrochioride salt;
{(3R, 5S)-l-[(2-Hydroxy-l-raethyl-ethyiarnino)-methyl]-3>5-dinicthy]-cyclohexyl}-acetic
acid, hydrochioride salt;
{(3R, 5S)-1 -[(2,2-Dimethoxy-ethylamino)-methyl]-3,5-dimethyl-cyclohexyl}-acetic acid,
hydrochioride salt;
{(3R,5S)-l-[(Cyclopentylniethyl-ami4ip)-methyI]-3,5-dimethyl-cyc]ohexyI}-acetic acid,
hydrochioride salt;
{(3R,5S)-l-((Cyclohexylmethyl-amino)-methy]]-3,5-dimethyl-cyc]ohexy]}-aceticacid.
hydrochioride salt;
((3R. 5S)-l-Cyclohexylaminomethyl-3,5-dimethyl-cyclohexyl)-acetic acid, hydrochioride
salt;
((3R,5S)-l-Carboxymethyl-3.5-dimethyl-cyclohexyl)-aceticacid;
trans--((3R, 5S)-]-Hydroxymethyl-3.5-dimethyl-cyclohexyl-acetic acid, hydrochioride
salt;
cis--((3R. 5S)-]-Hydroxyrnethyl-3,5-dimcthyl-cyclohexyI)-acetic acid, hydrochioride salt:
(l-Dimethylaminomcthy!-cyc!ohexYl)-acetic acid, hydrochioride salt;
(l-Butylaminomethyl-cyclohcxyl)-acetic acid, hydrochioride salt;
(1-[2.2-Dtmethoxy-ethylamino)-methyI]-cyclohexyl}-acetic acid, hydrochioride salt;
(l-methylaminomethyl-cyclohexyl)-acetic acid, hydrochioride salt;
(1-[(Benzyl-methyl-amino)-methyl]-cyclohexyl}-acetic acid, hydrochioride salt;
[]-(Phenethylamino-methyl)-cyclohexyl]-acetic acid, hydrochioride salt;
(1-[(3-Phenyl-propylamino)-methyl]-cyclohexyl}-acctic acid, hydrochioride salt;
((3R, 5S)-l-Hydroxymethyl-3,5-dimethyl-cyclohexyl)-acetic acid, sodium salt;
((3R, 5S)-l-Ethylaminomethyl-3.5-dirnethyl-cyclohexyI)-acctic acid, hydrochloride salt;
(l-Aminomethyl-4-ethyl-cyclohexyl)-acetic acid, hydrochloric salt;
(l-Aminomethyl-4-propyl-cyclohexyl)-acetic acid, hydrochloric salt;
((3R, 5S)-3.5-Dimelhyl-l-propylaminomethyl-cyclohexyl)-acetic acid, hydrochioride
salt;
[(1R, 3R)-l-(Benzylamino-methyl)-3-methyl-cyclohexyl]-acetic acid, hydrochioride salt;
{(1R, 3R)-1 -[(Ben2yl-methyl-amino)-methyl]-3-methyl-cyclohexyl}-acetic acid,
hydrochioride salt; and
((1R, 3R)-3-Methyl-l-methylaminomethyl-cyclohexyl)-acetic acid, hydrochioride salt;
Several of the compounds described by Formulas I, II, and III are known. Compounds of Formula A:
(Formula Removed)
wherein R is hydrogen or a lower alkyt radical and n is 4. 5, or 6 are known in United
States Patent Number 4,024,175 and its divisional United States Patent Number 4,087,544. The uses disclosed are: protective effect against cramp induced by thiosemicarbazide; protective action against cardiazole cramp: the cerebral diseases, epilepsy, faintness attacks, hypokinesia. and cranial traumas: and improvement in cerebral functions. The compounds are useful in geriatric patients. The patents arc hereby incorporated by reference.
Compounds of Formula B:
(Formula Removed)
or a pharmaceutically' acceptable salt thereof wherein R1 is a straighi or branched alkyl
group having from I to 6 carbon atoms, phenyl or cycloalkyl having from 3 to 6 carbon atoms; R2 is hydrogen or methyl; and R3 is hydrogen, or carboxyl are known in United States Patent Number 5.563,175 and its various divisional. These patents are hereby incorporated by reference.
The present invention also discloses novel compounds of the Formula IV
(Formula Removed)
R1-R4 are hydrogen or alkyl;
X is NR5 or O;
R5 is hydrogen or alkyi;
R6 is hydrogen, alkyl. benzyl, alkanoyl. alkoxyalkanoyl. arylalkyl. alkoxy, cycloalkyl,
allyl, alkylcycloalkyl. alkoxy, cycloalkyl, alkylcycloalkyl, trisubstituted halogenalkyl.
and wherein R1-R4 are each hydrogen then R6, is no' hydrogen or methyl; or a
pharmaceutically acceptable salt, ester, prodrug, or amide thereof. Like the comounds
of Formulas 1, II. and 111, the compounds are useful for the prophylactic and therapeutic
treatment of diabetic rctinopathy. including treatment at the prc-diabetic rctinopathy
stage, the nonprolifcrativc diabetic retinopathy stage, and the prolifcrativc diabetic
retinopathy stage.
Preferred compounds are those of Formula IV wherein R2 and R4arc hydrogen and R1 and R3 are alkyl; R2 and R4arc hydrogen and R, and R3 arc methyl; R1,R4are hydrogen; R1 is alkyl and R2-R4 are hydrogen; R1 is methyl and R:-R4arc hydrogen; R5 is hydrogen; X is NR6; X is O; R6, is alkyl; R6 is benzyl; R6, is acetyl; R6, is phenylalkyl; R6, is cycloalkyl; R6, is trifluoroalkyl; R6 is alkylcycloalkyl; R6, is alkoxy; and R6, is allyl.
Other preferred compounds are those of Formula IV wherein R3 and R4are hydrogen and R1 and R3 are methyl; R1-R4 are hydrogen; R1 is methyl and R:-R4arc hydrogen: R5 is hydrogen; X is NR6; R6, is alkyl; R6 is benzyl: R6 is acetyl; R6 is phenylalkyl; R6 is cycloalkyl; R6 is trifluoroalkyl; R6, is alkylcycloalkyl; R6, is alkoxy; and R6, is allyl.
Especially preferred compounds are selected from: (l-Allylaminomethyl-cyclohexyl)-acetic acid;
(1 -Prop-2-ynylaminornethyl-cyclohexyl)-acetic acid;
{1 -[(2,2,2-Trifluoro-ethylamino)-methyl]-cyclohexyl}-acetic acid;
(1-[(3,3,3-Trifluoro-propyIamino)-methyl]-cyclohexyl}-acetic acid;
1α,3ß,5ß- (l-AHylaminomethyl-3,5-dimethyl-cyclohexyl)-acetic acid;
1α,3ß,5ß-(3,5-DimethyI-l-prop-2-ynylaminomethyl-cyclohexyl)-acetic acid;
1α,3ß,5ß-{3,5-Dimethyl-l-[(2,2,2-trifluoro-ethylamino)-methyl]-cyclohexyl}-acetic
acid;
1α,3ß,5ß-{3,5-Dimethyl-l-[(3,3,3-trifluoro-propylamino)-methyl]-cyclohexyl}-acetic
acid;
trans-((lR,3R)-l-Allylaminomethyl-3-methyl-cyclohexyl)-aceticacid:
trans-((lR.3R)-3-Methyl-l-prop-2-ynylaminomethyl-cyclohexyl)-acetic acid:
trans-{(lR,3R)-3-Methyl-l-[(2.2,2-trifluoro-ethylamino)-mcthyI]-cyclohexyl}-acetic
acid;
trans-{(lR,3R)-3-Methyl-l-[(3,3.3-trifluoro-propylamino)-methyI]-cyclohexyl}-acetic
acid;
trans- {(lR.3R)-3-Mcthyl-l -[(4.4.4-trifluoro-butylamino)-methy I j-cyclohexyl[ -acetic
acid;
1α,3ß,5ß-(3,5-Dimethyl-]-[(4.4.4-trifluoro-butylamino)-mcthyl]-cyclohcxyl}-acetic
acid;
1α,3ß,5ß-{ I-[(Cyclopropylmcthyl-amino)-methyl]-3.5-dimetliyl-cyclohexyl}-acetic acid:
trans-{(lR.3R)-l-[(Cyclopropylmethyl-amino)-mcthy!]-3-methyl-cyclohexyl{ -acetic
acid;
trans-((lR.3R)-3-Methyl-]-methylaminomethyl-cyclohe.xyl)-acctic acid;
trans-((lR,3R)-l-Ethylaminomethyl-3-methyl-cyclohexyl)-acctic acid:
trans-((lR.3R)-3-Methyl-l-propylaminomethyl-cyclohexyl)-acctic acid:
trans-((lR.3R)-l-Butylaminomethyl-3-methyl-cyclohexyI)-acciic acid;
trans-((lR,3R)-l-Hydroxymethyl-3-methyl-cyclohexyl)-acetic acid;
1α,3ß,5ß-(1-[(Hydroxymethyl-amino)-methyl]-3.5-dimethyl-cyclohexyl}-acctic acid;
1α,3ß,5ß-(l-Aminomethyl-3.5-diethyl-cyclohexyI)-acetic acid, hydrochloride;
1α,3ß,5ß-(3,5-Dimethyl-1 -propylaminomethyl-cyclohexyl)-acetic acid, hydrochloride
salt;
Ja,3ß,5ß-(l-Ethylarninomethyl-3.5-dimethyl-cyclohexyl)-acetic acid, hydrochloride salt;
lα,3ß,5ß-(I-Benzylamino-methyl)-3,5-dimethyl-cyclohexyl]-acetic acid, hydrochloride
salt;
1α,3ß,5ß-(l-Dimethylaminomethyl-3,5-dimethyl-cyclohexyl)-acetic acid, hydrochloride
salt;
1α,3ß,5ß-(l-Butylaminomethyl-3,5-dimethyl-cyclohexyl)-acetic acid, hydrochloride salt;
1α,3ß,5ß-(1-[(Benzyl-methyl-amino)-methyl]-3,5-dimethyl-cyclohexyl}-acetic acid,
hydrochloride salt;
1α,3ß,5ß-(3,5-Dimethyl-l-methylaminomethyl-cyclohexyl)-acetic acid, hydrochloride
salt;
1α.3ß.5ß-[l-(Acetylamino-methyl)-3.5-dimethyl-cyclohexyl]-acetic acid:
1α,3ß.5ß-[]-(lsobutylamino-methyl)-3.5-dimethyl-cyclohexyI]-acetic acid, hydrochloride
salt;
1α,3ß,5ß-[3,5-Dimethyl-l-(phenethylamino-methyl)-cyclohexyl]-acetic acid.
hydrochloride salt;
lα,3ß.5ß- [3.5-Dimcthyl-l-[(3-phenyl-propylamino)-methyl]-cyclohexyl}-acetic acid.
hydrochloride salt:
[ 1 -[(Cyclobutylmethyl-amino)-methylJ-cyclohexyl J-acetic acid, hydrochioridc salt:
1α,3ß.5ß-[I-(Isopropylamino-methyl)-3.5-dirnethyl-cyclohexyI]-acctic acid.
hydrochloride salt;
(1-[(2-Methyl-butylamino)-methyl]-cyclohexyl}-acetic acid, hydrochioride salt:
(1-[(4.4.4-Trifluoro-butylamino)-methyl]-cyclohexyl}-acetic acid, hydrochloride salt:
(l-Ethylaminomethyl-cyclohexyl)-acctic acid, hydrochloride salt;
(1-[(Cyclopropylmethyl-amino)-methyl]-cyclohexyl}-acetic acid, hydrochloride salt;
(1-[(2-Hydroxy-l-methy!-ethylamino)-methyl]-cyclohexyl}-acctic acid, hydrochloride
salt;
[l-(Isobutylamino-methyl)-cyclohexyl]-acetic acid, hydrochioridc salt;
(l-Propylaminomethyl-cyclohexyl)-acetic acid, hydrochloride salt;
[l-(lsopropylamino-methyl)-cyclohcxy]]-acctic acid, hydrochloride salt;
(l-Cyclohexylaminomethyl-cyclohexyl)-acetic acid, hydrochloride salt;
[l-(Benzylamino-methyl)-cyclohexyl]-acelic acid, hydrochloride salt;
((lR,3R)-3-Methyl-l-propylaminomethyI-cycIohexyI)-acetic acid, hydrochloride salt;
{I-[Cyclopentylmethyl-amino)-mcthy]]-cyclohexyI}-acetic acid, hydrochloric salt;
(1-[(Cyclohexylmethyl-amino)-methyl]-cyclohexyl}-acetic acid, hydrochloric salt;
[ 1 -(tert-Butoxycarbonylamino-methyl)-cyclohexyl]-acetic acid;
[1 -(Acetylamino-methyl)-cyclohexyl]-acetic acid;
((3R, 5S)-1 -Cyclobutylaminomethyl-3,5-dimethyl-cyclohexyl)-acetic acid, hydrochloride salt;
{(3R, 5S)-3,5-Dimethyl-l-[(2-methyl-butylamino)-methyl]-cyclohexyl}-acetic acid,
hydrochloride salt; ':
{(3R, 5S)-l-[(2-Hydroxy-l-methyl-ethylamino)-methyl]-3.5-dimethy]-cyclohexyl}-acetic acid, hydrochloride salt;
{(3R, 5S)-1-[(2,2-Dimethoxy-ethylamino)-methyl]-3.5-dimcthyl-cyclohexyl}-acetic acid, hydrochloride salt;
{(3R, 5S)-1 -[(Cyclopentylmethyl-amino)-methyI]-3.5-dimethyl-cyclohexyi}-acetic acid, hydrochloride salt;
{(3R,5S) 1 -[(Cyclohexylmethyl-amino)-methyl]-3.5-dimethy 1-cyclohexy!} -acetic acid, hjdrochloridc salt;
((3R, 5S)-l-Cyclohexylaminomethyl-3.5-dimethyl-cyclohexyl)-acetic acid. hydrochloride salt;
((3R,5S)-l-Carboxymcthyl-3.5-dimethyl-cyclohexyl)-aceiic acid: lram-((3R. 5SH -Hydroxymethyl-3,5-dimethyl-cyciohexyl-acelic acid, hydrochloride salt;
cis-((3R. 5S)-I-Hydroxymethyl-3.5-dimethyl-cyclohexyl-acelic acid, hydrochloride salt; (l-Dimethylaminomethyl-cyclohexyl)-acetic acid, hydrochioridc salt: (l-Butylaminomethyl-cycIohexyl)-acetic acid, hydrochloride salt; (1-[2,2-Dimethoxy-eihylarnino)-mcthyl]-cyclohexyl}-acetic acid, hydrochloride sail: (l-methylaminomethyl-cyclohexyl)-acctic acid, hydrochioridc salt; (1-[(Benzyl-methyl-amino)-mcthy!]-cyclohexy!}-acetic acid, hydrochloride salt: [l-(Phenethylamino-methyI)-cvclohcxyl]-acetic acid, hvdrochloride salt; (1-[(3-Phenyl-propylamino)-methyl]-cyclohexyl}-acetic acid, hydrochioridc salt; ((3R. 5S)-l-Hydroxymethyl-3,5-dimethyl-eyclohexyl)-acetic acid, sodium salt; ((3R, 5S)-l-Ethylaminomethyl-3.5-dimethyl-cyclohexyl)-acetic acid, hydrochloride salt: (l-Aminomethyl-4-ethyl-cyclohexyl)-acetic acid, hydrochloric salt; (l-AminomethyI-4-propyI-cyclohcxyI)-acetic acid, hydrochloric salt;
((3R, 5S)-3,5-Dimethyl-l-p'ropylaminomethyl-cyclohexyl)-acetic acid, hydrochloride
salt;
[(1R, 3R)-l-(Benzylamino-methyl)-3-methyl-cycIohexyI]-acetic acid, hydrochloride salt:
{(1R, 3R)-l-[(Benzyl-methyl-amino)-methyl]-3-methyl-cyclohexyl}-acetic acid.
hydrochloride salt; and
((1R, 3R)-3-Methyl-l-methylaminorrtelhyl-cycIohexyl)-acetic acid, hydrochioride salt;
The present invention is also a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds of Formula IV and a pharmaceutically acceptable carrier.
Compounds of Formula 11. such as gabapentin. are useful for treating various conditions such as neurodegenerative disorder (US 5.084.479). depression (US 5,025.035). anxiety/panic (US 5.792,796). mania/bipolar disorders (US 5.510.381). anti-inflammatory- diseases (WO 98 158641), gastrointestinal damage (WO 99 108670). glaucoma, and pain. Because the compounds of Formula 11 and Formula IV both operate to affect the BCAT pathway and affect the synthesis of glutamatc. which is involved in each of the aforementioned indications, the compounds of Formula IV will also be useful for treating the indications. Thus, the present invention is also directed to the use of the compounds of Formula IV to treat each or more than one of neurodegenerative disorders (including but not limited to ALS. Parkinson's disease and Alzheimer's disease), depression, anxiety/panic, mania/bipolar disorders, anti-inflammatory diseases, glaucoma pain, or gastrointestinal damage.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Scheme showing the BCAT shuttle in the retina. The shuttle proper is highlighted and shows how branched chain amino acids in the glia can transaminale a-ketoglutarate to form glutamate. The BCKr/5 product diffuses across the intcrstitium (shown as grey) to the neurons where it is converted back to the branched chain amino acid by transamination with glutamate via BCATc. Abbreviations are as follows: BCAA=branched chain amino acid; GLU=glutamate; GLN=glutamine:
Gln'ase=glutaminase; pyr=pyruvate; PC=pyruvate carboxylase. aKG=α-ketoglutarate. BCATm=mitochondrial branched chain aminotransferase; BCATc=cytosoIic branched chain aminotransferase; BCKA=branched chain keto acid; GDH=glutamate dehydrogenase.
Figure 2. Effect of pyruvate concentration on incorporation of H14CO3 into glutamate plus glutamine (A) and pyruvate plus lactate (B) by ex vivo retinas. Hemi-retinas were incubated at 37°C under standard conditions with 25 mM HI4CO:, tor 20 minutes. Pyruvate in the medium was either 0.2 or 5 mM. Values shown are mean ± SEM (n=4, * p Figure3. Expression of BCAT isoenzymes in rat retina. Tissue extraction. SDS-PAGE. and immunoblotting were performed as described using the ECL detection kit. Immunoaffinity purified rat anti-BCATc peptide antibody (1:1000) and human anti-BCATm antibody (1:1000) were used.
Figure 4. The influence of gabapentin (GBP) and branched chain amino acids on incorporation of H14CO3 into glutamate plus glutamine by ex retinas. Half retinas were incubated at 37° C under standard conditions with 25 mM HI4CO; and 0.2 mM pyruvate for 20 minutes. In some instances gabapentin (1 mM) was included in the incubation medium and in others GBP (1 mM) plus all three branched amino acids (0.2 mM) each were included. Values shown are means ± SEM of llCO3 incorporated into glutamate plus glutamine in 20 minutes (n=4, * p Figure 5. Influence of gabapentin (GBP) concentration on incorporation of H14CO3 into glutamate and glutamine in ex vivo retinas. Conditions were similar to those of Figure 4 except for the concentration of GBP. Values shown are means ± SEM (n=6-7. * p DETAILED DESCRIPTION OF THE INVENTION
The present invention is predicated on the discovery that intraocular glutamate injection causes ganglion cell loss which is related to the early stages of diabetic retinopathy (Vorwerk et al. IOVS 37:1618-1624 (1996)) and that an inhibitor of the branched chain amino acid-dependent aminotransferase pathway, such as gabapentin for example, is effective in inhibiting the synthesis of glutamate. thus preventing diabetic retinopathy. Accordingly, the present invention provides a method for the prophylactic and therapeutic treatment of diabetic retinopathy, including treatment at the pre-diabetic retinopathy stage, the nonproliferative diabetic retinopathy stage, and the proliterative diabetic retinopathy stage. By "prophylactic" is meant the protection, in whole or in pan. against diabetic retinopathy. in particular diabetic macular edema. By "therapeutic" is meant the amelioration of diabetic retinopathy, itself, and the protection, in whole or in part, against further diabetic retinopathy, in particular diabetic macular edema.
The method comprises the administration of an inhibitor of the branched chain amino acid-dependent aminotransferase pathway in an amount sufficient to treat the retina for retinopathy prophylactically or therapcutically. Any inhibitor of the branched chain amino acid-dependeni aminotransferase pathway can be used in the method of the present invention as long as it is safe and efficacious. Herein, "branch chain amino acid-dependent aminotransferase (BCAT) inhibitor" will be used to refer to such compounds and is intended to encompass all compounds that ailect the branch chain amino acid-dependent aminotransferase pathway at any and all points in the pathway.
Preferably, ihc BCAT inhibitor is a compound of Formulas 1.11. and/or III. as described above, or a pharmaceutical ly acceptable BCAT pathway-inhibiting analogue or prodrug thereof, or a pharmaceutically acceptable salt, ester, or amide of any ol the foregoing.
Unless otherwise expressly stated, the following definitions are adhered to throughout this disclosure.
'"AlkyF means a straight or branched hydrocarbon radical having from 1 to 10 carbon atoms (unless stated otherwise) and includes, for example, methyl, ethyl, n-propyl. isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl. iso-pentyl, n-hexyl, and the like.
"Halogen" includes fluoro, chloro, bromo. and iodo.
"Alkenyl" means straight and branched hydrocarbon radicals having from 2 to 6 carbon atoms and one double bond and includes ethenyl, 3-buten-l-yl, 2-ethenylbutyl, 3-hexen-l-yl, and the like.
"Alkynyl" means straight and branched hydrocarbon radicals having from 2 to 6 carbon atoms and one triple bond and includes ethynyl, 3-butyn-l-yl. propynyl, 2-butyn-1 -yl, 3-pentyn-1 -y 1, and the like.
"Cycloalkyl" means a monocyelic or polycyclic hydrocarbyl group such as cyclopropyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclobutyl. adamantyl. norpinanyl. decalinyl, norbomyl, cyclohexyl. and cyclopentyl. Such groups can be substituted with groups such as hydroxy, keto. and the like. Also included are rings in which 1 to 3 heteroatoms replace carbons. Such groups are termed "heterocyclyl". which means a cycloalkyl group also bearing at least one heteroatom selected from O, S. or NR2.
examples being substituted or unsubstituted oxiranyl. pyrrolidinyl. pipcridyl. tetrahydropyran, piperazinyl. acylpiperazinyl, pyrrolidinyl. and morpholine.
'Alkoxy" refers to the alkyl groups mentioned above bound through oxygen, examples of which include methoxy. ethoxv, isopropoxy. ten-butoxy. and the like. In addition, alkoxy refers to polyethers such as -O-(CH2)2-0-CH3- and the like.
"Alkanoyl" groups arc alkyl linked through a carbonyl. ie. C1-C5-C(O)-. Such groups include formyl. acetyl. propionyl. butyryl. and isobuiyn I.
"Acyl" means an alkyl or aryl (Ar) group bonded through a carbonyl group, ie. R-C(O)-. For example, acyl includes a C1-C6 alkanoyl. including substituted alkanoyl,
wherein the alkyl portion can be substituted by NR4R5 or a carboxylic or hctcrocyclic group. Typical acyl groups include acetyl. benzoyl. and the like.
The alkyl, alkenyl. aikoxy. and alkynyl groups described above arc optionally substituted, preferably by 1 to 3 groups selected from NR4R\ phcnyl. substituted phenyl. thio C1-C6 alkyl, C1-C6 alkoxy, hydroxy. carboxy. C1-C6 alkoxycarbonyl. halo, nitrilc. cycloalkyl, and a 5- or 6-membered carbocyclic ring or heterocyclic ring having 1 or 2 heteroatoms selected from nitrogen, substituted nitrogen, oxygen, and sulfur. "Substituted nitrogen" means nitrogen bearing C1-C6 alkyl or (CH2)nPh where n is 1. 2, or 3. Perhalo and polyhalo substitution is also embraced.
Examples of substituted alkyl groups include 2-aminoethyl, pentachloroethyl, trifluoromethyl, 2-diethylaminoethyl, 2-dimethylaminopropyl, ethoxycarbonylmethyl, 3-phenylbutyl, methanylsulfanylmethyl, methoxymethyl, 3-hydroxypentyl, 2-carboxybutyl, 4-chlorobutyl, 3-cyclopropylpropyl, pentafluoroethyl, 3-morpholinopropyl, piperazinylmethyl, and 2-(4-methylpiperazinyl)ethyl.
Examples of substituted alkynyl groups include 2-methoxyethynyl, 2-ethylsuIfanyethynyl, 4-( 1 -piperazirtyI)-3-(butynyl), 3-phenyI-5-hexynyl, 3-diethylamino-3-butynyl, 4-chloro-3-butynyl, 4-cyclobutyl-4-hexenyl, and the like.
Typical substituted alkoxy groups include aminomethoxy. trifluoromcthoxy. 2-diethylaminoethoxy, 2-ethoxycarbonylethoxy, 3-hydroxypropoxy, 6-carboxhexyloxy. and the like.
Further, examples of substituted alkyl. alkenyl, and alkynyl groups include dimethylaminomethyl, carboxymethyl, 4-dimethylamino-3-buten-l -yl. 5-ethylmethylamino-3-pentyn-] -yl. 4-morpholinobutyl. 4-tetrahydropyrinidylbutyl, 3-imidazolidin-l -ylpropyl, 4-tetrahydrothiazol-3-yl-butyl, phcnylmethyl. 3-chlorophenylmethyl. and the like.
The terms "Ar" and "aryl" refer to unsubstituted and substituted aromatic groups. Heteroaryl groups have from 4 to 9 ring atoms, from 1 to 4 of which are independently selected from the group consisting of 0. S. and N. Preferred heteroaryl groups have 1 or 2 heteroatoms in a 5- or 6-membered aromatic ring. Mono and bicyclic aromatic ring systems are included in the definition of aryl and heteroaryl. Typical ary I and heteroaryl groups include phenyl, 3-chlorophenyl. 2.6-dibromophenyl, pyridyl, 3-methylpyridyl. benzothienyl, 2,4.6-tribromophcnyl. 4-ethylbenzothienyl, furanyl. 3.4-diethylfuranyl. naphthyl, 4,7-dichloronaphthyl, pyrrole, pyrazole. imidazole. thiazolc. and the like.
Preferred Ar groups are phenyl and phenyl substituted by 1. 2. or 3 groups independently selected from the group consisting of alkyl. alkoxy. thio. thioalkyl.
hydroxy, carbonyl groups, amino of the formula -NR4R5, and T{CH2)mQR4 or
T(CH2)mCO2R4 wherein m is 1 to 6, T is O. S, NR4. N(O)R4, NR4R6y, or CR4R5, Q
is O, S, NR5, N(O)R5, or NR5R6Y wherein R4 and R5 are as described above, and R7 is alkyl or substituted alkyl, for example, methyl, trichloroethyl. diphenylmethyl. and the like. The alkyl and alkoxy groups can be substituted as defined above. For example.
typical groups are carboxyalkyl, alkoxycarbonylalkyl, hydroxyalkyl, hydroxyalkoxy, and alkoxyalkyl.
The symbol"-" means a bond.
The term "patient" means all animals including humans. Examples of patients include humans, cows, dogs, cats, goats, sheep, and pigs.
The compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated fgrms. In general, the solvated forms, including hydrated forms, are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention.
Certain of the compounds of the present invention possess one or more chiral centers and each center may exist in the R(D) or S(L) configuration. The present invention includes all enantiomeric and epimeric forms as well as the appiopriate mixtures thereof.
The compounds of Formulas I, II, and III are capable of further forming both pharmaceutically acceptable analogs comprising salts, esters, amides, and prodrugs As used herein, the term "pharmaceutically acceptable salts, esters, amides, and prodrugs" refers to those carboxylate salts, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention which are, within the scope o!" sound medical judgment, suitable for use in contact with the tissues ot patients without undue loxicity. irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use. as well as the zwittcrionic forms, where possible, of the compounds of the invention. The term "salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed and including, but no! limited to, acid addition and/or base salts, solvents and N-oxides of a compound of Formulas 1, II. and III. This invention also provides pharmaceutical formulations comprising a compound of Formulas 1.11, pr HI together with a pharmaceutically acceptable carrier, diluent, or excipient therefor. All of these forms are within the present invention.
Pharmaceuticaliy acceptable acid addition salts of the compounds of Formulas I, II and III include salts derived form inorganic acids such as hydrochloric, nitric,
phosphoric, sulfuric, hydrobromic, hydriodic, phosphorus, and the like, as well as the salts derived from organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Such salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate. caprylate, isobutyrate, oxlate, malonate, succinate. suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbcnzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like. Also contemplated are the salts of amino acids such as arginate, gluconate, galacturonatc. and the like; see, for example. Berge. et al., "Pharmaceutical Salts," J. of Pharmaceutical Science. 1977;66:1-19.
The acid addition salts of the basic compounds are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in the conventional manner. The free base form may be regenerated by contacting the salt form with a base and isolating the free base in the conventional manniT. The free base forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free base for purposes of the present invention.
Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metal hydroxides, or of organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N.N'-dibenzylethylenediaminc. chloroprocaine, choline. diethanolamine, ethylenediamine, N-mcthylglucaminc. and procaine: see, for example, Berge. et al., supra.
The base addition salts of acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in a.conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention.
Examples of pharmaceutically acceptable, non-toxic esters of the compounds of this invention include C1-C6alley 1 esters wherein the alkyl group is a straight or branched chain. Acceptable esters also include C5-C7cycloalkyl esters as well as arylalkyl esters such as. but not limited to benzyl. C1-C4lkyl esters are preferred. Esters of the
compounds of the present invention may be prepared according to conventional methods.
Examples of pharmaceutically acceptable, non-toxic amides of the compounds of
this invention include amides derived from ammonia, primary C1-C6alkyl amines and
secondary C1-C6dialkyl amines wherein the alkyl groups are straight or branched chain. In the case of secondary amines the amine may also be in the form of a 5- or 6-membered heterocycle containing one nitrogen atom. Amides derived from ammonia. C1-C3alkyl primary amines, and C1-C2dialkyl secondary amines are preferred. Amides of the compounds of the invention may be prepared according to conventional methods.
The term "prodrug" refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above Formulae, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stel1α, "Pro-drugs as Novel Delivery Systems." Vol 14 of the A.C.S. Symposium Series, and in Biorcvcrxihle Carrier* in Drug Design, ed. Edward B. Roche. American Pharmaceutical Association and Pergamon Press, 1987. both of which are incorporated herein by reference. In general, a prodrug is a drug which has been chemically modified and may be biologically inactive at its site oi" action, but which may be degraded or modified by one or more enzymatic or other in vivo processes to the parent bioactive form.
A therapeutically effective amount is an amount of a compound of Formulas 1. II. or III, that when administered to a patient, ameliorates a symptom of the disease.
The BCAT inhibitor, which is preferably a compound of Formulas I. II. and/or III. a BCAT pathway-inhibiting analogue of Formulas I, II. and/or III. a BCAT pathway-inhibiting prodrug of Formulas I. II, and/or III. or a pharmaceutically acceptable salt of any of the foregoing, can be administered in accordance with the present inventive method by any suitable route. Suitable routes of administration include systemic, such as orally or by injection, topical, intraocular, periocular (e.g.. subTenon's). subconjunctival, subretinal, suprachoroidal and retrobulbar. The manner in which the BCAT inhibitor is administered is dependent, in part, upon whether the treatment of retinopathy is prophylactic or therapeutic. The manner in which the BCAT inhibitor is administered for
therapeutic treatment of reti.iopathy is dependent, in part, upon the cause of the retinopathy.
For example, given that diabetes is the leading cause of retinopathy, the BCAT inhibitor can be administered prophylactically as soon as the pre-diabetic retinopathy state is detected. For the prophylactic treatment of retinopathy that can result from diabetes, the BCAT inhibitor is preferably administered systemically, e.g., orally or by injection. For the therapeutic treatmentof nonproliferative diabetic retinopathy, the BCAT inhibitor can be administered systemically, e.g.. orally or by injection, or intraocuiarly. Proliferative diabetic retinopathy can be therapeutically' treated by the administration of the BCAT inhibitor intraocularly, topically, subconjunctivally or periocularly (e.g., subTenon's), for example. The BCAT inhibitor is preferably administered intraocularly, topically, subconjunctivally or periocularly (e.g.. subTenon's) for the prophylactic or therapeutic treatment of retinopathy before, during or after surgical removal from an eye of scar tissue generated during neovascularization during the proliferative diabetic stage.
The BCAT inhibitor is preferably administered as soon as possible after it has been determined that an animal, such as a mammal, specifically a human, is ai risk for retinopathy (prophylactic treatment) or has begun to develop retinopathy (therapeutic treatment). Treatment will depend, in pan. upon the particular BCAT inhibitor used, the amount of the BCAT inhibitor administered, the route of administration, and the cause and extent, if any. of retinopathy realized.
One skilled in the art will appreciate that suitable methods of adminisiering a BCAT inhibitor, which is useful in the present inventive method, are available. Although more than one route can be used to administer a particular BCAT inhibitor, a particular route can provide a more immediate and more effective reaction than another route. Accordingly, the described routes of administration are merely exemplary and are in no way limiting.
The dose administered to an animal, particularly a human, in accordance with the present invention should be sufficient to effect the desired response in the animal over a reasonable time frame. One skilled in the art will recognize that dosage will depend upon a variety of factors, including the strength of the particular BCAT inhibitor employed, the age, species, condition or disease state, and body weight of the animal, as well as the amount of the retina about to be affected or actually affected by retinopathy. The size of
the dose also will be determined by the route, timing and frequency of administration as well as the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular BCAT inhibitor and the desired physiological effect. It will be appreciated by one of ordinary skill in the art that various conditions or disease states, in particular, chronic conditions or disease states, may require prolonged treatment involving multiple administrations.
Suitable doses and dosage regimens can be determined by conventional range-finding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. The present inventive method will typically involve the administration of from about 1 mg/kg/day to about 100 mg/kg/day, preferably from about 15 mg/kg/day to about 50 mg/kg/day. if administered systemically. Intraocular administration typically will involve the administration of from about 0.1 mg total to about 5 mg total, preferably from about 0.5 mg total to about 1 mg total. A preferred concentration for topical administration is 100 mu M.
Compositions for use in the present inventive method preferably comprise a pharmaceutically acceptable carrier and an amount of a BCAT inhibitor sufficient to treat retinopathy prophylactically or therapcutically. The carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the compound, and by the route of administration, it will be appreciated by one of ordinary skill in the art that, in addition to the following described pharmaceutical compositions, the BCAT inhibitor can be formulated as polymeric compositions, inclusion complexes, such as cyclodextrin inclusion complexes, liposomes, microspheres. microcapsules and the like (see, e.g.. U.S. Pat. Nos. -t. 997.652. 5.185.152 and 5.718.922).
The BCAT inhibitor can be formulated as a pharmaceutically acceptable acid addition salt. Examples of pharmaceutically acceptable acid addition salts for use in the pharmaceutical composition include those.drived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric. nitric and sulfuric acids, and organic acids, such as tartaric. acetic, citric, malic, lactic, fumaric. benzoic, glycolic, gluconic. succinic. and arylsulphonic, for example p-toluenesulphonic, acids.
The pharmaceutically acceptable excipients described herein, for example, vehicles, adjuvants, carriers or diluents, are well-known to those who are skilled in the an and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the BCAT inhibitor and one which has no detrimental side effects or toxicity under the conditions of use.
The choice of excipient will be determined in part by the particular BCAT inhibitor, as well as by the particular jnethod used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following formulations are merely exemplar,' and are in no way limiting.
Injectable formulations are among those that are preferred in accordance with the present inventive method. The requirements for effective pharmaceutically carriers for injectable compositions are well-known to those of ordinary skill in the art (see Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia. Pa., Banker and Chalmers, eds.. pages 238-250 (1982). and ASHP Handbook on Injectable Drugs. Toissel. 4th ed., paces 622-630 (1986)). It is preferred that such injectable compositions be administered intramuscularly, intravenously, or intrapcritoneally.
Topical formulations are well-known to those of skill in the an. Such formulations arc suitable in the context of the present invention for application 10 the skin. The use of patches, comeal shields (see. e.g.. U.S. Pat. No. 5.185.152). and ophthalmic solutions (see. e.g.. U.S. Pat. No. 5.710.182) and ointments, e.g.. eye drops, is also within the skill in the art.
Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol. benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, com starch, potato
starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscannellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as galatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
The effectiveness of an orally administered drug is dependent upon the drug's efficient transport across the mucosal epithelium and its stability in entero-hepatic circulation. Drugs that are effective after parenteral administration but less effective orally, or whose plasma half-life is considered too shon. may be chemically modified into a prodrug form.
Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidams. buffers, bacteriostats. and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers. thickening agents, stabilizers, and preservatives. The inhibitor can be administered in a physiologically acceptable dilucni in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol. isopropanol. or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals. such as 2.2-dimethyl-1.3-dioxolane-4-mcthanol. ethers, such as poly(ethylencglycol) 400. an oil. a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride, with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent. such as pectin, carbomers, methylcellulosc. hydroxypropylmcthylccllulose. or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants. Oils, which can be used in parenteral formulations include petroleum. animaL vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, com, olive, petrolatum, and mineral.
Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
Suitable soaps for use in parenteral formulations include fatty alkali metals, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides. and alkyl pyridinium halides, (b) anionic determents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates. and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides. and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as. for example, alkyl-p-aminopropionates. and 2-alkyl-imidazoIine quaternary ammonium salts, and (e) mixtures thereof.
The parenteral formulations will typically contain from about 0.5 to about 25% by weight of the active ingredient in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophilc-lipophile balance (HLB) of from about 12 to about 17.
The quantify of surfactant in such formulations will typically range from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monoolcate and the high molecular weight adducts of cthylene oxide with a hydrophobic base, formed by the condensation of propylcne oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid cxcipicnt. for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
Such compositions can be formulated as intraocular formulations, sustained-release formulations or devices (see, e.g.. U.S. Pat. No. 5.378.475). For example, gelantin, chondroitin sulfate, a polyphosphoester, such as bis-2-hydroxyethyl-terephthalate (BHET), or a polylactic-glycolic acid (in various proportions) can be used to formulate sustained-release formulations. Implants (see, e.g.. U.S. Pat. Nos. 5.443.505, 4.853.224 and 4.997.652), devices (see, e.g.. U.S. Pat. Nos. 5.554.187. 4.863.457,
5,098,443 and 5,725,493), such as an implantable device, e.g., a mechanical reservoir, an intraocular device or an extraocular device with an intraocular conduit (e.g., 100 mu -1 mm in diameter), or an implant or a device comprised of a polymeric composition as described above, can be used.
The present inventive method also can involve the co-administration of other pharmaceutically active compounds. By "co-administration" is meant administration before, concurrently with, e.g., in conlBination with the BCAT inhibitor in the same formulation or in separate formulations, or after administration of a BCAT inhibitor as described above. For example, corticosteroids, e.g., prednisone. methylprednisolone. dexamethasone, or triamcinalone acetinide, or noncorticosteroid anti-inflammatory compounds, such as ibuprofen or flubiproben. can be co-administered. Similarly. vitamins and minerals, e.g.. zinc, anti-oxidants. e.g.. carotcnoids (such as a xanthophyll carotenoid like zeaxanthin or lutein), and micronutrients can be co-adminisiercd.
General synthetic schemes for preparing compounds of Formula I- IV.
EXAMPLE 1: trans-(lR3R)(l-AininomethyI-3-methyl-cycIohexyl)-acetic acid hydrocbioride
(Formula Removed)
Step i: 2-Cyano-((R)-3-methyl-cycIohexyIidene)-acetic acid ethyl ester
A mixture of 3-(R)-Methylcyclohexanone (125mmol), Ethyl cyanoacetaie (124mmol). Ammonium acetate (12.5mmol) and glacial Acetic acid (24mmnol) were rcfluxed with a Dean Stark trap for 24 Hours. The mixture was cooled and washed with H2O. The H2O washes were extracted with Toluene. The Tdluene extracts were combined with the original organic layer, dried over MgSO4 and the solvent evaporated. The crude oil was purified by Kugelrohr distillation to give an oil. (Bpt oven temp 1 50-1 60°C). Yield 86%
1HNMR (CDCl3) 400MHz δ: 1.01-1.05 (3H, m), 1.17-1.32 (1H, m), 1.35 (3H. t. J=7Hz), 1.42-2.30 (6H, m), 2.98 (1H, d, J=13Hz), 3.74 (1H, d, J=13Hz), 4.27 (2H, q, J=7Hz)
MS (CI) m/z: 208 (MH+)
Analysis: C12H17NO2 Calc: C 69.54 H 8.27 N 6.76;
Found: C 69.44 H 8.22 N 6.76
Step ii: trans-(lR3R)-l-Cyanomethyl-3-methyl-cyclohexanccarbonitrile
To a solution of NaCN (40mmol) in 6mL H20 and 160mL Ethanol (95%) was added 2-Cyano-((R)-3-methyl-cyciohexylidene)-acetic acid ethyl ester (40mmol). After 22 Hours at reflux the cooled solution was filtered, the filtrate acidified with gaseous HCI and Tillered again. The solvent was removed and the crude oil was purified by column chromatography to give a pale yellow crystalline solid. Yield 88%
1HNMR (CDCl3) 400MHz δ: 0.90 (IH. m). 0.9S (3H. d. J=6Hz). 1.11 (111.1. J=12Hz). 1.38 (1 H, dt. J=4. 9Hz). 1.60-1.90 (4H, m). 2.07 (2H, m). 2.68 (2H. s) MS (CI) m'z: 163 (MH*)
Analysis: C10H14N2 Calc: C 74.04 H 8.70 N 17.27
Found: C 74.05 H 8.71 N 17.25
Step iii: trans-2-((lR,3R)-l-Cyano-3-mcthyl-cyclohexyI)-acctimidic acid ethyl ester
To a solution of trans-(lR,3R)-l-Cyanomethyl-3-methyl-cyclohexanecarbonitrile (6.2mmol) in 30mL Ethanol (absolute) was added 30 mL dried Toluene. The solution was chilled in ice whilst saturating with gaseous HCI. The stoppered solution was then left to stand at room temperature for 24 Hours. The solvent was removed and the solid residue was triturated with Diethyl Ether to obtain a ppt which was dried to give a white crystalline solid. Yield 50%.
Mpll8-120°C
1HNMR (DMSO) 400MHz δ: 0.8-0.89 (IH, m), 0.91 (3H, d, J=6.3Hz), 1.06-1.12 (IH, m), 124-1.35 (IH, m), 1.37 (3H, t, J=7Hz), 1.41-1.95 (6H, m), 3.02 (2H, s), 4.49 (2H, q, J=7Hz) MS (Cl) m/z: 195 (MPT)
Analysis: Cl2H2ON2O . 1.08M HCI Calc: C 58.19 H 8.58 N 11.31
Found: C 58.25 H 8.59 N 11.59
Step iv: trans-((lR3R)-l-Cyano-3-methyl-cyclohexyl)-acetic acid ethyl ester
trans-2-((lR,3R)-l-Cyano-3-methyl-cyclohexyl)-acetimidic acid ethyl ester (1.1 mmol) was dissolved in ice cold H2O (40mL) and the pH adjusted with 1N HCI to pH 1.5. The solution was stirred at room temperature for 20 Hours. Ethylacetate was added (30mL) and the organic layer was washed with H2O. dried and the solvent removed to leave a clear oil. Yield 82%
1HNMR (CDCI3) 400MHz5: 0.78-0.90 (IH. m). 0.93 (3H. d. J=6Hz). 0.97-1.00 (111. m).
1.23-1.25 (IH. m). 1.29 (3H. t. J=7.2Hz). 1.59-1.80 (4H. m). 2.05-2.08 (211. Brt). 2.54 (2H.
s),4.20(2H.q.J=7.2Hz)
MS(CI)mz:210(MH)
Analysis: C12H19NO2: Calc: C 68.87 H 9.15 N 6.69
Found: C 68.87 H 9.11 N 6.90
Step v: trans-(5R,7R)-7-Methyl-2-aza-spiro|4.5]dccan-3-one
trans-((lR,3R)-l-Cyano-3-mcthyl-cyclohcxyI)-acetic acid ethyl ester (8.9mmol) was dissolved in NH3/EtOH (7%. 40mL) along with prewashed Rancy Nickel (H2O followed by EtOH) in a 250mL Parr flask. The solution was hydrogenated at 30 C. 46psi for 24 Hours. The cooled solution was filtered through a pad of celite. washing with Ethyl acetate. The solvent was removed from the filtrate to leave a white solid. Yield 30%.
Mp 92-98°C
1HNMR (DMSO) 400MHzδ: 0.75-0.82 (IH, m), 0.84 (3H, d, J=6.4Hz), 0.88-0.94 (IH, m), 1.14-1.19 (IH, m), 120-1.50 (2H, m), 1.50-1.63 (4H, m), 1.91 (2H, s), 3.03 (2H. s). 7.42
Analysis: C10HI7NO Calc: C 71.81 H 10.25 N 8.37
Found: C 71.80 H 10.29 N 8.31
Step vi: trans-((lR3R)-l-Aminomethyl-3-rnethyl-cycIohexyl)-acctic acid hydrochloridc
trans-(5R.7R)-7-Methyl-2-aza-spiro[4.5]decan-3-one (2.17mmol) was dissolved in a solution of 10M HCI (5mL) and H2O (5mL) and the mixture was relluxed at approx 140"C for 5 Hours. The cooled solution was diluted with 10 mL H2O and 10 mL DCM and the aqueous layer was further washed with 2x15 mL DCM. The aqueous layer was then reduced to dryncss to leave a white solid. Yield 76%.
Mpl48-155°C. [α]D = -2.5 (T=20°C.c=l. MeOH). One isomer (RR). 1HNMR (CDCl3) 400MHzδ: 0.69-0.79 (IH. m). 0.82 (3H. d. J=6Hz). 0.87-0.90 (IH. m). 1.12-1.20 (IH. dt. J=4.5. 13.31 Iz). 1.34-1.50 (3H. m). 1.60-1.63 (3H. m). 2.30 (2H. s). 3.01 (2H.s).7.93(3H. Brs) MS(Cl)m/z: 186(MH*)
Analysis: C,0H,oNO2. 1.1 HCI Calc: C 53.29 H 8.99 N 6.21
Found: C 53.23 H 8.99 N 6.45
EXAMPLE 2: (l-Aminomethyl-2-methyl-cyclohexyl)-acctic acid hydrochloridc
(Formula Removed)
Step i: 2-Cyano-(2-mcthyl-cycIohcxylidcnc)-acetic acid ethyl ester
(+/-) 2-Methyl cyclohexanone (SOmmol). Ethyl cyanoacetate (SOmmol). Ammonium acetate (Smmol) and glacial Acetic acid (16mmol) were reacted as in the General method (Example 1 ) step i to give a clear oil. Yield 76%.
1 HNMR (CDCl3) 400MHz 5: 1.23 (3H,dd. J=7. lOHz), 1.35 (3H. t. J=7Hz). 1.55-1.82 (5H, m), 1.93-2.05 (IH, m), 2.17 (IH. dt. J=5. MHz). 2.47 (IH, dt, J=5. 9Hz). 2.92-2.97 (IH, Br d, J=15Hz). 3.30-3.35 (IH, m), 3.81-3.86 (IH, Brd, J=15 Hz), 4.06-4.14 (IH, m). 4.23^.30 (3H,dq,J=l,6Hz) MS (Cl) m/z: 208 (MH+)
Analysis: C12HI7NO2 Calc: C 69.54 H 8.27 N 6.76
Found: C 69.26 H 8.26 N 6.66
Step ii: l-Cyanomethyl-2-rnethyl-cyclohexanecarbonitrile
2-Cyano-{2-methy]-cyclohexylidene)-acetic acid ethyl ester (37mmol), NaCN (37mmol) were reacted as in the General method (Example 1) step ii. The crude solid was purified
by column chromatography (3:1, Heptane : Ethylacetate), to give a clear oii. Yield 76%
1HNMR(CDCl3) 400MHz 5: 1.06(3H, d, J=6.8Hz), 1.11 (3H, d. J=6.8Hz). 1.20-2.20 (18H, m), 2.77 (2H, dd, J=16.8Hz). 2.63 (2H. dd. J=16.8Hz) MS(CI)m/z: 163 (MH+)
Analysis: C|0H14N2 .0.1 H2O Calc: C 73.49 H 8.69 N 16.86
Found: C 73.24 H 8.73 N 17.08
Step iii: 2-(1-Cvano-2-mcthyl-cyclohexyl)-acetimidic acid ethyl ester
l-Cyanomethyl-2-methyl-cyclohexanecarbonitrile(7.3mmol) was reacted as in the General method (Example 1) Step (iii) to give a white solid. Yield 70%.
Mpt 107-114°C.
1HNMR (DMSO) 400MHz 6: 1 .OO-1.06 (3H. 2xt. J=6.4Hz). 1.10-1.38 (2H. m). 1.38 (3H. i, J=6.8Hz), 1.40-2.10 (7H. m), 2.86. 2.92, 3.10. 3.28 (2H. 4xd. .1=14.14.4. 14.8. 14H?. resp.), 4.48 (2H, q, J=6.8Hz) MS (CI) m/z: 209 (MH*)
Analysis: CI2H2ON2 O . 1.06 HCi Calc: C 58.37 H 8.60 N 11.34
Found: C 58.15 H 8.63 N 11.60
Step iv: (l-Cyano-2-mcthyl-cyclohexyl)-altetic acid ethyl ester
2-(l-Cyano-2-methyl-cyclohexyl)-acetimidic acid ethyl ester (4.1mmol) was reacted as in the Genera] method (Example 1) step (iv) to give a clear oil. Yield 82%
1HNMR (CDC13) 400MHzo: 1.03, 1.09 (3R 2xd, J=7Hz), 1.27-1.30 (3H, m), 1.32-2.00 (8H, m) 2.10-2.20 (IH, m), 2.44, 2.82 (3H, 2xd, J=14.8Hz), 2.54 (IH. m), 4.16-4.22 (2H. m)
Analysis: C^H^NO, Calc: C 68.87 H 9.15 N 6.69
Found: C 68.84 H 9.13 N 6.75
Step v: 6-MethyI-2-aza-spiro|4.5)decan-3-one
(l-Cyano-2-methy!-cyclohexyl)-acetic acid ethyl ester (8.4mmol) was reacted as in the General method (Example 1) Step (v) for 24 Hours at 10°C. 50 psi. The crude oil was purified by column chromatography (Ethylacetate), to give a while solid. Yield 34%.
Mp: 85-90 °C
1HNMR (CDCl3) 400MHz 5: 0.88-0.91 (3H. dd, J=4. 6.8Hz). 1A1 -1.78 (911. m). 2.0U-2.30 (2H, m). 3.06-3.23 (2H. m). 7.27 (IH. Br s) MS(Cl)m'z: 168(MH*)
Analysis: C,0H,7NO Calc: C 71.81 H 10.24 N 8.37
Found: C 71.83 H 10.19 N 8.27
Step vi: (l-AminomcthyI-2-mcthyl-cyclohcxyI)-acetic acid hydrochloridc
6-Methyl-2-a7a-spiro[4.5jdecan-3-one (2.5mmol) was reacted as in the Genera! method (Example 1) step vi to give a white solid. Yield 42%.
Mp:108-110°C.
[cc]D = 0 (T=20.5° C. 01. MeOH). Two Diastereomers 3:1
1HNMR (DMSO + D2O) 400MHz5: 0.79, 0.85 (3H, 2xd, J=6.8Hz), 1.21-1.65 (9H, m), 2.22,2.43 (IH, 2xd, J=15Hz), 2.46,2.49 (IH, 2xd, J=15Hz), 2.83-2.92 (IH, 2xd. J=13.6Hz), 3.05,3.15 (lH,2xd,J=13.6Hz). MS(CI)m/z:186(MH+)
Analysis: C,oHl9NO2. 1.3 HC1 Calc: C 51.64 H 8.79 N 6.02
Found: C 51.66 H 8.91 N 6.16
EXAMPLE 3 (l-AminomethyI-33-dimethyl-cyclohexyl)-acctic acid hydrochloridc
(Formula Removed)
Step i: 33-Dimcthyl-cyclohcxanone:
Synthesised via the method outlined by Pelletier. S.W.. Mody. N.V., J. Or». Chart.. 1976. 41,1069.
A solution of lithium dimethyl cuprate was prepared by the addition of methyl lithium (1.4M in ether, 7725mL, 2.45mol) to copper (I) iodide (8.8g, 0.046mol) under argon. The solution was cooled to 0°C, and 3-methyl-cyclohexen-l-one (5mL, 0.044mol) was added dropwise, with stirring, and a deep yellow precipitate was formed. The suspension was stirred at room temperature for one hour before being poured into a solution of aqueous ammonia (lOOmL) and ammonium acetate (ca. 5g). The layers were separated and the aqueous layer washed with diethyl ether (3x 50mL). The combined organics were washed with saturated brine (3x lOOmL), dried (MgSO.0, and the solvent removed in vacuo to leave a dark yellow liquid.
'H NMR (400MHz. CDC13) 0.98 (6H, s). 1.59 (2H. m). 1.88 (2H. m). 2.14 (2H. m). 2.26
(2H, m).
IR (film) vnmcirf' 2956.1711 (CO), 1457,1368. 1292.1226. 1076.
Step ii: 2-Cyano-5.5-dimcthyl-3-propyl-hex-2-enoic acid ethyl ester
To a solution of 3.3-dimethylcyclohcxanonc (4g. 0.032mol) in toluene (25mL) was added ethyl cyanoacctatc (3.3~mL 0.032mol. 1 eq.). ammonium acetate (0.2-4;:. O.OOSmnl. O.leq.). and acetic acid (0.36mL. 0.006mol. 0.2eq.). The yellow solution was heated to reflux whilst attached to a Dean-Stark trap and heating was continued until no more water condensed in the trap. After cooling, the now orange solulion was washed with water (3x 25mL) and the organic layer dried (MgSO-t). Filtration and removal of the solvent in vacua gave the crude product as a deep orange liquid. Purification was achieved by Kuuelrohr distillation to leave the mixture of els and tram products as a pale yellow liquid, bp 160-170°C. 4 mbar (5.83g. 83%).
'H NMR (400MHz. CDC13) 0.96 (6H. s. 2x Me). 0.99 (6H. s. 2x Me).l.34 (6H. m. 2x Me of
ester). 1.49 (4H, m). 1.75 (2H, quin. 76.4), ;.82 (2H. quin, ./6.4). 2.46 (2H. s). 2.60 (2H. t. J
= 6.4 Hz). 2.80 (2H. s). 2.93 (2I-I, tT J = 6.4 Hz). 4.27 (4H. m).
MS(CI)z/e222(M+l)
IR (film) Vroxcm'1 2958, 2870. 2224 (CN). 1731 (C=O). 1606 (C=C), 1277. 1223.
Analysis: C11H17O2N Calc: C 70.56 H 8.65 N 6.32

Found: C 70.35 H8.79 N 6.25 Step Hi: l-CyanomethyI-33-din'ethyl-cyclohexanecarbonitrile
To a solution of the 2-Cyano-5.5-dimethyl-3-propyl-hex-2-enoic acid ethyl ester (1.26g.
0.006mol) in ethanol (100ml) and water (4mL) was added sodium cyanide (0.28g,
0.006mol, 1 eq.). The yellowish solution was heated to reflux for 8 hours and then cooled.
during which time an off-white precipitate was formed.
The suspension was filtered under vacuum and the filtrate acidified with HCI gas until the
pH was approximately 2. The mixture was then filtered a second time and then the solvent
removed in vacua to leave the crude product as a pale green solid.
Flash column chromatography. after adsorption of the crude product on to silica, eluting
with 0-50% EtOAc in heptane gave the binitrile as a colourless solid (0.57g. 57%).
'H NMR (400MHz, CDC13) 0.99 (3H, s, Me). 1.13 (1H. td. J 13.2. 4.2). 1.21 (3H. s. Me). 1.32 (2H. m). 1.54 (1H, m). 1.82 (3H. m). 2.15 (1H. m). 2.65 (2H. s. CH,CN). I3C NMR (400MHz, CDC13) 19.61. 25.17. 30.79. 31.18. 33.77. 34.79. 35.37. 37.92. 46.26. 115.06, 122.19. MS(CI)z/cI77(M+l)
IR (film) vwcm'1 2988. 2937. 2869. 2237 (2x CN). 1749. 1456. 1423. 1369. 1202. 1180. 1031,972.
Analysis: C11H16N2 Calc: C 74.96 H9.15 N 15.89
Found: C 75.08 H 9.32 N 15.80
Step iv: (l-Cyano-3,3-dimethyl-cyclohcxyI)-acetic acid ethyl ester:
l-Cyanomethyl-3,3-dimethyl-cyclohexanecarbonitrile (0.50g. 2.84mmol) was dissolved in absolute ethanol (20mL) at room temperature and then cooled to 0 C. Toluene (20mL) was added to the solution and then the reaction mixture was acidified by passing HCI gas through it at a gentle rate for ca. 45 mins. The flask was then stoppered and left to stand at room temperature for 24 hours.
The yellow solution was partitioned between ethyl acetate and water and the layers separated. The aqueous layer was extracted with ethyl acetate (3x 30mL), and the combined organics washed with aq. sat. sodium hydrogen carbonate solution (3x 50mL), brine (3x 50mL), dried (MgSO4), and the solvent removed under reduced pressure to leave a pale yellow liquid (0.59g, 93%).
1HNMR (400MHz, CDC13) 0.94 (3H s, Me), 1.16 (3H,m), 1.21 (3H, s, Me), 1.29 (3H, t, J
7.2, CH2CH3), 1.50 (IH, m), 1.65 (IH, dt, J 14.4, 7.6), 1.84 (IH, qt. J 13.3. 3.2).1.96 (IH,
dt, J 13.7. 2.2), 2.16 (IH, m), 2.48, (IH, d, J 15.6, C-2H), 2.54 (IH, d, J 15.6, C-2H). 4.20
(2H,q,J7.2, CH2CH3).
13C NMR (400MHz, CDC13) 14.21, 19.65, 25.42. 31.03. 34.04. 34.14. 36.08. 38.44. 46.14.
46.80,61.02,123.67, 169.00.
MS(Cl)z/e224(MVl).
IR (film) vmaxcm' 2998, 2937. 2868, 2234 (CN). 1738 (CO). 1457, 1372. 1217. 1181, 1154, 1026.
Analysis: C13H21NO2 Calc: C 69.92 H 9.48 N 6.27
Found: C 69.63 H 9.45 N6.15
Step v 7,7-Dimcthvl-2-aza-spiro[4.5]dccan-3-onc:
n-Cyano-3.3-dimethvl-cvclohcxyl)-acetic acid ethyl ester (0.5g. 2.23mmo!) was
hydrogenatcd in ethanolic ammonia (60mL) with Raney nickel as catalyst (cu. 0.25g) at
50°C and 50psi for 48 hours.
The catalyst was then removed by filtration through Celite, and the solvent removed in
vacua to leave a greenish crystalline solid.
Flash column chromatography. cluting with 0-100% ethyl acetate in heptane, gave the pure
lactam as a colourless solid (340 mg. 84%).
1HNMR (400MHz. CDC13) 0.89 (3H, s. Me), 0.92 (3H, s. Me). 1.25 (211. m), 1.36 (2H. m). 1.51(3H,m), 1.68 (lH,s), 2.18 (lH,d,y 16.4, CH2NH), 2.24 (lH,d,./16.8, CH:NH), 3.15 (2H, s, CH2CO).
I3C NMR (400MHz, CDCl3) 19.16, 29.88, 30.36, 3128, 36.57, 39.05, 39.61, 44.58, 49.54, 54.79,177.72. MS(CI)z/el82(M++l).
IR (film) vmax cm'1 3203, 3100 (NH), 2914, 2860, 1698 (C=O), 1486, 1374. 1317. 1289. 1257,1076.
Analysis: C11H19NO Calc: C 72.88 H 10.56 N 7.73
Found: C 72.38 H 10.47 N 7.56
Step vi (I-Aminomethyl-3,3-dimethyl-cycIohexyl)-acetic acid hydrochloride:
7,7-Dimethyl-2-aza-spiro[4.5]decan-3-one (0.3g, 1.66mmol) was dissolved in a mixture of HO (cone.. 5mL) and water (5mL). and the resultant colourless solution heated to reflux for 20 hours. The solution was cooled and then partitioned between water and dichloromcthane, and the layers separated. The aqueous layer was washed with dichloromethane (3x 20mL) and the water/HCl removed by rotary evaporation to leave ;he crude product as an off-white solid. Triruration of this solid with ethyl acetate and filtration of the product gave (I -Aminomcthyl-3.3-dimethyl-cyclohexyl)-acetic acid, hydrochloride sail as a colourless solid (140mg. 42%. 64% based on recovered starting material).
1HNMR (400MHz. DMSO) 0.90 (3H. s. Me). 0.92 (3H, s. Me). 1.15-1.-49 (SI I. m). 2.45 (2H, s. CH2CO21S). 2.90 (2H. br.q,./ 13.5. CH2NH3). 7.96 (3H. br.s. NH,). 12.36 (111. br.s. OH).
IR (film) vmaxcm-1' 2930. 1728 (C=O). 1272, 1123.
Analysis: C11H22NO2C1 Calc: C 56.04 119.41 N 5.94
Found: C 55.79 H9.61 N 6.23
EXAMPLE 4: (±Mtrans)-(l-AminomethyI-3,4-dimethyl-cyclopcntyl)-acctic acid hydrochloride
(Formula Removed)
Reagents: (i) Triethylphosphonoacetate, NaH; (ii) MeNO2.Bu4N+F; (iii) H2- Ni; (iv) HC1
Step i: synthesis of (±) (trans)-(3,4-Dimcthyl-cyclopenrylidcne)-acetic acid ethyl ester (4b)
NaH (60% dispersion in oil. 737 mg. 18.42 mmol) was suspended in dry tetrahydrofuran (50 mL) and cooled to 0°C. Tricthylphosphonoacetatc (3.S3 mL. 19.30 mmol) was added and the mixture stirred at 0°C for 15 minutes. The ketone (+/-)-(4a) (1.965 g. 17.54 mmol) in THF (10 mL) was then added and the mixture allowed to warm to room temperature. After 2 hours, the mixture was partitioned between dicthyl ether (200 mL) and water (150 mL). The orcanic phase was separated, washed with brine. dried (MgSO4) and the solvent removed in vacuo. The residue was purified by Hash
chromatography (silica, ethyl acetate:heptane 1:9) to give 3.01 g (94%) of (4b) as a colorless oil.
lHNMR400MHz(CDCl3):δ 1.01 (3H, d, J = 6 Hz). 1.03 (3H. d. .1 = 6 Hz). 1.26(3H, t.
J = 7 Hz), 1.49 (2H. m), 2.07 (IH. m), 2.24 (IH. m), 2.61 (1 H. m), 4.13 (2H. q, .1 = 7 Hz), 5.72 (1H,s). MS(CI+)m/e: 183 (M+l).
Step ii: synthesis of (±)(trans)-(3,4-Dimethyl-l-nitromethyl-cyclopentyl)-acctic acid ethyl ester (4c)
(±)(trans)-(3,4-Dirnethyl-cyclopentylidene)-acetic acid ethyl ester (4b) (2.95 g. 16.2 mmol) was dissolved in tetrahydrofuran (10 mL) and stirred at 70°C with nitromethane (1.9 mL, 35.2 mmol) and tetrabutylammonium fluoride (1.0 M in tetrahydrofuran, 22 mL, 22.0 mmol) After 6 hours, the mixture was cooled to room temperature, diluted with ethyl acetate (50 mL), and washed with 2N HC1 (30 mL) followed by brine (50 mL). The organic phase was collected, dried (MgSO4) and the
solvent removed in vacuo. The residue was purified by flash chromatography (silica, ethyl acetate.'heptane 1:9) to give 1.152 g (29%) of a clear oil.
1HNMR 400 MHz (CDC13): δ 0.98 (6H. d, J = 6 Hz). 1.10-1.39 (5H. m). 1.47 (211. m).
1.87 (1H. m), 2.03 (1H, m), 2.57 (2H, ABq, J = 16, 38 Hz). 4.14 (2H, q. J = 7 Hz). 4.61 (2H.ABq. .1= 12. 60 Hz). MS(ES+)m/e: 244 (M+1).
IR(film) cm-1: 1186. 1376. 1549. 1732.2956.
Step iii: Synthesis of (±)-(trans)-7,8-Dimcthyl-spiro[4.4]nonan-2-onc (4d)
(±)(trans)-(3.4-Dimethyl-]-nitromethyl-cyclopentyl)-acetic acid ethyl ester (4c) (1.14 g. 4.7 mmol) was dissolved in methanol (50 mL) and shaken over Raney nickel catalyst under an atmosphere of hydrogen (40 psi) at 30°C. Alter 5 hours, the catalyst was removed by filtration through celile. The solvent was removed in vacuo to uive 746 mg (95%) of a pale yellow oil which solidified on standing.
1H NMR 400 MHz (CDC13): δ 0.98 (6H, d. J = 6 Hz). 1.32 (2H. m). 1.46 (2H. m). 1.97
(2H, m), 2.27 (2H. ABq. J = 16, 27 Hz), 3.23 (2H, s). 5.62 (1 H. br s). MS(ES+)m/e: 168(M+1).
IR(film)vcm-l: 1451, 1681, 1715.2948,3196.
Step iv: Synthesis of (±HtransHl-Aininoniethyl-3,4-dimethyl-cyclopentyI)-acetJc acid hydrochloride (4)
(±)-(trans)-7.8-Dimethyl-spiro[4.4]nonan-2-one (4d) (734 mg. 4.40 mmol) was heated to reflux in a mixture ofl,4-dioxan (5 mL) and 6N HCI (15 mL). After 4 hours, the mixture was cooled to room temperature, diluted with water (20 mL), and washed with dichloromethane (3 x 30 mL). The aqueous phase was collected and the solvent removed in vacuo. The residue was inn/rated with ethyl acetate to give 675 mg (69%) of a white solid after collection and drying.
lHNMR400MHz(d6-DMSO): δ0.91 (6H. d. J = 6 Hz). 1.18(2H.m). 1.42(2H.m).
1.72 (1H. m). 1.87(1 H. m). 2.42 (2H. ABq. J = 16. 24Hz). 2.90 (2H. ABq. .1=12. 34
Hz), 8.00 (3H, br s). 12.34 (1 H. br s).
MS (ES+) m/e: 186 (M+l for the free base).
EXAMPLE 5 (cis/transH3R)-(l-Aminomethyl-3-mcthyl-cyciopcntyl)-:icetic acid hydrochloride (5)
(Formula Removed)
Reagents: (i) Triethylphosphonoacetate, NaH; (ii) MeNO2, Bu4N+F; (iii) H2. Ni; (iv) HCI
Step i: Synthesis of (R)-(3-Methyl-cyclopentylidene)-acetic acid ethyl ester (5b)
NaH (60% dispersion in oil, 1.86 g, 46.5 mmol) was suspended in dry tetrahydrofiiran (40 mL) and cooled to 0°C. Triethylphosphonoacetate (9.69 tnL, 48.8 mmol) was added and the mixture stirred at 0°C for 15 minutes. (R)-3-Methyl-cyclopemanone (5a) (5 ml, 46.5 mmol) in THF (10 mL) was then added and the mixture allowed to warm to room temperature. After 2 hours, the mixture was partitioned between diethyl ether (200 mL) and water (150 mL). The organic phase was separated, washed with brine, dried (MgSO4) and?the solvent removed in vacuo. The residue was
purified by flash chromatography (silica, ethyl acetatc:hcptane 1:9) to give 5.45 g (70%) of (2) as a colorless oil.
1H NMR 400 MHz (CDC13): δ 1.04 (3H, m). 1.27 (3H. t. J = 7 Hz). 1.80-2.74 (711. m).
2.90-3.15 (1H, m). 4.13 (2H. q, .1 = 7 Hz). 5.76 (1H. s).
MS(Cl+)m/e: 169(M+1).
IR(film) vcm-': 1205, 1371, 1653, 1716.2955.
Step ii: Synthesis of (cis/trans)-(3RH3-Methyl-l-nitromcthyl-cvclopcntyl)-iicctic acid ethyl ester (5c)
(RM3-Methyl-cyclopemylidene)-accuc acid ethyl esier (5b) (3.0 g. 17.8 mmol) was dissolved in tetrahydrofuran (20 mL) and stirred at 70°C with nitromcihanc (1.92 mL, 35.6 mmol) and tetrabutylammonium fluoride (1.0 M in ictruhydroluran. 25 mL. 25.0 mmol). After 18 hours, the mixture was cooled to room temperature, diluted with ethyl acetate (50 mL), and washed with 2N HCI (30 mL) followed by brine (50 mL). The organic phase was collected, dried (MgSO4). and the solvent removed in vacuo. The
residue was purified by flash chromatography (silica, ethyl acctatc:heptane 1:9) to iiive 2.00 g (49%) of a clear oil.
1HNMR 400 MHz (CDC13): δ 1.02 (3H. d. J = 6 Hz). 1.08-1.37 (5H. m). 1.59-2.! 7 (5H.
m), 2.64 (2H. m), 4.15 (2H, q. J = 7 Hz). 4.64 (2H, m). MS(ES+)m/e:230(M+l).
IR (film) v cm1: 1183, 1377. 1548, 1732,2956.
Step HhSynthesis of (cis/trans)-(7R)-7-MelhyI-spiro[4.4Jnonan-2-one (5d)
(cis/trans)-(3R)-(3-Methyl-l-nitromethyl-cycIopentyl)-acetic acid ethyl ester (5c) (1.98 g, 8.66 mmol) was dissolved in methanol (50 mL) and shaken over Raney nickel catalyst under an atmosphere of hydrogen (40 psi) at 30°C. After 18 hours, the catalyst was removed by filtration through celite. The solvent was removed in vacuo and the residue purified by flash chromatography (silica, ethyl acctate:heptane 1:1) to give 1.05 g (79%) of a white solid.
IH NMR 400 MHz (CDC13): δ 1.03 (3H, m). 1.22 (2H, m). 1.60-2.15 (5H, m). 2.22 (2H.
m), 3.20 and 3.27 (2H total, 2 x s, cis. and trans), 6.18 (IH. brs). MS(ES+)m/e: 154 (M+l). IR(film)vcm-l: 1695,2949.3231.
Step iv: Synthesis of (cis/trans)-(3R)-(l-Aminomcthyl'3-methyl-cyclopcnn'l)-;»cctic acid hydrochloridc (5)
(cis.'trans)-(7R)-7-Methyl-spiro[4.4jnonan-2-one (5d) (7-46 my. 4.88 mmol) was heated to reflux in a mixture of L4-dioxan (5 mL) and 6N HC1 (15 mL). After 4 hours, the mixture was cooled to room temperature, diluted with water (20 mL). and washed with dichloromethanc (3 x 30 mL). The aqueous phase was collected and the solvent removed in vacuo. The residue was triturated with ethyl acetate to give a white solid which was collected and dried. This was recrystallizcd from ethyl acctate/mcthanol to give 656 mg (65%) of (5) alter collection and drying.
1HNMR 400 MHz(d6-DMSO): δ 0.96 (3H. m). 1.01-1.24 (211. m). 1.42-2.10 (511. m),
2.41 and 2.44 (2H total. 2 x s, cis/trans), 2.94 (2H. m). 7.96 (3H. br s), 12.35 (1H. br s). MS (ES+) m/e: 172 (M+l for the free base).
EXAMPLE 6: (+)-(trans)-(l-AminomethyI-3,4-dimethyl-cyclopentyl)-acetic acid hydrochloride
(Formula Removed)
Reagents: (i) Trieihylphosphonoacetate. Nail; (ii) MeNO2. Bu4N' F(iii)H2(iv) HC1
Step i: Synthesis of (cis)-(3,4-l)inicthyl-cyclopentylidcnii)- ethyl i-stcr (fib)
NaH (60% dispersion in oil. 519 mg. 12.96 mmol) was suspended in dn tetrahydrofuran (30 niL) and cooled to 0°C. Triethylphosphonouceiaic (2.68 ml.. 13.5 mmol) was added and the mixture stirred at 0°C lor 15 minutes. (3S.4R)-3.4-Uimcthy|-cyclopentanone (6a) (1.21 g. 10.80 mmol) in THF (10 mL) was then added and the mixture allowed to warm to room temperature. After 2 hours, the mixture was partitioned between diethyl ether (200 mL) and water (150 mL). The organic phase was separated. washed with brine, dried (MgSO4l and the solvent removed in vacuo 'I he residue was
purified by flash chromatography (siliea. ethyl acetate:heptane 5:95) to give 1.40 g (7!%) of (2) as a colorless oil.
1HNMR 400 MHz (CDC1 13): δ 0.84 (3H. d, J = 6 Hz), 0.91 (3H, d. J = 6 Hz). 1.26 (3H. t, J = 7 Hz), 2.01-2.95 (6H. m). 4.13 (2H, q. J = 7 Hz), 5.76 (1H, s).
MS (CH-) m/e: 183(M+1).
IR (film) v cm'1: 1043,1125,1200,1658, 1715,2959.
Step ii: Synthesis of (trans)-(3,4-Dimethyl-l-nitromethyl-cyclopentyl)-acetic acid ethyl ester (6c)
(cis)-(3,4-Dimethyl-cyclopentylidene)-acetic acid ethyl ester (6b) (1.384 g. 7.60 mmol) was dissolved in tetrahydrofuian (10 mL) and stirred at 70°C with nitromethane (0.82 mL, 15.2 mmol) and tetrabutylammonium fluoride (1 .OM in tetrahydrofuran. 1 1.4 mL, 11.4 mmol). After 6 hours, the mixture was cooled to room temperature, diluted with ethyl acetate (50 mL) and washed with 2N HCI (30 mL) followed by brine (50 mL). The organic phase was collected, dried (MgSO4). and the solvent removed in vacuo. The residue was purified by flash chromatography (silica, ethyl acctale:heptanc 5:95) to give 0.837 g (45%) of a clear oil.
1HNMR 400 MHz (CDCI3): δ 0.91 (6H. d, .1 = 6 Hz), 1.21-1.39 (5H. m). 1.9S (211. m).
2.18 (2H. m). 2.64 (2H. s). 4.15 (2H. q. J = 7 Hz). 4.61 (211. s). MS(ES-)rae:2-44(M+l).
IR(film) v cm1: 1184. 1377. 1548. 1732.2961.
Step iii: Synthesis of (trans)-7,8-Dimethyl-spiro(4.4|nonan-2-onc (6d)
(tr3n.s)-(3.4-DimcthyI-l-nitromcthyl-cyclopcntyl)-acctic acid ethyl ester (6c) (0.83 g, 3.4 mmo!) was dissolved in methano! (30 mL) and shaken over Rancy nickei catalyst under an atmosphere of hydrogen (40 psi) at 30°C. After 4 hours, the catalyst was removed by filtration through celite. The solvent was removed in vacuo to give 567 mg (99%) of a pale yellow oil which solidified on standing.
1HNMR 400 MHz (CDCl3):δ 0.89 (6H.d. J=6Hz). 1.38(2H. m). 1.91 (211. m). 2.10
(2H,m). 2.32 (2H.s). 3.18(2H, s). 5.61 (IH. brs).
MS(ES+)m/e: 168 (M+l).
IR(film) vcm1: 1304. 1450. 1699.2871,9186.
Step iv: Synthesis of (trans)-(l-Aminomethyl-3,4-diraethyI-cyclopentyl)-acetic acid hydrochloride (6)
(trans)-7,8-Dimethyl-spirof4.4]nonan-2-one (6d) (563 mg, 4.40 mmol) was heated to reflux in a mixture of 1,4-dioxan (5 mL) and 6N HC1 (15 mL). After 4 hours, the mixture was cooled to room temperature, diluted with water (20 mL), and washed with dichloromethane (3 x 30 mL). The aqueous phase was collected and the solvent removed in vacuo. The residue was triturated with ethyl acetate to give a white solid which was collected and dried. This was recrystallized from ethyl acetate/methanol to give 440 mg (59%) of (6) after collection and drying.
1HNMR400MHz(d6-DMSO):δ0.84(6H.dJ = 6Hz). 1.21 (2H. m). 1.81 (2H. m). 2.06 (2H. m), 2.47 (2H, s), 2.89 (2H. s). 7.94 (3H. br s). 12.30(1 H. br s). MS (ES+) m/e: 186 (M+l for the free base).
EXAMPLE 7 (+Htrans)-(I-AminomethyI-3,4-dimethyl-cyclopcntvl)-acetic acid hydrochloride
(Formula Removed)
Reagents: (i) Triethylphosphonoacetate, NaH; (ii) MeNO2, BU4N+F"; (iii) H2, Ni; (iv) Hcl.
(3S,4S)-3,4-Dimethyl-cyclopentanone (7a) is known in the literature and can be synthesized by the methods outlined therein: Y. Kato, Chem. Pharm. Bull., 1966;14:1438-1439 and related references: W. C. M. C. Kokke, F. A. Varkevisser, J. Org. Chem., 1974;39:1535; R. Baker,*D. C. Billington, N. Eranayake, JCS Chem. Comm., 1981:1234; K. Furuta, K. Iwanaga, H. Yamamoto, Tel. Lett., 1986;27:4507; G. Solladie. O. Lohse, Tet. Asymm., ]993;4:1547; A. Rosenquist. I. Kvarnstrom. S. C. T. Svensson, B. Classon. B. Samuelsson. Ada Chem. Scant!.. 1992;46:1127; E. J. Corey, W. Su, Tci. Lett.. 1988;29:3423; D. W. Knight. B. Ojhara. TCI. Lett.. 1981;22:5101.
Step i: Synthesis of (trans)-(3.4-DimethyI-cyclopenrylidene)-acetic acid ethyl ester (7b)
To a suspension of sodium hydride (1.3 g. 32.5 mmol) in THF (60 ml) under nitrogen at 0°C was added triethyiphosphonoacetate (6.5 mL. 32.7 mmol) over 5 minutes. After stirring for a further 10 minutes, a solution of (3S.4S)-3.4-Dimethyl-cyclopentanonc (7a) (approx. 2.68 g. approx. 30 mmol) in THF (2x10 mL) was added 10 the now clear solution and the ice bath removed. After 4 hours the reaction was quenched by pouring into water (100 mL) and the mixture exuacted with ether (400 ml,). The organic phase was washed with saturated brine (100 mL). dried and concentrated in vacuo. Column chromatography (10:1 heptane/ethyl acetate) gave the product as an oil
(4.53 g). (approx. 91% yield)
1HNMR 400 MHz (CDC13): δ 1.01 (3H, d. J = 6 ilz). 1.03 (311. d. J = 6 Hz). 1.26 (3H. t.
.1 = 7 Hz). 1.49 (2H. m). 2.07 (1H, m), 2.24 (1H. m), 2.61 (111. m). 4.13 (2H. q. J = 7 Hz).
5.72 (lH,s).
MS (CI+) m/e: 183(M+1).
Step ii: Synthesis of (trans)-(3,4-DimethyI-l-nitromethyI-cycIopentyl)-acetic acid ethyl ester (7c)
To a solution of (trans)-(3,4-Dimethyl-cyclopentylidene)-acetic acid ethyl ester (7b) (4.24 g, 23.3 mmol) in THF (15 mL) was added TBAF (32 mL of a 1 M solution in THF, 32 mmol) followed by nitromethane (3 mL) and the reaction heated at 60°C for 8 hours. After cooling, the reaction mixture was diluted with ethyl acetate (150 mL) and washed with 2N HC1 (40 mL) then saturated brine (50 mL). Column chromatography (10:1 heptane/ethyl acetate) gave the product as an oil (2.24 g). (40% yield)
1HNMR 400 MHz (CDC13): δ 0.98 (6H, d, J = 6 Hz). 1.10-1.39 (5H. m). 1.47 (2H. m).
1.87 (1H, m). 2.03 (1H, m), 2.57 (2H. ABq. J = 16. 38 Hz). 4.14 (2H. q. J - 7 Hz).
4.61 (2H. ABq,J = 12.60Hz).
MS(ES+)m/e:244(M+l).
IR (film) vorr1: 1186, 1376. 1549, 1732,2956.
Step iii: Synthesis of (+)-(trans)-(l-Aminomcthyl-3,4-dimcthyl-cyclopcntyl)-acetic acid hydrochloride (7)
A solution of (trans H3.4-Dimcthyl-l-nitromcthyl-cyclopeniyl)-acctic acid ethyl ester (7c) (3.5 g, 14.4 mmol) in methanol (100 mL) in the presence of Ni sponge was hydrogenated at 30°C and 50 psi for 4 hours. Filtering off the catalyst and concentrating in vacuo gave a 2:1 mixture oflactam and aminoestcr. 2.53 g. calculated as 96%. which was used without purification. This mixture (2.53 g. 13.8 mmol) in dioxane (15 mL) and
6N HC1 (45 mL) was heated under reflux (oil bath = 1 ! 0°C) lor 4 hours. After cooling and diluting with water (60 mL), the mixture was washed with dichloromelhane (3 x 50 mL) and then concentrated in vacuo. The resulting oil was washed with ethyl acetate then dichloromethane to give a sticky foam which was dried to give the product as a white powder (2.32 g). (76% yield)
cq) (23°C) (H2O) (c = 1.002) = +28.2°.
1H NMR 400 MHz (d6-DMSO): δ 0.91 (6H, d, J = 6 Hz), 1.18 (2H, m), 1.42 (2H, m),
1.72 (1H, m), 1.87 (1H, m). 2.42 (2H, ABq, J = 16,24 Hz), 2.90 (2H, ABq, J = 12, 34 Hz), 8.00 (3H, br s), 12.34 (1H, br s). MS (ES+) m/e: 186 (M+l for the free base).
EXAMPLE 8 lα,3ß,5ß-(l-Aminomethyl-3,5-dimethyl-cyclohexyl)-acetic acid hydrocH2Oride
(Formula Removed)
Step i: Synthesis of 2-Cyano-((3R,5S)-3,5-dimcthyJ-cyclohexylidcnc)-acetic acid ethyl ester (8b): Cis-3.5-dimcthylcyclohexanone (8a, 15.78 g. 125.0 mmol) was dissolved in toluene (150 mL), and treated with ammonium acetate (0.96 g, 0.099 mmol). acetic acid (1.4 mL, 0.195 mmo!). and ethyl cyanoacetate (14.04 g. 99.2 mmol). The reaction was heated to reflux overnight with 9 Dean-Stark trap, then cooled to room temperature and washed with H2O (3 x 50 mL). The aqueous layer was backwashed with toluene (2 x 50 mL). The organic layers were combined, dried over Na2SO4, and concentrated. The residue was chromatographed on silica gel eluting with 5 % EtOAc/hexane to give 22.0 g (80%) of the desired product as a clear oil.
MS: 222.1 (M+l for C13H19N1O2); an oik.TLC: Si02, Rr0.40 (9:1 hexane/EtOAc): Analysis (C13H19N1O2) (Cal.) C: 70.56, H: 8.68, N: 6.33 (found) C: 70.74, H: 8.68, N: 6.33.1HNMR(CDC13) δ 0.83-0.95 (m,1 H), 1.01 (t, 6 H, J = 6.1 Hz), 1.32(t, 3 H. J = 7.3 Hz), 1.48-1.85 (m, 5 H), 2.97-3.02 (m, 1 H), 3.92 (dd, 1 H. J = 12.2, 2.2 Hz), 4.24 (q. 2H,J = 7.1Hz).
Step ii: Synthesis of 1α,3ß,5ß-l-Cyanomethyl-3,5-dimethyl-cycIohexanccarbonitrilc
(8c): The 2-Cyano-((3R,5S)-3,5-dimethyl-cyclohexylidene)-acetic acid ethyl ester (8b. 6.64 g. 30.0 mmol) was dissolved in EtOH (60 mL) and added to a suspension of NaCN (1.47 g. 30.0 mmol) in EtOH / H2O (100 mL / 8 mL). The reaction was heated to reflux overnight, then cooled to room temperature, filtered, and concentrated in vacuo to give a clear oil which solidified under vacuum. The residue was chromatographed on silica gel eluting with 4:1 hexane/EtOAc to give 4.05 g (77%) of the desired product as a white, sticky solid.
MS: no M+l ion observed (C11H16N2): an oil: TLC: SiO2. R, 0.31 (4:1 hexane/tlOAc); Analysis (C17HiftN;) (Cal.) C: 74.96. H: 9.15. N: 15.89 (found) C: 74.81. H: 9.25. N: 15.65. 1H NMR (CDCl3) δ 0.58 (dd. 1 H. J = 24.9. 12.2 Hz). 0.96 (d. 6 H. J = 6.6 Hz), 1.03(t.2H.J= 12.9 Hz). 1.75-1.90 (m. 3 H). 2.01 (d. 2 H. J = 11.5 Hz). 2.65 (s. 2 H).
Step iii: Synthesis of 1α,3ß,5ß-(l-Aminomethyl-3,5-dimcthyl-cyclohexyl)-acctic acid hydrochloride (8d): 1α,3ß,5ß-l -Cyanomethyl-3.5-dimcthyl-cyclohexanccarbonilrilc (8c, 5.51 g. 31.26 mmol) was dissolved in EtOH (80 ml.) and toluene (80 mL). cooled to 0 °C. and saturated with HC1 gas. The solution was stoppered and stirred overnight at
room temperature, then concentrated in vacuo to give the crude imino ester as a white
solid. This crude material was triturated with Et2O (150 mL) and dried under vacuum,
then treated with H3O (100 mL) and 1 N HC1 (4 mL). The reaction was stirred overnight at room temperature, then extracted with EtOAc (3 x 100 mL). The organic layers were
combined, washed with brine, dried over Na2SO4, and concentrated. The residue was
chromatographed on silica gel eluting with 6:1 hexane/EtOAc to give 5.78 g (83%) of the desired product as a clear oil.
MS: 224.1 (M+l for C13H21Nl02); an oil; TLC: Si02, Rf 0.32 (6:1 hexane/EtOAc); Analysis (C13H21N1O2) (Cal.) C: 69.92 H: 9.48, N: 6.27 (found) C: 70.03, H: 9.45. N: 6.30. 1HNMR (CDCl3) δ 0.53 (dd, 1 H, J = 24.9, 12.0 Hz), 0.91 (d. 6 H, J = 6.6 Hz), 1.27 (t, 3 H, J = 7.1 Hz), 1.70-1.76 (m. 1 H), 1.80-1.90 (m, 2 H). 2.02 (d, 2 H, J = 12.9 Hz), 2.51 (s, 2 H), 4.18 (q, 2 H, J = 7.1 Hz).
Step iv: Synthesis of 5a,7ß,9ß-7,9-DimethyI-2-aza-spiro|4.5)decan-3-one (8c):
1α,3ß,5ß-(1-Cyano-3.5-dimethyl-cyciohexyl)-acetic acid ethyl ester (8d, 5.82 g. 26.1 mmol) was dissolved in MeOH (150 mL), treated with 10% Rh / 2% Pd/C (1.0 g). and shaken under an atmosphere of H: (50 psi) for 28 h. The reaction was filtered and concentrated in vacuo. The residue was chromatographed on silica eel eluting with a gradient of 2:3 hexane/EiO.Ac. EtOAc, 5% MeOH/F.tOAc to give 2.21 g (47%) of the desired product.
MS: 182.0 (M+l for C11H10N1O1); mp 116-118 °C; TLC: SiO2 Rf 0.36 (6% MeOH/CH2Cl2); Analysis (C11H19N1O1) (Cal.) C: 72.88, H: 10.56. N: 7.73 (found) C: 72.84, H: 10.67, N: 7.63. 1HNMR (CDC13) δ 0.49 (dd, 1 H. J = 24.4. 12.0 Hz). 0.86-0.93 (m, 8H), 1.38-1.48 (m. 2 H). 1.60-1.71 (m, 3 H). 2.09 (s. 2 H). 3.16 (s. 2 H), 5.79 (br. 1 H).
Step v: Synthesis of 1α,3ß,5ß-(l-AminornethyI-3,5-dirnethyl-cycloricxyl)-acetic acid; hydrochloride salt (8): 5α,7ß,9ß-7,9-Dimethyl-2-aza-spirol4.5]decan-3-one (8c. 0.52 g. 2.87 mmol) was treated with 6 N HC1 (19 m,L) and heated to reflux overnight. The reaction was cooled to room temperature, upon which a white precipitate formed. The solid was filtered away, and the filtrate was cooled to 0 °C to afford a second crop of crystals, then filtered again. The solids were combined and dried under vacuum to give 0.46 g (68%) of the desired product.
MS: 200.1 (M+l for C11H21N1O2); mp 179-180 °C; Analysis (C11H21N1O2.HCl) (Cal.) C: 56.04, H: 9.41, N: 5.94 (found) C: 56.16, H: 9.42, N: 5.73. 1HNMR (CD3OD) δ 0.46-0.53 (m, 1 H), 0.77 (t, 1 H, J = 12.5 Hz), 0.85-0.91 (m, 7 H), 1.55-1.67 (m. 5 H). 2.11 (d. 2 H, J = 13.4 Hz), 2.36 (s, 2 H), 2.44 (s, 1 H), 3.14 (s, 2 H).
EXAMPLE 9 1α,3ß,5ß-(3,5-Dimethyl-l-propylaminomethyl-cyclohexyl)-acctic acid; hydrochloridesalt:
(Formula Removed)
1α,3ß.5ß-(1-Aminomcthyl-3.5-dimelhyl-cyclohc-xyl)-acetic acid: hydrochloridc sail (8. 1.0 g, 4.2 mmol) was dissolved in EtOH (100 mL). treated with sodium hydroxide solution (1 N, 5 mL). propionaldehyde (0.9 mL, 12.6 mmol). and 10 % Pd/C (0.3 g). and shaken under an atmosphere of H2 (50 psi) for 3 h. The reaction was filtered, treated with HCl/Et2O (1 M, 10 mL), and concentrated in vacuo. The residue was treated with hot isopropanol (100 mL), the NaCI was filtered away, and the clear solution was concentrated in vacuo. The crude product was recrystallized from i-PrOH/acetone/hexane (14: 200: 225 mL) and dried under vacuum to give 0.1 36 g (12%) of the desired product.
MS: 242.1 (M+l for C14H27N1O2); mp 178-180 °C; Analysis (C14H17N1O2.HC1.0.27
H2O) (Cal.) C: 59.48, H: 10.18, N: 4.95 (found) C: 59.53. H: 10.21. N: 4.78. 1HNMR (CD3OD) δ 0.52 (dd, 1 H, J = 24.2, 12.0 Hz). 0.83-0.90 (m, 8 H), 1.00 (t. 3 H. J = 7.6 Hz), 1.61-1.80 (m, 7 H), 2.43 (s, 2 H), 2.98 (t, 2 H. J = 7.8 Hz). 3.20 (s. 2 H).
EXAMPLE 10 1α,3ß,5ß-(l-EthyIaminoraethyI-3,5-dimethyl-cyclohexyI)-acetic acid
hydrochloride salt
(Formula Removed)
Example 10 was synthesized in accordance with the method of Example 9. except that acetaldehvde was used instead of propionaldehyde and ethyl acetate/heptane was used for the recrvstallization (30% yield).
MS:228(M+1 for C15H29N1O2): mp 188-190 °C: 1HNMR (DMSO) δ 0.85 (SH. br d). 1.24 (3 H. t. J = 7.1 Hz). 1.60 (6 H. m). 2.38 (d. s). 2.98 (2 11. m). 3.10 (2 11. br s). 8.33 (2 H, brs). 12.42(1 H.brs).
EXAMPLE 11 lα,3ß,5ß-(l-Bcnzylamtno-mcthyi)-3,5-(limcthyl-cyclohcxyl|-;jcetit acid; hydrochloride salt:
(Formula Removed)
Example 11 was synthesized in accordance with the method of Example 9, except that benzaldehyde was used instead of propionaldehyde and EtjO was used for the recrystallization (30% yield).
MS: 290.1 (M+l for C18H27N1O2); mp 162-163 °C: Analysis (C18H27N1O2.HC1.0.20H2O) (Cal.) C: 65.62, H: 8.69, N: 4.25 (found) C: 65.60, H: 8.71, N: 4.31. 1HNMR (CD3OD) δ 0.48 (dd, 1 H, J = 12.5 Hz), 0.79-0.96 '(m, 8 H), 1.46 (br, 2 H), 1.64 (d, 3 H, J = 13.4 Hz), 2.42 (s, 2 H), 3.19 (s, 2 H), 4.23 (s, 2 H), 7.43-7.50 (m, 5 H).
EXAMPLE 12 lα,3ß,5ß-(l-Dimethylaminomcthyl-3,5-dimcthyl-cvclohcxyl)-acctic acid; hydrochloridesalt:

(Formula Removed)
A solution of 1α,3ß,5ß-(l-Arninomethyl-3.5-dimcthyl-cyclohcxyl)-acetic acid: hydrochloride salt (8, 0.20 g. 0.59 mmol) in EtOH (15 mL). sodium hydroxide solution (I N, 0.59 mmol), and H2O (1 mL) was treated with 20% Pd/C (50 mg) and shaken under an atmosphere of112 (50 psi) for 15 hours. The reaction was filtered, treated with 1 N HCl/Et20 (1 mL). and concentrated in vacuo. The residue was dissolved in hot i-PrOI I (4 mL), diluted with acetone (100 mL) and Et20 (350 mL) and cooled 10 0 °C. The solid was filtered and dried under vacuum to give 103 mg (70% yield) of the desired product.
MS: 228.1 (M+l forC12H21N1O2); mp 195-196 °C; Analysis (CI2H23N1O2.HCI.0.41H2O) (Cal.) C: 57.58, H: 9.97. N: 5.16 (found) C: 57.73. H: 9.71. N: 4.91. 1HNMR (CD3OD) δ
(Formula Removed)
0.56 (dd, 1 H, J = 24.4,12.0 Hz), 0.89-0.92 (m, 8 H), 1.59-1.75 (m, 8 H), 2.51 (s, 2 H), 2.95 (s, 6 H), 3.44 (s, 2 H).
EXAMPLE 13 lo3ß,5ß-(l-ButyIaminomethyl-3,5-dimethyl-cyclohcxyl)-acetic acid; hydrochloride salt:
(Formula Removed)
Example 13 was synthesized in accordance with the method of Example 9. except butyraldehyde was used instead of propionaldchyde (48% yield).
MS: 256.! (M+l for: mp 184-185 °C: Analysis (C15H29N1O2.HCl) (Cal.) C: 61.73. H: 10.36. N: 4.80 (found) C: 61.71,11: 10.21. N: 4.72. 'll NMR (CD.OD) δ 0.53 (dd. 1 H. J = 24.4. 12.0 Hz). 0.86-0.99 (m. 11 H). 1.37-1.47 (m. 2 H). 1.60-1.75 (m. 7 H). 2.43 (s. 2 11), 3.02 (t. 2 H. J = 8.1 Hz). 3.20 (s. 2 H).
EXAMPLE 14 1α,3ß,5ß-(1-((Bcnzyl-mcthyI-amino)-methyl]-3,5-dimcthyl-
cyclohexyl)-acetic acid; hydrochloride salt:
(Formula Removed)
Example 14 was prepared in accordance with the method of Example 9, except that the N-benzyl analog (11) and formaldehyde were used as the starting material and PtO2 was used as the catalyst instead of Pd/C (70% yield).
MS: 304.1 (M+l for C19H29N1O2); mp 209-210 °C; Analysis (CI9H29N1O2.HC1.0.21H2O) (Cal.) C: 66.40, H: 8.92, N: 4.08 (foufid) C: 66.40, H: 9.05, N: 4.06. 1HNMR (CD,OD) δ 0..51 (dd, 1 H,J = 24.4, 12.2 Hz), 0.79-0.92 (m, 9 H), 1.29(br. 1 H). 1.56-1.82 (m. 4 H). 2.51 (dd. 2 H, J = 18.8, 16.3 Hz) 2.85 (s, 2 H). 3.23-3.49 (m. 2 H). 4.37 (dd. 2 H, .1 = 31.7, 12.7 Hz), 7.45-7.54 (m. 5 H).
EXAMPLE 15 1α,3ß,5ß-(3,5-Dimethyl-l-mcthyIaminomcthyl-cyclohcxyl)-acctic acid; hydrochloridc salt:
(Formula Removed)
Example 14 (0.14 g. 1.21 mmol) was dissolved in 95% EtOH (50 mL). treated with sodium hydroxide solution (I N, 1.21 mmol) as well as 20% Pd/C (0.1 g). and shaken under an atmosphere of Hi (50 psi) for 45 minutes. The reaction was filtered, treated with HCl/Et20 (1 M, 1 mL). and concentrated in vacuo. The residue was treated with hot iPrOH, the NaCl was filtered away, and the solution was concentrated in vacuo. The
crude product was recryslallized from iPrOH/acetonc/hcxane (7: 150: 110 mL) and dried
under vacuum to give 0.189 (63%) of the desired product.
MS: 214.1 (M+l for C12H11N1O2); mp 182-183 °C: Analysis (Cl2H23N1O2.HCL) (Cal.) C: 57.70, H: 9.68, N: 5.61 (found) C: 57.47, H: 9.58. N: 5.35. 1HNMR (CD.,OD) δ 0.52 (dd.
(Formula Removed)
1 H, J = 24.4,12.2 Hz), 0.8-0.98 (m, 8 H), 1.57-1.72 (m, 5 H), 2.38 (s, 2 H), 2.72 (s, 3 H), 3.21 (s, 2 H).
EXAMPLE 16 lα,P,5ß-[1-(Acetylamino-methyl)-3,5-dimethyl-cyclohexyI]-acetic acid:
(Formula Removed)
Example 8 (0.5 g, 2.1 mmol) was treated with H2O (10 mL) and NaOH (0.56 g, 14.0 mmol). cooled to 0 °C. and treated with acetic anhydride. The reaction was stirred overnight at room temperature, then diluted with EtOAc (100 mL) and washed with 1 N HCI (100 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The crude material was chromatographed on silica gel cluting with 4% MeOH/Cl I-CN to give 0.138 g (27%) of the desired product as an oil which solidified under vacuum.
MS: 242.1 (M+l for Cl3H23N1O3): mp 122-124 °C: Analysis (C13H23N1O3) (Cal.) C: 64.70, H: 9.61, N: 5.80 (found) C: 64.65, H: 9.59. N: 5.63. 1HNMR (CD3OD) δ 0.46 (dd, 1 H,J = 23.7, 1 1.7 Hz), 0.8 1-0.87 (m. 9 H). 1.56 (d. 2 H. J= 13.4 Hz). 1.65-1.76 (m. 3 H), 1 .98 (s, 3 H), 2. 1 8 (s. 2 H), 3.30 (s, 3 H).
EXAMPLE 17 1α,3ß,5ß-(l-(lsobufylamino-methyl)-3,5-dimcthyl-cyclohexyl]-accfic acid hydrochloridc salt
(Formula Removed)
Example 17 was synthesized in accordance with the method of Example 9, except isobutyraldehyde was used instead of propionaldehyde (53% yield).
MS: 256 (M+l for C15H29N1O2); mp 193-194 °C: 1HNMR (CD.-,OD) δ 0.53 (dd. 1 H. J 24.3, 12.5 Hz), 0.81 -0.90 (m. 2 H), 0.89 (d. 6H. J = 6.4 Hz). 1.03 (d. 6H. J = 6.8 Hz). 1.57-1.71 (m, 5H). 2.09 (m. 1H). 2.49 (s, 2 H), 2.88 (d. 2H. J = 7.1 Hz). 3.19 js. 2 H).
EXAMPLE 18 1α,3ß,5ß-[3,5-Dimcthyl-l-iphcncthvlamino-mcthy])-
cyclohex\ Ij-acctic acid hydrochloride salt:
(Formula Removed)
Example 18 was synthesized in accordance with the method of Example 9. c.xccpi phenylacctaldehyde was used instead of propionaldehyde (16% yield).
MS: 304 (M+l for C19H29N1O2): mp 188-18,9 °C; Analysis (C19H29N1O2.HC1) (Cal.) C: 67.14, H: 8.90, N: 4.12 (found) C: 66.94. H: 8.91, N: 3.98. 1HNMR (CD3OD) δ 0.46 (dd, 1 H, J = 24.6, 12.3 Hz), 0.81-0.91 (m, 8 H). 1.58-1.72 (m, 5 H). 2.50 (s, 2H). 3.04-3.08 (m, 2H), 3.26-3.30 (m, 4H), 7.23-7.75 (m, 5H).

EXAMPLE 19 lα,3ß,5ß-{3,5-Dimethyl-l-[(3-phenyl-propyIamino)-methylj-cyclohexyl}-acetic acid hydrochloride salt:
(Formula Removed)
Example 19 was synthesized in accordance with the method of Example 9. except hydrocinnamaldehyde was used instead of propionaldehyde (50% yield).
MS: 318 (M+l for C20H31N1O2): mp 171-172 °C: Analysis (C20H31N1O2.HCI) (Cal.) C: 67.87, H: 9.1 1. N: 3.96 (found) C: 67.83. H: 9.13. N: 3. 78. 1HNMR (CD3OD) R 0.53 (dd, 1 H. J = 24.6. 1 2. 1 1 Iz). 0.80-0.90 (m. 2 H). 0.90 (d. 61 1. .1 = 6.0 Hz). 1 .60- 1 .72 ( m. 5 H), 2.03-2.1 1 (m. 211). 2.45 (s. 2H). 2.73 (t. 2H. J = 7.5 Hz). 3.06 (t. 2H. .1 - 7.7 Hz). 3.3 (s,2H), 7.20-7. 18 (m.5H).
EXAMPLE 20 (1-[(Cycloburylmcthvl-amino)-mcthvl]-cyclohcxvl}-acetic acid hydrochloride salt:
(Formula Removed)
Example 20 was synthesized in accordance with the method of Example 26, except cyclobutanone was used instead of 2-methylbutyraldehyde (70% yield).
MS: 226 (M+1 forC13H23N1O2); mp 176-177 °C; Analysis (C13H23N1O2.HC1) (Cal.) C: 59.64, H: 9.24, N: 5.35 (found) C: 59.88, H: 9.18, N: 5.19. 1HNMR (CD3OD) δ1.16-1.60 (br., 10 H), 1.80-1.96 (m, 2 H), 2.21-2.40 (m, 4 H), 2.53 (s. 2H), 2.97 (s. 2H), 3.73 (m, 1H).
EXAMPLE 21 1α,3ß,5ß-(l-(Isopropylamino-methy])-3,5-dimethyl-cyclohexyl]-acetic acid hvdrochloride salt:
(Formula Removed)
Example 21 was synthesized in accordance with the method of Example 9. except acetone was used instead of propionaldehyde (50% yield).
MS: 242 (M+1 for C14H27N1O2); mp 206-207 °C; 1HNMR (CD..OD) δ 0.53 (dd. I H. J 24.8, 12.5 Hz), 0.80-0.90 (m.2 H), 0.99 (d, 6H. J = 6.1 Hz). 1.35 (d. 611. J - 6.6 Hz), 1.55-1.78 (m, 5H), 2.45 (s. 2H). 3.18 (s. 2 H), 3.41 (m. 1H).
EXAMPLE 22 l-Aminomcthyl-1-cyclohcxane-acctic acid was previously reported in
Satzingcr et al. US 4087544. May 2, 1978.
EXAMPLE 23 1-AminomefhyI-l-cyclopentane-accdc acid was previously reported in Satzinger et al. US 4087544. May 2, 1978.
EXAMPLE 24 Sodium salt of 1-aminomethyl-l-cyclopentane-acetic acid was
previously reported in Satzinger ec al. US 4087544, May 2,1978.
EXAMPLE 25 Sodium salt of l-(hydroxymethyl)cyclohexane-acetic acid was
previously reported in Satzinger et al. US 2960441,1960.
EXAMPLE 26 Synthesis of (1-[(2-MethyI-burylamino)-methyl]-cyclohexyl}-acetic acid; hydrochloride salt:
(Formula Removed)
A solution of (l-aminomethyl-cyclohexyl)-acetic acid (Example 22. 2.0 g. 11.7 mmol) in EtOH (100 mL) was treated with 2-mcthyl-butyraldehydc (4 mL. 35.1 mmol) and Pi03 (0.5 g). and shaken under at atmosphere of H? (50 psi) for 2 h. The reaction was filtered, treated with I N HCI/Et2O (12 mL). concentrated in vacuo. dissolved in hot i-PrOM (10 mL). diluted with acetone (600 mL). cooled to 0 °C. and filtered to give 1.58 g (49%) of the desired product.
MS: 242.1 (M+l for C14H27N1O2); mp 162-163 °C: Analysis (C141O2.HCL) (Cal.) C: 60.52, H: 10.16, N: 5.04 (found) C: 60.41. H: 10.26, N: 4.95. 1HNMR (CD3OD) δ 0.95 (t, 3 H, J = 7.3 Hz). 1.02(d,3H.J = 6.6Hz). 1.20-1.31 (m. 1 H). 1.42-1.56 (m. H H), 1.81-1.89 (m, 1 H), 2.63 (dd. 2 HJ = 18.3, 16.1 Hz). 2.82 (dd. 1 H, J = 12.5. 8.3 Hz), 2.99(12.5,6.1 Hz),3.07(dd.2H.J = 25.6, 12.9 Hz).
EXAMPLE 27 (1-[(4,4,4-Trifluoro-butylamino)-methyl]-cyclohexyl}-acetic acid;
hydrochloride salt was synthesized in accordance with the method of Example 26, except that 4-trifluorobutyraldehyde was used instead of 2-methyIbutyraldchyde (19% yield).
MS: 282.1 (M+l forC13H22N1O2F3)mp: 163-164 °C; Analysis (C13H22N1O2F3.HC1.0.05H2O) (Cal.) C: 49.00. H: 7.31, N: 4.40 (found) C: 48.60. H: 7.29: N: 4.19. 1HNMR (CD3OD) δ 1.40-1.54 (m. 1 1 H). 1.96-2.04 (m, 2 H). 2.26-2.38 (m, 2 H), 2.57 (s. 2H), 3.09-3.13 (m, 4 H).
EXAMPLE 28 (l-Ethylaminomcthyl-cyclohexyl)-acetic acid hydrochloride salt
Example 28 was synthesized in accordance with the method of Example 26. except that acetaldehyde was used instead of 2-methylbutyraldehyde (30% yield).
MS: 200.1 (M+l for C11H21N1O2): mp: 160-166 °C; Analysis (C11H20N1O2.HC1.) (Cal.) C: 56.04. H: 9.41, N: 5.94 (found) C: 55.98. H: 9.36, N: 5.79. 1HNMR (CD,OD) δ 1.31-
1.54 (m. 13 H), 2.54-2.56 (m, 2 H), 3.06-3.13 (m. 4 H).
EXAMPLE 29 (1-[(Cyclopropylmethyl-amino)-methyl|-cyclohexyl}-acetic acid hydrochloride salt:
Example 29 was synthesized in accordance with the method of Example 26, except that cyclopropanecarboxaldehyde was used instead of 2-methylbutyraldehyde (62% yield).
(Formula Removed)
MS: 226 (M+l for C11H23N1O2); mp 177-178 °C; Analysis (C17H23N1O:.HCl) (Cal.) C: 59.64, H: 9.24, N: 5.35 (found) C: 59.72, H: 9.15. N: 5.27.1HNMR (CD,OD) δ 0.50-0.56 (m, 2 H). 0.71-0.74 (m. 2H), 1.05-1.30 (m. IH). 1.42-1.54 (m. 10H). 2.52 (s. 2H). 2.94 (d,2H.J = 7.3Hz).3.10(s.2H).
EXAMPLE 30 (1-[(2-Hydroxy-l-methyl-elhylamino)-methyl|-cyclohcxyl}-acctic acid hvdrochloride sale
(Formula Removed)
Example 30 was synthesized in accordance with the method of Example 26. except that 1-hydroxy-2-propanone was used instead of 2-methylbutyraldchydc (60% yield).
MS: 230 (M+l for CI2H23N1O3); mp 164-165 °C; Analysis (C,2H23N1Oj.HCl) (Cal.) C: 54.23, H: 9.10, N: 5.27 (found) C: 54.23, H: 9.01, N: 5.18,1HNMR (CD3OD) δ 1.30-1.32 (m, 3 H), 1.44-1.55 (m, 1QH), 2.60 (s, 2H), 3.11 (m, 2H), 3.28 (br., 2H), 3.37 (br., 1 H), 3.57-3.61 (m, IH), 3.78-3.81 (m, IH).
EXAMPLE 31 [l-(Isobutylamin(Mnethyl)-cycIohexyl]-acetic acid hydrochloridc salt:
(Formula Removed)
Example 31 was synthesized in accordance with the method of Example 26. except that cyclopropanccarboxaldehyde was used instead of 2-mcthylbutyraldehyde (.15% yield).
MS: 228 (M+l for C13H25N1O2); mp 175-176 °C: Analysis (C13H25N1O2.HCI) (Cal.) C: 59.19, H: 9.93. N: 5.51, (found) C: 59.28, H: 9.98. N: 5.24.1HNMR (CD,OD) δ 1.03 (d, 6H, J = 6.9 Hz), 1.37-1.60 (m. 10H). 2.10-2.17 (m. IH). 2.63 (s. 2H). 2.87 (d. 2H. J = 7.4 Hz), 3.07 (s, 2H).
EXAMPLE 32 (l-Propylaminomethyl-cyclohexyl)-acctic acid hydrochloridc salt:
(Formula Removed)
Example 32 was synthesized in accordance with the method of Example 26, except that propionaldehyde was used instead of 2-methylbutyraldehyde.
MS: 214.1 (M+l forC12H23N1O2); mp: 174-177 °C; Analysis (CI2H23N1O2.HC1) (Cal.) C: 57.70. H: 9.69, N: 5.61 (found) C: 57.55. H: 9.77, N: 5.48. 1HNMR (CD3OD) δ 0.93 (t, 1 H, .1 = 6.3 Hz). 0.99-1.03 (m. 4 H). 1.40-1.54 (m. 13 H). 1.72-1.78 (m. 2 H). 2.96-3.29 (m. 2 H).
EXAMPLE 33 [l-(lsopropylamino-mcthyl)-cyclohexyl]-acetic acid hydrochloridc salt:
(Formula Removed)
Example 33 was synthesized in accordance with the method of Example 26. except that acetone was used instead of 2-methylbutyraldehyde.
MS: 214.1 (M+l forC12H23N1O2); mp: 193-194 °C; 1HNMR (CD3OD) δ 1.29-1.57 (m, 18 H), 2.59 (s, 2 H), 3.07 (s, 2 H), 3.38-3.42 (m, 1 H).
-EXAMPLE 34 (l-Cyclohexylaminomethyl-cycIohexyl)-acetic acid hydrochloridc
salt:
(Formula Removed)

Example 34 was synthesized in accordance with the method of Example 26. except that cyclohexanone was used instead of 2-methylbutyraldehyde (48% yield).
MS: 254 (M+l for C15H27N1O2); mp 198-199 °C: Analysis (C15H23N1O2..HCl) (Cal.) C: 62.12, H: 9.74. N: 4.83 (found) C: 62.18. H: 9.84. N: 4.75.1HNMR (CD,OD) δ 1.20-1.60 (m. 16 H), 1.63-1.76 (m. 1H). 1.88 (br.. 2H). 2.13 (br.. 2H). 2.60 (m.. 2H). 3.09 (m.. 3H).
EXAMPLE 35 [1-(Benzylamino-mcthyI)-cyclohcxyl]-acclic acid hydrochloridc salt:
(Formula Removed)
Example 35 was synthesized in accordance with the method of Example 26, except that benzaldehyde was used instead of 2-methylbutyraldehyde.
MS: 262.0 (M+l for C16H23N1O2); mp: 125-135 °C; 1HNMR (CD,OD) δ 1.38-1.49 (m. 11 H), 2.55 (s, 2 H), 3.07 (s, 2 H). 4.23 (s, 2 H), 7.43-7.49 (m, 5 H).
EXAMPLE 36. ((lR,3R)-3-Methyl-l-propylaminomethvl-cyc\ohexyl)-acetk acid; hydrochloride salt
(Formula Removed)
((1R, 3R)-l-Aminomethyl-3-methyl-cyclohexyl)-acetic acid; hydrochloride salt (Example 1, 1.1 g. 4.9 mmol) was dissolved in ethano! (95ML) and treated with 1 N NaOH (5 ml). Propionaldehyde (4.9 mL) was added followed by 1 0% Pd/C (0.5 c). and the reaction was shaken under an atmosphere of H: (50 psi) for 1 5.75 h. The reaction was filtered. I M HCI (20 mL) in ether was added, and the solution was concentrated in vacuo. The residue was then dissolved in hot isopropanol (100 mL). filtered, and concentrated in vacuo. The residue was then dissolved in isopropanol ( 1 0 mL) and recrystallized from acetone. The solid was filtered and the filtrate was cooled overnight to yield a second crop of crystals. The solids were combined to give the desired product (0.78 g, 66%).
MS: 228.1 (M+l forC13H25NO2); mp: 179-179.5 °C: Analysis (C13H23NO2.HCl) (Cal.) C: 59.19, H: 9.93, N: 5.31 ; (found) C: 59.2y,H: 9.97, N: 5.07. 1R (KBr. cm'1 ): 2929 (br). 1717, 1188. 1HNMR(CD3OD)δ0.84-1.03(m, 8H), 1.12-1. 17 (m, 1H) 1.46-1.79 (m. 8H), 2.43 (s, 2H), 3.00 (t, 2H, J = 7.8 Hz) 3.22 (s, 2H).
EXAMPLE 37. (1-[Cyclopentylmethyl-amino)-methyl]-cyclohexyI}-acetic acid;
hydrochloric salt
(Formula Removed)
(l-Aminomethyl-cyc]ohexyl)-acetic acid (3.0 g. 17.5 mmol) was dissolved in eihanol (45 mL). Cyclopentanecarbaldehyde (5 mL. 3 eq) was added and the reaction was treated with PtOi (0.25 g). The reaction was then shaken under an atmosphere of Hi (50 psi) tor 16.5 h. The reaction was filtered, 1N.HCI (20 mL) in ether was added, and the reaction was concentrated in vacuo. The residue was recrystailized from hoi isopropanol and acetone to give the desired product (2.32 g. 52%).
MS: 254.1 (M+1 for C15H27NO2); mp 172-173 °C: Analysis (Cal.) C: 62.16. H: 9.74, N: 4.83; (found) C: 61.92. H: 9.88. N: 4.86. IR (KBr. cm'1): 2930. 2854. 2509. 1689. 1455. 1211. 1HNMR (CD,OD) δ 1.23-1.32 (m. 2H). 1.41-1.73 (m. 16H). 1.90 (m. 1H). 2.63 (s. 2H). 3.01 (d. 2H, .1 = 7.6 Hz). 3.09 (s. 2H).
EXAMP7.E 38. {1-[(CycIohexylmethyl-amino)-methyl]-cyclohcxyl]-acctic acid; hydrochloric salt
(Formula Removed)
(l-Aminomethyl-cyclohexyl)-acelic acid (3.0 g, 17.5 mmol) was dissolved in ethanol (45 mL). Cyclohexanecarbaldehyde (5 mL, 3 eq) were added and the reaction was treated with PtO2 (0.25 g). The reaction was then shaken under an atmosphere of Hi (50 psi) for 16.5 h. The reaction was filtered, IN HC1 (20 mL) in ether was added, and the reaction was concentrated in vacuo. The residue was recrystallized from hot isopropanol and ether to give the desired product (3.57 g, 67%).
MS: 268.1 (M+l for CI6H29NO2); mp 167.5-168 °C; Analysis (CI5H29NO2.HC1) (Cal.) C: 63.24, H: 9.95, N: 4.61; (found) C: 63.14, H: 10.01, N: 4.45. 1R (KBr. cm'1): 2926. 2851. 1718, 1684. 1HNMR (CD3OD) δ 1.03 (m. 1H), 1.20-1.85 (m, 20H), 2.63 (s. 2H). 2.88 (d. 2H, J = 6.8 Hz). 3.07 (s, 2H).
EXAMPLE 39: [l-(tert-Butoxycarbonylarnino-rnethyI)-cyclohexyl]-acetic acid;
(Formula Removed)
(l-Aminomethyl-cyclohexyl)-acetic acid (10.0 g. 58.4 mmol) was dissolved in THF (280 mL), and it was treated with 1 N NaOH (128.5 mL). The reaction was cooled to 0 °C and 14.0 g (1.1 eq) of di-tert-butyldicarbonate were added portionwise with vigorous stirring. The reaction was allowed to warm to room temperature and stir overnight. The THF was then evaporated, and the aqueous layer was washed with ethyl acetate (3x50 mL). The aqueous layer was acidified with KH2PO4 (44 g) and foam was observed. Gradually the
foam was replaced with a white ppt. The mixture was stirred for 2h while of KH2O4 (1 g) was added slowly over time. The precipitate was filtered off and washed with water to yield the desired product (15.2 g. 96%).
MS: 272.1 (M+l for C14H25NO4); mp: 181 °C (dec); Analysis (C14H25NO4) (Cal.) C: 59.52, H: 9.36, N: 4.76; (found) C: 59.51, H: 9.03, N: 4.87. IR (KBr, cm'1): 3413. 3054, 2933 (br), 1709, 1666, 1531, 1248; 1HNMR(CDC13) δ 1.27-1.58 (m, 19H), 2.29 (s. 2H). 3.12 (d, 2H, J = 6.8 Hz), 4.97 (br, 1 H)..
EXAMPLE 40: [l-(Acetylamino-methyl)-cyclohexyl]-acetic acid O
(Formula Removed)
(l-Aminomethyl-cyclohexyl)-acetic acid (2.0 g. 1 1.7 mmol) was dissolved in FNO (20 mL), then NaOH (3.1 g. 77.5 mmol. 6.6 eq) and acetic anhydride (1.8 mL. 19.3 mmol, 1.7 eq) were added. The reaction was allowed to stir at room temperature for 2 days. IN HCI (125 mL) was added and the reaction extracted with EtOAc (125 ml.). The organic layer was separated, dried overNa2SO4. and concentrated in vacuo. The residue was dissolved in H2O ( 1 00 mL). treated with NaOH (0. 12 g), and concentrated in vacuo. The sample was then flash chromatographed with 6% MeOH/ CH2Cl3 10 give the desired product (0.3 1 g. 1 2 %).
MS: 214.1 (M+l C11H10NO3): mp: 107-108°C: Analysis (C11NO3.) (cai) C: 61.95 H: 8.98 N: 6.57 (found) C: 61.96 H: 9.03 N: 6.54; TLC 0.31 6% McOH/ CH:CK: IR (KBr. cm'1): 3286, 3120.2932. 24401714. 1576, 1419. 1313. 1208. 1HNMR (CDCI,) δ 1.25-1.51 (m. 10 H). 2.06 (s. 3H). 2.28 (s. 2H). 3.26-3.29 (m. 2H). 6.32 (br. 1 H).
EXAMPLE 41: ((3R, 5S)-l-Cycloburylaminomethyl-3,5-dimethyl-cyclohcxyI)-acctic acid; hydrochloride salt
(Formula Removed)
((3R, 5S)-l-Aminomethyl-3,5-dimethyl-cyclohexyl)-acetic acid; hydrochoride salt (0.76 g, 3.2 mmol) was dissolved in ethanol (45 mL) and treated with IN NaOH (3.2 mL). Cyclobutanone (1 mL, 6 eq) was added followed by PtO2 (0.2 g). The reaction was shaken under an atmosphere of H2 (50 psi) for 16 h. The reaction was then filtered. 1N HC1 in ether (35 mL) was added, and the reaction was concentrated in vacuo. The residue was dissolved in hot isopropanol (150 mL) and filtered. Then acetone (100 mL) and ether (600 mL) that had been cooled to 0 °C were added to the solution. The solution was allowed to sit at 0 °C for 2 h at which time crystals were observed. The crystals were collected and washed with ether and acetone (0.38 g, 41%).
MS: 254.1 (M-H for C15H27NO2); mp 200-201 °C; Analysis (C15H27NO2.HCl) (Cal.) C: 62.16. H: 9.74. N: 4.83; (found) C: 62.09. H: 9.72, N: 4.68. 1R (RBr. cm'1): 2926. 1720. 1189. 1HNMR (CD3OD) δ 0.48-0.57 (m. 1 H). 0.85-0.92 (m. 8H). 1.60-1.73 EXAMPLE 42: {(3R, 5S)-3,5-Dimcthyl-l-[(2-mcthyl-butylamino)-mcthyl)-cyclohexylj-acetic acid; hydrochloride salt
(Formula Removed)
((3R. 5S)-l-Aminomethyl-3,5-dimethyl-cyclohcxyl)-acetic acid: hydrochoride salt (1.0 g. 4.4 mmol) was dissolved in ethanol (47 mL) and treated with IN NaOH (4.4 mL). Isobutyraldehyde (1 mL, 3 eq) was added followed by Pt02 (0.17 g). Reaction was shaken under an atmosphere of H2 (50 psi) for 3 h. The reaction was then filtered, IN HC1 (10 mL) in ether was added, and the reaction was concentrated in vacuo. The
residue was dissolved in hot isopropanol (100 mL) and filtered. Then it was concentrated in vacuo and recrystallized from acetone and hexane. The crystals were collected and washed with hexane to yield the desired product (0.89 g, 75%).
MS: 270.1 (M+l for C16H31NO2); mp 171-173 °C; Analysis (C16H3iNO2.HCl) (Cal.) C: 62.82, H: 10.54, N: 4.58; (found) C: 62.59, H: 10.43, N: 4.52. IR (KBr, cm'1): 2964 (br). 1721, 1485, 1186, 840. 1HNMR (CD3OD) δ 0.48-0.57 (m, 1 H), 0.81 -1.07 (m, 12H), 1.23-1.27 (m, IH), 1.45 -1.72 (m, 6H), 1.83-1.91 (m, IH), 2.50 (s. 2H), 2.83 (dd. IH. .1 = 8.3, 12.5 Hz), 3.00 (dd, IH, J = 6.1. 12.5 Hz), 3.13-3.29 (m. 4H).
EXAMPLE 43: {(3R, 5S)-l-[(2-Hydroxy-l-mcthyl-ethylamino)-mcthyll-3,5-dimethyl-cyclohexyl}-acetic acid; hydrochloride salt
(Formula Removed)
((3R, 5S)-l-Aminomethyl-3.5-dimcthyl-cyclohexyl)-acetic acid; hydrochoride salt (1.0 g. 4.3 mmol) was dissolved in cthanol (45 mL) and treated with IN NaOH (4.3 mL). 1-Hydroxy-propan-2-one (I mL. 3 eq) was added followed by PiO2 (0.15 g). Reaclion was shaken under an atmosphere of Hi (50 psi) for 82 h. PtO2 (0.1 g) and l-Hydroxy-propan-2-one was added (1 mL), and the reaction was shaken under an H2 atmosphere for another 24h. The reaction was then filtered, 1 N HCI in ether (35 mL) was added, and the reaction was concentrated in vacuo. The residue was dissolved in hot isopropanol (150 mL) and filtered. Then it was concentrated in vacuo and recrystallizcd from hot isopropanol, acetone, and ether. The crystals were collected, washed with ether as well as acetone, and dried. (0.30 g, 1 .04 mmol, 24%)
MS: 258.1 (M+l for C14H27NO3); mp 178-179 °C; Analysis (C14H27NO3.HCl) (Cal.) C: 57.23. H: 9.60, N: 4.77; (found) C: 56.77, H: 9.39, N: 4.45. IR (KBr. cm'1): 3349, 2897, 1704, 1419, 1194. 1HNMR (CD3OD) δ 0.48-0.57 (m, IH), 0.82-0.90 (m, 8H). 1.25-1.32
(m, 4H), 1.60-1.75 (m, 5H): 2.47 (s, 2H), 3.23 (d, 2H, J = 5.1 Hz), 3.38 (m, 1H), 3.58 (dd, 1H, J = 12.0, 7.1 Hz), 3.79 (dd, 1H, J = 12.0,4.2 Hz)
EXAMPLE 44: {(3R, 5S)-l-[(2,2-Dimethoxy-ethylamino)-methylj-3,5-dirncthyI-cyclohexyl}-acetic acid; hydrochloride salt
(Formula Removed)
((3R, 5S)-l-AminomethyI-3,5-dimethyl-cyclohexyl)-acetic acid; hydrochloride salt (1.0 g, 4.3 mmol) was dissolved in ethanol (47 mL). Glyoxal 1,1-dimethyl acetal (3.7 mL. 3.0 eq) was added and the reaction was treated with PtOi (0. 1 7 g). The reaction was then shaken under an atmosphere of H2 (50 psi) for 3 h. The reaction was filtered. IN MCI in ether (10 mL) was added, and the reaction was concentrated in vacuo. The residue was dissolved in hot isopropanol (100 mL) and filtered. Then, the filtrate was concentrated. and the residue was recrystallizcd from acetone and hexanes to give the desired product (0.92 g, 75 %).
MS: 288.1 (M+1] for C15H29NO4); mp 125 °C (dec); Analysis (Cl5H29NO4.HCl) (Cal.) C: 55.63, H: 9.34. N: 4.32: (found) C: 55.29. H: 9.48. N: 4.18. 1R (KBr. cm-1): 2949. 1715, 1457.1191, 1134. 1077. 1HNMR (CD3OD) δ0.47-0.56 (m. IH). 0.81-0.90 (m. 8H). 1.59-1.71 (m. 5H), 2.46 (s. 2H), 3.18 (d. 2H. J = 5.1 Hz). 3.26-3.29 (m. 2H). 3.45-3.47 (m, 6H), 4.73 (t, 1H,J = 5.1 Hz).
EXAMPLE 45: {(3R, 5S)-l-[(CyclopenryImcthyl-amino)-mclhyI|-3,5-dimcthyl-cyclohexyl}-acctic acid; hydrochioride salt
(Formula Removed)
((3R, 5S)-l-Arninomethyl-3,5-dimethyl-cyclohexyl)-acetic acid; hydrochloride salt (1.0 g, 4.1 mmol) was dissolved in ethanol (45 mL) and treated with IN NaOH (4.2 mL).
Cyclopentanecarbaldehyde (1.5 mL, 3 eq) were added followed by Pt02 (0.2 g). Reaction was shaken under an atmosphere of H (50 psi) for 12 h. The reaction was then filtered, IN HCI in ether (35 mL) were added, and the reaction was concentrated in vacuo. The residue was dissolved in hot isopropanol (150 mL) and filtered. Then the solution was concentrated in vacuo and recrystallized from hot isopropanol, acetone, and ether. The crystals were collected and washed with ether and acetone. (0.40 g. 27%)
MS: 282.1 (M+l for C17H31NO2); mp 187-188 °C; Analysis (C17H31NO2.HC1) (Cal.) C: 64.23, H: 10.15, N: 4.41; (found) C: 64.1 1, H: 10.04. N: 4.34. IR (KBr, cm'1): 2952, 1720, 1452, 1 185. 1HNMR (CD3OD).δ 0.48-0.57 (m. 1 H). 0.81-0.90 (m. 8H). 1.25-1.32 (m,2H) 1.60-1. 71 (m, 9H), 1.89-1.93 (m. 2H). 2.25-2.29 (m. 1H). 2.49 (s, 2H). 3.01 (d. 2H, J = 7.6 Hz), 3.20 (s, 2H).
EXAMPLE -46: {(3R, 5S)-l-[(Cyclohcxylmethyl-amino)-mcthyl]-3,5-dimethvl-cyclohcxvlj-acetic acid; hydrochloride salt
(Formula Removed)
((3R, 5S)-l-Aminomethyl-3,5-dirnethyl-cyclohexyl)-aceiic acid: hydrochloride salt (1.0 g, 4.2 mmol) was dissolved in ethanol (45 mL) and treated with 1N NaOH (4.3 mL). Cyclohexanecarbaldehyde (1.5 mL. 3 eq) were added followed by Pt02 (0.15 g). Reaction was shaken under an atmosphere of 112 (50 psi) for 17 h. The reaction was then filtered. IN HCI in ether (35 mL) was added, and the reaction was concentrated in vacuo. The residue was dissolved in hot isopropanol (150 mL) and filtered. Then the solution was concentrated in vacuo and recrystalliaed from hot isopropanol. acetone, and ether. The crystals were collected and washed with ether and acetone. (0.46 g. 34%)
MS: 296.2 (M-H for C18H31NO2); mp 176-177 °C; Analysis (C18H33NO2.HCl) (Cal.) C:
65.13, H: 10.32, N: 4.22; (found) C: 64.89, H: 10.36, N: 4.09. IR (KBr, cm'1): 2919. 2851 1721,1453,1192.1HNMR(CD3OD) δ 0.48-0.57 (m, 1H), 0.81-0.90 (m, 8H), 1.00-1.36 (m, 5H), 1.60-1.71 (m, 11H), 2.49 (s, 2H), 2.88 (d, 2H, J = 6.8 Hz), 3.18 (s. 2H).
EXAMPLE 47: ((3R, 5S)-1-CyclohexylaminomethyI-3,5-dimethyI-cyclohexyI)-acetic acid; hydrochloride salt
(Formula Removed)
((3R. 5S)-l-Aminomethyl-3,5-dimcthyl-cyclohexyl)-acetic acid: hvdrochloridc salt (0.99 g, 4.2 mmol) was dissolved in ethanol (45 mL) and treated with IN NaOH (4.2 mL). Cyclohcxanone ( 1 .5 mL. 3 cq) was added followed by PtO2 (0.2 gl Reaction was shaken under an atmosphere of Hi (50 psi) for 17 h. The reaction was then filtered. 1N HCI in ether (35 mL) was added, and the reaction was concentrated in vacuo. The residue was dissolved in hot isopropanol (150 mL) and filtered. Then the solution was concentrated in vacuo and rccrystallized from hot isopropanol. acetone, and ether. The crystals were collected and washed with ether and acetone. (0.33 g. 25%)
MS: 282.1 (M+l for C17H31NO2); mp 210 °C; Analysis (C17H31NO2. HCI) (Cal.) C: 64.23. H: 10.15, N: 4.41; (found) C: 63.89. H: 9.98, N: 4.33. IR (KBr. cm-1):2921. 1691. 1452. 1216. 1HNMR (CD3OD) δ 0.47-0.57 (m, 1H). 0.82-0.90 (m. 8H). 1.30-1.46 (m. 5H), 1.57-1.63 (m, 2H). 1.69 (d. 411, J = 12.9.Hz). 1.86 (d, 2H. J = 12.5 Hz) 2.13 (d. 211. J = 10.3 Hz), 2.46 (s. 2H), 3.04-3.10 (m 1H), 3.21 (s, 2H).
EXAMPLE 48: ((3R,5S)-l-Carboxymethyl-3,5-dimethyI-cyclohexyl)-acetic acid
(Formula Removed)
Step i: Synthesis of (8R,10S)-8,10-DimethyI-2,4-dioxo-3-aza-spiro|5.5|undccane-l,5-dicarbonitrile; ammonium salt (48a): A solution of absolute ethanol (40 mL) was cooled to 0 °C and saturated with NH3 gas, then treated with cis-3.5-dimethyl-cyclohexanone (5.0 g, 39.62 mmol) and ethyl cyanoacetatc (8.43 mL. 79.24 mmol). The flask was stoppered, allowed to warm to room temperature, and stirred overnight. The precipitate was filtered, rinsed with EtiO and dried under vacuum to give 6.04 g (59%) ol the desired product as a pale solid.
MS: 260.1 (M+l for C14H17N3O2): Analysis (C14H17N3O2.NH3.0.88 H2O) (Cai.) C: 57.55. H: 7.51, N: 19.17 (found) C: 57.67. II: 7.45. N: 19.06: IR (KBr. cm'1) 3298. 2954. 2172. 1700, 1575. 1HNMR(CDCl3) δ 0.46-0.50 (m. 1 H). 0.82-1. 17 (m. 1 1 H). 1.65-1.9S (m. 3 H), 3.58 (dd, 2 H. J = 13.9. 6.8 Hz).
Step ii: Synthesis of Example 48: (8S. 10R)-8.10-Dimethyl-2.4-dioxo-3-aza-spiro[5.5]undecane-l,5-dicarbonitrile (48a. 25.36 g. 97.8 mmol) was treated with concentrated H2SO4 (102.mL)and water (30 mL). The reaction was heated to 150 °C for 5 h; then allowed to cool to room temperature and stir overnight. The reaction was filtered and the solid was collected and dissolved in 1 N NaOH. This solution was filtered, then the filtrate was acidified with concentrated HC1 (40 mL). The precipitate was collected and dried overnight under high vacuum. The compound was then dissolved in hot isopropanol (100 mL) and recrystallized from water yielding the desired product (13.21 g, 59%).
MS: 229.1 (M+1 for C12H20O4); Analysis (CI2H2O4) (Cal.) C: 63.14, H: 8.83; (found) C: 63.36, H: 8.92. IR (KBr, cm'1): 2950,2913,1698. 1HNMR (CD3OD) δ 0.40-0.48 (m, 1H), 0.77-0.85 (m, 8H) 1.57-1.77 (m, 5H) 2.41 (s, 2H), 2.56 (s. 2H).
EXAMPLE 49: trans-((3R, 5S)-l-Hydroxymethyl-3,5-dimethyl-cyclohexyl-acetic acid; hydrochioride salt
(Formula Removed)
Step i & ii: Synthesis of (cis/trans)-(7S,9R)-7,9-Dimcthy]-2-oxa-spiro|4.5|dec;in-3-one (49a):
(i). The di-acid (48. 6.36 g. 27.9 mmol) was suspended in H:0 (93 mL). treated with ammonium hydroxide (3.5 mL) and silver nitrate (4.73 g. 27.9 mmol). and stirred for 20 minutes at room temperature. The reaction was filtered and the solid re-slurried three times with H2O, then dried under vacuum in the dark for two days at 50 °C to give 6.41 g of the silver di-salt, which was carried on immediately, (ii). The silver di-salt (1.24 g, 2.81 mmol) was placed in an amber flask,,ated with CC14 (50 mL) and iodine (0.72 g, 2.81 mmol), and heated to 75 °C overnight. The reaction was filtered, concentrated in vacuo, and chromatographed on silica gel eluting with 4:1 hexanes/EtOAc to give 0.37 g (72%) of the desired product as a pale oil (49a).
MS: 183.0(M+l forC11H18O2); Analysis(C11H18O2.0.05H2O) (Cal.) C: 72.13, H: 9.96 (found) C: 71.77, H: 9.69; IR(KBr, cm'1) 3298.2954. 2172, 1700, 1575, 1450, 1337.
1HNMR (CDC13, ~1:1 mixture of cis/trans diastereomers) δ 0.49-0.59 (m. 1 H). 0.82-1.02 (m, 10 H), 1.42-1.53 (m, 2 H), 1.58-1.72 (m, 3 H), 2.30 (s, 1 H, for one isomer), 2.39 (s. 1 H, for the other isomer 2), 3.96 (s, 1 H, for one isomer), 4.10 (s, 1 H. for the other isomer).
Step iii: Synthesis of (cis/trans)-/V-Benz}'l-2-((3R,5S)-l-hydroxymcthyl-3,5-dimcthyl-cyclohexyl)-acetamide (49b and 49c): The lactone (49a. 0.85 g. 4.66 mmol) was dissolved in THF (18 mL). treated with benzvlamine (0.61 mL. 5.60 mmol). and heated to reflux for two days. The reaction was cooled to room temperature, diluted with EtOAc, washed with saturated sodium bicarbonate solution and brine, dried over NaiSOj. and concentrated in vacuo. The residue was chromatographed on silica gel cluting with 1:1 hexane/EtOAc to give 0.79 g (59%) of the desired product as a clear oil (mixture of cis and trans isomers, 49b and 49c).
MS: 290.1 (M-H for C18H27N1O2): an oil; TLC: SiO2. R, 0.16(2:1 hcxane/ElOAc): Analysis (C1H27N1O2) (Cal.) C: 74.70. H: 9.40. N: 4.84 (foundI C: 74.41. H: 9.35. N: 4.57.
Step iv: Separation of 49b and 49c: The mixture of cis- and trans-isomers (49b and 49c) was separated by preparative HPLC (C18 column eluting with a gradient of 60% CH3CN / 40% H2O for 5 minutes, then 45% CH3CN / 55% H,O for 35 minutes, all mobile phases with 0.05% TFA).
The trans-isomer (49b): RT = 23.600 min. 54 mg yield. 1HNMR (CDCl3) δ 0.36-0.62 (m. 3 H). 0.82 (d. 6 H, J = 6.3 Hz). 1.39-1.70 (m. 5 H). 2.14 (s. 2 H), 2.58 (br. 1 H). 3.60 (s, 2 H), 4.43 (d, 2 H, J = 5.9 Hz). 5.97 (br. 1 H), 7.26-7.37 (m. 5 H).
The cis-isomer (49c): RT = 25.828 min, 87 mg yield. 1HNMR (CDC13) δ 0.47-0.56 (m, 1 H), 0.65 (t, 2 H, J = 13.2 Hz), 0.85 (d, 6 H, J = 6.3 Hz), 1.47-1.68 (m, 5 H), 2.38 (s. 2 H). 2.58 (br, 1 H), 3.40 (s, 2 H), 4.45 (d, 2 H, J = 5.9 Hz), 6.03 (br, 1 H), 7.28-7.36 (m. 5 H).
Step v: The synthesis of Example 49: The trans isomer of N-Benzy!-2-( 1 -hydroxymethyl-cyclohexyl)-acetamidtei(49b, 87 mg, 0.303 mmol) was taken from HPLC, dissolved in 5 mL of water, and treated with 24 mg of NaOH. The reaction was heated to reflux for 3 d. Then it was cooled to room temperature, diluted with 1 N NaOH, and extracted with EtOAc. The aqueous layer was acidified with cone. HC1 (5 mL). and then diluted with 1 N HC1, and the product was extracted with EtOAc. The organic layer was washed with brine and dried over Na2SO4. The solution was concentrated to yield 23 mg (61%) of the desired product.
MS: 201 (M-H for C11H20O3):1HNMR(CD3OD) δ 0.48-0.57 (m. 1 II). 0.88-1.00 (m, 8H), 1.40-1.49 (m.2H). 1.68-1.70 (m. 3H). 2.28 (s. 2H). 4.11 (s. 211).
EXAMPLE 50: cis-((3R, 5S)-l-Hydroxymc(hyl-3,5-dimcthyl-cyclohcxyl-acctic acid; hydrochloridc salt
(Formula Removed)
The cis isomer of N-Benzyl-2-(l-hydroxymethyl-cyclohexyl)-acetamide (49c, 87 mg, 0.303 mmol) was taken from HPLC, disspjved in 5 mL of water, and treated with 24 mg of NaOH. The reaction was heated to reflux for 3 d, then cooled to room temperature, diluted with 1 N NaOH, and extracted with EtOAc. The aqueous layer was acidified with
concentrated HCI (5 mL), and then diluted with 1 N HCI, and the product was extracted
with EtOAc. The organic layer was washed with brine and dried over NajSO-t. The solution was concentrated to yield 43 mg (71%) of the desired product.
MS: 201.1 (M+l for C11H20O3); 1H NMR (CD3OD) δ 0.48-0.57 (m, 1H), 0.88-0.96 (m, 8H), 1.42-1.50 (m, 2H), 1.59-1.69 (m, 3H), 2.37 (s. 2H), 3.94 (s. 2H).
EXAMPLE 51: (l-Dimethylaminomethyl-cyclohexyl)-acetic acid; hydrochloride salt
(Formula Removed)
(l-Aminomethyl-cvclohexyl)-acetic acid (0.51 g. 1.0 mmol) was dissolved in ethanol, and formaldehyde (3 mL. 2 eq) was added. 10% Pd/C (1.6 g) was added and the reaction was shaken at room temperature under a hydrogen atmosphere of 760 Torr for 12 h. Then the reaction was treated with HCI until the pH was about 2. and ii was recrysiallized from acetone and ether to give 1.62 g (87%) of the desired product.
MS: 200.! (M+l for C,,H21NO2); mp: !40-142°C; Analysis (C11H21NO2.HCl) (Cal.) C: 54.99, H: 9.44 N: 5.83; (found) C: 54.90, H: 9.36, N: 6.22. 1HNMR (CD3OD) δ 1.42-1.59 (m. 10H), 2.63 (s, 2H). 2.96 (s. 6H). 3.26-3.29 (m. 2H).
EXAMPLE 52 (1-Butylaminomethyl-cyclohexyl)-acctic acid; hydrochloride salt
(l-Aminomethy]-cyclohexyl)-acetic acid (0.86 g, 5.0 mmol) was dissolved in ethanol
(100 mL), then Pd/C (10%. 0.86 g) and butyraldehyde (1.44 g. 3 eq) were added. The reaction was placed under 760 Torr of hydrogen at room temperature until the reaction was complete. It was then filtered, acidified, and concentrated. The residue was dissolved in isopropanol, filtered, and recrystallized from acetone and ether to yield the final product.
MS: 228.1 (M+l for C13H25NO2); mp: 142-1 54°C; Analysis (C14H25NO2.HCl) (Cal.) C: 59.19, H: 9.93, N: 5.31; (found) C: 58.85. H: 9.86. N: 5.05. 1HNMR (CD3OD) δ 0.98 (t, 3H, J = 7.3 Hz), 1.39-1.54 (m, 12H), 1.66-1.72 (m, 2H), 2.57 (s, 2H). 3.01 (t. 2H. J = 9.3 Hz), 3.09 (s. 2H).
EXAMPLE 53: (1-[2,2-Dimethoxy-ethylamino)-methyI|-eyclohcxyl}-acctic acid; hydrochloridc salt
(Formula Removed)
(l-Aminomethyl-cyclohexyl)-acctic acid (2.0 g. 11.7 mmol) was dissolved in ethanol (100 mL). Glyoxal 1,1-dimcthyl acetal (3.7 mL, 3 eq) were added and the reaction was treated with PtO2 (0.5 g). The reaction was then shaken under an atmosphere of H2 (50 psi) for 4 h. The reaction was filtered, 1N HC1 in ether (15 mL) was added, and the reaction was concentrated in vacuo. The residue was recrystallized from hot isopropanol (25 mL) and acetone (700 mL) to give 0.44 g (52%) of the desired product.
MS: 260.1 (M+l for C13H25NO4; mp 128-129 °C; Analysis (C14HisNOj-HCI) (Cal.) C:
52.79, H: 8.86, N: 4.74; (found) C: 52.84, H: 8.94. N: 4.67. IR (KBr, cm'1): 2926, 1693,1457, 1208, 1087. 1HNMR (CD3OD) δ 1.40-1.55 (m, 10H), 2.60 (s, 2H), 3.13 (s, 2H), 3.17 (d, 2H, J = 4.9 Hz), 3.45 (s, 6H).4.72 (t. IH, J = 5.1 Hz)
EXAMPLE 54: (1-merhvlaminoniethy]-cyclohexyl)-acetic acid; hvdrochloride salt
(Bcnzyl-methyl-amino)-methyl]-cyclohcxyl} -acetic acid: hvdrochloride salt (Example 55. 4.1 g. 13.2 mmol) was dissolved in 95% ethane! (250 mL). 10% Pd/C (1 g) was added and the reaction was shaken at room temperature under a hydrogen atmosphere of 760 Torr for i h. The reaction was filtered and concentrated, then recrystallized from acetone and ether to yield 2.69 g (92%) of desired product.
MS: 186.0 (M+l for C10H19NO2): mp: 158-162T; Analysis (C10H17NO2.HCl) (Cal.) C: 53.09, H: 9.13, N: 6.19: (found) C: 53.14. H: 8.96. N: 6.34; 'lH NMR (CD3OD) δ 1.42-1.54 (m, 10H), 2.50-2.53 (m. 2H), 2.71-2.74 (m. 3H). 3.09-3.1 1 (m. 2H).
EXAMPLE 55: (1-[(Benzyl-methyI-amino)-mefhyl]-cyclohcxyI}-acetic acid;
hydrochloride salt
(Formula Removed)
[l-(Benzylamino-methyI)-cyc]ohexyl]-acetic acid; hydrochloride salt (0.49 g, 1.7 mmol) was dissolved in ethanol (45 mL) and treated with IN NaOH (1.8 mL). Formaldehyde (1 ml, 5 eq) was added and the reaction was treated with PtO2 (0.2 g). The reaction was then shaken under an atmosphere of H2 (50 psi) for 30 h. The reaction was filtered, IN HC1 in ether (10 mL) was added, and the reaction was concentrated in vacuo. To give 0.31 g (1.14 mmol, 67%) of the desired product.
MS: 274.1 (M-l for CI7H23NO2); an oil; IR (KBr, cm'1): 3205, 2930, 2261, 1684, 1451, 1198. HRMS: (calc) 276.1963 (found) 276.1973 (M+l for CI7H25NO2): 1HNMR (CD.,00) δ 1.33-1.63 (m, 13H), 2.28 (s, 2H), 3.06 (s, 2H), 4.40 (s. 2H), 7.21-7.34 (m, 5H).
EXAMPLE 56: [1-(Phenethylamino-methyl)-cyclohexyI|-acetic acid; hydrochloridc salt
(Formula Removed)
(l-Aminomcthyl-cyclohcxyl)-acetic acid (2.0 g, 11.7 mmol) was dissolved in ethanoi (100 mL). Phenyl-acetaldehydc (4 mL. 3 eq) was added and the reaction was treated with 10% Pd/C (0.5 g). The reaclion was then shaken under an atmosphere of H2 (50 psi) for 3 h. The reaction was filtered, IN HCI in ether (20 mL) was added, and the reaction was concentrated in vacuo. The residue was rccrystallized from hot isopropanol and ether to give 0.15 g (4.1%) of the desired product.
MS: 276.1 (M+l for C17H25NO2); mp 182-183 °C: Analysis (C17H25NO3.HC1) (Cal.) C: 64.54, H: 8.46, N: 4.43; (found) C: 64.54,H: 8.35. N: 4.34. IR (KBr, cm-1):2929. 2855. 2813, 1693, 1455, 1220. 1HNMR (CD3OD) δ 1.38-1.58 (m. 10H), 2.56 (s, 2H), 3.01-3.06 (m, 2H), 3.14 (s, 2H), 3.23-3.29 (m, 2H), 7.21-7.34 (m. 5H)

EXAMPLE 57: (1-[(3-Phenyl-propylamino)-methyl)-cyclohexyl}-acetic acid; hydrochloride salt
(Formula Removed)
(l-Aminomethyl-cyclohexyl)-acetic acid (2.00 g. 11.7 mmol) was dissolved in ethanol (100 mL). 3-Phenyl-propionaldehyde (4.6 mL. 3 cq) were added and the reaction was treated with 10% Pd/C (0.5 g). The reaction was then shaken under an atmosphere of" 11: (50 psi) for 3 h. The reaction was filtered. IN HCI (20 mL) in ether was added, and the reaction was concentrated in vacuo. The residue was recrystallizcd from hot isopropanoi and ether to give 0.46 g (12%) of the desired product.
MS: 290.1 (M-l for C18H27NO2); mp 168-169 °C: Analysis (Cal.) C: 66.34. H: 8.66. N: 4.40; (found) C: 66.48. H: 8.65. N: 4.17. 1R (KBr. cm'1): 2932. 2854. 1686, 1454. 1217. 1HNMR (CD3OD) δ 1.38-1.58 (m. 10H). 2.00-2.04 (m. 211). 2.56 (s. 2H), 2.71 (t. 2H. J = 7.6 Hz) 3.01-3.08 (m. 4H). 7.21-7.34 (m. 5H).
EXAMPLE 58: ((3R, 5S)-l-Hydroxymethyl-3,5-dimethy]-cyclohcxyl)-acctic acid; sodium salt
(Formula Removed)
(7S, 9R)-7,9-DimethyI-2-oxa-spiro[4.5]decan-3-one (49a, 1.04 g. 5.71 mmol) was dissolved in water (25 mL) and treated with NaOH (2.5 g). The reaction was heated to
reflux (115°C) for 4h. The solution was then cooled to room temperature, washed with EtjO, and the aqueous layer was concentrated in vacuo to give a solid (8 g). The residue was washed with IN acetic acid, and it was extracted with EtOAc, which was then concentrated. The residue was then recrystallized from EtOH, MeOH. and hexanes to yield 0.75 g (3.75 mmol, 66%).
MS: 199.0 (M-l forC11H20O3); HRMS: (caic) 199.1334 (found) 199.1333 (M-l for C11H20O3);mp>2500C;lR(KBr, cm'1): 2953, 2910, 1777, 1706, 1458, 1168. 1012.
EXAMPLE 59: ((3R, 5S)-l-Ethylaminomethyl-3,5-dimethyl-cyclohcxyl)-acctic acid; hydrochloridesalt
(Formula Removed)
((3R. 5S)-l-Aminomcthyl-3.5-dimcthyl-cycIohexyI)-acctic acid: hydrochloridc salt (59a, 0.418 g. 1.8 mmol. Compound 59a was prepared according to the literature procedure: Bryans. J. Bioorganic &. Medicinal Chemistry Letters. 1997. 7(19). 2481.) was dissolved methanol (45 mL) and treated with IN NaOH (1.8 mL). Acetaldchyde (0.5 mL. 3 cq) was added and the reaction was treated with PtCK (0.2 g). The reaction was then shaken under an atmosphere of H2 (50 psi) for 3 h. The reaction was filtered. IN HCI (2 mL) in ether was added, and the reaction was concentrated in vacuo. The residue was dissolved in hot isopropanol (100 mL) and filtered. Hexanes (100 mL) was added to the filtrate at 0 °C, and the precipitate was collected to give 31 mg (0.14 mmol. 8%) of the desired product.
MS: 228.1 (M+l for C13H25NO2); mp 167-168 °C; 1R (KBr, cm'1): 2954 (br). 2502. 1685. 1459, 1223. Analysis (C13H25NO2.HCI) (Cal.) C: 56.60, H: 9.98. N: 5.11; (found) C: 56.60, H: 9.85, N: 4.77. 1HNMR (CD3OD) δ 0.47-0.56 (m, IH). 0.78-0.89 (m. 8H). 1.30-1.36 (m. 3H), 1.61-1.79 (m, 5H), 2.58 (s, 2H), 2.99-3.10 (m, 4H).
EXAMPLE60: (l-Aminomethyl-4-ethyl-cyclohexyI)-aceticacid; hydrochloric salt

(Formula Removed)
Step i: Synthesis of 2-Cyano-(4-ethyl-cyclohexylidene)-acetic acid ethyl eslcr: (60a): 4-EthyIcyclohexanone (13.0g. 103.0 mmol) was dissolved in toluene (150 mL). and treated with ammonium acetate (0.79 g, 10.3 mmol), glacial acetic acid (1.2 mL). and ethyl cyanoacetate (11.65 g, 103.0 mmol). The reaction was heated to reflux for 56 h with a Dean-Stark trap: then it was cooled room temperature and washed with H2O (2x100mL). The aqueous layer was backwashed wiih toluene (2) 150mL). The organic layers were combined and dried over Na2SOj. and concentrated in vacuo. The residue was chromatographcd on silica gel eluting with 5:1 hexanes/ethyl acetate 10 give 19.81 g (87%) of the desired product as a clear yellow oil (60a).
MS: 222.1 (MH for C13H19N1O2): an oil: TLC: SiO2 R, 0.93 (1:1 hcxanc/EiOAc):. IR (KBr, cm'1): 2931,2225. 1728. 1602, 1233. 1HNMR (CDCl3) δ 0.82 (t. 3H. J - 7.4 Hz). 1.11-1.38 (m,7H), 1.39-1.45 (m. 1H), 1.90-2.09 (m. 3H). 2.20-2.28 (m. 1H). 2.91-2.98 (m. 1H), 3.75-3.79 (m. 1 H)) 4.15-4.20 (m. 2H).
Step ii: Synthesis of cis-l-Cyanomethyl-4-ethyI-cyclohexanecarbonitrile: (60b): 2-Cyano-(4-ethyl-cyclohexylidene)-acetic acid ethyl ester (60a. 16.57 g. 74.86 mmol) was dissolved in EtOH (100 mL)and added to a suspension of NaCN (3.67 g. 74.86 mmol) in EtOH / H2O (300 mL /16 mL). The reaction was heated to reflux overnight, then cooled to room temperature and filtered to remove salt that had precipitated (4.58 g). The filtrate was concentrated in vacuo to yield a clear, highly viscous, yellow oil. The residue was flash chromatographed on silica gel eluting with 16:1 hexanes/EtOAc to give 11.82 g
(77%) (a mixture of 81% of cis- and 19% of trans-isomers) of product as a yellow oil (60c).
MS: no M+l ion observed (Ci,H16N2); an oil; TLC: Si02) Rf0.55 (2:1 hexane/EtOAc); Analysis (C11Hl6N2) (Cal.) C: 74.96, H: 9.15, N: 15.89 (found) C: 74.84, H: 8.97, N: 15.74. IR (KBr, cm'1): 2934, 2240,1453. 1HNMR (CD3OD) δ 0.90 (t, 3H, J = 7.6 Hz), 1.18-1.47 (m, 7H) 1.83-1.87 (m, 2H) 107-2.10 (m, 2H), 2.85 (s. 2H).
Step iii. Synthesis of m-(l-Cyano-4-ethyl-cyclohexyl)-acetic acid ethyl ester ((60c):
m-l-Cyanomethyl-4-ethyl-cyclohexanecarbonitrile (60b. 6.16 g, 34.95 mmol) was dissolved in EtOH (92 mL) and toluene (92 mL). cooled to 0 °C. and saturated with HCI gas. The solution was stoppered and allowed to stir overnight at room temperature. Jt was then concentrated in vacuo to give a yellowish white solid. The residue was triturated with ethyl ether (200 mL) and dried under vacuum, then treated with H2O (90 mL) and 1 N HCI (4 mL) was added. The reaction was allowed to stir overnight at room temperature and yellow oil droplets were observed in solution after 18 h. The compound was extracted with EtOAc (3 x 100 mL): the organic layers were combined, washed with brine, dried over Na2SO4, and concentrated. The residue was flash chromatocraphed with 16:1 hexanes/EtOAc to give 4.40 g (56%) of the desired product (60c).
MS: 224.1 (M+l for C13H:,NO2); oil; TLC: SiO2. Rr0.38 (CH2Cl2); Analysis (C13H21NO2) (Cal.) C: 69.92, II: 9.48, N: 6.27 (found) C: 69.97, H: 9.49. N: 6.17. IR (KBr, cm-'): 2930, 2236, 1736. 1187. 1HNMR (CD,OD) δ 0.87-0.90 (m. 3H). 1.18-1.46 (m. 10H), 1.77-1.80 (m, 2H), 2.05-2.09 (m, 2H).2.59 (s. 2H), 4.13-4.15 (m. 2H).
Step iv: Synthesis of 8-Ethyl-2-aza-spiro|4.5Jdecan-3-one: (60d): cis-( I -Cyano-4-ethyl-cyclohexyl)-acetic acid ethyl ester (60c.2.7 g, 12.1 mmol) was dissolved in MeOH (250 mL) and treated with 10% Rh/ 2% Pd/C (0.5 g) and shaken under an atmosphere of H2 (50 psi) for 3 h. The reaction was then filtered, concentrated in vacuo, and
chromatographed on silica gel eluting with 3% MeOH/CH2CN to give 1.13 g (52%) of the desired product as a white solid (60d).
MS: 181.0 (M+l for Ci,H,9N1O,);mp 131-133 °C;TLC:SiO2,Rr0.22 (4% MeOH/CH2Cl2); Analysis (C11H,sN|Oi) (Cal.) C: 72.28, H: 10.08, N: 7.66 (found) C: 72.66, H: 10.45, N: 7.26. IR (KBr, cm'1): 3197,2918, 1680. 1HNMR (CDC13) δ 0.83-0.97 (m, 5 H), 1.06-1.23 (m, 3 H), 1.31 -1.39 (m. 2 H). 1.63-1.67 (m, 4 H). 2.09-2.11 (m, 2 H), 3.15-3.17 (m, 2 H), 5.73 (br, 1 H).
Step v: Synthesis of Example 60: 8-Ethyl-2-aza-spiro[4.5]decan-3-one (60d. 3.8 g, 21.1 mmol) was treated with concentrated HCI (141 mL) and healed to 110 °C for 24 h. The reaction was then concentrated, and recrystallized from hot isopropanol (20 mL) and acetone to yield 3.3 g (16.6 mmol, 79%) of the product.
MS: 200.1 (M+l for C,|H21NO2); Analysis (C,|H21NO2.HCI) (Cal.) C: 56.04, II: 9.41, N: 5.94; (found) C: 56.06. H: 9.46, N: 5.88. IR (KBr. cm'1): 2916 (br). 1698. 1526. 1281.
1HNMR (CD3OD) δ 0.89 (!. 3H, J = 7.3 Hz). 1.02-1.40 (m. 8H). 1.68 (t. 4H. .1 = 16.1 Hz). 2.38 (s.2H), 3.13(s. 2H).
EXAMPLE 61: (l-Aminomethyl-4-propyl-cyclohexyl)-acetic acid; hydrochloric salt
(Formula Removed)
Step i. Synthesis of 2-Cyano-(4-nropvl-cvclohexvlidene)-acetic acid ethvl ester:
(61 a): 4-w-Propyl-cvclohexanone (14.0 g. 99.8 mmol) was dissolved in toluene (145 mL). and treated with ammonium acetate (0.77 g. 9.98 mmol). glacial acetic acid (1.1 mL). and ethvl cyanoacetate (11.3 g. 99.8 mmol). The reaction was heated to reflux overnight with a Dean-Stark trap: then it was cooled room temperature and washed with H2O (2 x 200 mL). The aqueous laver was backwashed with toluene (3 x 200 mU. The organic layers were combined and dritd over Na?SQj. and concentrated in vacuo. The residue was chromatographed on silica gel elating, with 8:1 hexane/ethvl acetate to give 21.58 g (92%) of the desired product as a clear yellow oil (6 la).
MS: 236.1 (M+l for C14H2|NO2); an oil; TLC: SiO2, Rf 0.57 (8:1 hexane/EtOAc): Analysis (C14H21NO2) (Cal.) C: 71.46. H: 8.99. N: 5.95 (found) C: 71.38. H: 8.92. N: 6.10. IR(KBr. cm'1): 2929. 2225. 1728. 1603. 1227. 1HNMR (CDC13) δ 0.83 (t. 3H. J = 7.3 Hz). 1.07-1.30 (m.9H). 1.51-1.56 (m, 1H). 1.91-2.00 (m, 2H). 2.04-2.12 (m. 1H). 2.23-2.30 (m, 1 H). 2.95-3.01 (m, 1 H). 3.77-3.83 (m. 1 H). 4.20 (q. 2H. J = 7.1 Hz).
Step ii: Synthesis of m-l-Cyanomcthyl-4-propyl-cycIohexanccarbonitrilc: (61 b): 2-
Cyano-(4-n-propyl-cycIohcxylidene)-acctic acid ethyl ester (6la. 19.46 g. 82.70 mmol) was dissolved in EtOH (100 mL) and added to a suspension of NaCN (4.053 g. 82.70 mmol) in EtOH / H2O (340 mL / 20 mL). The reaction was heated 10 reflux overnight, then cooled to room temperature and filtered to remove salt that had precipitated. The filtrate was concentrated in vacuo to yield a clear, highly viscous, yellow oil. This oil was redissolved in EtOAc and washed with brine (2 x 150 mL). dried over Na2SO4. and concentrated. The residue was Hash chromatographed on silica gel eluting with 19:1 hexane/EtOAc to give 4.69 g of 61b. (74% yield).
MS: no M+l ion observed (C12H18N2); an oil; TLC: Si02. Rf 0.38 (3:1 hcxanc/ElOAc); Analysis (C12HI8N2) (Cal.) C: 75.74. H: 9.53, N: 14.72 (found) C: 76.05. H: 9.58. N: 14.90. IR (KBr, cm'1): 2931. 2239. 1453, 75 8. 1HNMR (CDC13) δ 0.86 (t. 3H, .1 = 7.1 Hz), 1.18-1.34 (m, 7H). 1.42-1.61 (m. 2H). 1.81-1.83 (m, 2H), 2.06-2.09 (m, 2H), 2.65 (s, 2H).
Step iii. Synthesis of ro-(l-Cyano-4-propyl-cyclohexyl)-acetic acid ethyl (61 c): cis-\-CyanornethyI-4-rt-propyl-cyclohexanecarbonitrile (61b, 3.87 g, 20.34 mmol) was dissolved in EtOH (50 mL) and toluene (50 mL), cooled to 0 °C, and saturated with HC1 gas over 20 minutes. The solution was stoppered and allowed to stir
overnight at room temperature. It was then concentrated in vacuo to give a yellowish white solid. The residue was triturated with ethyl ether (200 mL) and dried under vacuum, then dissolved in H2O (80 mL). 1 N HC1 (4 mL) was added. The reaction became clear after 4 hours of stirring and it was allowed to continue to stir overnight at room temperature. Yellow oil droplets were observed in solution after 18 h. The compound was extracted with ElOAc (4 x 200 mL); the organic layers were combined, washed with brine (2 x 200 mL). dried over Na2SO4. and concentrated. The residue was flash chromatographcd with 19:1 hexanes/EtOAc and 3.27 g of product (61c. 68%) was obtained.
MS: 238.1 (M+l for C13H21NO2): oil; TLC: Si02. Rr0.38 (CH2Cl2); Analysis (C13H21NO2) (Cal.) C: 70.85, H: 9.77. N: 5.90 (found) C: 70.65. H: 9.72. N: 5.84. 1R (KBr, cm'1): 2929. 2236. 1736. 1185. 1HNMR (CDCl3) δ 0.85 (t. 3R J = 7.! Hz). 1.19-1.55 (m. 10H). 1.71-1.74 (m. 2H), 2.08-2.10 (m. 2H). 2.59 (s. 2H). 4.16 (q. 2H. J = 7.1 Hz).
Step iv. Synthesis of cis-8-propyI-2-aza-spiro[4.5]dccan-3-one (61d): cis-(1-Cyano-4-propyl-cyclohexyl)-acetic acid ethyl ester (61c. 8.27 g. 34.8 mmol) was dissolved in methanol (90 mL), treated with triethylamine and 10% Rh / 2% Pd/C. and shaken under an atmosphere of H2 (50 psi) for 55 h. The reaction was filtered and concentrated in vacuo. The residue was chromarographed on silica gel eluting with 5: i hexanes/EtOAc to give 6.41 g (61 d, 94%) of the desired product.
MS: 196.1 (M+l for C12H21NO); an oil; TLC: SiO2, Rr 0.38 (hexane/EtOAc); Analysis (C12H21NO) (Cal.) C: 73.80, H: 10.84, N: 7.17; (found) C: 73.86. H: 10.76, N: 7.11. IR
(KBr, cm'1): 3192, 2924, 1704, 1678. 1HNMR (CDC13) δ 0.83-1.17 (m, 5H), 1.18-1.39
(m, 7H). 1.61-1.65 (m, 2H), 1.71-1.74 (m, 2H) 2.11 (s, 2H). 3.16 (s. 2H), 5.85 (br, 1H).
Step v: Synthesis of Example 61: (l-Aminomethy!-4-propyI-cyclohexyl)-acetic acid;
hydrochloric salt: 8-Propyl-2-aza-spiro[4.5]decan-3-one (61 d, 5.8 g, 29.75 mmol) was
treated with concentrated HC1 (150 mL) and heated to 115°C overnight. The reaction was then allowed to cool to room temperature and precipitate was observed. This was filtered to obtain 3.47 g of a white solid, and the filtrate was cooled and re-filtered to give a total of 6.24 g (99%) of the desired product.
MS: 214.1 (M+l for C12H23NO2); mp 142-144°C: Analysis (C121!23NO2.HC1.0.86H2O) (Cal.) C: 58.91, H: 9.83. N: 5.72; (found) C: 59.27. H: 9.60, N: 5.71. 1HNMR (CD,OD)
δ 0.86-0.90 (m, 3H). 0.97-1.41 (m. 10H). 1.61-1.75 (m. 4H). 2.37 (s. 211). 3.13 (s. 2H). IR(KBr. cm')2919. 1702. 1525.1461.1188.
EXAMPLE 62: ((3R, 5S)-3,5-Dimethyl-l-propylaminomelhyl-cyclohe.xyl)-acelic acid; hydrochloride salt
(Formula Removed)
((3R, 5S)-l-Aminomethyl-3.5-dimethyl-cyflohexyi)-acetic acid; hydrochloride salt (59a, 0.50 g. 2.12 mmol) was dissolved in ethanol (45 mL) and treated with 1.8 mL of IN NaOH. Propionaldehyde (0.5 mL, 3 eq) was added and the reaction was treated with PtO2 (0.2 g). The reaction was then shaken under an atmosphere of H2 (50 psi) for 3 h. The reaction was filtered, IN HCI in ether (5 mL) was added, and the reaction was
concentrated in vacuo. The residue was dissolved in hot isopropanol (100 mL) and filtered. The filtrate was concentrated and recrystallized from hot isopropanol (4 mL), Et20 (225 mL), and hexanes (150 mL) to give 200 mg (0.83 mmol, 39%) of product.
MS: 242.1 (M+l for C14H27NO2); IR (KBr, cm'1): 2952 (br), 1686, 1457, 1218. Analysis (C14H27NO2 HCI 0.24H2O) (Cal.) C: 5ft60, H: 10.17, N: 4.96; (found) C: 59.68, H: 10.09, N: 4.64. 1HNMR (CD3OD) δ 0.48-0.57 (m, 1 H), 0.78-1.05 (m. 11 H), 1.61 -1.78 (m, 7H), 2.60 (s, 2H), 2.93-3.00 (m, 4H).
EXAMPLE 63: [(IR, 3R)-l-(Bcnzylamino-methyl)-3-mcthyI-cycIohcxyl]-acetic acid; hydrochloride salt
(Formula Removed)
((IR, 3R)-l-Aminorncthyl-3-rnethyl-cyclohcxyI)-acetic acid: hydrochloride sal! (Example 1.3.0g. 13.5 mmol) was dissolved in ethanol (80 mL) and treated with 1 N NaOH (14 mL. 1+ equivalents). Benzaldehyde (4.5 mL) was added followed h\ 10% Pd/C (1.0 g), and the reaction was shaken under an atmosphere of H? (50 psi) lor 2.75 h. The reaction was filtered, I M HCI in ether (20 mL) was added, and the solution was concentrated in vacuo. The residue was then dissolved in hot isopropanol ( 1 00 mL). filtered, and concentrated in vacuo. The residue was then dissolved in the minimum amount of hot isopropanol necessary and recrystallized from ether. The solid was Tillered and the filtrate was cooled overnight to yield a second crop of crystals. The solids were combined to give 1 .35 g (36%) of the desired product.
MS: 276.1 (M+l for C11H25NO2); HRMS: (calc) 276.1963 (found) 276.1962 (M-H for C17H25NO2); mp: 157-158 °C; IR (KBr, cm'1): 2932 (br), 1691, 1450. 1201; Analysis (C17H2JNO2 HCI 0.24H2O) (Cal.) C: 64.58, H: 8.44, N: 4.43; (found) C: 64.57. H: 8.44,
N: 4.28. 1HNMR (CD3OD) δ 0.77-1.69 (m, 12H), 2.42 (s, 2H), 3.21 (s, 2H), 4.25 (s, 2H), 7.43-7.52 (m, 5H).
EXAMPLE 64: {(1R, 3R)-l-[(Benzyl-methyI-amino)-methyl]-3-methyI-cyclohcxyl}-acetic acid; hydrochloride salt
(Formula Removed)
[(1R. 3R)-l-(Benzylamino-methyl)-3-methyl-cycIohexyl]-acetic acid: hydrochloride salt (Example 63. 1.02 g. 3.27 mmol) was dissolved in ethanol (40 mL) and treated with 1 N NaOH (3.3 mL, 1 + eq). Formaldehyde (2.5 mL) was added followed by Pt02 (0. 1 g). and the reaction was shaken under an atmosphere of H; (50 psi) for 8 h. The reaction was filtered. 1 M HCI in ether (10 mL) was added, and the solution was concentrated in vacuo. The residue was then dissolved in hot isopropanol ( 1 00 mL). filtered, and concentrated in vacuo. The residue was then dissolved in the minimum amount of hot isopropanol necessary and rccrystallized from ether and hexanc. The solid was collected to give 0.704 g (2.16 mmol. 66%) of the desired product.
MS: 290.1 (C18H27NO2): HRMS: 290.2128 (C18H27NO2): mp: 204-205 °C: 1R (KBr. cm" '):2926(br). 1720. 1457. 1387. 1203: :H NMR (CD3OD) δ 0.82-0.98 (m. 5H). 1.24-1.91 (m, 7H). 2.38-2.58 (m. 2H), 2.91 (d, 3H. J = 1 1.2 Hz). 3.43-3.54 (m. 2H). 4.33-4.49 (m, 2H), 7.50-7.56 (m,5H).
EXAMPLE 65: ((lR,3R)-3-Methyl-l-methylaminomcthyl-cyciohcxy[)-acetic acid; hydrochloride salt
(Formula Removed)
{(1R, 3R)-l-[(Benzyl-methyl-amino)-methyl]-3-methyl-cyclohexyl}-acetic acid:
hydrochloride salt (Example 64, 0.60 g, 1.84 mmol) was dissolved in ethanol (50 mL). 20% PoYC (0.1 g), and the reaction was shaken under an atmosphere of H2 (50 psi) for 7 min.. The reaction was filtered, 1 M HC1 in ether (5 mL) was added, and the solution was concentrated in vacuo. The resiflile was then dissolved in hot isopropanol (100 mL). filtered, and concentrated in vacuo. The residue was then dissolved in me minimum amount of hot isopropanol necessary and recrystallized from ether and hexane. The crystals were to give 0.15 g (35%) of the desired product.
MS: 200.1 (M+l for C11H21NO2): HRMS: (calc) 200.1650. (found) 200.1646 (M + l for C11H21NO2):mp: 160-161 °C; IR (KBr. cm'1): 2925 (br). 1719. 1457; Analysis (C11H21NO2HCI 0.40 H:0)(Cal.)C: 54.38. H: 9.46. N: 5.77: (found) C: 54.27. !!: 9.36. N:5.65. 1HNMR (CD.-.OD) δ 0.83-0.97 (m. 5H). 1.20-1.28 (m. Ill) 1.42-1.74 im. 6il) 2.38 (s. 2H). 2.73(s.3I I). 3.23 (s. 2H).
EXAMPLE 66: Summary
Using an ex vivo model of freshly dissected rat retinas, the mechanisms bv which brain glutamate levels are maintained within normal limits in neural tissue was investigated. Anaplerotic glutamate synthesis may be important lo replenish ciutamatc oxidized in the glia following neurotransmission. Points of rate control through this dc novo pathway were identified as pyruvatc carboxylation and branched chain amino transamination. To test the relationship between glutamate synthesis and pyruvatc carboxylase activity, synthesis of 14C-glutamate plus 14C-glutamine from H14CO3 was measured as a function of medium pyruvate. Increasing pyruvate from 0.2 to 5 mM
increased the HC-amino acid synthesis by 66 ± 15%. The specific radioactivity of
glutamine relative to that of HI4CO3 after 20 minutes of incubation indicated that =30% of glutamine in the retina is derived from bicarbonate fixation. To test the dependence of glutamate synthesis on availability of branched chain amino acids (BC.AA) as a source of nitroeen, the effect of inhibiting the ieuronal branched chain aminotransferase (BCATc)
was measured. Gabapentin (GBP) (1 mM), a selective inhibitor of BCATc in neurons. inhibited synthesis of 14C-glutamate plus 14C-glutamine from H14CO2, by 31% without slowing H14CO3 incorporation into lactate and citric acid cycle intermediates. Inhibition of da nova glutamate synthesis was reversed by addition of 0.6 mM BCAA. The data indicated that pyruvate carboxylasc and branched chain aminotransferase share rate control of cle nnvo glutamate synthesis in the retina, and that dc novo glutamate synthesis is quantitatively important for maintenance of retinal glutamate levels.
Methods
Experimental animals
Sprague Duwley rats (2l)0-400g) were used for all experiments. Rats were housed under a 12 hour light/dark cycle, and fed and watered adlibidwn. After anaesthetizing the rats with-nembuta! (IP), eyes were enucleated and retinas dissected in ice-cold buffer. COi fixation
Freshly isolated half-retinas were prcincubatcd at 37°C for 3 minutes in buffer A (pH 7.4) containing 118 mM NaCl, 4.7 mM KC1,2.5 mM Cad. 1.1 mM KH2P04. 1.17 mM MgSO4, 20 mM HEPES. 5 mM glucose, 0.05 mM NH4CI and 25 mM NaH14CO3,. Pyruvate (either 0.2 or 5mM) was also included in the buffer. 1 ml buffer as well as the gas phase within 20 ml vials were equilibrated with 95% O2, 5% CO2. Incubations were initiated by addition of 10 µCi/ml HI4CO3. The vessels were then immediately closed to
the atmosphere. Most incubations continued for 20 minutes, a/though in some
preliminary studies, times were varied from 10-60 minutes. Reactions were stopped by removing the half retinas from the buffer and placing them in 2% perchloric acid. The buffer was separately acidified with perchloric acid (final concentration 2%) and
unreacted 14CO2 was allowed to diffuse out of the acidified samples.
The total amount of product (non-volatile carbon 14 metabolites) was determined by measuring the radioactivity in aliquots of the medium and tissue samples. Metabolites in the extracts were separated by Dowex-1 acetate chromatography as previously described: Williamson, J.R.. and Corkey. B. (1969) "Methods in Enzymology". Vol. 13. ed. Lowsnsic'm, J.M. (Academic Press) pp 434-513. and quantified by scintillation counting. In most instances, individual metabolite carbon 14 data is reported as the sum of the radioactivity in the medium plus tissue extracts. Complete quantification of "T-metabolite amounts in both the retina and in the medium can be found below in Tables I and II.
Data not shown in the results and discussion sections where collected in the course of carrying out the evaluation of the influences of pyruvate on H14CO3 fixation in the retina. Figure 2 shows only the effect of pyruvate on the sum glutamate plus glutamine and the sum pyruvate plus lactate. Table I provides values for all metabolites that became labeled with H CO3 and their companmentation.
Likewise additional data, not shown in the results and discussion sections, were collected in evaluating the influence of gabapentin. Thus Table II is an expansion of the data shown in Figure 4, and provides values for all metabolites labeled with H14CO3 and their compartmentation.
Table I
(Table Removed)
Table II

(Table Removed)
The chromatographic procedure employed in this example separates clutamine. glutamate, aspartate. and lactic acid. The citric acid cycle intermediates remain on the Dowex columns and are estimated as the difference between the total counts in the sample and the counts that are elutcd. Evidence of the validity of this procedure has been reported previously. Before subjecting samples to chromatograph), 14C-pyruvic acid in the samples was converted stoichiometrically to lactate by treating the samples with excess NADH and lactate dehydrogenase. Therefore the single lactale peak reported
contains all carbon 14 in both lactate and pyruvate. To determine specific activity of glutamate and glutamine in the retinal samples, the mass amount of the glutamate was measured fluorometrically using a standard enzymatic procedure: Patel, M.S. (1989) in Neuromethods: Carbohydrate and Energy Metabolism, Vol II. eds. Boulton. A.A and Baker G.D. (The Humana Press. Inc.) pp 309-340. The mass amount of glutamine was measured after chromatographic separation from glutamate followed by a luminometric determination previously described. AJP and creatine phosphate were measured fluorometrically using an enzymatic assay.
Retinal protein was determined using the DC protein assay (Bio-Rad. Richmond, CA, USA). Each half retina contained about 0.4 mg protein.
Pyruvate Carboxylase Assay
Pyruvate carboxylase activity was assayed in fresh samples of whole brain and excised retinas. The method described by Patel Patel. M.S. (1989) in Neurumcihndx: Carbohydrate and Encrg Mciaholism, Vol II, cdc. Boulton. A.A and Baker G.D. (The Humana Press. Inc.J pp 309-340. was used and involves measuring 14CO2 fixation under optimal conditions in sonicated tissue.
Western Blot
SDS-PAGH was performed as described Patel. M.S. (1989) in Xcwomc thuds: Carbohydrate und Energy Metabolism. Vol 11. eds. Boulton. A.A and Baker G.D. (The Humana Press. Inc.) pp 309-340. using 10% gels. For immunobloumg. protein were transferred to Immobilon P membranes. Membranes were blocked with 5% BSA and incubated with immunoaffinity purified rabbit anti-rat BCATc peptide antibody (1:1000 dilution) or immunoaffinity purified rabbit anti-human BCATni peptide antibody (1:1000 dilution). The immunoreactive protein bands were visualized using the ECL system according to manufacturer's instructions (Amersham. Arlington Heights. 1L. USA).
Calculations and Statistics
Data are reported in text and figures by determining the dpm/mg in a particular metabolite and dividing by the specific activity of medium HCO). Therefore all data arc reported as nmoles of H14CO3/mg protein for any given metabolite.
Results are reported as the mean ± standard error of the mean. Statistical significance was judged by paired t-test and a p RESULTS
Pyruvate carboxylase levels are similar in retina and whole brain
Patcl demonstrated that pyruvate carboxylase is the major CO: fixing enzyme in the adult rodent brain. To judge whether this might also be true in the retina, the activity of pyruvate carboxylase at 37°C in sonicates from ncocortcx was compared with those from the retina. The ncoconcx levels (2.9 - 0.1 nmoles H14CO3. fixcd/min/mg) were similar to those found in sonicated retinal tissues (3.4 ± 0.4 nmoles H14CO3 fixed/mi n/mg). indicating that pyruvate carboxylasc was just as active in retina as whole brain.
Rates of both CO: fixation and C-glutamatc appearance are a function of pyruvate levels in the retina
In preliminary experiments retinas were incubated with 0.2 mM pyruvate as described in methods for 10. 20, 40 and 60 minute periods. Total UCO: was monitored as well as ATP and creatine phosphate levels. ATP and creatine phosphate levels remained constant as a function of time, demonstrating that oxygenation and pH control were adequate throughout. Total CO, fixation (5.50 ±0.14 nmolcs/mg protein) peaked
between 20 and 40 minutes, but was already 76% of maximal (4.2 ± 0.1 nmoles/mg) at
10 minutes. Since ATP (20 ± 0.2 nmoles/mg) and creatine phosphate (16 ± 1.1 nmoles/mg) levels were high and did not change with time, this peak indicated that the rate of CO2 fixation was constant, but that continuous breakdown of non-volatile carbon 14 products to CO2 + H2O produced near steady state level of glutamate and glutamine specific radioactivity by 20 minutes.
To assess the control of pyruvate carboxylation over glutamate synthesis, retinas were incubated with either 0.2 or 5 mM pyruvate in the medium and intracellular and extracellular metabolites analyzed. The total "CO: fixed in 20 minutes in the presence ol 0.2 mM pyruvate was 5.27 ± 0.45 nmoles H COy'mg. whereas that fixed in the presence of 5 mM pyruvate was 9.50 ± 0.74 nmoles H14CO3/mg. Figure 2 shows that increasing unlabelied pyruvate from 0.2 to 5 mM increases the sum l4C-gIutamate plus '"C-glutamme (from 1.74 ± 0.19 to2.90± 0.31 nmoles/mg. p These data show that an increase in flux through pyruvate carboxylase induces an increase in de novo synthesis of retinal glutamate. A somewhat larger increase in pyruvate recycling occurs simultaneously.
A large fraction of total glutamine synthesis is derived from pyruvate carboxylation
The importance of pyruvate carboxylation as a source for replenishing the giutamate released from retinal nerve endings during neuronal transmission was assessed
by determining the specific radioactivity of glutamine in retinal tissue incubated with
medium containing H14CO3. A fixed proportion of glutamine synthesis in retina derives
from unlabelled giutamate released by neurons and taken up by Miiller glia. The remainder comes from glia] α-ketoglutarate synthesized anaplerotically from pyruvate and 14CO2. Therefore, the specific radioactivity of glutamine relative to the specific activity of Hl4CO3 provides a minimal value for the proportion of glutamine derived from anaplerosis. The product of pyruvate carboxylase in the glia is [ 1.4-'4C]-oxaloacetate. Both the 1 and 4 positions are labelled because the carbon 14 initially incorporated into the 4-C position of oxaloacetate is randomized between the 1 and 4 positions by equilibration of oxalacetatc with symmetrical fumarate. The labelled oxaloacetate condenses with pyruvate to form citrate, which is then isomerizcd to isocitratc. f 1.4-14C]-isocitrate is oxidized and decarboxylated to [5-14C] α-ketoglutarate by isocitrate dehydrogenase. Therefore the α-ketoglutarate synthesized anaplerotically has approximately half of the specific radioactivity of the original substrate H14CO), depending on the degree of randomization of the label in oxaloacetate. This is also true of the glutamine synthesized from the [5-'1C]α-ketoglutaratc.
The specific activity of glutamine was measured in the retinal tissue extracts incubated with 0.2 mM pyruvate and H14CO3 for 10. 20. 40 and 60 minutes. Only the tissue glutamine (not medium) is reported. Specific radioactivity of glutamine in the tissue was constant between 10 and 60 minutes. After 20 minutes of incubation the mass

amount of glutamine in the tissue was 4.50 ± 0.44 nmoles/mg, and its specific
radioactivity was 1970 ±115 dpm/nmol or 0.158 ± 0.009 dpm/nmoi of the bicarbonate specific activity. This indicates that about 32% of the glutamine was produced by anaplerosis and pyruvate carboxylase.
The specific radioactivity ofVetinal glutamate (269 ± 8 dpm/nmol) was lower than
that of glutamine and also constant as a function of time from 10-60 min. It was only 14% of the specific activity of glutamine. This suggests that gluiamaic metabolic turnover in the neurons is rapid with respect to anaplerotic synthesis. However, since neuronal glutamate turnover cannot replenish the glutamate carbon skeleton lost during neurotransmission. the anaplerotic pathway is of prime importance in maintaining neuronal glutamate levels. The present data show unequivocally ihat the anaplerotic pathway provides a large (=32%) proportion of the glutamine required for glutarnaic replenishment.
Synthesis of retinal plutamatc and glutamine is partially dependent on branched chain amino acid transamination
Recent studies of glutamate synthesis in cultured astrocytes suggested that the conversion of α-ketoglutarate to glutamate could act as a rate-controlling step in anaplerotic synthesis of glutamate. Several groups have indicated that the a-amino jj:roup of glutamate cannot be supplied as ammonia and must therefore be supplied by transamination of α-ketoglutarate with another amino acid: Kanamori. K... [loss. B.D., and Kondrat. R.W. (1998) J. Newochcm. 70. 1304-1315, Lacmmli. U.K. (1970) Xatttrc 221, 680-885, including our own Haberg. A., Qu. H.. Bakken. I.J.. Sandc. L.M.. White. L.R.. Haraldseth, O.. Unsgard, G.. Assley. J.. Sonnewald. U. (1998) Dcv. Ncitmsci. 20. 389-398. Branched chain amino acids are likely nitrogen contributors since they can cross the blood-brain and blood-retinal barriers efficiently. Hutson and coworkers have proposed that a nitrogen shuttle between neurons and glia which involves branched chain
amino acids and two SCAT isoenzymes, one in neuronal cytosol (BCATc) and one in
glial mitochondria (BCATm) can also provide the needed nitrogen: Hutson, S.M.,
Berkich, D., Down, P., Xu, B., Aschner, M., and LaNoue, K.F. (1998; J. Neurochem. 71
863-874. According to this hypothetical shuttle, neuronal BCATc facilitates glial
BCATm conversion of α-ketoglutarate to glutamate by providing BCAA regenerated in
the neurons (cf Figure 1).
As a first step toward investigating the role of the BCAT isoenzymes in de nova glutamate synthesis in the retina, immunoblotting with BCATm and BCATc specific antibodies was used to determine whether the BCAT isoenzymes are expressed in rat retina. As shown in Figure 3. both BCATc and BCATm are expressed in the retina at levels that are comparable to those found in whole brain homogenates. Therefore freshlv dissected rat retina is an appropriate intact neural system in which to test the concept of nitrogen shuttling between neurons and glia.
YO
The Icucinc analog gabapentin (GBP) is a neuroactive drug which inhibits BCATc. but not BCATm: Su. T.-Z.. Lunney. E.. Campbell. G.. Oxender. D.L. (1995) J Neurochem. 64, 2125-2131. It was confirmed that GBP inhibits the synthesis of Icucine in freshly dissected rat retinas (data not shown). The effect of GBP on the synthesis of 14C-glutamate plus l4C-glutaminc from HI4CO, in ex vivo rat retinas was then measured. As shown in Figure 4, 1 mM GBP reduced formation of l4C-glutamatc plus 14C-glutaminc to 71% of control values (2.26 ± 0.05 versus 3.18 ± 0.12 nmol/mg. p mM GBP was not significant. Incorporation of the label into all of the metabolites is shown in Table II.
Further experiments were carried out to investigate dose/response properties of GBP. Freshly dissected retinas were incubated for 20 minutes in buffer A containing
either 0, 0.2, 1, or 5 mM GBP. As shown in Figure 5, increasing the concentration of GBP over 1 mM did not significantly increase the extent of its inhibition of the synthesis of 14C-slutamate plus 14C-glutamine from H CO3. Again GBP has no influence on formation of citric acid cycle intermediates, lactate and pyruvatc (Figure 5). This simoons the proposal that the site of GBP inhibition of glutamate synthesis is the BCAT mediated conversion of α-ketoglutarate to glutamate.
Discussion
The studies described evaluated mechanisms for maintenance ot CNS uluiarnate levels. Previous studies showed that glutamine synthetase is required in retinas and hippocampal slices to maintain levels of neuronal glutamate: Su. T.-Z.. Lunncy. L.. Campbell. G.. Oxcndcr. D.L. (1995)7. hcurochvm. 6-4. 2125-213 1. Immunocytochemical measurements indicate that 90% or more of retinal and whole brain glutamate is neuronal and that inhibition of glutamine synthetasc for 90 minutes depletes the neuronal glutamate pool to negligible levels: Ottersen. O.P.. Zhang. N.. and Walbcrg. F. (1992) Neurosci. 46, 519-534. This demonstrated that glutamine synthetasc is required for maintenance of ncurona! glutamate. Although glutamate can be synthesized in neurons from α-ketoglutarate. lack of pyruvate carboxylase or another anaplerotic enzyme prevents replenishment of α-ketoglutarale, and thus, precludes net glutamale synthesis within the neurons except from giutamine via glutaminase.
In this example the sources and control of glutamine synthesis was investigated by incubating excised retinas with HI4C03 and measuring the appearance and specific radioactivity of 14C-glutaminc and 14C-glutamate. More 14C-glutamine is synthesized in the presence of 5 mM pyruvate than in the presence of 0.2 mM. This indicates that pyruvate carboxylase activity influences the rate of glutamine synthesis. However, lack
of sensitivity of this control is demonstrated by the observation that when pyruvate is increased from 0.2 mM to 5 mM the magnitude of the increase in pyruvate carboxvlase flux (80.3%) is not directly translated into an equivalent increase in l4C-ulutamatc plus 14C-gIutamine (69.0%). Flexibility of the control is provided by the potential for citric acid cycle decarboxylation and pyruvate recycling. The pyruvate dependence of total HI4CO3 fixation in retinal tissue was similar to that seen in cultured isolated astrocvtes Gambcnno. W.C.. Berkich, D.A.. Lynch. C.J.. Xu, B.. and LaNoue. K..F (1997)7. Neurochcm 69. 2312-2325. However, in astrocvtes. which have no way to regenerate endogenous BCAA used to transaminate α-ketoglutarate. the pyruvatc-induccd increases in C03 fixation were not translated into increases in 14C-glulamaic und 14C-ulutamine levels. Instead, the carbon 14 accumulated solely in citric acid cycle intermediates and into "recycled" pyruvate.
In the retinal studies reported here, the specific radioactivity of clutaminc at steady state was 0.158 of the bicarbonate specific activity. Since, approximately half of the I4C incorporated into oxalacetate is retained in α-kctoglutaratc. the results show that = 32% of the glutamine is derived from H14CO3. This is the first direct estimate of'lhc contribution of anaplerosis to glutamine synthesis in a neuronal tissue. Previous estimates have been indirect, and involved isotopomer analysis of NMR spectra following infusion of I3C glucose (3-5, 11-13) into intact brains. Estimates of the contribution of pyruvate carfaoxylation to glutamine synthesis using these NMR methods have been lower than the present one, and range from less than 1% (3) to 5-10% (4,5) to 20-25%: Shank. R.P., Bennet, G.S.. Freytag. S.O., and Campbell. G.L. (1985) Brain Res. 329, 364-367,
Griffen, J.L., Rae, C., Radda, O.K., and Matthews, P.M. (1999) Biochim. Biophys. Ada 1450, 297-307, depending largely on initial assumptions of the mathematical models employed. The present results suggest that glial pyruvate carboxylation and the conversion of citric acid cycle intermediates to glutamate and glutaminc arc likely to be quantitiatively important for retinal function and possibly for whole brain function
The observation that GBP slows the conversion of citric acid cycle iniermediates to glutamine and glutamate strongly suggests that neuronal synthesis of BCAA by BCATc is necessary for optimal rates of amination of α-ketoglutarate to glutamate. A reasonable scheme for their involvement in this process is shown in Figure 1.
The results, which demonstrate inhibition of 14C-clutamatc and 14C-glutammc synthesis from H14CO3 bv GBP and reversal of the inhibition by BCAA. arc consistent with the described shuttle. The hypothesis also provides a possible explanation lor the anti-epileptic action of GBP: Taylor. C.P.. Gee. N.S.. Su. T.Z.. Kocsis. J.D.. \Vdty. D.F.. Brown, J.P.. Dooley, D.J.. Boden, P., Singh. L. (1998) Epilepsy Res. 29. 233-249. Indeed, decreased glutamatc synthesis is a common action of GBP and the anti-epileptic carbonic anhydrase inhibitors: Hazcn. S.A.. Wahecd. A.. Sly, Vv'.S.. LaNoue. K.F.. Lynch, CJ. (1997) Dev. Neurosci 19. 162-171. Also. Maple Syrup Urine Disease, the hereditary ailment caused by a mutation of the gene for branched chain keto acid dehydrogenase, if untreated induces high serum BCAA in patients and subsequent neurological abnormalities: Chuang. D.I. and Shih. V.E. (1995) in The Metabolic Buxix of Inherited Disease, eds. Schriver. C.R.. Beaudet. A.L.. Sly. S.. and Vailc. D. (McGraw-Hill) pp. 1239-1277, which may be related to abnormal regulation of glutamatc synthesis. Finally, the implications of nitrogen shuttling during glutamate synthesis must be studied further in light of the efficacy of GBP in the treatment of neuropathic pain: Ottersen. O.P., Zhang, N., and Walbcrg, F. (1992) Neurosci. 46, 519-534 and neurodegeneration. Chuang, D.I. and Shih, V.E. (1995) in The Metabolic Basis of Inherited Disease, eds.
Schriver, C.R., Beaudet, A.L., Sly, S., and Vaile, D. (McGraw-Hill) pp. 1239-1277. Rothsetin. J.D. and Kuncl, R.W. (1995)7. Neurochem. 65, 643-651.
EXAMPLE 67
Enzyme preparation: Frozen rat bVains were thawed on ice and minced into fine pieces. For each brain 5 ml of isotonic buffer [225 mM mannitol, 75 mM sucrose. 5 mM MOPS (pH 7.1), 1 mM EDTA, 1 mM EGTA, 1 mM DTT containing leupeptin, pepstatin and PMSF] was added. The samples were homogenized in Potters homogenizer (B. Bran Biotech International) at 500 rpm for ten strokes, followed by additional ten strokes at 800 rpm. The homogenates were centnfuged in Bcckman J2-M1 centrifuge with rotor JA-20 at 19, 000 rpm for 1 hr. The supernatant was dialyzed with at least three changes of dialysis buffer (225 mM mannitol. 75 mM sucrose, 5 mM MOPS pH 7.1, ImM EDTA, 1 mM EGTA. and 1 mM DTT) for 24 his. The resulting enzyme was stored at -SO °C for further assavs.
Assay of BCAT: The reaction was done in 1.5 ml Eppendorf tubes in a volume of !00 µl containing 50 mM Tris-HCL (pH 8.3). 25 µM [14C] L-leucinc. 10 mM 2-oxoglutarate, 15 µM pyridoxal phosphate. 1 mM DTT. and enzyme. The solutions for the reaction mixture were made in 10-fold concentrations. After 10 min incubation at room temperature (22 °C). the reaction was stopped by adding 100 ul of 15 mM p-nitrophenyl hydrazine in 2 M HCL. After a few minutes, 1 mL of a toluene-based scintillation liquid (4.5 g of PPO and 0.5 g of bis-MSB in 1 liter of toluene) was added, the tube was capped and mixed thoroughly with a Vortex mixer. Then the tubes were ccntrifugcd at 13, 500 rpm (Eppendorf centrifuge 5415C) for 10 min. Six hundreds ul of organic layer from each tube was placed into scintillation vials, followed by adding 10 ml of scintillation cocktail for counting.

(Table Removed)


WE CLAIM:
1. A branched chain amino acid-dependent aminotransferase inhibitor
compound of Formula IV.
(Formula Removed)
R1-R4 are hydrogen or alkyl;
X is NR5 or O;
R5 is hydrogen or alkyl,
R6 is hydrogen, alkyl, benzyl, alkanoyl. alkoxyalkanoyl, arylalkyl. alkoxy. cycloalkyl, allyl, alkylcycloalkyl, alkoxy, cycloalkyl, alkylcycloalkyl. trisubstituted halogenalkyl, and wherein R1-R4are each hydrogen the R6, is not hydrogen or methyl; or a pharmaceutically acceptable salt, ester, prodrug. or amide thereof.
2.A compound as claimed in claim 1, wherein R2 and R4 are hydrogen and R1 and R3 are alkyl; R4and R4 are hydrogen and R1 and R3 are methyl; R1-R4are hydrogen; R1, is alkyl and R2-R4 are hydrogen; R1 is methyl and R2-R4are hydrogen; R5 is hydrogen; X is NR6; X is O; R6 is alkyl; R6 is benzyl; R6 is acetyl; R6 is phenylalkyl; R6, is cycloalkyl; R6, is trifluoroalkyl; R6, is alkvJcycloalkvl: R6 is alkoxy; and R6, is allyl.
3.A compound as claimed in claim 1, wherein R2 and R4 are hydrogen and R1 and R3 are methyl: R1-R4 are hydrogen; R1 is methyl and R1-R4 are hydrogen; R5 is
hydrogen; X is NR6; R6 is alkyl; R6, is benzyl; R6, is acetyl; R6, is phenylalkyl; R6 is cycloalkyl; R6 is trifluoroalkyl; R6 is alkylcycloalkyl; RA is alkoxy; and R6 is allyl.
4. A compound as daimed in claim 1, wherein R3 and it, are hydrogen and R1, and R3,
are methyl; R1-R6 are hydrogen.
5. A compound as claimed in claim 1 and selected from the group consisting of:
(1-Allylaminomethyl-cyclohexyl)-acclic acid;
(1 -Prop-2-ynylaminomethyl-cyclohexyl)-acetic acid:
{ l-[(2,2.2-Trifluoro-ethyIamino)-methyl]-cvclohexyl}-acetic acid;
{1 -[(3.3,3-Trifluoro-propylamino)-methyl]-cyclohexy]}-acetic acid:
1α,3ß,5ß- (l-AIlylaminomethyl-3,5-dimelhyl-cyclohcxyl)-acetic acid; 1α.3ß,5ß-(3,5-Dimethyl-l-prop-2-ynylaminomclhyi-cyclohexly)-acetic acid:
•1α.3ß,5ß-{3.5-Dimcthyl-I-[(2,2.2-trifluoro-ethy!amino)-methy[1-cy(.lohexyl}-acetic acid;
1α.3ß.5ß-{3.5-Dimelhyl-l-[(3,3,3-irifluoro-propylamino)-mcthyl]-cyciohcxyl}-acetic acid;
trans-((l R.3R)-l-Allylaminomethyl-3-mcthyl-cyclohexyli-aceiic acid; trans-((lR.3R)-3-MelhyI-I-prop-2-ynyiaminomethyl-cyclohexyl)-acctic acid; trans-{(1 R,3R)-3-Methy!-l-[(2,2.2-trinuoro-ethylamino)-meihylj-cyc]ohcxyl}-acetic acid;
trans-{(lR,3R)-3-Methyl-l.-[(3,3.3-trifluoro-propylamino)-mcthyl]-cyclohcxy!}-acetic acid;
trans-{(lR,3R)-3-MethyI-l-[(4,4,4-trifiuoro-butylamino)-mcthyl]-cyclohexyl}-acetic acid;
1α,3ß,5ß-{3,5-Dimethyl-l-[(4,4,4-trifluoro-butylamino)-mcthylJ-cyclohexy!}-acetic acid;
lα,3ß,5ß-{I-[(Cyclopropylmethyl-amino)-methylj-3.5-dimcthyl-cyclohexyl}-acetic acid;
trans- {(1 R,3R)-1 -[(Cyclopropylmethyl-amino)-methyI]-3-methy 1-cyclohexyl} -acetic acid;
trans-((1R,3R)-3-Methyl-l-methylammomethyl-cyclohexyl)-acetic acid
trans-((lR,3R)-l-Ethylaminomethyl-3-methyl-cyclohexyl)-acetic acid
trans-((lR,3R)-3-Methyl-l-propylaminomethyl-cyclohexyl)-acetic acid
traas-((lR,3R)-l-Butvlaminomethy]-3-methvl-cyclohexyl)-acetic acid
trans-((lR,3R)-l-Hydroxymethyl-3-methvl-cyclohcxyl)-acetic acid
1α,3ß,5ß-(1-[(Hydroxymethyi-amino)-methyl]-3,5-dimethvl-cy,lohexyl}-acetic
acid; lα,3ß,5ß-(l.Aminomeihyl-3,5-dicthyl-cyclohexyl)-acetic acid.hydrochloride:
1α,3ß,5ß-(3.5-DimethvI-l-pn)pylaminomcihy|cyclohexyl)-acetic acid
hydrochloride salt; 1α.3ß,5ß-(l-Ethylaminomethyl-3,5-dimcthyl-cyclohexyl)-acetic acid
hydrochloride salt; 1α.3ß.5ß-(l-enzylamiNO-methyl)-3.5-d,methvl-cyclohexyl)-acetic acid
hydrochloride salt; 1α.3ß.5ß-(l-Diincthylaminomcthyl-3.5-dimcthyl-ccyclohexyl)-acetic acid
hydrochloride salt;
1α,3ß,5ß-( 1 -Dulvlam.nomethyl-3.5-d.methyl-cyclohexyl)-acetic acid hydrochloride salt;
1α,3ß,5ß-! 1 -[(Bcnzy l-mcthyl-amino)-methvl]-3.5-d,mcthyl-cyclohexyl)-acetic acid
acid, hydrochloride salt; 1α.3ß.5ß-(3.5-Dimcthyl-l-mcthylaminomcthvl-cyclohexyl)-acetic acid
hydrochloride salt; 1α.3ß,5ß-[l-(Acetylamino-methyl)-3.5-dimethyl-cyclohexyl)-acetic acid
1α.3ß,5ß-[l-(lsobutylamino-methyl)-3.5-dimcthyl-cyclohexyl)-acetic acid
hydrochloride salt; 1α,3ß,5ß-[3.5-Dimcthyl-l-(phenethylamino-methyl)-cyclohexyl)-acetic acid
hydrochloride salt; 1α.3ß,5ß-{3,5-Dimethyl-l-[(3-phenyl-propylamino)-mcthyl]-cyclohexyl)-acetic
acid, hydrochloride salt;
(1-[(Cyclobutvlmethyl-amino)-methyl]-cyclohexyl}-acetic acid, hydrochloride
1α,3ß,5ß-[l-(Isopropylamono-methyl)-3,5-dimethyl-cyclohexyl]-acetic acid,
hydrochloride salt;
(1-[(2-MethyI-butylamino)-methyl]-cyclohexyl}-acetic acid, hydrochloride salt; (1-[(4,4,4-Trifluoro-buryIamino)-methyl]-cyclohexyl}-aceticacid. hydrochloride salt;
(1-Ethylarninornethy]-cyc]ohexyl)-acetic acid, hydrochloride salt; (1-[(Cyclopropylmethyl-ammo)-methyl]-cyclohexyI}-acetic acid, hydrochloride salt;
(1-[(2-Hydroxy-l-methyl-etbylamino)-methyl]-cyclohexyl}-acetic acid, hydrochloride salt;
[ l-(lsobutylamino-methyl)-cyclohexyl]-acctic acid, hydrochloride salt: (1-Propylaminomethyl-cyclohexyI)-acetic acid, hydrochloride sah; [l-(lsopropylamino-methyl)-cyclohexy!]-acetic acid, hydrochloride salt: (1 -Cyclohexylarninornethyl-cyclohexyl)-acet!c acid, hydrochloride sah. [l-(Benzylamino-methyl)-cyciohexylj-acetic acid, hydrocliloridc salt: ((1 R.3R)-3-Mcthyl-l-propylaminomethyl-cyclohe.xyl)-acetic acid, hydrochloride salt:
{1-[Cyclopentylmethyl-amino)-methyI]-cyclohexyl J-acciic acid, hydrochloric salt:
(1-[(Cyclohexylmelhyi-amino)-methyl]-cyclohexyl}-aceiic acid, hydrochloric salt;
[l-(tert-Butoxycarbonylamino-melhyl)-cyc!ohcxyl]-aceiic acid; [ 1 -(Acctylamino-methyl)-cyclohexyl]-acetic acid:
((3R, 5S)-l-Cyclobutylaminomethyl-3,5-dimethyl-cyclohexyl)-acetic acid, hydrochloride salt;
{(3R, 5S}-3,5-Dimethyl-l-[(2-methyI-bmylamino)-melhyl]-cyclohexyl}-acetic acid, hydrochloride salt;
{(3R, 5S)-I-[(2-Hydroxy-l-melhyI-ethyIamino)-methyl]-3.5-dimethyl-cyclohexyl}-acetic acid, hydrochloride salt;
{(3R,5S)-l-[(2.2-Dimethoxy-ethylamino)-methyl]-3,5-dimcthyl-cyclohcxyl}-acetic acid, hydrochloride salt;

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Patent Number 240383
Indian Patent Application Number IN/PCT/2001/00708/DEL
PG Journal Number 20/2010
Publication Date 14-May-2010
Grant Date 06-May-2010
Date of Filing 08-Aug-2001
Name of Patentee WARNER-LAMBERT COMPANY
Applicant Address 201 TABOR ROAD, MORRIS PLAINS, NEW JERSEY 07950, U.S.A
Inventors:
# Inventor's Name Inventor's Address
1 JUSTIN STEPHEN BRYANS DEAN COTTAGE, 3W.WICKHAM ROAD, BALSHAM CB1 6DZ, ENGLAND
2 LAIN -YEN HU 4881 S.RIDGESIDE CIRCLE, ANN ARBOR, MICHIGAN 48105, U.S.A
3 SUSAN M. HUTSON 1123 W.4TH STREET, WINSTON-SALEM, NORTH CAROLINA 27101, U.S.A
4 KATHRYN FOLEY LANOUE 417 HILLTOP ROAD, HUMMELSTOWN, PENNSYLVANIA 17036, U.S.A
5 ERICH LIETH 123 ANN LANE, CHAPEL HILL, NORTH CAROLINA 27516, U.S.A
6 MICHAEL FRANCIS RAFFERTY 3711 ROLLING RIDGE COURT, ANN ARBOR, MICHIGAN 48105, U.S.A
7 TODD ROBERT RYDER 91 CORNHILL PLACE, ROCHESTER, NEW YORK 14608, U.S.A
8 TI-ZHI SU 2519 MEADE COURT, ANN ARBOR, MICHIGAN 48105, U.S.A
9 DEVIN FRANKLIN WELTY 37 TESSERA DRIVE, FOOTHILLS RANCH, CALIFORNIA 92612, U.S.A
10 DAVID JUERGEN WUSTROW 5101 JOHN HOLMES ROAD, ANN ARBOR, MICHIGAN 48103, U.S.A
PCT International Classification Number C07C 229/29
PCT International Application Number PCT/US2000/30769
PCT International Filing date 2000-11-08
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/169,635 1999-12-08 U.S.A.
2 60/175,399 2000-01-11 U.S.A.
3 60/230,020 2000-09-05 U.S.A.