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Title:
CALCIUM RECEPTOR-ACTIVE COMPOUNDS
Document Type and Number:
WIPO Patent Application WO/1996/012697
Kind Code:
A2
Abstract:
The present invention features compounds of general formulae a), b), c), able to modulate one or more activities of an inorganic ion receptor and methods for treating diseases or disorders by modulating inorganic ion receptor activity. Preferably, the compound can mimic or block the effect of extracellular Ca2+ on a calcium receptor.

Inventors:
Van Wagenen, Bradford C.
Moe, Scott T.
Balandrin, Manuel F.
Delmar, Eric G.
Nemeth, Edward F.
Application Number:
PCT/US1995/013704
Publication Date:
May 02, 1996
Filing Date:
October 23, 1995
Export Citation:
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Assignee:
NPS PHARMACEUTICALS, INC.
International Classes:
C07C211/27; A61K31/135; A61K31/137; A61K31/165; A61K31/215; A61K31/34; A61K31/343; A61K31/357; A61K31/36; A61K31/38; A61K31/381; A61K31/40; A61K31/445; A61K31/4468; A61P3/00; A61P3/14; A61P9/08; A61P9/12; A61P13/12; A61P19/00; A61P19/08; A61P19/10; A61P35/00; A61P43/00; C07C211/28; C07C211/29; C07C211/30; C07C211/31; C07C211/35; C07C211/38; C07C211/42; C07C215/08; C07C215/48; C07C217/16; C07C217/54; C07C217/56; C07C217/58; C07C217/60; C07C217/62; C07C219/28; C07C223/02; C07C225/16; C07C229/38; C07C255/58; C07C317/32; C07C323/32; C07D207/08; C07D207/323; C07D211/58; C07D215/14; C07D307/79; C07D317/58; C07D317/64; C07D319/18; C07D333/54; G01N33/566; G01N33/68; (IPC1-7): C07C211/27; C07C211/30; C07C217/58; C07C211/28; A61K31/135
Domestic Patent References:
WO1994018959A11994-09-01
WO1993004373A11993-03-04
WO1995011221A11995-04-27
WO1995021815A11995-08-17
WO1995018134A11995-07-06
Foreign References:
EP0508307A21992-10-14
DE2541184A11976-04-15
US4000197A1976-12-28
DE1231690B1967-01-05
Other References:
CHEMICAL ABSTRACTS, vol. 121, no. 19, 7 November 1994 Columbus, Ohio, US; abstract no. 230462, KOMEYOSHI, YUKIO ET AL 'Optically active amines and their manufacture, intermediates, and uses' & JP,A,06 116 214 (SUMITOMO CHEMICAL CO, JAPAN)
JOURNAL OF MEDICINAL CHEMISTRY, vol. 25, no. 6, 1982 WASHINGTON US, pages 670-679, J. E. CLIFTON ET AL. 'Arylethanolamines derived from Salicylamide with .alpha. and .beta.-adrenoceptor blocking activity.'
JOURNAL OF THE CHEMICAL SOCIETY, CHEMICAL COMMUNICATIONS, 1992 LETCHWORTH GB, pages 980-2, G.-Z. WANG ET AL. 'Ruthenium-catalysed transfer hydrogenation of imines by propan-2-ol'
TETRAHEDRON: ASYMMETRY, vol. 2, no. 3, 1991 OXFORD GB, pages 183-186, S. G. DAVIES ET AL. 'Asymmetric synthesis of R-.beta.-amino butanoic acid and S-.beta.tyrosine'
CAN. J. CHEM. (1994), 72(7), 1699-704 , July 1994 MAJEWSKI, MAREK ET AL. 'Enantioselective deprotonation of protected 4-hydroxycyclohexanones'
TETRAHEDRON, vol. 41, no. 24, 1985 OXFORD GB, pages 6005-11, J. C. G. VAN NIEL ET AL. 'NADH models XXI. Stereoselective reduction of chireal imines with hantzsch ester'
SYNLETT, no. 9, 1995 STUTTGART DE, pages 961-2, YUKIHIKO HASHIMOTO ET AL. 'Highly diastereoselective addition of organometallic reagents to chiral imines derived from 1-(2-methoxyphenyl)ethylamine'
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Claims:
Claims
1. An inorganic ion receptor modulating compound having the formula: wherein Art is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CHZOH, CONH2, CN, acetoxy, N (CH3) 2, phenyl, phenoxy, benzyl, benzyloxy, a, a dimethylbenzyl, NO2, CHO, CH3CH (OH), acetyl, ethylene dioxy; Ar2 is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CHZOH, CONH2, CN, and acetoxy; q is 0,1, 2, or 3; and R is either H, lower alkyl; and pharmaceutically salts and complexes thereof; wherein said compound modulates one or more inorganic ion receptor activities.
2. The compound of claim 1, said Arl phenyl, if present, has 1 to 5 substituents each independently selected from the group consisting of, isopropyl, CH30, CF3 CH3S, CF30, I, Cl, F, and CH3 ; said Ar2 phenyl, if present, has 1 to 5 substituents each independently selected from the group consisting of, isopropyl, CH30, CH3S, CF30, I, Cl, F, CF3, and CH3; said compound is a calcimimetic; and said inorganic ion receptor activity is calcium receptor activity.
3. The compound of claim 2, wherein q is 2, said Arl phenyl having 1 to 5 substituents is present, and said Ar2 phenyl having 1 to 5 substituents is present.
4. Compound of claim 3, said Ar2 phenyl is a meta methoxy phenyl.
5. The compound of claim 2, wherein q is 0 and said Ar2 naphthyl is present.
6. The compound of claim 5, wherein said Arl phenyl having 1 to 5 substituents is present.
7. The compound of claim 2, wherein q is 2, said Ar phenyl having 1 to 5 substituents is present, and said Ar2 naphthyl.
8. The compound of claim 2, wherein said Arl phenyl, if present, has 1 to 5 substituents each independently selected from the group consisting of, CF30, I, C1, F, and CF3 ; and said Ar2 phenyl, if present, has 1 to 5 substituents each independently selected from the group consisting of, CF30, I, C1, F, CH30, and CF3.
9. The compound of claim 3, wherein said Arl phenyl has 1 to 5 substituents each independently selected from the group consisting of, CF30, I, C1, F, and CF3; and said Ar2 phenyl has 1 to 5 substituents each independently selected from the group consisting of, CF30, I, Cl, F, CH30, and CF3.
10. The compound of claim 9, wherein said Ar2 phenyl is a metamethoxy phenyl.
11. The compound of claim 2, wherein R is CH3.
12. The compound of claim 3, wherein R is CH3.
13. The compound of claim 4, wherein R is CH3.
14. The compound of claim 7, wherein R is CH3.
15. The compound of claim 11, wherein said compound has the formula: or pharmaceutically acceptable salts and complexes thereof.
16. An inorganic ion receptor modulating compound having the formula: wherein Ar3 is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CHZOH, CONH2, CN, acetoxy, benzyl, benzyloxy, dimethylbenzyl, NO2, CHO, CH3CH (OH), N (CH3) 2, acetyl, ethylene dioxy; Ar4 is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH2OH, CONH2, CN, and acetoxy; R8 is either hydrogen or phenyl; Rg is either hydrogen or methyl; and Ri. is either hydrogen, methyl, or phenyl; or pharmaceutically acceptable salts and complexes thereof.
17. An inorganic ion receptor modulating compound having the formula: wherein Ars is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CHZOH, CONH2, CN, acetoxy, benzyl, benzyloxy, a, adimethylbenzyl, NO2, CHO, CH3CH (OH), acetyl, ethylene dioxy,CH=CHphenyl; Ar6 is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, acetyl, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH2OH, CONH2, CN, carbomethoxy, OCH2C (0) C2Hs and acetoxy; Ril is hydrogen or methyl; and R12 is hydrogen or methyl.
18. A pharmaceutical composition comprising a compound of any of claims 117 and a pharmaceutical acceptable carrier.
19. A method for treating a patient in need of such treatment comprising the step of administering to said patient a therapeutically effective amount of the pharmaceutical composition of claim 18.
20. The method of claim 19, wherein said patient is a human, said disease is characterized by either, or both, of: (1) abnormal calcium homeostasis, and (2) an abnormal amount of an extracellular or intracellular messenger whose production can be affected by calcium receptor activity; and said compound is a calcimimetic.
21. The method of claim 19, wherein said patient is a human and said disease selected from the group consisting of primary and secondary hyperparathyroidism, Paget's disease, hypercalcemia malignancy, osteoporosis, hypertension, and renal osteodystrophy.
Description:
DESCRIPTION Calcium Receptor-Active Compounds Field of the Invention This invention relates to the design, development, composition and use of compounds able to modulate one or more inorganic ion receptor activities.

Background of the Invention Certain cells in the body respond not only to chemical signals, but also to ions such as extracellular calcium ions (Ca2+). Changes in the concentration of extracellular Ca 2 * (referred to herein as" [Ca2']") alter the functional responses of these cells. One such specialized cell is the parathyroid cell which secretes parathyroid hormone (PTH). PTH is the principal endocrine factor regulating Ca2+ homeostasis in the blood and extracellular fluids.

PTH, by acting on bone and kidney cells, increases the level of Catin the blood. This increase in [Ca2+] then acts as a negative feedback signal, depressing PTH secretion. The reciprocal relationship between [Ca2] and PTH secretion forms the essential mechanism maintaining bodily Ca"homeostasis.

Extracellular Ca2+ acts directly on parathyroid cells to regulate PTH secretion. The existence of a parathyroid cell surface protein which detects changes in [Ca2"] has been confirmed. Brown et al., 366 Nature 574,1993. In parathyroid cells, this protein acts as a receptor for extracellular Ca2" ("the calcium receptor"), and detects changes in [Ca2"] and to initiate a functional cellular response, PTH secretion.

Extracellular Carcan exert effects on different cell functions, reviewed in Nemeth et al., 11 Cell Calcium 319, 1990. The role of extracellular Ca2* in parafollicular (C- cells) and parathyroid cells is discussed in Nemeth, 11

Cell Calcium 323,1990. These cells have been shown to express similar Ca 2. receptor. Brown et al., 366 Nature 574,1993; Mithal et al., 9 Suppl. 1 J. Bone and Mineral Res. s282,1994; Rogers et al., 9 Suppl. 1 J. Bone and Mineral Res. s409,1994; Garrett et al., 9 Suppl. 1 J.

Bone and Mineral Res. s409,1994. The role of extra- cellular Ca2+ on bone osteoclasts is discussed by Zaidi, 10 Bioscience Reports 493,1990. In addition keratinocytes, juxtaglomerular cells, trophoblasts, pancreatic beta cells and fat/adipose cells all respond to increases in extra- cellular calcium which likely reflects activation of calcium receptors of these cells.

The ability of various compounds to mimic extra- cellular Ca2+ in vi tro is discussed by Nemeth et al., (spermine and spermidine) in"Calcium-Binding Proteins in Health and Disease,"1987, Academic Press, Inc., pp. 33-35; Brown et al., (e. g., neomycin) 128 Endocrin- ology 3047,1991; Chen et al., (diltiazem and its analog, TA-3090) 5 J. Bone and Mineral Res. 581, 1990; and Zaidi et al., (verapamil) 167 Biochem. Biophys. Res. Commun.

807,1990. Nemeth et al., PCT/US93/01642, International Publication Number WO 94/18959, and Nemeth et al., PCT/US92/07175, International Publication Number WO 93/04373, describe various compounds which can modulate the effect of an inorganic ion on a cell having an inorganic ion receptor.

The references provided in the background are not admitted to be prior art.

Summary of the Invention The present invention features compounds able to modulate one or more activities of an inorganic ion receptor and methods for treating diseases or disorders by modulating inorganic ion receptor activity. Preferred compounds can mimic or block the effect of extracellular calcium on a cell surface calcium receptor.

Diseases or disorders which can be treated by modulating inorganic ion receptor activity include one or more of the following types: (1) those characterized by abnormal inorganic ion homeostasis, preferably calcium homeostasis; (2) those characterized by an abnormal amount of an extracellular or intracellular messenger whose production can be affected by inorganic ion receptor activity, preferably calcium receptor activity; (3) those characterized by an abnormal effect (e. g., a different effect in kind or magnitude) of an intracellular or extra- cellular messenger which can itself be ameliorated by inorganic ion receptor activity, preferably calcium receptor activity; and (4) other diseases or disorders in which modulation of inorganic ion receptor activity, preferably calcium receptor activity will exert a bene- ficial effect, for example, in diseases or disorders where the production of an intracellular or extracellular messenger stimulated by receptor activity compensates for an abnormal amount of a different messenger. Examples of extracellular messengers whose secretion and/or effect can be affected by modulating inorganic ion receptor activity include inorganic ions, hormones, neurotransmitters, growth factors, and chemokines. Examples of intracellular messengers include cAMP, cGMP, IP3, and diacylglycerol.

Thus, a compound of this invention preferably modulates calcium receptor activity and is used in the treatment of diseases or disorders which can be affected by modulating one or more activities of a calcium receptor. Calcium receptor proteins enable certain specialized cells to respond to changes in extracellular Ca2+ concentration. For example, extracellular Ca2+ inhibits the secretion of parathyroid hormone from para- thyroid cells, inhibits bone resorption by osteoclasts, and stimulates secretion of calcitonin from C-cells.

In a preferred embodiment, the compound is used to treat a disease or disorder characterized by abnormal bone and mineral homeostasis, more preferably calcium homeo-

stasis. Extracellular Catis under tight homeostatic control and controls various processes such as blood clotting, nerve and muscle excitability, and proper bone formation. Abnormal calcium homeostasis is characterized by one or more of the following activities: (1) an abnormal increase or decrease in serum calcium; (2) an abnormal increase or decrease in urinary excretion of calcium; (3) an abnormal increase or decrease in bone calcium levels, for example, as assessed by bone mineral density measurements; (4) an abnormal absorption of dietary calcium; (5) an abnormal increase or decrease in the production and/or release of messengers which affect serum calcium levels such as parathyroid hormone and calcitonin; and (6) an abnormal change in the response elicited by messengers which affect serum calcium levels.

The abnormal increase or decrease in these different aspects of calcium homeostasis is relative to that occurring in the general population and is generally associated with a disease or disorder.

Diseases and disorders characterized by abnormal calcium homeostasis can be due to different cellular defects such as a defective calcium receptor activity, a defective number of calcium receptors, or a defective intracellular protein acted on by a calcium receptor. For example, in parathyroid cells, the calcium receptor is coupled to the Gi protein which in turn inhibits cyclic AMP production. Defects in Gi protein can affect its ability to inhibit cyclic AMP production.

Thus, a first aspect the invention features an inorganic ion receptor modulating compound having the formula: STRUCTURE I

where Ar1 is either naphthyl or phenyl optionally sub- stituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH20H, CONH2, CN, acetoxy, N (CH3) 2, phenyl, phenoxy, benzyl, benzyloxy, a, a- dimethylbenzyl, NO2, CHO, CH3CH (OH), acetyl, ethylene dioxy; Ar2 is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH20H, CONH2, CN, and acetoxy; q is 0,1, 2, or 3; and R is either H, or lower alkyl; and pharmaceutically salts and complexes thereof.

Compounds of this invention have preferred stereo- chemistry. The CH3 shown in Structure I is at a chiral center and provides an a- (R)-methyl structure. When R is CH3, the R shown in Structure I is also at chiral center which provides an (R)-methyl structure. Thus, when R is CH3, the Structure I compound has (R, R) stereochemistry.

Inorganic ion receptor activities are those processes brought about as a result of inorganic ion receptor acti- vation. Such processes include the production of mole- cules which can act as intracellular or extracellular messengers.

Inorganic ion receptor-modulating compound include ionomimetics, ionolytics, calcimimetics, and calcilytics.

Ionomimetics are compounds which bind to an inorganic ion receptor and mimic (i. e., evoke or potentiate) the effects of an inorganic ion at an inorganic ion receptor. Prefer- ably, the compound affects one or more calcium receptor activities. Calcimimetics are ionomimetics which effects one or more calcium receptor activities and bind to a calcium receptor.

Ionolytics are compounds which bind to an inorganic ion receptor and block (i. e., inhibit or diminish) one or more activities caused by an inorganic ion at an inorganic ion receptor. Preferably, the compound affects one or more calcium receptor activities. Calcilytics are iono- lytics which block one or more calcium receptor activities evoked by extracellular calcium and bind to a calcium receptor.

Ionomimetics and ionolytics may bind at the same receptor site as the native inorganic ion ligand binds or can bind at a different site (e. g., allosteric site). For example, NPS R-467 binding to a calcium receptor results in calcium receptor activity and, thus, NPS R-467 is classified as a calcimimetic. However, NPS R-467 binds to the calcium receptor at a different site (i. e., an allosteric site) than extracellular calcium.

A measure of a compounds effectiveness can be deter- mined by calculating the ECso or ICso for that compound.

The ECso is the concentration of a compound which causes a half maximal mimicking effect. The ICso is the concentra- tion of compound which causes a half-maximal blocking effect. ECso and ICso for compounds at a calcium receptor can be determined by assaying one or more of the activi- ties of extracellular calcium at a calcium receptor.

Examples of assays for measuring EC50, and ICso are described Nemeth et al., PCT/US93/01642, International Publication Number WO 94/18959, and Nemeth et al., PCT/US92/07175, International Publication Number WO

93/04373, (both of these publications are hereby incorpor- ated by reference here) and below. Such assays include oocyte expression assays and measuring increases in intra- cellular calcium ion concentration ( [Ca"I i) due to calcium receptor activity. Preferably, such assays measure the release or inhibition of a particular hormone associated with activity of a calcium receptor.

An inorganic ion receptor-modulating compound prefer- ably selectively targets inorganic ion receptor activity in a particular cell. For example, selective targeting of a calcium receptor activity is achieved by a compound exerting a greater effect on a calcium receptor activity in one cell type than at another cell type for a given concentration of compound. Preferably, the differential effect is 10-fold or greater as measured in vivo or in vitro. More preferably, the differential effect is mea- sured in vivo and the compound concentration is measured as the plasma concentration or extracellular fluid con- centration and the measured effect is the production of extracellular messengers such as plasma calcitonin, parathyroid hormone, or plasma calcium. For example, in a preferred embodiment, the compound selectively targets PTH secretion over calcitonin secretion.

Preferably, the compound is either a calcimimetic or calcilytic having an ECso or ICso at a calcium receptor of less than or equal to 5 yM, and even more preferably less than or equal to 1 yM, 100 nmolar, 10 nmolar, or 1 nmolar using one of the assays described below. More preferably, the assay measures intracellular Catin HEK 293 cells transformed with nucleic acid expressing the human para- thyroid calcium receptor and loaded with fura-2. Lower ECso's or ICso's are advantageous since they allow lower concentrations of compounds to be used in vivo or in vitro. The discovery of compounds with low ECso's and ICso's enables the design and synthesis of additional compounds having similar or improved potency, effect- iveness, and/or selectivity.

Another aspect of the present invention features an inorganic ion receptor modulating compound having the formula: STRUCTURE II

where Ar3 is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH2OH, CONH2, CN, acetoxy, benzyl, benzyloxy, u, u-dimethylbenzyl, N02, CHO, CH3CH (OH), N (CH3) acetyl, ethylene dioxy.

Ar4 is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH2OH, CONH,, CN, and acetoxy; R8 is either hydrogen or phenyl; Rg is either hydrogen or methyl; and R1o is either hydrogen, methyl, or phenyl; or pharmaceutically acceptable salts and complexes thereof.

Another aspect of the present invention features an inorganic ion receptor modulating compound having the formula: STRUCTURE III

where Ars is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH2OH, CONH2, CN, acetoxy, benzyl, benzyloxy, a, dimethylbenzyl, NO2, CHO, CH3CH (OH), acetyl, ethylene dioxy,-CH=CH-phenyl; Ar6 is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, acetyl, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CHZOH, CONH2, CN, carbomethoxy, OCH2C (O) C2Hs and acetoxy; Rll is hydrogen or methyl; and R12 is hydrogen or methyl.

Another aspect of the present invention features a pharmaceutical composition made up of an inorganic ion receptor-modulating compound described herein and a physiologically acceptable carrier. A"pharmacological composition"refers to a composition in a form suitable for administration into a mammal, preferably a human.

Preferably, the pharmaceutical composition contains a sufficient amount of a calcium receptor modulating compound in a proper pharmaceutical form to exert a therapeutic effect on a human.

Considerations concerning forms suitable for admin- istration are known in the art and include toxic effects, solubility, route of administration, and maintaining

activity. For example, pharmacological compositions injected into the blood stream should be soluble.

Pharmaceutical compositions can also be formulated as pharmaceutically acceptable salts (e. g., acid addition salts) and complexes thereof. The preparation of such salts can facilitate the pharmacological use of a compound by altering its physical characteristics without prevent- ing it from exerting a physiological effect.

Another aspect the present invention features a method for treating a patient by modulating inorganic ion receptor activity using inorganic ion receptor modulating compounds described herein. The method involves adminis- tering to the patient a pharmaceutical composition con- taining a therapeutically effective amount of an inorganic ion receptor-modulating compound. In a preferred embodi- ment, the disease or disorder is treated by modulating calcium receptor activity by administering to the patient a therapeutically effective amount of a calcium receptor- modulating compound.

Inorganic ion receptor-modulating compounds, and compositions containing the compounds, can be used to treat patients. A"patient"refers to a mammal in which modulation of an inorganic ion receptor will have a bene- ficial effect. Patients in need of treatment involving modulation of inorganic ion receptors can be identified using standard techniques known to those in the medical profession.

Preferably, a patient is a human having a disease or disorder characterized by one more of the following: (1) abnormal inorganic ion homeostasis, more preferably abnormal calcium homeostasis; (2) an abnormal level of a messenger whose production or secretion is affected by inorganic ion receptor activity, more preferably affected by calcium receptor activity; and (3) an abnormal level or activity of a messenger whose function is affected by inorganic ion receptor activity, more preferably affected by calcium receptor activity.

Diseases characterized by abnormal calcium homeo- stasis include hyperparathyroidism, osteoporosis and other bone and mineral-related disorders, and the like (as described, e. g., in standard medical text books, such as "Harrison's Principles of Internal Medicine"). Such diseases are treated using calcium receptor-modulating compounds which mimic or block one or more of the effects of extracellular Cas'on a calcium receptor and, thereby, directly or indirectly affect the levels of proteins or other compounds in the body of the patient.

By"therapeutically effective amount"is meant an amount of a compound which relieves to some extent one or more symptoms of the disease or disorder in the patient; or returns to normal either partially or completely one or more physiological or biochemical parameters associated with or causative of the disease or disorder.

In a preferred embodiment, the patient has a disease or disorder characterized by an abnormal level of one or more calcium receptor-regulated components and the com- pound is active on a calcium receptor of a cell selected from the group consisting of: parathyroid cell, bone osteoclast, juxtaglomerular kidney cell, proximal tubule kidney cell, distal tubule kidney cell, central nervous system cell, peripheral nervous system cell, cell of the thick ascending limb of Henle's loop and/or collecting duct, keratinocyte in the epidermis, parafollicular cell in the thyroid (C-cell), intestinal cell, platelet, vascular smooth muscle cell, cardiac atrial cell, gastrin- secreting cell, glucagon-secreting cell, kidney mesangial cell, mammary cell, beta cell, fat/adipose cell, immune cell, GI tract cell, skin cell, adrenal cell, pituitary cell, hypothalamic cell and cell of the subfornical organ.

More preferably, the cells are chosen from the group consisting of: parathyroid cell, central nervous system cell, peripheral nervous system cell, cell of the thick ascending limb of Henle's loop and/or collecting duct in the kidney, parafollicular cell in the thyroid (C-cell),

intestinal cell, GI tract cell, pituitary cell, hypothalamic cell and cell of the subfornical organ.

In a preferred embodiment, the compound is a calcimimetic acting on a parathyroid cell calcium receptor and reduces the level of parathyroid hormone in the serum of the patient. More preferably, the level is reduced to a degree sufficient to cause a decrease in plasma Ca2'.

Most preferably, the parathyroid hormone level is reduced to that present in a normal individual.

In another preferred embodiment, the compound is a calcilytic acting on a parathyroid cell calcium receptor and increases the level of parathyroid hormone in the serum of the patient. More preferably, the level is increased to a degree sufficient to cause an increase in bone mineral density of a patient.

Patients in need of such treatments can be identified by standard medical techniques, such as blood or urine analysis. For example, by detecting a deficiency of protein whose production or secretion is affected by changes in inorganic ion concentrations, or by detecting abnormal levels of inorganic ions or hormones which effect inorganic ion homeostasis.

Various examples are used throughout the application.

These examples are not intended in any way to limit the invention.

Other features and advantages of the invention will be apparent from the following figures, detailed des- cription of the invention, examples, and the claims.

Brief Description of the Drawings Figs. la-lr, show the chemical structures of different compounds.

Figs. 2-131 provided physical data for representative compounds herein described.

Description of the Preferred Embodiments The present invention features compounds able to modulate one or more inorganic ion receptor activities, preferably the compound can mimic or block an effect of an extracellular ion on a cell having an inorganic ion receptor, more preferably the extracellular ion is Cal+ and the effect is on a cell having a calcium receptor.

Publications concerned with the calcium activity, calcium receptor and/or calcium receptor modulating compounds include the following: Brown et al., Nature 366: 574, 1993; Nemeth et al., PCT/US93/01642, International Publication Number WO 94/18959; Nemeth et al., PCT/US92/07175, International Publication Number WO 93/04373; Shoback and Chen, J. Bone Mineral Res. 9: 293 (1994); and Racke et al., FEBS Lett. 333: 132, (1993).

These publications are not admitted to be prior art to the claimed invention.

I. Calcium Receptors Calcium receptors are present on different cell types and can have different activities in different cell types.

The pharmacological effects of the following cells, in response to calcium, is consistent with the presence of a calcium receptor: parathyroid cell, bone osteoclast, juxtaglomerular kidney cell, proximal tubule kidney cell, distal tubule kidney cell, central nervous system cell, peripheral nervous system cell, cell of the thick ascend- ing limb of Henle's loop and/or collecting duct, keratino- cyte in the epidermis, parafollicular cell in the thyroid (C-cell), intestinal cell, platelet, vascular smooth muscle cell, cardiac atrial cell, gastrin-secreting cell, glucagon-secreting cell, kidney mesangial cell, mammary cell, beta cell, fat/adipose cell, immune cell, GI tract cell, skin cell, adrenal cell, pituitary cell, hypothala- mic cell and cell of the subfornical organ. In addition, the presence of calcium receptors on parathyroid cell, central nervous system cell, peripheral nervous system

cell, cell of the thick ascending limb of Henle's loop and/or collecting duct in the kidney, parafollicular cell in the thyroid (C-cell), intestinal cell, GI tract cell, pituitary cell, hypothalamic cell and cell of the sub- fornical organ, has been confirmed by physical data.

The calcium receptor on these different cell types may be different. It is also possible that a cell can have more than one type of calcium receptor. Comparison of calcium receptor activities and amino acid sequences from different cells indicate that distinct calcium receptor types exist. For example, calcium receptors can respond to a variety of di-and trivalent cations. The parathyroid calcium receptor responds to calcium and Gd while osteoclasts respond to divalent cations such as calcium, but do not respond to Gd3+. Thus, the parathyroid calcium receptor is pharmacologically distinct from the calcium receptor on the osteoclast.

On the other hand, the nucleic acid sequences encoding calcium receptors present in parathyroid cells and C-cells indicate that these receptors have a very similar amino acid structure. Nevertheless, calcimimetic compounds exhibit differential pharmacology and regulate different activities at parathyroid cells and C-cells.

Thus, pharmacological properties of calcium receptors may vary significantly depending upon the cell type or organ in which they are expressed even though the calcium receptors may have similar or even identical structures.

Calcium receptors, in general, have a low affinity for extracellular Ca2+ (apparent Kd generally greater than about 0.5 mM). Calcium receptors may include a free or bound effector mechanism as defined by Cooper, Bloom and Roth,"The Biochemical Basis of Neuropharmacology", Ch. 4, and are thus distinct from intracellular calcium receptors, e. g., calmodulin and the troponins.

Calcium receptors respond to changes in extracellular calcium levels. The exact changes depend on the particu- lar receptor and cell line containing the receptor. For

example, the in vitro effect of calcium on the calcium receptor in a parathyroid cell includes the following: 1. An increase in internal calcium. The increase is due to the influx of external calcium and/or to mobilization of internal calcium. Characteristics of the increase in internal calcium include the following: (a) A rapid (time to peak < 5 seconds) and transient increase in [Ca2+] i that is refractory to inhibition by 1 UM La3+ or 1 yM Gd3+ and is abolished by pretreatment with ionomycin (in the absence of extracellular Ca2+); (b) The increase is not inhibited by dihydro- pyridines; (c) The transient increase is abolished by pre- treatment for 10 minutes with 10 mM sodium fluoride; (d) The transient increase is diminished by pretreatment with an activator of protein kinase C (PKC), such as phorbol myristate acetate (PMA), mezerein or (-)- indolactam V. The overall effect of the protein kinase C activator is to shift the concentration-response curve of calcium to the right without affecting the maximal response; and (e) Pretreatment with pertussis toxin (100 ng/ml for > 4 hours) does not affect the increase.

2. A rapid (< 30 seconds) increase in the formation of inositol-1, 4,5-triphosphate or diacylglycerol. Pre- treatment with pertussis toxin (100 ng/ml for > 4 hours) does not affect this increase; 3. The inhibition of dopamine-and isoproterenol- stimulated cyclic AMP formation. This effect is blocked by pretreatment with pertussis toxin (100 ng/ml for > 4 hours); and 4. The inhibition of PTH secretion. Pretreatment with pertussis toxin (100 ng/ml for > 4 hours) does not affect the inhibition in PTH secretion.

Using techniques known in the art, the effect of calcium on other calcium receptors in different cells can

be readily determined. Such effects may be similar in regard to the increase in internal calcium observed in parathyroid cells. However, the effect is expected to differ in other aspects, such as causing or inhibiting the release of a hormone other than parathyroid hormone.

II. Inorganic Ion Receptor Modulatina Compounds Inorganic ion receptor modulating compounds modulate one or more inorganic ion receptor activities. Preferred calcium receptor modulating compounds are calcimimetics and calcilytics. Inorganic ion receptor modulating com- pounds can be identified by screening compounds which are modelled after a compound shown to have a particular activity (i. e., a lead compound).

A preferred method of measuring calcium receptor activity is to measure changes in [Ca2+] i. Changes in [Ca2+] i can be measured using different techniques such by using HEK 293 cells transduced with nucleic acid express- ing the human parathyroid calcium receptor and loaded with fura-2; and by measuring an increase in Cl-current in a Xenopus oocyte injected with nucleic acid coding for a calcium receptor. (See Nemeth et al., PCT/US93/01642, International Publication Number WO 94/18959.) For example, poly (A)' mRNA can be obtained from cells express- ing a calcium receptor, such as a parathyroid cell, bone osteoclast, juxtaglomerular kidney cell, proximal tubule kidney cell, distal tubule kidney cell, cell of the thick ascending limb of Henle's loop and/or collecting duct, keratinocyte in the epidermis, parafollicular cell in the thyroid (C-cell), intestinal cell, central nervous cell, peripheral nervous system cell, platelet, vascular smooth muscle cell, cardiac atrial cell, gastrin-secreting cell, glucagon-secreting cell, kidney mesangial cell, mammary cell, beta cell, fat/adipose cell, immune cell, and GI tract cell. Preferably, the nucleic acid is from a parathyroid cell, C-cell, or osteoclast. More preferably,

the nucleic acid encodes a calcium receptor and is present on a plasmid or vector.

In preferred embodiments the calcium receptor modulating compound is a calcimimetic which inhibits bone resorption in vivo by an osteoclast; inhibits bone resorp- tion in vitro by an osteoclast; stimulates calcitonin secretion in vitro or in vivo from a c-cell; inhibits parathyroid hormone secretion from a parathyroid cell in vitro and decreases PTH secretion in vivo; elevates calcitonin levels in vivo; or blocks osteoclastic bone resorption in vitro and inhibits bone resorption in vivo.

In another preferred embodiment the calcium receptor modulating compound is a calcilytic which evokes the secre- tion of parathyroid hormone from parathyroid cells in vitro and elevates the level of parathyroid hormone in vivo.

Preferably, the compound selectively targets inorganic ion receptor activity, more preferably calcium receptor activity, in a particular cell. By"selectively" is meant that the compound exerts a greater effect on inorganic ion receptor activity in one cell type than at another cell type for a given concentration of compound.

Preferably, the differential effect is 10-fold or greater.

Preferably, the concentration refers to blood plasma concentration and the measured effect is the production of extracellular messengers such as plasma calcitonin, para- thyroid hormone or plasma calcium. For example, in a preferred embodiment, the compound selectively targets PTH secretion over calcitonin secretion.

In another preferred embodiment, the compound has an ECso or ICso less than or equal to 5 yM at one or more, but not all cells chosen from the group consisting of: para- thyroid cell, bone osteoclast, juxtaglomerular kidney cell, proximal tubule kidney cell, distal tubule kidney cell, central nervous system cell, peripheral nervous system cell, cell of the thick ascending limb of Henle's loop and/or collecting duct, keratinocyte in the epi-

dermis, parafollicular cell in the thyroid (C-cell), intestinal cell, platelet, vascular smooth muscle cell, cardiac atrial cell, gastrin-secreting cell, glucagon- secreting cell, kidney mesangial cell, mammary cell, beta cell, fat/adipose cell, immune cell, GI tract cell, skin cell, adrenal cell, pituitary cell, hypothalamic cell and cell of the subfornical organ. More preferably, the cells are chosen from the group consisting of parathyroid cell, central nervous system cell, peripheral nervous system cell, cell of the thick ascending limb of Henle's loop and/or collecting duct in the kidney, parafollicular cell in the thyroid (C-cell), intestinal cell, GI tract cell, pituitary cell, hypothalamic cell and cell of the sub- fornical organ. The presence of a calcium receptor in this group of cells has been confirmed by physical data such as in situ hybridization and antibody staining.

Preferably, inorganic ion receptor modulating com- pounds mimic or block the effects of an extracellular ion on a cell having an inorganic ion receptor, such that the compounds achieve a therapeutic effect. Inorganic ion receptor modulating compounds may have the same, or dif- ferent, effects on cells having different types of inor- ganic ion receptor morphology (e. g., such as cells having normal inorganic ion receptors, a normal number of inor- ganic ion receptor, an abnormal inorganic ion receptor, and an abnormal number of inorganic ion receptors).

Calcium receptor modulating compounds preferably mimic or block all of the effects of extracellular ion in a cell having a calcium receptor. However, calcimimetics need not possess all the biological activities of extra- cellular Ca2'. Similarly, calcilytics need not block all of the activities caused by extracellular calcium. Addi- tionally, different calcimimetics and different calci- lytics do not need to bind to the same site on the calcium receptor as does extracellular Ca2+ to exert their effects.

Inorganic modulating compounds need not effect inor- ganic receptor activity to the same extent or in exactly

the same manner as the natural ligand. For example, a calcimimetic may effect calcium receptor activity to a different extent, to a different duration, by binding to a different binding site, or by having a different affin- ity, compared to calcium acting at a calcium receptor.

A. Calcimimetics 1. Structure I Compounds Structure I compounds able to modulate calcium receptor activity have the following formula: where, Arl is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH2OH, CONH2,CONH2, CN, acetoxy, N (CH3) 2, phenyl, phenoxy, benzyl, benzyloxy, a, a- dimethylbenzyl, NO2, CHO, CH3CH (OH), acetyl, ethylene dioxy, preferably each substituent is independently selected from the group consisting of, CH3, CH30, CH3CH20, methylene dioxy, Br, C1, F, I, CF3, CHF2, CH2F, CF30, CF3CH20, CH3S, OH, CH20H, CONH2, CN, NO2, CH3CH2, propyl, isopropyl, butyl, isobutyl, t-butyl, and acetoxy. More preferably, Arl is either a naphthyl or a phenyl having 1-5 substituents each independently selected from the group consisting of isopropyl, CH30, CH3S, CF30, I, C1, F, CF3, and CH3, more pref erably CF30, I, C1, F, and CF3; Ar2 is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl,

halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH20H, CONH2, CN and acetoxy, preferably each substituent is independently selected from the group consisting of, CH3, CH30, CH3CH20, methylene dioxy, Br, C1, F, I, CF3, CHF2, CH2F, CF30, CF3CH20, CH3S, OH, CH2OH, CONH2, CN, NO2, CH3CH2, propyl, isopropyl, butyl, isobutyl, t-butyl, and acetoxy. More preferably, Ar2 is either a naphthyl or a phenyl having 1-5 substituents each independently selected from the group consisting of isopropyl, CH30, CH3S, CF30, I, C1, F, CF3,. and CH3, more preferably CF30, I, C1, F, CH30, and CF3. q is 0,1, 2, or 3; and R is either H, or CH3; and pharmaceutically salts and complexes thereof. "Lower alkyl"refers to a saturated hydrocarbon having 1-4 carbons, preferably 1-3 carbon atoms, which may be straight chain or branched.

"Lower alkoxy"refers to"O-lower alkyl". Where"O" is an oxygen joined to a lower alkyl.

"Lower thioalkyl"refers to"S-lower alkyl". Where "S"is a sulfur joined to a lower alkyl.

"Lower haloalkyl"refers to a lower alkyl substituted with at least one halogen. Preferably, only the terminal carbon of the lower haloalkyl is substituted with a halogen and 1 to 3 halogens are present. More preferably, the lower haloalkyl contains 1 carbon. Preferably, the halogen substitutions are either Cl or F.

"Lower haloalkoxy"refers to"O-lower haloalkyl".

Where"O"is an oxygen joined to a lower haloalkyl. a. Ar1 and Ar2 are Both Optionally Substituted Phenvls In a preferred embodiment both Arl and Ar2 are optionally substituted phenyls and the compound has following formula:

where R is hydrogen or methyl m and n are each independently 0,1, 2,3, 4, or 5; each X is independently selected from the group con- sisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower halo- alkoxy, OH, CH2OH, CONH2, CN, acetoxy, N (CH3) 2, phenyl, Phenoxy, benzyl, benzyloxy, α,α-dimethylbenzyl, NO2, CHO, CH3CH (OH), acetyl, ethylene dioxy. Preferably each X is independently selected from the group consisting of, Ch3, CH30, CH3CH2O, methylene dioxy, Br, Cl, F, I, CF3, CHF2, <BR> <BR> <BR> <BR> CH2F, CF30, CF3CH20, CH3S, OH, CH2OH, CONH2, CN, NO2, CH3CH2, propyl, isopropyl, butyl, isobutyl, t-butyl, and acetoxy.

More preferably, each X is independently selected from the group consisting of isopropyl, CH30, CH3S, CF30, I, Cl, F, CF3, and CH3, more preferably CF30, I, Cl, F, and CF3; each Z is independently selected from the group con- sisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower halo- alkoxy, OH, CH20H, CONH2, CN, and acetoxy. Preferably each Z is independently selected from the group consisting of, CH3, CH30, CH3CH2O, methylene dioxy, Br, Cl, F, I, CF3, CHF2, CH2F, CF30, CF3CH20, CH3S, OH, CH20H, CONH2, CN, CH3CH2, propyl, isopropyl, butyl, isobutyl, t-butyl, and acetoxy.

More preferably, each Z is independently selected from the group consisting of, isopropyl, CH30, CH3S, CF30, CF3, I, Cl, F, and CH3.

In a more preferred embodiment, at least one of the Z substituents is in the meta position. More preferably, the compound has the following formula:

where R is either hydrogen or methyl; m is 0,1, 2,3, 4, or 5, preferably 1 or 2; and each X is independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower halo- alkoxy, OH, CH20H, CONH2, CN, acetoxy, N (CH3) 2t phenyl, phenoxy, benzyl, benzyloxy, a,-dimethylbenzyl, NO2, CHO, CH3CH (OH), acetyl, ethylene dioxy, preferably each substi- tuent is independently selected from the group consisting of, CH3, CH30, CH3CH2O, methylene dioxy, Br, Cl, F, I, CF3, CHF2, CH2F, CF3O, CF3CH2O, CH3S, OH, CH2OH, CONH2, CH, NO2 CH3CH2, propyl, isopropyl, butyl, isobutyl, t-butyl, and acetoxy, more preferably, isopropyl, CH30, CH3S, CF30, CF3, I, Cl, F, and CH3.

More preferably, the compound has the formula: where R is either hydrogen or methyl; Ri is either halogen or hydrogen, preferably Ri is either F, or hydrogen;

R2 is either hydrogen, halogen, lower alkyl, lower haloalkyl, or lower haloalkoxy, preferably, R2 is either hydrogen, CF3, CH3, OCF3, or F, and R3 is either hydrogen, halogen, or alkoxy, preferably, R3 is either Cl, F, hydrogen, or methoxy, more preferably methoxy.

In alternative more preferred combinations; at least two of Rl, R2, and R3 is halogen, preferably F and R is hydrogen or CH3; R is hydrogen or CH3, R2 is either lower haloalkyl, or lower haloalkoxy, preferably OCF3 or CF3, and R1 and R3 is hydrogen; and R is CH3, R3 is halogen, prefer- ably Cl, R, is either halogen or hydrogen, preferably F or hydrogen, and R2 is either hydrogen, lower alkyl, lower haloalkyl, or lower haloalkoxy, preferably, hydrogen, CF3, CH3, OCF3, or F. b. Ar2 is Naphthyl and q is 0 In another preferred embodiment, Ar2 is naphthyl, q is 0, and the compound has the formula: where Arl is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH2OH, CONH2, CN, acetoxy, N (CH3) phenyl, phenoxy, benzyl, benzyloxy, a, a- dimethylbenzyl, NO2, CHO, CH3CH (OH), acetyl, ethylene dioxy, preferably each substituent is independently selected from the group consisting of, CH3, CH30, CH3CH2O,

methylene dioxy, Br, Cl, F, I, CF3, CHF2, CH2F, CF30, CF3CH20, CH3S, OH, CH2OH, CONH2, CN, NO2, CH3CH2, propyl, isopropyl, butyl, isobutyl, t-butyl, and acetoxy. More preferably, Arl is either a naphthyl or a phenyl having 1-5 substituents each independently selected from the group consisting of isopropyl, CH30, CH3S, CF3, CF30 I, Cl, F, and CH3 More preferably, Arl is an optional substituted phenyl where the compound has the formula:

where Xn represents the optional substituents for the optionally substituted phenyl as described above (with the preferred substituents and number of substituents as described above).

Even more preferably the compound has the formula:

where R is either CH3 or hydrogen; R4 is either lower alkyl, halogen, or alkoxy, preferably isopropyl, chlorine, or methoxy; and R. is either hydrogen, lower alkyl, or halogen, preferably methyl, CH3, Br, or Cl. c. Ar2 is Naphthyl and q is 2 In another preferred embodiment, Arl is a substituted phenyl, Ar2 is naphthyl, q is 2 and the compound has the formula:

where R is either hydrogen or CH3 ; n is 0,1, 2,3, 4, or 5, preferably 1 or 2; and each X is independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH20H, CONH2, CN, acetoxy, N (CH3) phenyl, phenoxy, benzyl, benzyloxy, a,-dimethylbenzyl, NO2, CHO, CH3CH (OH), acetyl, ethylene dioxy, preferably each substituent is independently selected from the group consisting of, Ch3, CH3O, CH3CH2O, methylene dioxy, Br, Cl, F, I, CF3, CHF2, CH2F, CF30, CF3CH20, CH3S, OH, CH2OH, CONH2, CN, NO2, CH3CH2, propyl, isopropyl, butyl, isobutyl, t-butyl, and acetoxy, more preferably, isopropyl, CH30, CH3S, CF30, CF3, I, C1, F, and CH3.

More preferably, the compound has the formula:

where R6 is either is either hydrogen, lower haloalkyl, or lower haloalkoxy, preferably hydrogen, OCF3 or CF3; and R, is either halogen or hydrogen, preferably chlorine or hydrogen.

In other embodiments R, R6 and R, are as described above (with the preferred substituents as described above), provided that when both R and R6 are hydrogen, R, is not Cl; and R is CH3, and R6 and R, is as described above (with the preferred substituents as described above).

2. Structure II Compounds Structure II compounds have the formula: where Ar3 is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CHZOH, CONH2, CN, acetoxy, benzyl, benzyloxy, a, a-dimethylbenzyl, N02, CHO, CH3CH (OH), N (CH3) 2, acetyl, ethylene dioxy, preferably N (CH3) lower alkoxy, or lower alkyl; Ar4 is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CHZOH, CONH2, CN, and acetoxy, preferably lower alkoxy, more preferably methoxy; Ra is either hydrogen or phenyl, preferably hydrogen;

Rg is either hydrogen or methyl; and Rlo is either hydrogen, methyl, or phenyl, more preferably when RI, is methyl the chiral carbon it is attached to is the (R) stereoisomer.

Preferably, the a-methyl in Structure II is an (R)-a- methyl.

3. Structure III Compounds Structure III compounds have the formula: where Ars is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH20H, CONH2, CN, acetoxy, benzyl, benzyloxy, a, a-dimethylbenzyl, NO2, CHO, CH3CH (OH), acetyl, ethylene dioxy,-CH=CH-phenyl, prefer- ably, lower alkyl, phenoxy,-CH=CH-phenyl, dimethylbenzyl, methoxy, methylene, or ethylene; Arg is either naphthyl or phenyl optionally substi- tuted with 0 to 5 substituents each independently selected from the group consisting of, acetyl, lower alkyl, halo- gen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH20H, CONH2, CN, carbo- methoxy, OCH2C (O) C2H5 and acetoxy, preferably methoxy, lower alkyl, phenyl, halogen, CF3, CN, carbomethoxy or, OCH2C (0) C2H5 ; Rll is hydrogen or methyl, preferably when Rll is methyl the carbon to which it is attached is an (R) stereoisomer; and

R12 is hydrogen or methyl, preferably when Rl, is methyl the carbon to which it is attached is an (R) stereoisomer.

4. Calcimimetic Activity The ability of compounds to mimic the activity of Ca2+ at calcium receptors can be determined using procedures known in the art and described by Nemeth et al., PCT/US93/ 01642, International Publication Number WO 94/18959. For example, calcimimetics possess one or more and preferably all of the following activities when tested on parathyroid cells in vitro: 1. The compound causes a rapid (time to peak < 5 seconds) and transient increase in intracellular calcium concentration that is refractory to inhibition by 1 jUM La or 1 µM Gd3+,Gd3+, The increase in [Ca2+] i persists in the absence of extracellular Ca2+, but is abolished by pre- treatment with ionomycin (in the absence of extracellular Cl2+) 2. The compound potentiates increases in [Ca2+] ; elicited by submaximal concentrations of extracellular Ca2+; 3. The increase in [Ca 2+1 elicited by extracellular Ca 2, is not inhibited by dihydropyridines; 4. The transient increase in [Ca2+] i caused by the compound is abolished by pretreatment for 10 minutes with 10 mM sodium fluoride; 5. The transient increase in [Ca2+] i caused by the compound is diminished by pretreatment with an activator of protein kinase C (PKC), such as phorbol myristate acetate (PMA), mezerein or (-)-indolactam V.

The overall effect of the protein kinase C activator is to shift the concentration-response curve of the compound to the right without affecting the maximal response; 6. The compound causes a rapid (< 30 seconds) increase in the formation of inositol-1, 4,5-triphosphate and/or diacylglycerol;

7. The compound inhibits dopamine-or isopro- terenol-stimulated cyclic AMP formation; 8. The compound inhibits PTH secretion; 9. Pretreatment with pertussis toxin (100 ng/ml for > 4 hours) blocks the inhibitory effect of the compound on cyclic AMP formation, but does not effect increases in [Ca2'] i, inositol-1, 4,5-triphosphate, or diacylglycerol, nor decreases in PTH secretion; 10. The compound elicits increases in Cl- current in Xenopus oocytes injected with poly (A) +-enriched mRNA from bovine or human parathyroid cells, but is without effect in Xenopus oocytes injected with water, or liver mRNA; and 11. Similarly, using a cloned calcium receptor from a parathyroid cell, the compound will elicit a response in Xenopus oocytes injected with the specific cDNA or mRNA encoding the receptor.

Different calcium activities can be measured using available techniques. (See, Nemeth et al., PCT/US93/01642, International Publication Number WO 94/18959.) Parallel definitions of compounds mimicking Ca2+ activity on other calcium responsive cell, preferably at a calcium receptor, are evident from the examples provided herein and Nemeth et al., PCT/US93/01642, International Publication Number WO 94/18959.

Preferably, the compound as measured by the bioassays described herein, or by Nemeth et al., PCT/US93/01642, International Publication Number WO 94/18959, has one or more, more preferably all of the following activities: evokes a transient increase in internal calcium, having a duration of less that 30 seconds (preferably by mobilizing internal calcium); evokes a rapid increase in [Ca2+] i, occurring within thirty seconds; evokes a sustained increase (greater than thirty seconds) in [Ca2+] i (prefer- ably by causing an influx of external calcium); evokes an increase in inositol-1, 4,5-triphosphate or diacylglycerol levels, preferably within less than 60 seconds; and

inhibits dopamine-or isoproterenol-stimulated cyclic AMP formation.

The transient increase in [Ca2+] i is preferably abolished by pretreatment of the cell for ten minutes with 10 mM sodium fluoride, or the transient increase is dimin- ished by brief pretreatment (not more than ten minutes) of the cell with an activator of protein kinase C, prefer- <BR> <BR> <BR> ably, phorbol myristate acetate (PMA), mezerein or (-) indolactam V.

C. Calcilytics The ability of a compound to block the activity of extracellular calcium at a calcium receptor can be deter- mined using standard techniques based on the present disclosure. (See, also Nemeth et al., PCT/US93/01642, International Publication Number WO 94/18959.) For example, compounds which block the effect of extracellular calcium, when used in reference to a parathyroid cell, possess one or more, and preferably all of the following characteristics when tested on parathyroid cells in vitro: 1. The compound blocks, either partially or completely, the ability of increased concentrations of extracellular Ca2+ to: (a) increase [Ca2+] i, (b) mobilize intracellular Ca2', (c) increase the formation of inositol-1,4, 5- triphosphate, (d) decrease dopamine-or isoproterenol- stimulated cyclic AMP formation, and (e) inhibit PTH secretion; 2. The compound blocks increases in Cl-current in Xenopus oocytes injected with poly (A) '-mRNA from bovine or human parathyroid cells elicited by extracellular Cas'or calcimimetic compounds, but not in Xenopus oocytes injected with water or liver mRNA; 3. Similarly, using a cloned calcium receptor from a parathyroid cell, the compound will block a response in

Xenopus oocytes injected with the specific cDNA, mRNA or cRNA encoding the calcium receptor, elicited by extracellular Cas'or a calcimimetic compound.

Parallel definitions of compounds blocking Ca2+ activity on a calcium responsive cell, preferably at a calcium receptor, are evident from the examples provided herein and Nemeth et al., PCT/US93/01642, International Publication Number WO 94/18959.

III. TREATMENT OF DISEASES OR DISORDERS Diseases or disorders which can be treated by modulating calcium receptor activity are known in the art.

For example, diseases or disorders which can be treated by modulating calcium receptor activity can be identified based on the functional responses of cells regulated by calcium receptor activity. Functional responses of cells regulated by calcium receptor are know in the art, includ- ing PTH secretion by parathyroid cells, calcitonin secretion by C-cells, and bone resorption by osteoclasts.

Such functional responses are associated with differ- ent diseases or disorders. For example, hyperparathyroid- ism results in elevated levels of PTH in the plasma.

Decreasing the plasma levels of PTH offers an effective means of treating hyperparathyroidism. Likewise, increas- ing plasma levels of calcitonin is associated with an inhibition of bone resorption. Inhibiting bone resorption is an effective treatment for osteoporosis. Thus, modula- tion of calcium receptor activity can be used to treat diseases such as hyperparathyroidism, and osteoporosis.

Those compounds modulating inorganic ion receptor activity, preferably calcium receptor activity, can be used to confer beneficial effects to patients suffering from a variety of diseases or disorders. For example, osteoporosis is an age-related disorder characterized by loss of bone mass and increased risk of bone fracture.

Compounds can be used to block osteoclastic bone resorp- tion either directly (e. g., an osteoclast ionomimetic

compound) or indirectly by increasing endogenous calci- tonin levels (e. g., a C-cell calcimimetic). Alterna- tively, a calcilytic active on the parathyroid cell cal- cium receptor will increase circulating levels of para- thyroid hormone, stimulating bone formation. All three of these approaches will result in beneficial effects to patients suffering from osteoporosis.

In addition, it is known that intermittent low dosing with PTH results in an anabolic effect on bone mass and appropriate bone remodeling. Thus, compounds and dosing regimens evoking transient increases in parathyroid hor- mone (e. g., intermittent dosing with a parathyroid cell ionolytic) can increase bone mass in patients suffering from osteoporosis.

Additional diseases or disorders can be identified by identifying additional cellular functional responses, associated with a disease or disorder, which are regulated by calcium receptor activity. Diseases or disorder which can be treated by modulating other inorganic ion receptors can be identified in an analogous manner.

The inorganic ion receptor-modulating compounds of the present invention can exert an affect at an inorganic ion receptor causing one or more cellular effects ulti- mately producing a therapeutic effect. Calcium receptor- modulating compounds of the present invention can exert an effect on calcium receptor causing one or more cellular effects ultimately producing a therapeutic effect.

Different diseases can be treated by the present invention by targeting cells having a calcium receptor.

For example, primary hyperparathyroidism (HPT) is characterized by hypercalcemia and abnormal elevated levels of circulating PTH. A defect associated with the major type of HPT is a diminished sensitivity of para- thyroid cells to negative feedback regulation by extra- cellular Ca2+. Thus, in tissue from patients with primary HPT, the"set-point"for extracellular Ca2+ is shifted to the right so that higher than normal concentrations of

extracellular Ca2t are required to depress PTH secretion.

Moreover, in primary HPT, even high concentrations of extracellular Ca2+ often depress PTH secretion only partially. In secondary (uremic) HPT, a similar increase in the set-point for extracellular Catis observed even though the degree to which Ca2+ suppresses PTH secretion is normal. The changes in PTH secretion are paralleled by changes in [Ca2+] i: the set-point for extracellular Ca2+- induced increases in [Ca2+] i is shifted to the right and the magnitude of such increases is reduced.

Patients suffering from secondary HPT may also have renal osteodystrophy. Calcimimetics appear to be useful for treating both abnormal PTH secretion and osteodys- trophy in such patients.

Compounds that mimic the action of extracellular Ca2+ are beneficial in the long-term management of both primary and secondary HPT. Such compounds provide the added impe- tus required to suppress PTH secretion which the hypercal- cemic condition alone cannot achieve and, thereby, help to relieve the hypercalcemic condition. Compounds with greater efficacy than extracellular Ca2+ may overcome the apparent nonsuppressible component of PTH secretion which is particularly troublesome in the major form of primary HPT caused by adenoma of the parathyroid gland.

Alternatively or additionally, such compounds can depress synthesis of PTH, as prolonged hypercalcemia has been shown to depress the levels of preproPTH mRNA in bovine and human adenomatous parathyroid tissue. Prolonged hypercalcemia also depresses parathyroid cell prolifera- tion in vitro, so calcimimetics can also be effective in limiting the parathyroid cell hyperplasia characteristic of secondary HPT.

Cells other than parathyroid cells can respond directly to physiological changes in the concentration of extracellular Ca2+. For example, calcitonin secretion from parafollicular cells in the thyroid (C-cells) is regulated by changes in the concentration of extracellular Ca2+.

Isolated osteoclasts respond to increases in the concentration of extracellular Ca2t with corresponding increases in [Ca2] i that arise partly from the mobilization of intracellular Ca2+. Increases in [Ca2'] i in osteoclasts are associated with the inhibition of bone resorption.

Release of alkaline phosphatase from bone-forming osteo- blasts is directly stimulated by calcium.

Renin secretion from juxtaglomerular cells in the kidney, like PTH secretion, is depressed by increased concentrations of extracellular Ca2+. Extracellular Ca causes the mobilization of intracellular Cas'in these cells. Other kidney cells respond to calcium as follows: elevated Ca2+ inhibits formation of 1, 25 (OH) 2-vitamin D by proximal tubule cells, stimulates production of calcium- binding protein in distal tubule cells, and inhibits tubular reabsorption of Ca2'and Mg2'and the action of vasopressin on the thick ascending limb of Henle's loop (MTAL), reduces vasopressin action in the cortical collecting duct cells, and affects vascular smooth muscle cells in blood vessels of the renal glomerulus.

Calcium also promotes the differentiation of intestinal goblet cells, mammary cells, and skin cells; inhibits atrial natriuretic peptide secretion from cardiac atria; reduces cAMP accumulation in platelets; alters gastrin and glucagon secretion; acts on vascular smooth muscle cells to modify cell secretion of vasoactive factors; and affects cells of the central nervous system and peripheral nervous system.

Thus, there are sufficient indications to suggest that Ca2+, in addition to its ubiquitous role as an intracellular signal, also functions as an extracellular signal to regulate the responses of certain specialized cells. Compounds of this invention can be used in the treatment of diseases or disorders associated with disrupted Ca2+ responses in these cells.

Specific diseases and disorders which might be treated or prevented, based upon the affected cells, also

include those of the central nervous system such as seizures, stroke, head trauma, spinal cord injury, hypoxia-induced nerve cell damage such as in cardiac arrest or neonatal distress, epilepsy, neurodegenerative diseases such as Alzheimer's disease, Huntington's disease and Parkinson's disease, dementia, muscle tension, depres- sion, anxiety, panic disorder, obsessive-compulsive disorder, post-traumatic stress disorder, schizophrenia, neuroleptic malignant syndrome, and Tourette's syndrome; diseases involving excess water reabsorption by the kidney such as syndrome of inappropriate ADH secretion (SIADH), cirrhosis, congestive heart failure, and nephrosis; hypertension; preventing and/or decreasing renal toxicity from cationic antibiotics (e. g., aminoglycoside anti- biotics); gut motility disorders such as diarrhea, and spastic colon; GI ulcer diseases; GI diseases with excessive calcium absorption such as sarcoidosis; and autoimmune diseases and organ transplant rejection.

While calcium receptor-modulating compounds of the present invention will typically be used in therapy for human patients, they may also be used to treat similar or identical diseases in other warm-blooded animal species such as other primates, farm animals such as swine, cattle, and poultry; and sports animals and pets such as horses, dogs and cats.

IV. Administration The different compounds described by the present invention can be used to treat different diseases or disorders by modulating inorganic ion receptor activity, preferably calcium receptor activity. The compounds of the invention can be formulated for a variety of modes of administration, including systemic and topical or local- ized administration. Techniques and formulations gener- ally may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA. Administration of iono- mimetics and ionolytics is discussed by Nemeth et al.,

PCT/US93/01642, International Publication Number WO 94/18959.

Suitable dosage forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such dosage forms should allow the compound to reach a target cell whether the target cell is present in a multicellular host or in culture. For example, pharmacological compounds or compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and dosage form which retard the compound or composition from exerting its effect.

Compounds can also be formulated as pharmaceutically acceptable salts (e. g., acid addition salts) and complexes thereof. Pharmaceutically acceptable salts are non-toxic salts at the concentration at which they are administered.

The preparation of such salts can facilitate the pharmaco- logical use by altering the physical characteristic of the compound without preventing it from exerting its physio- logical effect. Useful alterations in physical properties include lowering the melting point to facilitate trans- mucosal administration and increasing the solubility to facilitate administering higher concentrations of the drug.

Pharmaceutically acceptable salts include acid addi- tion salts such as those containing sulfate, hydrochlor- ide, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate. (See e. g., PCT/US92/03736, hereby incorpor- ated by reference herein.) Pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethane- sulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid.

Pharmaceutically acceptable salts can be prepared by standard techniques. For example, the free base form of a compound is dissolved in a suitable solvent, such as an aqueous or aqueous-alcohol solution, containing the appro- priate acid and then isolated by evaporating the solution.

In another example, a salt is prepared by reacting the free base and acid in an organic solvent.

Carriers or excipients can also be used to facilitate administration of the compound. Examples of carriers and excipients include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents. The compositions or pharmaceutical composition can be administered by different routes including intrave- nously, intraperitoneal, subcutaneous, and intramuscular, orally, topically, or transmucosally.

For systemic administration, oral administration is preferred. Alternatively, injection may be used, e. g., intramuscular, intravenous, intraperitoneal, and sub- cutaneous. For injection, the compounds of the invention are formulated in liquid solutions, preferably in physio- logically compatible buffers such as Hank's solution or Ringer's solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms can also be produced.

Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for trans- mucosal administration, bile salts and fusidic acid deriv- atives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays, for example, or using suppositories. For

oral administration, the compounds can be formulated into conventional oral administration dosage forms such as capsules, tables, and liquid preparations.

For topical administration, the compounds of the invention can be formulated into ointments, salves, gels, or creams, as is generally known in the art.

The amounts of various compounds of this invention to be administered can be determined by standard procedures. Generally, a therapeutically effective amount is between about 1 nmole and 3 mole of the compound, preferably 0.1 nmole and 1 gmole depending on its ECso or ICso and on the age and size of the patient, and the disease or disorder associated with the patient. Generally, it is an amount between about 0.1 and 50 mg/kg, preferably 0.01 and 20 mg/kg of the animal to be treated.

V. Examples Examples are provided below illustrating different aspects and embodiments of the present invention. These examples are not intended to limit the claimed invention.

Example 1: Cloning of Human Parathyroid Calcium Receptor From a Human Parathyroid Gland Adenoma Tumor This example describes the cloning of a human para- thyroid calcium receptor from a human parathyroid gland adenoma tumor using pBoPCaRl as a hybridization probe (See, Nemeth et al., PCT/US93/01642, International Publication Number WO 94/18959). The probe was used to identify nucleic acid encoding human parathyroid gland calcium receptor by cross-hybridization at reduced stringency.

Messenger RNA was prepared from a human parathyroid gland adenoma tumor removed from a 39-year-old Caucasian male diagnosed with primary hyperparathyroidism. Northern blot analysis of this mRNA using pBoPCaRl as a hybridiza- tion probe identified calcium receptor transcripts of about 5 Kb and about 4 Kb. A cDNA library was constructed

from the mRNA. Double-stranded cDNA larger than 3 Kbp were size-selected on an agarose gel and ligated into the cloning vector lambda ZapII. Five hundred thousand primary recombinant phage were screened with the 5.2 Kbp cDNA insert of pBoPCaRl as a hybridization probe. The pBoPCaRl insert was labeled by random-primed synthesis using [32P]-dCTP to a specific activity of 1 x 109 cpm/yg.

Library screening was performed at a hybridization stringency of 400 mM Na+, 50% formamide at a temperature of 38°C. Plaque lift filters were hybridized at a probe concentration of 500,000 cpm/ml for 20 hours. Following hybridization, filters were washed in 1 x SSC at 40°C for 1 hr.

The primary screen identified about 250 positive clones identified by hybridization to pBoPCaRl. Seven of these clones were taken through secondary and tertiary screens to isolate single clones that hybridized to the pBoPCaRl probe. These seven clones were analyzed by restriction enzyme mapping and Southern blot analysis.

Three of the clones contained cDNA inserts of about 5 Kbp and appear to be full-length clones corresponding to the 5 Kb mRNA. Two of the clones contain cDNA inserts of about 4 Kbp and appear to be full-length clones corresponding to the 4 Kb mRNA.

Restriction enzyme mapping of the two different sized inserts indicate that they share regions of sequence simi- larity in their 5'ends, but diverge in their 3'end sequences. DNA sequence analyses indicate that the smaller insert may result from alternative polyadenylation upstream of the polyadenylation site used in the larger insert.

Representative cDNA inserts for both size classes were subcloned into the plasmid vector pBluescript SK.

Linearization followed by in vitro transcription using T7 RNA polymerase produced cRNA transcripts. The cRNA transcripts were injected into Xenopus oocytes (150 ng/pl RNA; 50 nl/oocyte) for functional analysis. Following

incubation periods of 2-4 days, the oocytes were assayed for the presence of functional calcium receptors. Both clone types gave rise to functional calcium receptors as assessed by the stimulation of calcium-activated chloride currents upon addition of appropriate calcium receptor agonists. Known calcium receptor agonists, including NPS R-467 and NPS R-568 (see, Nemeth et al., PCT/US93/01642, International Publication Number WO 94/18959), activated the oocyte-expressed receptor at about the same concen- trations known to be effective for the native parathyroid cell receptor. Thus, both clones encode a functional, human parathyroid cell calcium receptor.

Plasmids were prepared by subcloning each size class of insert into pBluescript thereby producing pHuPCaR 5.2 and pHuCaR 4.0. The nucleic acid sequence, and amino acid sequence, of the inserts are shown in SEQ. ID. Nos. 1 and 2.

Several differences were observed between the nucleic acid sequences of the two cDNA inserts. Sequence analyses of the two cDNA inserts indicate the existence of at least two sequence variants differing in the 3'untranslated region and which may result from alternative polyadenyla- tion. In addition, sequence variation exists at the 5' end of the inserts. These distinct sequences correspond to untranslated regions and may have arisen due to alternative transcriptional initiation and/or splicing.

Three additional sites of sequence variation are observed within the coding regions of cDNA clones pHuPCaR5.2 and pHuPCaR4.0 (see SEQ. ID. NOs. 1 and 2) demonstrating that these cDNA clones encode distinct proteins. Sequence analysis of the human CaR gene indicates that the additional 30 base pairs of DNA in cDNA clone pHuPCaR5. 2, as compared to the pHuPCaR 4.0 cDNA clone, results from alternative mRNA splicing. The alternative mRNA splicing is predicted to insert 10 additional amino acids into the CaR polypeptide encoded by the pHuPCaR5.2 cDNA at a site between aa#536 and aa#537 in

polypeptide encoded by pHuPCaR4.0 cDNA. In addition, pHuPCaR4.0 encodes glutamine (Gln) at aa&num 925 and glycine (Gly) at position 990 whereas pHuPCaR5.2 encodes arg (Arg) at both equivalent positions. The human CaR gene encodes for Gln and Arg, respectively, at these positions. The difference between the pHuPCaR4.0 cDNA compared to human DNA appears to represent a true sequence polymorphism within the human population while the single base change in pHuPCaR5.2 probably reflects a mutation which occurred during its cloning. Both cDNAs encode functional calcium receptors as demonstrated by the ability of Xenopus oocytes injected with cRNA prepared from these cDNA clones to respond to 10 mM extracellular calcium as ascertained by Cl-conductance. However, it is possible that these two receptor isoforms are functionally and/or pharmaco- logically distinct.

Example 2: Selection of Stable Recombinant Cells Expressinc the Calcium Receptor Clonal cell lines that stably express the two human and the bovine calcium receptors have been isolated.

Calcium receptor cDNAs were subcloned in two different, commercially available expression vectors; pMSG (obtained from Pharmacia) and Cep4B (obtained from Invitrogen). The first vector contains the selectable marker gene for xanthine-guanine phosphoribosyltransferase (gpt) allowing stably transfected cells to overcome the blockade of the purine biosynthetic pathway imposed by addition of 2 ßg/ml aminopterin and 25 Hg/ml mycophenolic acid. The second vector encodes a gene conferring resistance to the anti- biotic hygromycin (used at 200 Hg/ml). HuPCaR 5.2 and HuPCaR 4.0 cDNAs (SEQ. ID. NOs. 1 and 2, respectively) were removed from the parent bluescript plasmid with Not I and Hind III restriction enzymes and then either ligated directly into Not I + Hind III digested Cep4B or treated with the klenow fragment of DNA polymerase prior to blunt- end ligation into Sma I digested pMSG.

The pMSG subclone containing the HuPCaR 5.2 insert was transfected into CHO cells as discussed above.

Selection has resulted in 20 resistant clones which are being characterized. The Cep4B subclone containing the HuPCaR 5.2 insert was transfected into HEK 293 cells as described above. Selection with hygromycin resulted in a pool of stable clones. Clones expressing the HuPCaR 4.0 receptor isoform were prepared similarly.

Cells obtained from the pool of hygromycin selected HEK 293 cells transfected with Cep4B containing the HuPCaR 5.2 insert were plated on collagen coated Aklar squares which had been placed into individual wells of 12-well tissue culture plates. Two to six days later, medium was removed and the cells washed with balanced salt solution and 1 ml of buffer containing 1 yM fura2-AM, 1 mM CaCl2 and 0. 1t BSA and 1 mM CaCl2. Measurements of fluorescence in response to calcium receptor agonists were performed at 37°C in a spectrofluorimeter using excitation and emission wavelengths of 340 and 510 nm, respectively. For signal calibration, Fmax was determined after addition of iono- mycin (40 yM) and the apparent Fmin was determined by addition of 0.3 M EGTA, 2.5 M Tris-HC1; pH 10. Robust increases in [Ca2+] i were observed in response to the addition of the following calcium receptor agonists: Ca2+ (10 mM), Mg2+ (20 mM) and NPS R-467. Control cells expressing functional substance K receptors did not respond to these calcimimetic compounds.

Additional clonal isolates of HEK 293 cells trans- fected with pHuPCaR4.0 sequence were obtained. These were tested for responsiveness to calcimimetics as described above except that the cells were tested while in suspension.

Example 3: Using Fura-2 Loaded Parathyroid cells To Measure to Calcium Receptor Activity This section describes procedures used to obtain parathyroid cells from calves and humans, and to use the parathyroid cells to measure calcium receptor activity.

Parathyroid glands were obtained from freshly slaughtered calves (12-15 weeks old) at a local abattoir and transported to the laboratory in ice-cold parathyroid cell buffer (PCB) which contains (mM): NaCl, 126; KC1,4; MgC12, 1; Na-HEPES, 20; pH 7.4 ; glucose, 5.6, and variable amounts of Cal,, e. g., 1.25 mM. Human parathyroid glands, were obtained from patients undergoing surgical removal of parathyroid tissue for primary or uremic hyperparathyroid- ism (uremic HPT), and were treated similarly to bovine tissue.

Glands were trimmed of excess fat and connective tissue and then minced with fine scissors into cubes approximately 2-3 mm on a side. Dissociated parathyroid cells were prepared by collagenase digestion and then purified by centrifugation in Percoll buffer. The resultant parathyroid cell preparation was essentially devoid of red blood cells, adipocytes, and capillary tissue as assessed by phase contrast microscopy and Sudan black B staining. Dissociated and purified parathyroid cells were present as small clusters containing 5 to 20 cells. Cellular viability, as indexed by exclusion of trypan blue or ethidium bromide, was routinely 95%.

Although cells can be used for experimental purposes at this point, physiological responses (e. g., suppressi- bility of PTH secretion and resting levels of [Ca2+] i) should be determined after culturing the cells overnight.

Primary culture also has the advantage that cells can be labeled with isotopes to near isotopic equilibrium, as is necessary for studies involving measurements of inositol phosphate metabolism.

After purification on Percoll gradients, cells were washed several times in a 1: 1 mixture of Ham's F12-

Dulbecco's modified Eagle's medium (GIBCO) supplemented with 50 yg/ml streptomycin, 100 U/ml penicillin, 5 Hg/ml gentamicin and VITS. ITS+ is a premixed solution con- taining insulin, transferrin, selenium, and bovine serum albumin (BSA)-linolenic acid (Collaborative Research, Bedford, MA). The cells were then transferred to plastic flasks (75 or 150 cm2; Falcon) and incubated overnight at 37°C in a humid atmosphere of 5% CO2. No serum is added to these overnight cultures, since its presence allows the cells to attach to the plastic, undergo proliferation, and dedifferentiate. Cells cultured under the above condi- tions were readily removed from the flasks by decanting, and show the same viability as freshly prepared cells.

Purified parathyroid cells were resuspended in 1.25 mM CaCl2-2o BSA-PCB containing 1 UM fura-2-acetoxymethyl- ester and incubated at 37°C for 20 minutes. The cells were then pelleted, resuspended in the same buffer, but lacking the ester, and incubated a further 15 minutes at 37°C. The cells were subsequently washed twice with PCB containing 0.5 mM CaCl2 and 0.5% BSA and maintained at room temperature (about 20°C). Immediately before use, the cells were diluted five-fold with prewarmed 0.5 mM CaCl2- PCB to obtain a final BSA concentration of 0.1%. The con- centration of cells in the cuvette used for fluorescence recording was 1-2 x 106/ml.

The fluorescence of indicator-loaded cells was measured at 37oC in a spectrofluorimeter (Biomedical Instrumentation Group, University of Pennsylvania, Philadelphia, PA) equipped with a thermostated cuvette holder and magnetic stirrer using excitation and emission wavelengths of 340 and 510 nm, respectively. This fluorescence indicates the level of cytosolic Ca2+.

Fluorescence signals were calibrated using digitonin (50 g/ml, final) to obtain maximum fluorescence (FmaX), and EGTA (10 mM, pH 8.3, final) to obtain minimal fluorescence (Fmin), and a dissociation constant of 224 nM. Leakage of dye is dependent on temperature and most occurs within the

first 2 minutes after warming the cells in the cuvette.

Dye leakage increases only very slowly thereafter. To correct the calibration for dye leakage, cells were placed in the cuvette and stirred at 37°C for 2-3 minutes. The cell suspension was then removed, the cells pelleted, and the supernatant returned to a clean cuvette. The super- natant was then treated with digitonin and EGTA to esti- mate dye leakage, which is typically 10-15% of the total Ca2+-dependent fluorescent signal. This estimate was subtracted from the apparent Fmin.

Example 4: Usinq Fura-2 Loaded HEK 293/pHuPCaR4.0 Cells To Measure to Calcium Receptor Activity This section describes procedures used to assay calcium receptor activity using fura-2 loaded HEK 293/pHuPCaR4.0 cells. HEK 293 cells transfected with pHuPCaR4.0 were loaded with fura-2 by incubating the cells in Dulbecco's modified Eagle's media buffered with 20 mM HEPES containing about 5 UM fluo-3/AM for one hour at room temperature. Cell were then rinsed with Hank's balanced salt solution buffered with 20 mM HEPES containing 1 mM CaCl2 and 1 mM MgCl2. Compounds to be tested were then added to the cells and fluorescence was measured (excitation and emission wavelengths of 340 and 510 nm, respectively).

Example 5: Measurinq the Ability of Compounds to Modulate Calcium Receptor Activity The ability of different compounds to modulate cal- cium receptor activity was assayed by measuring increases in [Ca2+] in HEK 293 cells transfected with nucleic acid encoding pHuPCaR4.0 using fura-2 loaded cells or using parathyroid cells loaded with using fura-2 loaded cells.

Results of different experiments are summarized in Tables l. a, l. b. 1, l. b. 2, l. c., and 2. Tables l. a, l. b. 1, l. b. 2, and l. c summarizes the effects of compounds, at different concentrations, on calcium receptor activity assayed as

described in Example 4 (i. e., using HEK 293 cells trans- fected with nucleic acid encoding pHuPCaR4.0, which were loaded with fura-2).

Table 2, summarizes the results of different experi- ments where the ECso was calculated either parathyroid cells, or HEK 293/pHuPCaR4.0, loaded with fura-2. Cells were loaded with fura-2 and assayed as described in Example 2 (for parathyroid cells) or Example 3 (for HEK 293/pHuPCaR4.0 cells).

Table l. a. Calcimimetic compounds which-produce crreater than 40% response at 3.3 ng/mL in HEK-293 cells ex-pressing the human calcium receptor.

Compound % activity Code at four concentrations (ng/mL) 3300 330 33 3.3 Reference compounds R-568 95 69 24 17P 101 86 54 17X 105 93 51 24X 126 109 124 109 24Y 119 120 127 102 17J 116 118 122 102 25A 122 120 114 92 17E 116 110 110 92 24Z 138 138 135 90 14S 116 106 105 88 25E 132 129 122 85 17G 125 128 119 77 14T 126 125 117 77 17H 126 124 111 74 140 119 119 102 74 251 119 113 114 74 12J 131 130 113 68

Compound % activity Code at four concentrations (ng/mL) 3300 330 33 3. 3 <BR> <BR> <BR> <BR> <BR> 12I 115 111 93 68<BR> <BR> <BR> <BR> <BR> <BR> 25G 130 115 99 66 9R 108 101 64 <BR> <BR> <BR> 12F 118 110 101 63<BR> <BR> <BR> <BR> <BR> <BR> <BR> 120 110 117 94 62<BR> <BR> <BR> <BR> <BR> <BR> 23Z 129 126 100 61<BR> <BR> <BR> <BR> <BR> <BR> 17M 115 99 59<BR> <BR> <BR> <BR> <BR> <BR> <BR> 16V 114 102 58<BR> <BR> <BR> <BR> <BR> <BR> <BR> 250 126 115 96 57<BR> <BR> <BR> <BR> <BR> <BR> 25J 119 123 105 56<BR> <BR> <BR> <BR> <BR> <BR> <BR> 16L 146 138 98 56<BR> <BR> <BR> <BR> <BR> <BR> 12N 115 106 102 55<BR> <BR> <BR> <BR> <BR> <BR> <BR> 16T 97 88 55<BR> <BR> <BR> <BR> <BR> <BR> 25U 107 107 95 55<BR> <BR> <BR> <BR> <BR> <BR> <BR> 17P 101 86 54<BR> <BR> <BR> <BR> <BR> <BR> 16Q 110 88 53<BR> <BR> <BR> <BR> <BR> <BR> <BR> 23E 137 113 102 53<BR> <BR> <BR> <BR> <BR> <BR> 17C 113 120 99 52<BR> <BR> <BR> <BR> <BR> <BR> 25L 97 97 85 52<BR> <BR> <BR> <BR> <BR> <BR> <BR> 8Z 101 97 52<BR> <BR> <BR> <BR> <BR> <BR> 17X 105 93 51<BR> <BR> <BR> <BR> <BR> <BR> <BR> 13R 132 98 51<BR> <BR> <BR> <BR> <BR> <BR> 170 112 96 51<BR> <BR> <BR> <BR> <BR> <BR> <BR> 23Q 122 114 98 51<BR> <BR> <BR> <BR> <BR> <BR> 16X 111 96 51<BR> <BR> <BR> <BR> <BR> <BR> <BR> 24V 127 98 71 50<BR> <BR> <BR> <BR> <BR> <BR> 130 115 94 50<BR> <BR> <BR> <BR> <BR> <BR> <BR> 17N 108 86 49<BR> <BR> <BR> <BR> <BR> <BR> 21V 122 116 99 48<BR> <BR> <BR> <BR> <BR> <BR> <BR> 24M 132 134 99 48<BR> <BR> <BR> <BR> <BR> <BR> 13U 108 79 47

Compound % activity Code at four concentrations (ng/mL) 3300 330 33 3. 3 24P 140 138 110 46 17Y 109 94 79 46 11X 100 76 45 25H 115 107 89 45 22J 99 71 45 9C 104 82 45 13S 102 87 45 10Q 103 100 84 44 13P 110 83 44 8K 98 81 44 13N 114 88 43 10N 106 97 77 43 12H 114 115 94 43 25P 90 81 75 41 18A 111 88 40 14L 109 78 40 Table l. b. l. Calcimimetic compounds which produce greater than 40% response at 33 nq/mL in HEK-293 cells expressing- the human calcium receptor Compound activity Code at four concentrations (ng/mL) 3300 330 33 3. 3 Reference compounds R-568 95 69 24 17P 101 86 54 17X 105 93 51 12C 134 125 98 39 161 121 117 96 36

Compound %activity Code at four concentrations (ng/mL) 3300 330 33 3. 3 17D 108 91 38 <BR> <BR> <BR> 17F 111 90 28<BR> <BR> <BR> <BR> 24C 116 113 87 32<BR> <BR> <BR> <BR> 25K 124 107 86 35<BR> <BR> <BR> <BR> 13F 125 122 85 38<BR> <BR> <BR> <BR> 21F 109 85 36<BR> <BR> <BR> <BR> 21S 132 131 85 34<BR> <BR> <BR> <BR> 1OF 96 84 27<BR> <BR> <BR> <BR> 14R 106 107 84 37<BR> <BR> <BR> <BR> 13G 111 128 82 29 14Z 118 103 82 20 <BR> <BR> <BR> 16N 122 159 82 a 8U 123 129 82 11 <BR> <BR> <BR> 23W 117 97 81 25 12G 139 139 81 35 <BR> <BR> <BR> 15G 113 80 32<BR> <BR> <BR> <BR> 25M 118 100 79 25<BR> <BR> <BR> <BR> 13V 110 79 33<BR> <BR> <BR> <BR> 14P 112 103 78 30<BR> <BR> <BR> <BR> 6T 123 129 78 15<BR> <BR> <BR> <BR> 14Q 101 78 35 17L 111 104 78 31 <BR> <BR> <BR> 24K 106 78 30<BR> <BR> <BR> <BR> 24U 106 106 78 25<BR> <BR> <BR> <BR> 25Q 116 95 77 20 8J 104 77 39 23H 121 114 77 28 21C=4U 134 114 76 17 <BR> <BR> <BR> 25F 97 85 76 28 16R 100 76 25 17I 118 97 76 18

Compound I activity Code at four concentrations (ng/mL) 3300 330 33 3. 3 <BR> <BR> <BR> <BR> <BR> 24J 103 75 31<BR> <BR> <BR> <BR> <BR> <BR> <BR> 210 109 75 37<BR> <BR> <BR> <BR> <BR> <BR> <BR> 24G 109 94 75 22<BR> <BR> <BR> <BR> <BR> <BR> <BR> 15I 111 93 75 24<BR> <BR> <BR> <BR> <BR> <BR> <BR> 21D 104 75 17<BR> <BR> <BR> <BR> <BR> <BR> <BR> 20Y 117 95 74 24<BR> <BR> <BR> <BR> <BR> <BR> <BR> lOP 102 74 8<BR> <BR> <BR> <BR> <BR> <BR> <BR> 23M 113 97 74 26<BR> <BR> <BR> <BR> <BR> <BR> <BR> 14Y 109 73 17<BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> 17K 98 97 73 37<BR> <BR> <BR> <BR> <BR> <BR> <BR> 12E 117 121 73 23<BR> <BR> <BR> <BR> <BR> <BR> <BR> 17Z 99 73 37<BR> <BR> <BR> <BR> <BR> <BR> <BR> 16W 102 73 4 23K 106 107 72 24 <BR> <BR> <BR> <BR> <BR> 25X 96 94 72 22<BR> <BR> <BR> <BR> <BR> <BR> <BR> 13W 109 71 12<BR> <BR> <BR> <BR> <BR> <BR> <BR> 23P 125 99 70 22<BR> <BR> <BR> <BR> <BR> <BR> <BR> 18B 111 96 69 26 21Y 100 68 36 <BR> <BR> <BR> <BR> <BR> 17W 92 67 13 23A 103 67 24 <BR> <BR> <BR> <BR> <BR> 23G 127 93 67 13<BR> <BR> <BR> <BR> <BR> <BR> <BR> 13M 92 66 15<BR> <BR> <BR> <BR> <BR> <BR> <BR> 21U 104 104 66 18<BR> <BR> <BR> <BR> <BR> <BR> <BR> 21R 100 66 15 10S/10T 86 65 13 <BR> <BR> <BR> <BR> 17R 98 65 13<BR> <BR> <BR> <BR> <BR> <BR> <BR> 13X 102 65 13<BR> <BR> <BR> <BR> <BR> <BR> <BR> 4N 100 65 13<BR> <BR> <BR> <BR> <BR> <BR> <BR> 21E 94 64 4<BR> <BR> <BR> <BR> <BR> <BR> <BR> 15J 80 75 64 13

compound'-. activity Code at four concentrations (ng/mL) 3300 330 33 3. 3 22Y 114 64 28 21G 88 63 18 24L 105 62 10 10V 99 62 8 l0W/lOX 98 61 9 17B 92 61 19 23Y 106 87 61 16 11Y 103 61 20 Table l. b. 2 Calcimimetic compounds which produce greater than 40k response at 33 ng/mL in HEK-293 cells expressing the human calcium receptor Compound Code activity at four concentrations (ng/mL) 3300 330 33 3.3 reference compounds R568 95 69 24 17P 101 86 54 17X 105 93 51 18C 99 87 60 18 23T 102 74 60 31 4V 93 59 8G 84 59 6 231 102 58 3 21M 102 58 17 240 137 114 58 8 3U 89 57 9A 82 56 6 12M 98 86 56 11 12B 130 110 56 4

Compound Code % activity at four concentrations (ng/mL) 3300 330 33 3. 3 <BR> <BR> <BR> <BR> <BR> 21P 92 56 13<BR> <BR> <BR> <BR> <BR> <BR> 8T 85 55 13 10L/10M 99 55 4 24I 109 84 55 11 <BR> <BR> <BR> <BR> 14N 89 55 15<BR> <BR> <BR> <BR> <BR> <BR> 23R 104 86 54 13<BR> <BR> <BR> <BR> <BR> <BR> 23S 97 53 3<BR> <BR> <BR> <BR> <BR> <BR> 21T 133 112 53 3<BR> <BR> <BR> <BR> <BR> <BR> lOW/lOX 81 53 4 13% 90 53 6 <BR> <BR> <BR> <BR> <BR> 6R 94 52 7<BR> <BR> <BR> <BR> <BR> <BR> 20I 87 52 12<BR> <BR> <BR> <BR> <BR> <BR> 24A 122 85 52 9 12D 128 109 52 5 <BR> <BR> <BR> <BR> 6X 84 52 10<BR> <BR> <BR> <BR> <BR> <BR> 18T 99 74 52 14<BR> <BR> <BR> <BR> <BR> <BR> 21X 119 101 51 2<BR> <BR> <BR> <BR> <BR> <BR> 23J 102 61 si 29<BR> <BR> <BR> <BR> <BR> <BR> lOZ 96 51 5<BR> <BR> <BR> <BR> <BR> <BR> 16Z 88 51 9 23N 96 50 2 <BR> <BR> <BR> <BR> <BR> 16U 85 50 4 11D 96 50 4 <BR> <BR> <BR> 23X 94 49 1<BR> <BR> <BR> <BR> <BR> <BR> 17A 88 49 7<BR> <BR> <BR> <BR> <BR> <BR> 20J 80 48 8<BR> <BR> <BR> <BR> <BR> <BR> 22X 86 48 10<BR> <BR> <BR> <BR> <BR> <BR> 23U 87 48 3<BR> <BR> <BR> <BR> <BR> <BR> 9Z 74 48 4<BR> <BR> <BR> <BR> <BR> <BR> 16J 92 76 47 31<BR> <BR> <BR> <BR> <BR> <BR> 25N 94 73 46 8<BR> <BR> <BR> <BR> <BR> <BR> 4P 81 46 8

Compound Code % activity at four concentrations (ng/mL) 3300 330 33 3.3 230 111 79 46 13 13Q 95 46 5 4G 83 46 12Y 80 46 10 12L 88 45 10 23F 82 45 5 11W 81 44 2 8H 88 44 7 25V 89 59 43 26 25W 95 69 42 8 1OR 82 42 7 21N 124 98 42 4 8S 73 42 7 8X 75 40 19 13E 123 94 40 2 Table l. c. Calcimimetic compounds which produce greater than 40k response at 330 nq/mL in HEK-293 cells expressing the human calcium receptor Compound Code activity at four concentrations (ng/mL) 3300 330 33 3.3 reference compounds R568 95 69 24 17P 101 86 54 17X 105 93 51 7X 85 3H 84 3L 81 28 160 129 81 21 2 80/8Q 124 80 14 0

Compound Code % activity at four concentrations (ng/mL) 3300 330 33 3.3 14A 98 78 10 7 23L 107 77 37 9 1T 76 7W 76 4H 77 37 8D 75 5M 73 21 4U 72 24E 94 71 35 6 16M 130 68 11 4 4M 68 34 2S 67 29 17V 91 66 27-1 2X 66 15 23D 91 66 35 13 4P 65 32 5B/5C 65 20 3M 64 19 16K 78 62 36 8 5D 62 18 4D 61 13 24B 76 61 34 11 24H 81 60 32 13 5L 60 16 2Y 59 10 5G 58 16 3V 56 14 2Q 56 4 14B 75 55 11 4 13Z 93 54 22 5 8A 54 24D 87 53 34 39

Compound Code % activity at four concentrations (ng/mL) 3300 330 33 3.3 1D 53 131 85 52 3 1 3B 52 15 8C 51 14H 112 49 5 5 7U 49 5E 48 7 13H 88 48 36 12 13Y 106 47 2 4 4J 47 8 141 80 45 11 7 4B 45 8 3D 45 4 3R 45 2 3A 41 7 14J 55 41 6 5 41 40 9 TABLE 2 Arylalkylamine Calcimimetics from Figure 1 Active at the Parathyroid Cell Calcium Receptor In Vitro (ECso < 5 yM) CompoundCode EC50 Compound Code EC50 (from Fig. 1) (M) (from Fig. 1) | (yM) NPSR-467 2.0 11X 0.83 NPSR-568 0.60 11Y 2.8 3U 0.64 12L 1.7 3V 1.8 12U 1.2 4A 1.4 12V 0.42 4B 2.0 12W 3.2

4C 2. 0 12Y 2. 0 4D 4. 4 12Z 0. 11 4G 1. 8 13Q ca. 0.8 4H >3. 0 13R 0. 25 4J 2. 2 13S <0. 13 4M 2. 1 13U 0. 19 4N 0. 8 13X <0. 75 4P 1. 6 14L 0. 26 4R/6V 4.2 14Q 0.47 4S 3. 3 14U 0. 13 4T/4U 1. 6 14V 1. 7 4V 2. 5 14Y 0. 38 4W 2. 3 15G ca. 0.5 4Y 1. 3 16Q 0. 04 4Z/5A 4. 4 16R 0. 36 5B/5C 2. 8 16T 0. 04 5W/5Y 3.6 16V <0.13 6E 2.7 16W 0.59 6F (R, R-) 0. 83 16X 0. 10 6R 3. 4 17M 0. 15 6T 2. 9 170 0. 04 6X 2. 5 17P 0. 04 7W 3. 2 17R 0. 39 7X 1. 1 17W 0. 43 8D 2.5 17X 0.02 8J 0. 78 20F <1. 0 8K1. 3201>1. 0 8R 2. 6 20J >3. 0 8S 1. 7 20R 2. 4 8T 1. 8 20S 4. 2 8U 0. 44 21D 3. 0 8X 0.76 21F 0.38 8Z 0.40 21G 1.1

9C 0. 60 210 0. 26 9D 1.4 21P 0.43 9R 0.25 21Q 1.4 9S 4. 8 21R 0. 37 1OF 0. 89 25C > 2 11D1. 825D0. 019 Examples 6-17: Synthesis of Compounds The compounds described herein can be synthesized using standard techniques such as those described by Nemeth et al., PCT/US93/01642, International Publication Number WO 94/18959. Examples describing representative syntheses of compounds described in the text are provided below.

Synthesis of compounds 9R, 14U, and 17P were prepared by reductive amination of a commercially available aldehyde or ketone with a primary amine in the presence of sodium cyanoborohydride or sodium triacetoxyborohydride.

Compounds 11Y, 12H, 12K, 12M, 14S, 14T, 16L-O, 17E, 17G, 17J, 24X, 24Y, 25A, 25E-25K, and 250 were prepared in a similar manner.

It was found for the syntheses of these three compounds (9R, 14U, and 16P) that sodium triacetoxyboro- hydride afforded the desired diastereoisomers with greater diastereoselectivity than using sodium cyanoborohydride.

The enriched mixtures were further purified to a single diastereomer by normal-phase HPLC or by recystallization from organic solvents.

Compounds 8J, 8U, 11X, 17M, and 25Y were prepared from the condensation of a primary amine with an aldehyde or ketone in the presence of titanium (IV) isopropoxide.

The resulting intermediate imines were then reduced in situ by the action of sodium cyanoborohydride, sodium borohydride, or sodium triacetoxyborohydride. The intermediate enamine for the synthesis of compound 8U was

catalytically reduced using or palladium dihydroxide on carbon.

Compounds 12U, 12V and 12Z were prepared by a diisobutylaluminum hydride (DIBAL-H) mediated condensation of an amine with a nitrile. The resulting intermediate imine is reduced in situ by the action of sodium cyano- borohydride or sodium borohydride. The intermediate alkenes (compounds 12U and 12V) were reduced by catalytic hydrogenation in EtOH using palladium on carbon.

Compounds which were converted to their corresponding hydrochloride were done so by treatment of the free base with ethereal HCl to afford white solids.

The amines in these syntheses were purchased from Aldrich Chemical Co., Milwaukee, WI, or from Celgene Corp., Warren, NJ, or were prepared synthetically using standard techniques. All other reagent chemicals were purchased from Aldrich Chemical Co.

Example 6: Synthesis of Compound 25Y N- (3- (2-Phenyl) propyl)-1- (1-naphthyl) ethylamine A mixture of 3-phenyl-1-propylamine (135 mg, 1 mmol), 1'-acetonaphthone (170 mg, 1 mmol), and titanium (IV) iso- propoxide (355 mg, 1.3 mmol) was stirred at room tempera- ture for 1 hour. The reaction was treated with 1 M ethanolic sodium cyanoborohydride (1 mL) and stirred at room temperature for 16 hours. The reaction was diluted with ether and treated with water (0.1 mL). The reaction was centrifuged and the ether layer removed and concen- trated to a milky oil. A small portion of this material (10 mg) was purified by HPLC (Phenomenex, 1.0 x 25 cm, 5 yM silica) using a gradient of dichloromethane to 10% methanol in dichloromethane containing 0.1% isopropyl- amine. This afforded the product (free base) as a single component by GC/El-MS (Rt= 10.48 min) m/z (rel. int.) 289 (M+, 11), 274 (63), 184 (5), 162 (5), 155 (100), 141 (18), 115 (8), 91 (45), 77 (5).-

Example 7: Synthesis of Compound 8J <BR> <BR> <BR> <BR> N- (3-phenylpropyl)-1- (3-thiomethylphenyl) ethylamine hydrochloride 3'-Aminoacetophenone (2.7 g, 20 mmol) was dissolved in 4 mL of concentrated HC1, 4 g of ice and 8 mL of water.

The solution was cooled to 0°C, and sodium nitrite (1.45 g, 21 mmol) dissolved in 3-5 mL of water was added over 5 minutes while maintaining the temperature below 6°C.

Sodium thiomethoxide (1.75 g, 25 mmol) was dissolved in 5 mL of water and cooled to O°C. To this solution was added the diazonium salt over 10 minutes while maintaining the temperature below 10°C. The reaction was stirred for an additional hour while allowing the temperature to rise to ambient. The reaction mixture was partitioned between ether and water. The ether layer was separated and washed with sodium bicarbonate and sodium chloride, and dried over sodium sulfate. The ether was evaporated to give a 74% yield of 3'-thiomethylacetophenone. The crude material was purified by distillation at reduced pressure.

3-Phenylpropylamine (0.13 g, 1 mmol), 3'- thiomethylacetophenone (0.17 g, 1 mmol), and titanium (IV) isopropoxide (0.36 g, 1.25 mmol) were mixed together and allowed to stand for 4 hours. Ethanol (1 mL) and sodium cyanoborohydride (0.063 g, 1 mmol) were added and the reaction was stirred overnight. The reaction was worked up by the addition of 4 mL of ether and 200 yL of water.

The mixture was vortexed and then spun in a centrifuge to separate the solids. The ether layer was separated from the precipitate, and the solvent removed in vacuo. The oil was redissolved in dichloromethane and the compound purified by preparative TLC on silica gel eluted with 3% methanol/dichloromethane to yield the title compound as a pure oil: GC/EI-MS (Rt=7. 64 min) m/z (rel. int.) 285 (M+, 18), 270 (90), 180 (17), 151 (100), 136 (32), 104 (17), 91 (54), 77 (13).

Example 8: Synthesis of Compound 8U N-3- (2-me thoxyphenyl)-1-propyl- (R)-3-me thoxy-a- methylbenzylamine hydrochloride A mixture of (R)- (+)-3-methoxy-a-methylbenzylamine (3.02 g, 20 mmol), 2-methoxycinnamaldehyde (3.24 g, 20 mmol), and titanium (IV) isopropoxide (8.53 g, 30 mmol, 1.5 Eq.) was stirred 2 hours at room temperature and treated with 1 M (20 mL) ethanolic sodium cyanoboro- hydride. The reaction was stirred overnight (16 hours), diluted with diethylether, and treated with water (1.44 mL, 80 mmol, 4 Eq.). After mixing for 1 hour the reaction mixture was centrifuged and the ether layer removed and concentrated to an oil. This material was dissolved in glacial acetic acid, shaken with palladium hydroxide and hydrogenated under 60 p. s. i. hydrogen for 2 hours at room temperature. The catalyst was removed by filtration and the resulting solution concentrated to a thick oil. This material was dissolved in dichloromethane and neutralized with 1 N NaOH. The dichloromethane solution was separated from the aqueous phase, dried over anhydrous potassium carbonate and concentrated to an oil. This material was dissolved in ether and treated with 1 M HCl in diethyl- ether. The resulting precipitate (white solid) was collected, washed with diethylether, and air dried.

GC/El-MS (Rt = 9. 69 min) of this material (free base) showed a single component: m/z (rel. int.) 299 (M+, 21), 284 (100), 164 (17), 150 (8), 135 (81), 121 (40), 102 (17), 91 (43), 77 (18).

Example 9: Synthesis of Compound 9R <BR> <BR> <BR> (R)-N- (1- (2-naphthyl) ethyl)- (R)-1- (1-naphthyl) ethylamine hydrochloride A mixture of (R)- (+)-1- (1-naphthyl) ethylamine (10.0 g, 58 mmol), 2'-acetonaphthone (9.4 g, 56 mmol), titanium (IV) isopropoxide (20.7 g, 73.0 mmol), and EtOH (abs.) (100 mL) was heated to 60°C for 3 hours. Sodium cyano- borohydride (NaCNBH3) (3.67 g, 58.4 mmol) was then added.

The reaction mixture was stirred at room temperature for 18 hours. Ether (1 L) and H20 (10 mL) were added to the reaction mixture and the resulting precipitate was then removed by centrifugation. The supernatant was evaporated under vacuum and the crude product was recrystallized four times from hot hexane, to provide 1. 5 g of pure (98+W) diastereomer. The free base was dissolved in hexane, filtered, and then ethereal HC1 was added to precipitate the product as a white solid (1.1 g, 6 W yield), m. p. : softens 200-240°C (dec.).

Example 10: Synthesis of Compound 11X <BR> <BR> <BR> N- (4-Isopropylbenzyl)- (R)-1- (1-naphthyl) ethylamine hydrochloride A mixture of (R)- (+)-l- (l-naphthyl) ethylamine (1.06 g, 6.2 mmol), 4-isopropylbenzaldehyde (0.92 g, 6.2 mmol), and titanium (IV) isopropoxide (2.2 g, 7.7 mmol) was heated to 100°C for 5 min then allowed to stir at room temperature for 4 hours. Sodium cyanoborohydride (NaCNBH3) (0.39 g, 6.2 mmol) was then added followed by EtOH (1 mL).

The reaction mixture was stirred at room temperature for 18 hours. Ether (100 mL) and H20 (1 mL) were added to the reaction mixture and the resulting precipitate was then removed by centrifugation. The supernatant was evaporated under vacuum and the crude product was chromatographed on silica gel (50 mm X 30 cm column) (elution with li MeOH/ CHCl3). The chromatographed material was then dissolved in hexane and ethereal HC1 was added to precipitate the product as a white solid (0.67 g, 35 % yield), m. p.; 257- 259°C.

Example 11: Synthesis of Compound 12U N-3- (2-methylphenyl)-1-propyl- (R)-3-methoxy-a- methylbenzylamine hydrochloride A solution of 2-methylcinnamonitrile (1.43 g, 10 mmol) in dichloromethane (10 mL) was cooled to 0°C and treated dropwise (15 minutes) with 1 M diisobutylaluminum

hydride (10 mL, dichloromethane). The reaction was stirred at 0°C for 15 minutes and treated dropwise (15 minutes) with a 1 M solution of (R)- (+)-3-methoxy-a- methylbenzylamine (1.51 g, 10 mmol) in dichloromethane (10 mL). The reaction was stirred 1 hours at 0°C and poured into a solution of ethanol (100 mL) containing sodium cyanoborohydride (1 g, 16 mmol). The reaction mixture was stirred 48 hour at room temperature. The reaction was diluted with ether and neutralized with 1 N NaOH. The ether layer was removed, dried over anhydrous potassium. carbonate and concentrated to an oil. This material was chromatographed through silica using a gradient of dichloromethane to 5% methanol in dichloromethane to afford the unsaturated intermediate, a single component by GC/El-MS (Rt=10. 06 min) m/z (rel. int.) 281 (M+, 17), 266 (59), 176 (19), 146 (65), 135 (73), 131 (100), 91 (21), 77 (13).

The unsaturated intermediate in ethanol was hydrogenated (1 atm H2) in the presence of palladium on carbon for 16 hours at room temperature. The product from this reaction was converted to the hydrochloride salt by treatment with 1 M HC1 in diethylether. GC/El-MS (Rt = 9.31 min) of this material (free base) showed a single component: m/z (rel. int.) 283 (M+, 21), 268 (100), 164 (12), 148 (8), 135 (85), 121 (12), 105 (49), 91 (23), 77 (21).

Example 12: Synthesis of Compound 12V N-3- (3-methylphenyl)-1-propyl- (R)-3-methoxy-a- methylbenzylamine hydrochloride The compound was prepared following the procedure <BR> <BR> <BR> described in Example 11, but using 2-methylcinnamonitrile. * The unsaturated intermediate was a single component by GC/EI-MS (Rt = 10. 21 min) m/z (rel. int.) 281 (M+, 57), 266 (86), 146 (98), 135 (88), 131 (100), 115 (43), 102 (26), 91 (43), 77 (18). Reduction of this material and hydro- chloride formation using the procedure described Example

11 afforded the product. GC/EI-MS (Rt = 9.18 min) of this material (free base) showed a single component; m/z (rel. int.) 283 (M+, 19), 268 (100), 164 (11), 148 (8), 135 (76), 121 (16), 105 (45), 91 (23), 77 (21).

Example 13: Synthesis of Compound 12Z N-3- (2-chlorophenyl)-1-propyl- (R)-1- (1-naphthyl) ethylamine hydrochloride The compound was prepared following the procedures described in Example 11, but using 2-chlorohydrocinnamo- nitrile and (R)- (+)-1- (1-naphthyl) ethylamine on a 10 mmol scale. Chromatography through silica using a gradient of dichloromethane to 5% methanol in dichloromethane afforded the product as a single component by TLC analysis (5% methanol in dichloromethane). The hydrochloride was prepared by treatment with 1 M HC1 in diethylether.

Example 14: Synthesis of Compound 14U <BR> <BR> <BR> <BR> (R)-N- (1- (4-m e thoxyphenyl) e thyl)- (R)-1- (1- naphthyl) ethylamine hydrochloride A mixture of (R)- (+)-1- (1-naphthyl) ethylamine (1.1 g, 6.2 mmol), 4'-methoxyacetophenone (0.93 g, 6.2 mmol), titanium (IV) isopropoxide (2.2 g, 7.7 mmol), and EtOH (abs.) (1 mL) was heated to 60°C for 3 hours. Sodium cyanoborohydride (NaCNBH3) (0.39 g, 6.2 mmol) was then added, and the reaction mixture was stirred at room temperature for 18 hours. Ether (200 mL) and H2O (2 mL) were added to the reaction mixture and the resulting precipitate was then removed by centrifugation. The supernatant was evaporated under vacuum and the crude product was chromatographed on silica gel (25 mm X 25 cm column) (elution with 1% MeOH/CHCl3). A portion of this material was HPLC chromatographed [Selectosil, 5 UM silica gel; 25 cm x 10.0 mm (Phenomenex, Torrance, CA), 4 mL per minute; W det. 275 nM; 12% ethyl acetate-88% hexane (elution time 12.0 min)]. The HPLC purified diastereomer was then dissolved in hexanes and ethereal HC1 was added

to precipitate the product as a white solid (20 mg), m. p. : 209-210°C (dec.).

Example 15: Synthesis of Compound 17M <BR> <BR> <BR> N-(3-chloro-4-methoxybenzyl)-(R)-1-(1-naphthyl) ethylamine hydrochloride A mixture of (R)- (+)-1- (1-naphthyl) ethylamine (6.6 g, 39 mmol), 3'-chloro-4'-methoxybenzaldehyde (6.6 g, 39 mmol), and titanium (IV) isopropoxide (13.8 g, 48.8 mmol), and EtOH (abs.) (30 mL) was heated to 80°C for 30 minutes then allowed to stir at room temperature for 3 hours.

Sodium cyanoborohydride (NaCNBH3) (2.45 g, 39 mmol) was then added. The reaction mixture was stirred at room temperature for 18 hours. Ether (100 mL) and H20 (2 mL) were added to the reaction mixture and the resulting precipitate was-then removed by centrifugation. The supernatant was evaporated under vacuum and the crude product was chromatographed on silica gel (50 mm X 30 cm column) (elution with CH2Cl2). The chromatographed material was then dissolved in hexane (500 mL), decolor- ized with Norits filtered (0.2 yM), and then ethereal HC1 was added to precipitate the product as a while solid (10.2 g, 56 W yield), m. p. : 241-242°C (dec.).

Example 16: Synthesis of Compound 17P 4-Methoxy-3-methylacetophenone [17P Precursor] A mixture of 4'-hydroxy-3'-methylacetophenone (5.0 g, 33.3 mmol), iodomethane (5.7 g, 40.0 mmol), K2CO3 (granular, anhydrous) (23.0 g, 167 mmol), and acetone (250 mL) was refluxed for 3 hours. The reaction mixture was then cooled to room temperature, filtered to remove the inorganic salts, and evaporated under vacuum. The crude product was dissolved in ether (100 mL) and washed with H20 (2 x 20 mL). The organic layer was dried (Na2SO4) and evaporated to yield 4.5 g, 82.4% yield. The ketone was used in the following reaction without further purification.

(R)-N-(l-(4-Methoxy-3-methylphenyl) ethyl)-(R)-l-( naphthyl) ethylamine hydrochloride [Compound 17P] A mixture of (R)- (+)-l- (l-naphthyl) ethylamine (4.24 g, 24.8 mmol), 4'-methoxy-3'-methylacetophenone (4.06 g, 24.8 mmol), and titanium (IV) isopropoxide (8.8 g, 30.9 mmol), and EtOH (abs.) (1 mL) was heated to 100°C for 2 hours. Isopropanol (45 mL) was added and the reaction was then cooled to 10°C in an ice bath. Sodium triacetoxy- borohydride (NaHB (O2CCH3) 3) (10.5 g, 49.5 mmol) was then added in portions over 15 minutes. The reaction mixture was then heated to 70°C for 18 hourse The mixture was cooled to room temperature and poured into ether (400 mL).

The suspension was centrifuged, the supernatant was collected and the pellet was washed with ether (400 mL).

The combined organic washings were evaporated under vacuum. The residue was dissolved in ether (400 mL) and washed with 1 N NaOH (4 x 50 mL) and H20 (2 x 50 mL). The organic layer was dried (Na2SO4), filtered and evaporated under vacuum. EtOH (abs.) was added to the wet residue which was then dried thoroughly on a rotary evaporator to provide an oil. The mixture was then chromatographed on silica gel (50 mm x 30 cm) [elution with (1% MeOH: li IPA: CHC13) to give 4.8 g of an oil].

The desired diastereomer was further purified by HPLC chromatography [SUPELCOSIL'"PLC-Si, 18/iM silica gel; 25 cm x 21.2 mm (Supelco, Inc., Bellefonte, PA), 7 mL per minute; UV det. 275 nM: 20% EtOAc-80% hexane (elution time 9.5-11. 0 min)]. Injections (800 UL aliquots) of the mixture (100 mg/mL solution in eluent) provided 65 mg of the desired isomer. Multiple HPLC injections provided 1.0 g of purified material. The HPLC chromatographed material was dissolved in hexane (50 mL) and the hydrochloride salt was precipitated with ethereal HC1. The salt was collected on fritted glass and washed with hexane to provide 1.0 g of a white solid, mp 204-205°C.

Example 17: Svnthesis of Compound 17X 3-Chloro-4-methoxybenzaldehyde A mixture of 3-chloro-4-hydroxybenzaldehyde (25 g, 160 mmol), iodomethane (27.25 g, 192 mmol), K2CO3 (granu- lar, anhydrous) (110.6 g, 800 mmol), and acetone (300 mL) was refluxed for 3 hours. The reaction mixture was then cooled to room temperature. Diethyl ether (500 mL) was added and the mixture was filtered through paper to remove the inorganic solids. The filtrate was evaporated under reduced pressure, dissolved in diethyl ether (800 mL), and washed with 0.1 N NaOH (3 x 100 mL). The organic layer was dried (Na2SO4) and evaporated under vacuum to yield 24 g, 92% yield of crude product. This material was further purified by chromatography on silica gel (50 mm x 30 cm) (elution with hexane-EtOAc, 5: 1) to give 15.02 g, 56% yield of a white solid: TLC (hexane-EtOAc, 5: 1) Rf=0. 24; GC Rt=4. 75 min; MS (EI) m/z 170 (M'), 172 (M+2).

I-Methyl- (3'-chloro-4'-methoxybenzyl) alcohol A mixture of 3-chloro-4-methoxybenzaldehyde (13 g, 76.5 mmol), methylmagnesium chloride (52 g, 153 mmol), and THF (300 mL) was refluxed for 3 hours. The reaction mixture was cooled to room temperature. NH4Cl (satd. soln., 6 mL) was added dropwise followed by diethyl ether (500 mL) and the mixture was filtered through paper to remove the inorganic solids. The filtrate was evaporated under reduced pressure and the resulting solid was dissolved in diethyl ether (300 mL) and washed with water (4 x 25 mL). The organic layer was dried (Na2SO4) and evaporated under vacuum to yield 11.3 g, 80% yield of crude product. This material was further purified by chromatography on silica gel (50 mm x 30 cm) (elution with CH2Cl2) to yield 11.3 g, 63 ; yield of an oil; TLC (CH2Cl2) Rf=0.25; GC Rt=5. 30 min; MS (EI) m/z 186 (M+), 188 (M+2).

3'-Chloro-4'-methoxyacetophenone A mixture of 1-methyl- (3'-Chloro-4'-methoxybenzyl) alcohol (7.6 g, 41 mmol), pyridinium chlorochromate (PCC) (13.16 g, 61.5 mmol), and CHCl (300 mL) was allowed to stir at room temperature for 2 hours. Diethyl ether (1000 mL) was added and the resulting mixture was placed on a chromatography column of silica gel (50 mm x 30 cm) (elution with diethyl ether) to yield 7.3 g, 97% yield of crude solid product. GC analysis of this material showed it to be 99% pure and it was used in the following reac- tion without further purification. TLC (diethyl ether) Rf=1. 0; GC Rt=5. 3 min; MS (EI) m/z 184 (M'), 184 (M+2).

(R, R)-N- (1-Ethyl-4'-methoxy-3'-chlorophenyl)-1- (1- naphthylethyl) amine A mixture of 3'-chloro-4'-methoxyacetophenone (5.3 g, 29 mmol), (R)- (+)-l- (l-naphthyl) ethylamine (4.98 g, 29 mmol), titanium (IV) isopropoxide (10.2 g, 36 mmol), and isopropanol (20 mL) was heated to 100°C for 3 hours.

Sodium triacetoxyborohydride (NaB (O2CCH3) 3 ; 12. 29 g, 58 mmol) was added in portions over 10 minutes. The reaction mixture was heated to reflux for 30 minutes and was then allowed to stir at room temperature for 18 hours. The mixture was then poured into diethyl ether (500 mL); H2O (2 mL) was added and the suspension was centrifuged to remove the fine precipitate of titanium salts. The supernatant was collected and the pellet was washed with ether (500 mL). The combined organic layers were dried (Na2SO4) and evaporated under vacuum to yield 6.81 g, 70% of crude product.

This material was further purified by chromatography on silica gel (50 mm x 30 cm) (elution with 3% MeOH-97% CH2Cl2) to give 2.01 g of an oil. The diastereomer was further purified by recrystallization. The free base (1.98 g) was converted to its HC1 salt with ethereal HC1.

This salt was dissolved in hot isopropanol (65 mL) and the solution was filtered through paper. The filtrate was

evaporated under vacuum and the resulting solid dissolved in isopropanol (30 mL). After standing at room tempera- ture for 18 hours, the crystalline solid was collected, washed with cold isopropanol (20 mL), and dried to yield 0.87 g, 40% (from free base) of the diastereomerically pure hydrochloride salt: mp 236-237lit (dec); TLC (MeOH- CH2Cl2 [99: 1]) Rf=0.25; GC Rt=11. 06 min; FTIR (KBr pellet, cm-1) 3433,2950, 2931,2853, 2803,2659, 2608,2497, 1604, 1595,1504, 1461,1444, 1268,1260, 1067,1021, 802,781, 733; MS (EI) m/z 339 (M+), 341 (M+2).

Example 18: Additional Synthesis Protocol Preparation of 22Z and 23A A stirred solution of sodium hydride (2.173 g, 60% in oil, 54.325 mmol) in dimethylformamide (100ml) was treated dropwise with triethyl phosphonoacetate (12.47 g, 55.65 mmol) and stirred 30 min at rt. After this time, a solution of m-trifluoromethoxy benzaldehyde (10.0 g, 52.6 mmol) in dimethylformamide (50 ml) was added dropwise and the solution stirred 30 min at rt and 30 min at 100°C.

The reaction was quenched by the addition of water and transferred to a separatory funnel using diethyl ether (500 ml). The ether solution was washed with saturated ammonium chloride (4 x 500 ml), dried over anhydrous mag- nesium sulfate, filtered and concentrated to afford ethyl m-trifluoromethoxycinnamate as an oil; m/z (rel. int.) 260 (M+, 19), 232 (16), 215 (100), 187 (21), 101 (28).

The ethyl ester in ethanol (100 ml) was reduced under 60 p. s. i. hydrogen using a catalytic amount (10% by weight) palladium hydroxide. After reduction (2 hr, rt) the reaction was filtered and concentrated to afford ethyl m-trifluoromethoxyhydrocinnamate as an oil; m/z (rel. int.) 262 (M+, 16), 217 (7), 188 (100), 175 (28), 103 (31), 91 (18), 77 (23).

The saturated ethyl ester was hydrolyzed in a solution of ethanol-10 M sodium hydroxide (1: 1) for 16 hr at rt. After this time the solution was acidified and tho

product extracted into diethyl ether. The ether solution was dried over anhydrous magnesium sulfate and concen- trated to afford m-trifluoromethoxyhydrocinnamic acid as a solid; m/z (rel. int.) 234 (M+, 46), 188 (100), 174 (65), 103 (27), 91 (12), 77 (17).

The acid was stirred in excess thionyl chloride for 4 hr at rt. The excess thionyl chloride was evaporated at reduced pressure (100°C) to afford m-trifluoromethoxy- hydrocinnamyl chloride as an oil. The product was used without further purification.

A solution of m-trifluoromethoxyhydrocinnamyl chloride (9.8 g, 39 mmol) in tetrahydrofuran was cooled to -78°C and treated dropwise with a solution (13 ml of 3 M in tetrahydrofuran) of methylmagnesium bromide (39 mmol).

The reaction was stirred 4 hr at-78°C, 8 hr at rt, and quenched with dilute HC1. The reaction mixture was extracted with diethyl ether. The ether was dried over anhydrous magnesium sulfate, filtered and concentrated to an oil. Chromatography of this material through silica using a gradient of hexane to acetone afforded 4- (3- trifluoromethoxyphenyl)-2-butanone as an oil; m/z (rel. int.) 232 (M+, 68), 217 (7), 189 (59), 175 (31), 103 (28), 43 (100).

A solution of 4- (3-trifluoromethoxyphenyl)-2-butanone (2.32 g, 10 mmol), (R)-1- (3-methoxyphenyl) ethylamine (1.51 g, 10 mmol), and titanium (IV) isopropoxide (3.55 g, 12.5 mmol) were stirred 4 hr at rt. The reaction mixture was then treated with a solution (10 ml of 1 M) of ethanolic sodium cyanoborohydride (10 mmol) and stirred 16 hr at rt.

The reaction was diluted with diethyl ether (50 ml) and treated with water (0.72 ml, 40 mmol). After mixing thoroughly the solution was centrifuged and the ether layer decanted and concentrated to an oily solid. The solid was suspended in diethyl ether, filtered through 0.45 UM CR PTFE Acrodisc and concentrated to give a clear oil. Repetitive preparative thin-layer chromatography using 5% methanol in chloroform afforded the two

diasteriomers, (S, R)-N- [4- (3-trifluoromethoxyphenyl)-2- butyl]-1- (3-methoxyphenyl) ethylamine, 22Z [m/z (rel. int.) 367 (M+, 3), 352 (20), 232 (4), 178 (47), 135 (100), 105 (14), 91 (10), 77 (11)] and (R, R)-N- [4- (3-trifluoro- methoxyphenyl)-2-butyl]-1- (3-methoxyphenyl) ethylamine, 23A; m/z (rel. int.) 367 (M+, 3), 352 (19), 232 (7), 178 (43), 135 (100), 105 (19), 91 (10), 77 (11).

Preparation of 22X and 22Y In a similar fashion an equal molar amount of 4- (3- <BR> <BR> <BR> trifluoromethoxyphenyl)-2-butanone, (R)-1- (1-naphthyl) ethylamine and 1.25 equivalents titanium (IV) isopropoxide were mixed and the intermediate imine reduced with ethanolic sodium cyanoborohydride. Work-up and repetitive preparative thin-layer chromatography using 5% methanol in <BR> <BR> <BR> chloroformafforded (S, R)-N- [4-(3-trifluoromethoxyphenyl)- 2-butyl]-1-(l-naphthyl) (l-naphthyl) ethylamine, 22X; m/z (rel. int.) 387 (M+, 3), 372 (15), 198 (15), 176 (12), 155 (100), 128 (8), 115 (6), 109 (4), 103 (5), 77 (8) and (R, R)-N- [4- (3- trifluoromethoxyphenyl)-2-butyl]-1- (1-naphthyl) ethylamine, 22Y; mZz (rel. int.) 387 (M+, 2), 372 (12), 198 (16), 176 (11), 155 (100), 128 (8), 115 (6), 109 (4), 103 (5), 77 (8).

Preparation of 4T In a similar fashion an equal molar amount of 4- (2- chlorophenyl)-2-butanone, prepared from o-chlorobezalde- hyde, (R)-1 (3-methoxyphenyl) ethylamine and 1.25 equiva- lents titanium (IV) isopropoxide were mixed and the inter- mediate imine reduced with ethanolic sodium cyanoboro- hydride. Work-up and repetitive preparative thin-layer chromatography using 5k methanol in chloroform afforded <BR> <BR> <BR> (R, R)-N- [4- (2-chlorophenyl)-2-butyl]-1- (3-methoxyphenyl) ethylamine, 4T; m/z (rel. int.) 317 (M+, 3), 302 (16), 178 (62), 178 (62), 135 (100), 125 (15), 105 (10), 91 (6), 77 (8).

Preparation of 21Y In a similar fashion an equal molar amount of 4- (3- trifluoromethylphenyl)-2-butanone, prepared from m- trifluoromethylbezaldehyde, (R)-1- (3-methoxyphenyl) ethylamine and 1.25 equivalents titanium (IV) isopropoxide were mixed and the intermediate imine reduced with ethanolic sodium cyanoborohydride. Work-up and repetitive preparative thin-layer chromatography using 5 methanol in chloroform afforded (R, R)-N- [4- (3-trifluoromethylphenyl)- 2-butyl]-1- (3-methoxyphenyl) ethylamine, 21Y [m/z (rel. int.) 351 (M+, 2), 336 (18), 216 (4), 202 (3), 178 (45), 135 (100), 105 (13), 91 (9), 77 (8)] and (S, R)-N- [4- (3- trifluoromethylphenyl)-2-butyl]-1- (3-methoxyphenyl) ethylamine, 21X.

Preparation of 25C and 25D In a similar fashion an equal molar amount of 4- (3- trifluoromethylphenyl)-2-butanone, (R)-l- (l-naphthyl) ethylamine and 1.25 equivalents titanium (IV) isopropoxide were mixed and the intermediate imine reduced with ethanolic sodium cyanoborohydride. Work-up and repetitive preparative thin-layer chromatography using 5% methanol in chloroform afforded (S, R)-N- [4- (3-trifluoromethylphenyl)- 2-butyl]-1- (1-naphthyl) ethylamine, 25C [m/z (rel. int.) 371 (M+, 3), 356 (16), 198 (15), 155 (100), 129 (8), 115 (5), 109 (3), 77 (2)] and (R, R)-N- [4- (3-trifluoro- methylphenyl)-2-butyl]-l- (l-naphthyl) ethylamine, 25D; m/z (rel. int.) 371 (M', 3), 356 (16), 198 (15), 155 (100), 129 (8), 115 (5), 109 (3), 77 (2).

Preparation of 21D In a similar fashion an equal molar amount of 4- phenyl-2-butanone (Aldrich Chemical Co.), (R)-l- (3-meth- oxyphenyl) ethylamine and 1.25 equivalents titanium (IV) isopropoxide were mixed and the intermediate imine reduced with ethanolic sodium cyanoborohydride. Work-up and repetitive preparative thin-layer chromatography using Si

methanol in chloroform afforded (R, R)-N- (4-phenyl-2- butyl)-l- (3-methoxyphenyl) ethylamine, 21D [m/z (rel. int.) 283 (M', 4), 268 (13), 178 (45), 135 (100), 105 (15), 91 (43), 77 (11)] and (S, R)-N- (4-phenyl-2-butyl)-l- (3- methoxyphenyl) ethylamine, 21E.

Preparation of 21F In a similar fashion an equal molar amount of 4- phenyl-2-butanone (Aldrich Chemical Co.), (R)-1- (1- naphthyl) ethylamine and 1.25 equivalents titanium (IV) isopropoxide were mixed and the intermediate imine reduced with ethanolic sodium cyanoborohydride. Work-up and repetitive preparative thin-layer chromatography using 5% methanol in chloroform afforded (R, R)-N- (4-phenyl-2- butyl)-l- (l-naphthyl) ethylamine, 21F; m/z (rel. int.) 303 (M+, 6), 288 (14), 198 (22), 155 (100), 129 (8), 115 (5), 91 (19), 77 (4).

Preparation of 12Z A stirred solution of 2-chlorohydrocinnamonitrile (Aldrich Chemical Co., 1.66 g, 10 mmol) in dichloromethane (100 ml) was cooled to-78°C and treated dropwise with diisobutylaluminum hydride (1.42 g, 10 mmol). The reac- tion was stirred 1 hr at rt, cooled to-78 °C and treated with a solution of 1- (1-naphthyl) ethylamine (1.71 g, 10 mmol) in dichloromethane (25 ml). The reaction was trans- ferred to an ice bath and stirred 2 hr. After this time the reaction was poured directly into a stirred solution of ethanolic sodium borohydride (50 ml of 0.2 M, 10 mmol).

The mixture was stirred 30 min at rt and the excess sodium borohydride quenched by the addition of 10% HC1. The solution was then made basic by the addition of 10 N NaOH and transferred to a separatory funnel washing with diethyl ether (300 ml). The aqueous phase was removed and the remaining organic layer washed with 1 N NaOH (3 x 100 ml). The organic layer was dried over anhydrous magnesium sulfate, and concentrated to an oil. Chromatography of

this material through silica gel using a gradient of chloroform to 10% methanol-chloroform afforded 2.34g (72% yield) of (R)-N- [3- (2-chlorophenyl) propyl]-1- (1- naphthyl) ethylamine, 12Z, as a clear oil; m/z (rel. int.) 323 (M+, 2), 308 (63), 288 (7), 196 (5), 184 (5), 155 (100), 125 (24), 115 (8), 103 (4), 91 (3), 77 (7).

Preparation of 12B In a similar fashion, 4-methylcinnamonitrile was treated with diisobutyl aluminum hydride and the intermediate aluminum-imine complex treated with (R)-1- (3- methoxyphenyl) ethylamine. The intermediate imine was treated with ethanolic sodium borohydride. Work-up and chromatography yielded (R)-N- [3- (4-methylphenyl) prop-2- enyl]-1- (3-methoxyphenyl) ethylamine, 12B, as a clear, colorless oil; m/z (rel. int.) 281 (M+, 6), 266 (5), 176 (27), 146 (75), 135 (63), 131 (100), 115 (25), 105 (21), 91 (21), 77 (21).

Preparation of 12C In a similar fashion, 2-methylcinnamonitrile was treated with diisobutyl aluminum hydride and the intermediate aluminum-imine complex treated with (R)-1- (3- methoxyphenyl) ethylamine. The intermediate imine was treated with ethanolic sodium borohydride. Work-up and chromatography yielded (R)-N- [3- (2-methylphenyl) prop-2- enyl]-1- (3-methoxyphenyl) ethylamine, 12C, as a clear, colorless oil; m/z (rel. int.) 281 (M+, 4), 266 (15), 176 (18), 146 (62), 135 (58), 131 (100), 115 (23), 105 (19), 91 (38), 77 (17).

Preparation of 12D In a similar fashion, 2,4, 6-trimethylcinnamonitrile was treated with diisobutyl aluminum hydride and the intermediate aluminum-imine complex treated with (R)-1- (3- methoxyphenyl) ethylamine. The intermediate imine was treated with ethanolic sodium borohydride. Work-up and

chromatography yielded (R)-N- [3- (2, 4,6-trimethylphenyl) prop-2-enyl]-l- (3-methoxyphenyl) ethylamine, 12D, as a clear, colorless oil; m/z (rel. int.) 309 (M+, 8), 294 (25), 174 (82), 159 (100), 135 (52), 129 (29), 105 (21), 91 (17), 77 (14).

Preparation of 12E In a similar fashion, 4-isopropylcinnamonitrile was treated with diisobutyl aluminum hydride and the inter- mediate aluminum-imine complex treated with (R)-l- (3- methoxyphenyl) ethylamine. The intermediate imine was treated with ethanolic sodium borohydride. Work-up and chromatography yielded (R)-N- [3- (4-isopropylphenyl) prop-2- enyl]-1- (3-methoxyphenyl) ethylamine, 12E, as a clear, colorless oil; m/z (rel. int.) 309 (M+, 9), 294 (7), 174 (98), 159 (22), 135 (80), 117 (100), 105 (35), 91 (37), 77 (19).

Preparation of 12F In a similar fashion, 2,4-dimethylcinnamonitrile was treated with diisobutyl aluminum hydride and the inter- mediate aluminum-imine complex treated with (R)-l- (3- methoxyphenyl) ethylamine. The intermediate imine was treated with ethanolic sodium borohydride. Work-up and chromatography yielded (R)-N- [3- (2, 4-dimethylphenyl) prop- 2-enyl]-l- (3-methoxyphenyl) ethylamine, 12F, as a clear, colorless oil; m/z (rel. int.) 295 (M+, 8), 294 (15), 174 (29), 160 (75), 145 (100), 135 (68), 117 (21), 105 (30), 91 (26), 77 (19).

Preparation of 12G In a similar fashion, 3-methylcinnamonitrile was treated with diisobutyl aluminum hydride and the inter- mediate aluminum-imine complex treated with (R)-l- (3- methoxyphenyl) ethylamine. The intermediate imine was treated with ethanolic sodium borohydride. Work-up and chromatography yielded (R)-N- [3- (3-methylphenyl) prop-2-

enyl]-l- (3-methoxyphenyl) ethylamine, 12G, as a clear, colorless oil; m/z (rel. int.) 281 (M+, 5), 266 (9), 176 (24), 146 (71), 135 (62), 131 (100), 115 (23), 105 (19), 91 (41), 77 (18).

Preparation of 25E In a similar fashion, cinnamonitrile was treated with diisobutyl aluminum hydride and the intermediate aluminum- imine complex treated with (R)-1- (3-methoxyphenyl) ethyl- amine. The intermediate imine was treated with ethanolic sodium borohydride. Work-up and chromatography yielded (R)-N- (3-phenylprop-2-enyl)-1- (3-methoxyphenyl) ethylamine, 25E, as a clear colorless oil; m/z (rel. int.) 267 (M+, 3), 252 (14), 176 (17), 135 (62), 117 (100), 105 (28), 91 (56), 77 (33).

Preparation of 25G In a similar fashion, a-methylcinnamonitrile was treated with diisobutyl aluminum hydride and the inter- mediate aluminum-imine complex treated with (R)-1- (3- methoxyphenyl) ethylamine. The intermediate imine was treated with ethanolic sodium borohydride. Work-up and chromatography yielded (R)-N- (2-methyl-3-phenylprop-2- enyl)-l- (3-methoxyphenyl) ethylamine, 25G, as a clear, colorless oil; m/z (rel. int.) 281 (M+, 5), 266 (18), 190 (12), 146 (78), 135 (82), 131 (100), 115 (21), 105 (21), 91 (62), 77 (19).

Preparation of 6X A stirred solution of sodium hydride (1.8 g, 75 mmol) in dimethylformamide (150 ml) was treated with a solution of diethylcyanomethyl phosphonate (13.3 g, 75 mmol) in dimethylformamide (50 ml). The reaction was stirred 30 min at rt. After this time the reaction was treated with 3-chlorobenzaldehyde (10.54 g, 75 mmol) and stirred 1 hr at rt and 30 min at 60°C. The reaction was then quenched by the addition of water (200 ml). The reaction mixture

was transferred to a separatory funnel using diethyl ether (300 ml) and the resulting organic phase washed with water (5 x 300 ml) and brine. The organic layer was dried over anhydrous potassium carbonate and concentrated to yield 3- chlorocinnamonitrile (11.06 g) as a solid. The solid was dissolved in tetrahydrofuran (50 ml) and treated with excess diborane and stirred 30 min at rt. The reaction was poured over ice/10% HC1. The acidic aqueous phase was washed with diethyl ether (2 x 200 ml). The aqueous phase was made basic by the addition of 10 N NaOH and extracted with diethyl ether (200 ml). The ether extract was dried over anhydrous potassium carbonate and concentrated to afford 3- (3-chlorophenyl) propylamine as an oil (0.6 g, 3.54 mmol). The 3- (3-chlorophenyl) propylamine (0.60 g, 3.54 mmol), 3'-methoxyacetophenone (0.53 g, 3.54 mmol) and 1.25 molar equivalents titanium (IV) isopropoxide (1.26 g, 4.43 mmol) were mixed 4 hr at rt and the intermediate imine treated with an ethanolic sodium cyanoborohydride (5 ml of 1 M, 5 mmol). The reaction was stirred 16 hr at rt, diluted with diethyl ether (50 ml) and treated with water (0.32 ml, 17.7 mmol). After mixing thoroughly the solution was centrifuged and the ether layer concentrated to a milky solid. This material was suspended in diethyl ether and filtered through a 0.45 UM CR PTFE Acrodisc.

The ether wash was concentrated to an oil. Chromatography of this material (silica, preparative thin-layer chromato- graphy) using 3% methanol-dichloromethane (containing isopropylamine) afforded N- [3- (3-chlorophenyl) propyl]-l- (3-methoxyphenyl) ethylamine, 6X; m/z (rel. int.) 303 (M+, 3), 288 (40), 196 (3), 164 (8), 135 (100), 125 (46), 103 (26), 91 (29), 77 (29).

Preparation of 6V An equal molar amount of 3- (4-chlorophenyl) propylamine (prepared in a similar fashion from 4- chlorobenzaldehyde as above) 3'-methoxyacetophenone and 1.25 molar equivalents titanium (IV) isopropoxide were

mixed 4 hr at rt and the intermediate imine treated with an ethanolic sodium cyanoborohydride (5 ml of 1M, 5 mmol).

Work-up and chromatography afforded N- [3- (4-chlorophenyl) propyl]-1- (3-methoxyphenyl) ethylamine, 6V, as an oil; m/z (rel. int.) 303 (M+, 8), 288 (91), 196 (4), 164 (10), 135 (100), 125 (61), 103 (21), 91 (21), 77 (18).

Preparation of 20A In a similar fashion, an equal molar amount of 1- (1- methoxyphenyl) ethylamine, 4-t-butylacetophenone and 1.25 molar equivalents titanium (IV) isopropoxide were mixed 4 hr at rt and the intermediate imine treated with an ethan- olic sodium cyanoborohydride (5 ml of 1M, 5 mmol). Work- up and chromatography afforded (R)-N- [1- (4-t-butylphenyl) ethyl]-l- (1-naphthyl) ethylamine, 20A, as an oil; m/z (rel. int.) 331 (M+, 12), 316 (29), 161 (70), 155 (100), 131 (14), 127 (13), 115 (10), 105 (6), 91 (10), 77 (7).

Preparation of 25H and 25I In a similar fashion, an equal molar amount of (R)-1- <BR> <BR> <BR> (3-methoxyphenyl) ethylamine, trans-4-phenyl-3-butene-2-one and 1.25 molar equivalents titanium (IV) isopropoxide were mixed 4 hr at rt and the intermediate imine treated with an ethanolic sodium cyanoborohydride (5 ml of 1 M, 5 mmol). Work-up and chromatography afforded (R, R)-N- (2- methyl-4-phenybut-3-enyl)-1- (3-methoxyphenyl) ethylamine, 25H, as an oil; m/z (rel. int.) 283 (M+, 4), 268 (13), 178 (40), 135 (100), 105 (15), 91 (47), 77 (13) and (S, R)-N- <BR> <BR> <BR> (2-methyl-4-phenybut-3-enyl)-1- (3-methoxyphenyl) ethylamine, 25I, as an oil; m/z (rel. int.) 283 (M+, 4), 268 (13), 178 (40), 135 (100), 105 (15), 91 (47), 77 (13).

Preparation of 16L and 16M In a similar fashion, an equal molar amount of (R)-1- (3-methoxyphenyl) ethylamine, 3-methoxyacetophenone and 1.25 molar equivalents titanium (IV) isopropoxide were mixed 4 hr at rt and the intermediate imine treated with

an ethanolic sodium cyanoborohydride (5 ml of 1 M, 5 mmol). Work-up and chromatography afforded (R, R)-N- [1- (4- methoxyphenyl) ethyl]-1- (3-methoxyphenyl) ethylamine, 16L, as an oil; m/z (rel. int.) 284 (M-1, 1), 270 (85), 150 (83), 135 (100), 120 (12), 105 (28), 91 (25), 77 (23) and (S, R)-N- [1- (4-methoxyphenyl) ethyl]-l- (3-methoxyphenyl) ethylamine, 16M, as an oil; m/z (rel. int.) 284 (M-1, 1), 270 (53), 150 (98), 135 (100), 120 (11), 105 (33), 91 (25), 77 (23).

Preparation of 5B/5C In a similar fashion, 4-chloroacetophenone was used to prepare 3-methyl-3- (4-chlorophenyl) cinnamonitrile. The nitrile was catalytically reduced (palladium hydroxide, acetic acid, 60 p. s. i. hydrogen 2 hr) to generate 3- methyl-3- (4-chlorophenyl) propylamine. An equal molar amount of the amine, 3'-methoxyacetophenone and 1.25 molar equivalents titanium (IV) isopropoxide were mixed 4 hr at rt and the intermediate imine treated with an ethanolic sodium cyanoborohydride (5 ml of 1 M, 5 mmol). Work-up and chromatography afforded N- (3-methyl-3- (4- chlorophenyl) propyl]-1- (3-methoxyphenyl) ethylamine, 5B/5C as an oil; m/z (rel. int.) 317 (M+, 12), 302 (74), 210 (2), 182 (4), 164 (12), 135 (100), 121 (25), 103 (40), 91 (19), 77 (28).

Preparation of 4Z/5A In a similar fashion, 3-chloroacetophenone was used to prepare 3-methyl-3- (3-chlorophenyl) cinnamonitrile. The nitrile was catalytically reduced (palladium hydroxide, acetic acid, 60 p. s. i. hydrogen 2 hr) to generate 3- methyl-3- (3-chlorophenyl) propylamine. An equal molar amount of the amine, 3'-methoxyacetophenone and 1.25 molar equivalents titanium (IV) isopropoxide were mixed 4 hr at rt and the intermediate imine treated with an ethanolic sodium cyanoborohydride (5 ml of 1 M, 5 mmol). Work-up and chromatography afforded N- [3-methyl-3- (3-chlorophenyl)

propyl]-1- (3-methoxyphenyl) ethylamine, 4Z/5A, as an oil; m/z (rel. int.) 283 (M+, 17), 268 (71), 164 (13), 135 (100), 121 (21), 105 (27), 91 (26), 77 (14).

Preparation of 4Y In a similar fashion, 2-chloroacetophenone was used to prepare 3-methyl-3- (2-chlorophenyl) cinnamonitrile. The nitrile was catalytically reduced (palladium hydroxide, acetic acid, 60 p. s. i. hydrogen 2 hr) to generate 3- methyl-3- (2-chlorophenyl) propylamine. An equal molar amount of the amine, 3'-methoxyacetophenone and 1.25 molar equivalents titanium (IV) isopropoxide were mixed 4 hr at rt and the intermediate imine treated with an ethanolic sodium cyanoborohydride (5 ml of 1 M, 5 mmol). Work-up and chromatography afforded N- [3-methyl-3- (2-chloro- phenyl) propyl]-1- (3-methoxyphenyl) ethylamine, 4Y, as an oil; m/z (rel. int.) 283 (Mt, 17) 268 (71), 164 (13), 135 (100), 121 (21), 105 (27), 91 (26), 77 (14).

Preparation of 6T A solution of NPS R-568 (30.3 g 100 mmol) in dichloromethane at-78°C was treated dropwise with boron- tribromide (50 g, 200 mmol). The reaction was stirred 1 hr at rt and poured over ice. The hydrobromide was extracted from the aqueous phase with chloroform. The chloroform solubles were then washed (4 x 100 ml) with 50% HC1. The chloroform wash was dried over anhydrous magnesium sulfate and concentrated to afford (R)-N- [3- (2- chlorophenyl) propyl]-1- (3-hydroxyphenyl) ethylamine hydro- chloride as a solid. A solution of sodium hydride (0.48 g, 20 mmol) in dimethylformamide was treated with (R)-N- <BR> <BR> <BR> [3- (2-chlorophenyl) propyl]-1- (3-hydroxyphenyl) ethylamine hydrochloride (3.25 g, 10 mmol) and the reaction stirred 1 hr at rt. The reaction was treated with iodoethane (1.71 g, 11 mmol) and stirred 16 hr at rt. Work-up and chromatography through silica using 3% methanol in <BR> <BR> <BR> chloroformafforded (R)-N- [3- (2-chlorophenyl) propyl]-1- (3-

ethoxyphenyl) ethylamine, 6T, as an oil; m/z (rel. int.) 316 (M+, 1), 302 (100), 282 (11), 196 (5), 178 (7), 149 (74), 121 (34), 103 (25), 91 (28), 77 (29).

Preparation of 6R NPS R-467 was used in a similar fashion to prepare (R)-N- (3-phenylpropyl)-l- (3-ethoxyphenyl) ethylamine, 6R, as an oil; m/z (rel. int.) 283 (M+, 10), 268 (74), 178 (11), 162 (8), 149 (100), 121 (30), 103 (16), 91 (86), 77 (29).

Preparation of 3U An equal molar mixture of 3,3-diphenylpropylamine (2.11 g, 10 mmol), 1'-acetonaphthone (1.70 g, 10 mmol) and 1.25 equivalents of titanium (IV) isopropoxide (3.55 g, 12.5 mmol) were stirred 4 hr at rt. The reaction mixture was then treated with a 1 M solution of ethanolic sodium cyanoborohydride (12.5 ml, 12.5 mmol) and stirred 16 hr at rt. The reaction was diluted with diethyl ether (50 ml) and treated with water (0.72 ml, 40 mmol). After mixing thoroughly the mixture was centrifuged and the ether layer decanted and concentrated to a milky oil. The oil was suspended in diethyl ether and filtered through a 0.45 UM CR PTFE Acrodisc. The diethyl ether filtrate was concen- trated to afford N- ethylamine, 3U, as a clear, colorless oil; m/z (rel. int.) 365 (M+, 17), 350 (19), 181 (23), 155 (100), 141 (25), 115 (11), 91 (13), 77 (6).

Preparation of 6F In a similar fashion equal molar amounts 1- (3- methoxyphenyl) ethylamine (1.51 g, 10 mmol), 2'-acetonaph- thone (1.70 g, 10 mmol) and 1.25 equivalents of titanium (IV) isopropoxide (3.55 g, 12.5 mmol) were treated as above. Work-up yielded N- [1- (2-naphthyl) ethyl]-1- (3- methoxyphenyl) ethylamine, 6F, as a clear, colorless oil;

m/z (rel. int.) 305 (M+, 1), 290 (35), 170 (49), 155 (100), 135 (55), 115 (8), 105 (10), 91 (9), 77 (10).

Preparation of 4G In a similar fashion equal molar amounts of (R))-1- phenylethylamine,,'-acetonaphthone and 1.25 equivalents of titanium (IV) isopropoxide were mixed and the resulting intermediate imine was reduced with ethanolic sodium cyanoborohydride. Work-up and chromatography yielded N- [l- (l-naphthyl) ethyl)-1-phenylethylamine, 4G, as a clear, colorless oil; m/z (rel. int.) 275 (M+, 16), 260 (79), 155 (100), 127 (27), 105 (70), 77 (32).

Preparation of 4H In a similar fashion equal molar amounts of (R)-1- phenylethylamine, 2'-acetonaphthone and 1.25 equivalents of titanium (IV) isopropoxide were mixed and the resulting intermediate imine was reduced with ethanolic sodium cyanoborohydride. Work-up and chromatography yielded N- [l- (2-naphthyl) ethyl]-1-phenylethylamine, 4H, as a clear, colorless oil; m/z (rel. int.) 275 (M+, 1), 260 (61), 155 (100), 120 (36), 105 (55), 77 (15).

Preparation of 6E In a similar fashion equal molar amounts of 1- (3- methoxyphenyl) ethylamine, 1'-acetonaphthone and 1.25 equivalents of titanium (IV) isopropoxide were mixed and the resulting intermediate imine was reduced with ethan- olic sodium cyanoborohydride. Work-up and chromatography yielded N-1- (l-naphthyl) ethyl-l- (3-methoxyphenyl) ethyl- amine, 6E, as a clear, colorless oil; m/z (rel. int.) 305 (M+, 10), 290 (30), 170 (43), 155 (100), 135 (69), 115 (9), 105 (15), 91 (14), 77 (18).

Example 19: Pharmaceutical Formulation Preparation of a pharmaceutical formulation suitable for administering a calcimimetic into a human patient is shown in Table 3.

TABLE 3 Ingredient mg/capsule g/representative batch of 5,000 capsules NPS R-568 56. 0 280. 0 Pregelatinized 134.0 670.0 Starch NF Microcrystalline 34.0 170.0 Cellulose NF Colloidal Silicon 1.0 5.0 Dioxide Total 225 mg 1125 g Other examples of NPS (R)-568 hydrochloride formulations and dosage forms include those suitable for sustained or extended release, using standard techniques.

Proper dosing can also be carried out using standard techniques. For example, in one set of experiments, 10- 400 mg oral doses of NPS (R)-568 hydrochloride showed pharmacological activity in human subjects. Significant levels of the O-glucuronide conjugate of 17Q, a principal metabolite of NPS (R)-568, was observed in human plasma following oral administration of NPS (R)-568 hydro- chloride. Thus, the glucuronide conjugate of 17Q may be exerting some beneficial effect.

Using standard techniques other suitable dosage ranges for NPS (R)-568 can be determined.

Suitable dosage ranges, formulations, and dosage forms for other compounds described herein can also be determined by one skilled in art based on the teachings provided in the application.

Other embodiments are within the following claims.

Thus, while several embodiments have been shown and de- scribed, various modifications may be made, without departing from the spirit and scope of the present inven- tion.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: NPS Pharmaceuticals, Inc.

(ii) TITLE OF INVENTION: CALCIUM RECEPTOR-ACTIVE COMPOUNDS (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Lyon & Lyon (B) STREET: First Interstate World Center, Suite 4700 633 West Fifth Street (C) CITY: Los Angeles (D) STATE: California (E) COUNTRY: USA (F) ZIP: 90017 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: 3.5" Diskette, 1.44 Mb storage (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: FastSeq (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: Prior applications total, including application described below: 2 (A) APPLICATION NUMBER: U. S. 08/353,784 (B) FILING DATE: 8 December, 1994 (A) APPLICATION NUMBER: PCT/US/94/12117 (B) FILING DATE: 21 October, 1994 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Heber, Sheldon O.

(B) REGISTRATION NUMBER: 38,179 (C) REFERENCE/DOCKET NUMBER: 215/304 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (213) 489-1600 (B) TELEFAX: (213) 955-0440 (C) TELEX: 67-3510 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5006 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 436.. 3699 (D) OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GCTGCTGTGG CCGGACCCGA AGGCGGGCGC CGGGAGCGCA 40 GCGAGCCAGA CGCGCCTCTC CAAGACCGTG ACCTTGGCAT 80 AGGGAGCGGG GCTGCGCGCA GTCCTGAGAT CAGACCAGAG 120 CTCATCCTCG TGGAGACCCA CGGCCGAGGG GCCGGAGCTG 160 CCTCTGTGCG AGGGAGCCCT GGCCGCGGCG CAGAAGGCAT 200 CACAGGAGGC CTCTGCATGA TGTGGCTTCC AAAGACTCAA 240 GGACCACCCA CATTACAAGT CTGGATTGAG GAAGGCAGAA 280 ATGGAGATTC AAACACCACG TCTTCTATTA TTTTATTAAT 320 CAATCTGTAG ACATGTGTCC CCACTGCAGG GAGTGAACTG 360 CTCCAAGGGA GAAACTTCTG GGAGCCTCCA AACTCCTAGC 400 TGTCTCATCC CTTGCCCTGG AGAGACGGCA GAACC 435 ATG GCA TTT TAT AGC TGC TGC TGG GTC CTC TTG GCA 471 Met Ala Phe Tyr Ser Cys Cys Trp Val Leu Leu Ala 1 5 10 CTC ACC TGG CAC ACC TCT GCC TAC GGG CCA GAC CAG 507 Leu Thr Trp His Thr Ser Ala Tyr Gly Pro Asp Gln 15 20 CGA GCC CAA AAG AAG GGG GAC ATT ATC CTT GGG GGG 543 Arg Ala Gln Lys Lys Gly Asp Ile Ile Leu Gly Gly 25 30 35 CTC TTT CCT ATT CAT TTT GGA GTA GCA GCT AAA GAT 579 Leu Phe Pro Ile His Phe Gly Val Ala Ala Lys Asp 40 45 CAA GAT CTC AAA TCA AGG CCG GAG TCT GTG GAA TGT 615 Gln Asp Leu Lys Ser Arg Pro Glu Ser Val Glu Cys 50 55 60 ATC AGG TAT AAT TTC CGT GGG TTT CGC TGG TTA CAG 651 Ile Arg Tyr Asn Phe Arg Gly Phe Arg Trp Leu Gln 65 70 GCT ATG ATA TTT GCC ATA GAG GAG ATA AAC AGC AGC 687 Ala Met Ile Phe Ala Ile Glu Glu Ile Asn Ser Ser 75 80 CCA GCC CTT CTT CCC AAC TTG ACG CTG GGA TAC AGG 723 Pro Ala Leu Leu Pro Asn Leu Thr Leu Gly Tyr Arg 85 90 95 ATA TTT GAC ACT TGC AAC ACC GTT TCT AAG GCC TTG 759 Ile Phe Asp Thr Cys Asn Thr Val Ser Lys Ala Leu 100 105 GAA GCC ACC CTG AGT TTT GTT GCT CAA AAC AAA ATT 795 Glu Ala Thr Leu Ser Phe Val Ala Gln Asn Lys Ile 110 115 120 GAT TCT TTG AAC CTT GAT GAG TTC TGC AAC TGC TCA 831 Asp Ser Leu Asn Leu Asp Glu Phe Cys Asn Cys Ser 125 130 GAG CAC ATT CCC TCT ACG ATT GCT GTG GTG GGA GCA 867 Glu His Ile Pro Ser Thr Ile Ala Val Val Gly Ala 135 140 ACT GGC TCA GGC GTC TCC ACG GCA GTG GCA AAT CTG 903 Thr Gly Ser Gly Val Ser Thr Ala Val Ala Asn Leu 145 150 155 CTG GGG CTC TTC TAC ATT CCC CAG GTC AGT TAT GCC 939 Leu Gly Leu Phe Tyr Ile Pro Gln Val Ser Tyr Ala 160 165 TCC TCC AGC AGA CTC CTC AGC AAC AAG AAT CAA TTC 975 Ser Ser Ser Arg Leu Leu Ser Asn Lys Asn Gln Phe 170 175 180 AAG TCT TTC CTC CGA ACC ATC CCC AAT GAT GAG CAC 1011 Lys Ser Phe Leu Arg Thr Ile Pro Asn Asp Glu His 185 190 CAG GCC ACT GCC ATG GCA GAC ATC ATC GAG TAT TTC 1047 Gln Ala Thr Ala Met Ala Asp Ile Ile Glu Tyr Phe 195 200 CGC TGG AAC TGG GTG GGC ACA ATT GCA GCT GAT GAC 1083 Arg Trp Asn Trp Val Gly Thr Ile Ala Ala Asp Asp 205 210 215 GAC TAT GGG CGG CCG GGG ATT GAG AAA TTC CGA GAG 1119 Asp Tyr Gly Arg Pro Gly Ile Glu Lys Phe Arg Glu 220 225 GAA GCT GAG GAA AGG GAT ATC TGC ATC GAC TTC AGT 1155 Glu Ala Glu Glu Arg Asp Ile Cys Ile Asp Phe Ser 230 235 240 GAA CTC ATC TCC CAG TAC TCT GAT GAG GAA GAG ATC 1191 Glu Leu Ile Ser Gln Tyr Ser Asp Glu Glu Glu Ile 245 250 CAG CAT GTG GTA GAG GTG ATT CAA AAT TCC ACG GCC 1227 Gln His Val Val Glu Val Ile Gln Asn Ser Thr Ala 255 260 AAA GTC ATC GTG GTT TTC TCC AGT GGC CCA GAT CTT 1263 Lys Val Ile Val Val Phe Ser Ser Gly Pro Asp Leu 265 270 275 GAG CCC CTC ATC AAG GAG ATT GTC CGG CGC AAT ATC 1299 Glu Pro Leu Ile Lys Glu Ile Val Arg Arg Asn Ile 280 285 ACG GGC AAG ATC TGG CTG GCC AGC GAG GCC TGG GCC 1335 Thr Gly Lys Ile Trp Leu Ala Ser Glu Ala Trp Ala 290 295 300 AGC TCC TCC CTG ATC GCC ATG CCT CAG TAC TTC CAC 1371 Ser Ser Ser Leu Ile Ala Met Pro Gln Tyr Phe His 305 310 GTG GTT GGC GGC ACC ATT GGA TTC GCT CTG AAG GCT 1407 Val Val Gly Gly Thr Ile Gly Phe Ala Leu Lys Ala 315 320 GGG CAG ATC CCA GGC TTC CGG GAA TTC CTG AAG AAG 1443 Gly Gln Ile Pro Gly Phe Arg Glu Phe Leu Lys Lys 325 330 335 GTC CAT CCC AGG AAG TCT GTC CAC AAT GGT TTT GCC 1479 Val His Pro Arg Lys Ser Val His Asn Gly Phe Ala 340 345 AAG GAG TTT TGG GAA GAA ACA TTT AAC TGC CAC CTC 1515 Lys Glu Phe Trp Glu Glu Thr Phe Asn Cys His Leu 350 355 360 CAA GAA GGT GCA AAA GGA CCT TTA CCT GTG GAC ACC 1551 Gln Glu Gly Ala Lys Gly Pro Leu Pro Val Asp Thr 365 370 TTT CTG AGA GGT CAC GAA GAA AGT GGC GAC AGG TTT 1587 Phe Leu Arg Gly His Glu Glu Ser Gly Asp Arg Phe 375 380 AGC AAC AGC TCG ACA GCC TTC CGA CCC CTC TGT ACA 1623 Ser Asn Ser Ser Thr Ala Phe Arg Pro Leu Cys Thr 385 390 395 GGG GAT GAG AAC ATC AGC AGT GTC GAG ACC CCT TAC 1659 Gly Asp Glu Asn Ile Ser Ser Val Glu Thr Pro Tyr 400 405 ATA GAT TAC ACG CAT TTA CGG ATA TCC TAC AAT GTG 1695 Ile Asp Tyr Thr His Leu Arg Ile Ser Tyr Asn Val 410 415 420 TAC TTA GCA GTC TAC TCC ATT GCC CAC GCC TTG CAA 1731 Tyr Leu Ala Val Tyr Ser Ile Ala His Ala Leu Gln 425 430 GAT ATA TAT ACC TGC TTA CCT GGG AGA GGG CTC TTC 1767 Asp Ile Tyr Thr Cys Leu Pro Gly Arg Gly Leu Phe 435 440 ACC AAT GGC TCC TGT GCA GAC ATC AAG AAA GTT GAG 1803 Thr Asn Gly Ser Cys Ala Asp Ile Lys Lys Val Glu 445 450 455 GCG TGG CAG GTC CTG AAG CAC CTA CGG CAT CTA AAC 1839 Ala Trp Gln Val Leu Lys His Leu Arg His Leu Asn 460 465 TTT ACA AAC AAT ATG GGG GAG CAG GTG ACC TTT GAT 1875 Phe Thr Asn Asn Met Gly Glu Gln Val Thr Phe Asp 470 475 480 GAG TGT GGT GAC CTG GTG GGG AAC TAT TCC ATC ATC 1911 Glu Cys Gly Asp Leu Val Gly Asn Tyr Ser Ile Ile 485 490 AAC TGG CAC CTC TCC CCA GAG GAT GGC TCC ATC GTG 1947 Asn Trp His Leu Ser Pro Glu Asp Gly Ser Ile Val 495 500 TTT AAG GAA GTC GGG TAT TAC AAC GTC TAT GCC AAG 1983 Phe Lys Glu Val Gly Tyr Tyr Asn Val Tyr Ala Lys 505 510 515 AAG GGA GAA AGA CTC TTC ATC AAC GAG GAG AAA ATC 2019 Lys Gly Glu Arg Leu Phe Ile Asn Glu Glu Lys Ile 520 525 CTG TGG AGT GGG TTC TCC AGG GAG CCA CTC ACC TTT 2055 Leu Trp Ser Gly Phe Ser Arg Glu Pro Leu Thr Phe 530 535 540 GTG CTG TCT GTC CTC CAG GTG CCC TTC TCC AAC TGC 2091 Val Leu Ser Val Leu Gln Val Pro Phe Ser Asn Cys 545 550 AGC CGA GAC TGC CTG GCA GGG ACC AGG AAA GGG ATC 2127 Ser Arg Asp Cys Leu Ala Gly Thr Arg Lys Gly Ile 555 560 ATT GAG GGG GAG CCC ACC TGC TGC TTT GAG TGT GTG 2163 Ile Glu Gly Glu Pro Thr Cys Cys Phe Glu Cys Val 565 570 575 GAG TGT CCT GAT GGG GAG TAT AGT GAT GAG ACA GAT 2199 Glu Cys Pro Asp Gly Glu Tyr Ser Asp Glu Thr Asp 580.585 GCC AGT GCC TGT AAC AAG TGC CCA GAT GAC TTC TGG 2235 Ala Ser Ala Cys Asn Lys Cys Pro Asp Asp Phe Trp 590 595 600 TCC AAT GAG AAC CAC ACC TCC TGC ATT GCC AAG GAG 2271 Ser Asn Glu Asn His Thr Ser Cys Ile Ala Lys Glu 605 610 ATC GAG TTT CTG TCG TGG ACG GAG CCC TTT GGG ATC 2307 Ile Glu Phe Leu Ser Trp Thr Glu Pro Phe Gly lie 615 620 GCA CTC ACC CTC TTT GCC GTG CTG GGC ATT TTC CTG 2343 Ala Leu Thr Leu Phe Ala Val Leu Gly Ile Phe Leu 625 630 635 ACA GCC TTT GTG CTG GGT GTG TTT ATC AAG TTC CGC 2379 Thr Ala Phe Val Leu Gly Val Phe lie Lys Phe Arg 640 645 AAC ACA CCC ATT GTC AAG GCC ACC AAC CGA GAG CTC 2415 Asn Thr Pro Ile Val Lys Ala Thr Asn Arg Glu Leu 650 655 660 TCC TAC CTC CTC CTC TTC TCC CTG CTC TGC TGC TTC 2451 Ser Tyr Leu Leu Leu Phe Ser Leu Leu Cys Cys Phe 665 670 TCC AGC TCC CTG TTC TTC ATC GGG GAG CCC CAG GAC 2487 Ser Ser Ser Leu Phe Phe Ile Gly Glu Pro Gln Asp 675 680 TGG ACG TGC CGC CTG CGC CAG CCG GCC TTT GGC ATC 2523 Trp Thr Cys Arg Leu Arg Gln Pro Ala Phe Gly Ile 685 690 695 AGC TTC GTG CTC TGC ATC TCA TGC ATC CTG GTG AAA 2559 Ser Phe Val Leu Cys Ile Ser Cys Ile Leu Val Lys 700 705 ACC AAC CGT GTC CTC CTG GTG TTT GAG GCC AAG ATC 2595 Thr Asn Arg Val Leu Leu Val Phe Glu Ala Lys Ile 710 715 720 CCC ACC AGC TTC CAC CGC AAG TGG TGG GGG CTC AAC 2631 Pro Thr Ser Phe His Arg Lys Trp Trp Gly Leu Asn 725 730 CTG CAG TTC CTG CTG GTT TTC CTC TGC ACC TTC ATG 2667 Leu Gln Phe Leu Leu Val Phe Leu Cys Thr Phe Met 735 740 CAG ATT GTC ATC TGT GTG ATC TGG CTC TAC ACC GCG 2703 Gln Ile Val Ile Cys Val Ile Trp Leu Tyr Thr Ala 745 750 755 CCC CCC TCA AGC TAC CGC AAC CAG GAG CTG GAG GAT 2739 Pro Pro Ser Ser Tyr Arg Asn Gln Glu Leu Glu Asp 760 765 GAG ATC ATC TTC ATC ACG TGC CAC GAG GGC TCC CTC 2775 Glu Ile Ile Phe Ile Thr Cys His Glu Gly Ser Leu 770 775 780 ATG GCC CTG GGC TTC CTG ATC GGC TAC ACC TGC CTG 2811 Met Ala Leu Gly Phe Leu Ile Gly Tyr Thr Cys Leu 785 790 CTG GCT GCC ATC TGC TTC TTC TTT GCC TTC AAG TCC 2847 Leu Ala Ala Ile Cys Phe Phe Phe Ala Phe Lys Ser 795 800 CGG AAG CTG CCG GAG AAC TTC AAT GAA GCC AAG TTC 2883 Arg Lys Leu Pro Glu Asn Phe Asn Glu Ala Lys Phe 805 810 815 ATC ACC TTC AGC ATG CTC ATC TTC TTC ATC GTC TGG 2919 Ile Thr Phe Ser Met Leu Ile Phe Phe Ile Val Trp 820 825 ATC TCC TTC ATT CCA GCC TAT GCC AGC ACC TAT GGC 2955 Ile Ser Phe Ile Pro Ala Tyr Ala Ser Thr Tyr Gly 830 835 840 AAG TTT GTC TCT GCC GTA GAG GTG ATT GCC ATC CTG 2991 Lys Phe Val Ser Ala Val Glu Val Ile Ala Ile Leu 845 850 GCA GCC AGC TTT GGC TTG CTG GCG TGC ATC TTC TTC 3027 Ala Ala Ser Phe Gly Leu Leu Ala Cys Ile Phe Phe 855 860 AAC AAG ATC TAC ATC ATT CTC TTC AAG CCA TCC CGC 3063 Asn Lys Ile Tyr Ile Ile Leu Phe Lys Pro Ser Arg 865 870 875 AAC ACC ATC GAG GAG GTG CGT TGC AGC ACC GCA GCT 3099 Asn Thr Ile Glu Glu Val Arg Cys Ser Thr Ala Ala 880 885 CAC GCT TTC AAG GTG GCT GCC CGG GCC ACG CTG CGC 3135 His Ala Phe Lys Val Ala Ala Arg Ala Thr Leu Arg 890 895 900 CGC AGC AAC GTC TCC CGC AAG CGG TCC AGC AGC CTT 3171 Arg Ser Asn Val Ser Arg Lys Arg Ser Ser Ser Leu 905 910 GGA GGC TCC ACG GGA TCC ACC CCC TCC TCC TCC ATC 3207 Gly Gly Ser Thr Gly Ser Thr Pro Ser Ser Ser Ile 915 920 AGC AGC AAG AGC AAC AGC GAA GAC CCA TTC CCA CGG 3243 Ser Ser Lys Ser Asn Ser Glu Asp Pro Phe Pro Arg 925 930 935 CCC GAG AGG CAG AAG CAG CAG CAG CCG CTG GCC CTA 3279 Pro Glu Arg Gln Lys Gln Gln Gln Pro Leu Ala Leu 940 945 ACC CAG CAA GAG CAG CAG CAG CAG CCC CTG ACC CTC 3315 Thr Gln Gln Glu Gln Gln Gln Gln Pro Leu Thr Leu 950 955 960 CCA CAG CAG CAA CGA TCT CAG CAG CAG CCC AGA TGC 3351 Pro Gln Gln Gln Arg Ser Gln Gln Gln Pro Arg Cys 965 970 AAG CAG AAG GTC ATC TTT GGC AGC GGC ACG GTC ACC 3387 Lys Gln Lys Val Ile Phe Gly Ser Gly Thr Val Thr 975 980 TTC TCA CTG AGC TTT GAT GAG CCT CAG AAG AAC GCC 3423 Phe Ser Leu Ser Phe Asp Glu Pro Gln Lys Asn Ala 985 990 995 ATG GCC CAC AGG AAT TCT ACG CAC CAG AAC TCC CTG 3459 Met Ala His Arg Asn Ser Thr His Gln Asn Ser Leu 1000 1005 GAG GCC CAG AAA AGC AGC GAT ACG CTG ACC CGA CAC 3495 Glu Ala Gln Lys Ser Ser Asp Thr Leu Thr Arg His 1010 1015 1020 CAG CCA TTA CTC CCG CTG CAG TGC GGG GAA ACG GAC 3531 Gln Pro Leu Leu Pro Leu Gln Cys Gly Glu Thr Asp 1025 1030 TTA GAT CTG ACC GTC CAG GAA ACA GGT CTG CAA GGA 3567 Leu Asp Leu Thr Val Gln Glu Thr Gly Leu Gln Gly 1035 1040 CCT GTG GGT GGA GAC CAG CGG CCA GAG GTG GAG GAC 3603 Pro Val Gly Gly Asp Gln Arg Pro Glu Val Glu Asp 1045 1050 1055 CCT GAA GAG TTG TCC CCA GCA CTT GTA GTG TCC AGT 3639 Pro Glu Glu Leu Ser Pro Ala Leu Val Val Ser Ser 1060 1065 TCA CAG AGC TTT GTC ATC AGT GGT GGA GGC AGC ACT 3675 Ser Gln Ser Phe Val Ile Ser Gly Gly Gly Ser Thr 1070 1075 1080 GTT ACA GAA AAC GTA GTG AAT TCA TAAAATGGAA 3709 Val Thr Glu Asn Val Val Asn Ser 1085 GGAGAAGACT GGGCTAGGGA GAATGCAGAG AGGTTTCTTG 3749 GGGTCCCAGG GATGAGGAAT CGCCCCAGAC TCCTTTCCTC 3789 TGAGGAAGAA GGGATAATAG ACACATCAAA TGCCCCGAAT 3829 TTAGTCACAC CATCTTAAAT GACAGTGAAT TGACCCATGT 3869 TCCCTTTAAA ATTAAAAAAA AGAAGAGCCT TGTGTTTCTG 3909 TGGTTGCATT TGTCAAAGCA TTGAGATCTC CACGGTCAGA 3949 TTTGCTGTTC ACCCACATCT AATGTCTCTT CCTCTGTTCT 3989 ATCCCACCCA ACAGCTCAGA GATGAAACTA TGGCTTTAAA 4029 CTACCCTCCA GAGTGTGCAG ACTGATGGGA CATCAAATTT 4069 GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC 4109 CAGTCCTGCC CAATGCCTTG ACAACAGACT GAATTTTAAA 4149 TGTTCACAAC ATAAGGAGAA TGTATCTCCT CCTATTTATG 4189 AAAACCATAT GATATTTTGT CTCCTACCTG CTGCTGCTAT 4229 TATGTAACAT CCAGAAGGTT TGCACCCCTC CTATACCATA 4269 TGTCTGGTTC TGTCCAGGAC ATGATACTGA TGCCATGTTT 4309 AGATTCCAGG ATCACAAGAA TCACCTCAAA TTGTTAGGAA 4349 GGGACTGCAT AAACCAATGA GCTGTATCTG TAATTAATAT 4389 TCCTATATGT AGCTTTATCC TTAGGAAAAT GCTTCTGTTG 4429 TAATAGTCCA TGGACAATAT AAACTGAAAA ATGTCAGTCT 4469 GGTTTATATA AGGCAGTATT ATTGAGCTCT ATTTCCCCAC 4509 CCCACTATCC TCACTCCCAT AAGCTAAGCC TTATGTGAGC 4549 CCCTTCAGGG ACTCAAGGGT CCAGAAGTCC CTCCCATCTC 4589 TACCCCAAAG AATTCCTGAA GCCAGATCCA CCCTATCCCT 4629 GTACAGAGTA AGTTCTCAAT TATTGGCCTG CTAATAGCTG 4669 CTAGGGTAGG AAAGCGTGGT TCCAAGAAAG ATCCACCCTC 4709 AAATGTCGGA GCTATGTTCC CTCCAGCAGT GGTATTAATA 4749 CTGCCGGTCA CCCAGGCTCT GGAGCCAGAG AGACAGACCG 4789 GGGTTCAAGC CATGGCTTCG TCATTTGCAA GCTGAGTGAC 4829 TGTAGGCAGG GAACCTTAAC CTCTCTAAGC CACAGCTTCT 4869 TCATCTTTAA AATAAGGATA ATAATCATTC CTTCCCCTCA 4909 GAGCTCTTAT GTGGATTAAA CGAGATAATG TATATAAAGT 4949 ACTTTAGCCT GGTACCTAGC ACACAATAAG CATTCAATAA 4989 ATATTAGTTA ATATTAT 5006 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3809 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 373.. 3606 (D) OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CAACAGGCAC CTGGCTGCAG CCAGGAAGGA CCGCACGCCC 40 TTTCGCGCAG GAGAGTGGAA GGAGGGAGCT GTTTGCCAGC 80 ACCGAGGTCT TGCGGCACAG GCAACGCTTG ACCTGAGTCT 120 TGCAGAATGA AAGGCATCAC AGGAGGCCTC TGCATGATGT 160 GGCTTCCAAA GACTCAAGGA CCACCCACAT TACAAGTCTG 200 GATTGAGGAA GGCAGAAATG GAGATTCAAA CACCACGTCT 240 TCTATTATTT TATTAATCAA TCTGTAGACA TGTGTCCCCA 280 CTGCAGGGAG TGAACTGCTC CAAGGGAGAA ACTTCTGGGA 320 GCCTCCAAAC TCCTAGCTGT CTCATCCCTT GCCCTGGAGA 360 GACGGCAGAA CC ATG GCA TTT TAT AGC TGC TGC TGG 396 Met Ala Phe Tyr Ser Cys Cys Trp 1 5 GTC CTC TTG GCA CTC ACC TGG CAC ACC TCT GCC TAC 432 Val Leu Leu Ala Leu Thr Trp His Thr Ser Ala Tyr 10 15 20 GGG CCA GAC CAG CGA GCC CAA AAG AAG GGG GAC ATT 468 Gly Pro Asp Gln Arg Ala Gln Lys Lys Gly Asp Ile 25 30 ATC CTT GGG GGG CTC TTT CCT ATT CAT. TTT GGA GTA 504 Ile Leu Gly Gly Leu Phe Pro Ile His Phe Gly Val 35 40 GCA GCT AAA GAT CAA GAT CTC AAA TCA AGG CCG GAG 540 Ala Ala Lys Asp Gln Asp Leu Lys Ser Arg Pro Glu 45 50 55 TCT GTG GAA TGT ATC AGG TAT AAT TTC CGT GGG TTT 576 Ser Val Glu Cys Ile Arg Tyr Asn Phe Arg Gly Phe 60 65 CGC TGG TTA CAG GCT ATG ATA TTT GCC ATA GAG GAG 612 Arg Trp Leu Gln Ala Met Ile Phe Ala Ile Glu Glu 70 75 80 ATA AAC AGC AGC CCA GCC CTT CTT CCC AAC TTG ACG 648 Ile Asn Ser Ser Pro Ala Leu Leu Pro Asn Leu Thr 85 90 CTG GGA TAC AGG ATA TTT GAC ACT TGC AAC ACC GTT 684 Leu Gly Tyr Arg Ile Phe Asp Thr Cys Asn Thr Val 95 100 TCT AAG GCC TTG GAA GCC ACC CTG AGT TTT GTT GCT 720 Ser Lys Ala Leu Glu Ala Thr Leu Ser Phe Val Ala 105 110 115 CAA AAC AAA ATT GAT TCT TTG AAC CTT GAT GAG TTC 756 Gln Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe 120 125 TGC AAC TGC TCA GAG CAC ATT CCC TCT ACG ATT GCT 792 Cys Asn Cys Ser Glu His Ile Pro Ser Thr Ile Ala 130 135 140 GTG GTG GGA GCA ACT GGC TCA GGC GTC TCC ACG GCA 828 Val Val Gly Ala Thr Gly Ser Gly Val Ser Thr Ala 145 150 GTG GCA AAT CTG CTG GGG CTC TTC TAC ATT CCC CAG 864 Val Ala Asn Leu Leu Gly Leu Phe Tyr Ile Pro Gln 155 160 GTC AGT TAT GCC TCC TCC AGC AGA CTC CTC AGC AAC 900 Val Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn 165 170 175 AAG AAT CAA TTC AAG TCT TTC CTC CGA ACC ATC CCC 936 Lys Asn Gln Phe Lys Ser Phe Leu Arg Thr Ile Pro 180 185 AAT GAT GAG CAC CAG GCC ACT GCC ATG GCA GAC ATC 972 Asn Asp Glu His Gln Ala Thr Ala Met Ala Asp Ile 190 195 200 ATC GAG TAT TTC CGC TGG AAC TGG GTG GGC ACA ATT 1008 Ile Glu Tyr Phe Arg Trp Asn Trp Val Gly Thr Ile -205 210 GCA GCT GAT GAC GAC TAT GGG CGG CCG GGG ATT GAG 1044 Ala Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Glu 215 220 AAA TTC CGA GAG GAA GCT GAG GAA AGG GAT ATC TGC 1080 Lys Phe Arg Glu Glu Ala Glu Glu Arg Asp Ile Cys 225 230 235 ATC GAC TTC AGT GAA CTC ATC TCC CAG TAC TCT GAT 1116 Ile Asp Phe Ser Glu Leu Ile Ser Gln Tyr Ser Asp 240 245 GAG GAA GAG ATC CAG CAT GTG GTA GAG GTG ATT CAA 1152 Glu Glu Glu Ile Gln His Val Val Glu Val Ile Gln 250 255 260 AAT TCC ACG GCC AAA GTC ATC GTG GTT TTC TCC AGT 1188 Asn Ser Thr Ala Lys Val Ile Val Val Phe Ser Ser 265 270 GGC CCA GAT CTT GAG CCC CTC ATC AAG GAG ATT GTC 1224 Gly Pro Asp Leu Glu Pro Leu Ile Lys Glu Ile Val 275 280 CGG CGC AAT ATC ACG GGC AAG ATC TGG CTG GCC AGC 1260 Arg Arg Asn Ile Thr Gly Lys Ile Trp Leu Ala Ser 285 290 295 GAG GCC TGG GCC AGC TCC TCC CTG ATC GCC ATG CCT 1296 Glu Ala Trp Ala Ser Ser Ser Leu Ile Ala Met Pro 300 305 CAG TAC TTC CAC GTG GTT GGC GGC ACC ATT GGA TTC 1332 Gln Tyr Phe His Val Val Gly Gly Thr Ile Gly Phe 310 315 320 GCT CTG AAG GCT GGG CAG ATC CCA GGC TTC CGG GAA 1368 Ala Leu Lys Ala Gly Gln Ile Pro Gly Phe Arg Glu 325 330 TTC CTG AAG AAG GTC CAT CCC AGG AAG TCT GTC CAC 1404 Phe Leu Lys Lys Val His Pro Arg Lys Ser Val His 335 340 AAT GGT TTT GCC AAG GAG TTT TGG GAA GAA ACA TTT 1440 Asn Gly Phe Ala Lys Glu Phe Trp Glu Glu Thr Phe 345 350 355 AAC TGC CAC CTC CAA GAA GGT GCA AAA GGA CCT TTA 1476 Asn Cys His Leu Gln Glu Gly Ala Lys Gly Pro Leu 360 365 CCT GTG GAC ACC TTT CTG AGA GGT CAC GAA GAA AGT 1512 Pro Val Asp Thr Phe Leu Arg Gly His Glu Glu Ser 370 375 380 GGC GAC AGG TTT AGC AAC AGC TCG ACA GCC TTC CGA 1548 Gly Asp Arg Phe Ser Asn Ser Ser Thr Ala Phe Arg 385 390 CCC CTC TGT ACA GGG GAT GAG AAC ATC AGC AGT GTC 1584 Pro Leu Cys Thr Gly Asp Glu Asn Ile Ser Ser Val 395 400 GAG ACC CCT TAC ATA GAT TAC ACG CAT TTA CGG ATA 1620 Glu Thr Pro Tyr Ile Asp Tyr Thr His Leu Arg Ile 405 410 415 TCC TAC AAT GTG TAC TTA GCA GTC TAC TCC ATT GCC 1656 Ser Tyr Asn Val Tyr Leu Ala Val Tyr Ser Ile Ala 420 425 CAC GCC TTG CAA GAT ATA TAT ACC TGC TTA CCT GGG 1692 His Ala Leu Gln Asp Ile Tyr Thr Cys Leu Pro Gly 430 435 440 AGA GGG CTC TTC ACC AAT GGC TCC TGT GCA GAC ATC 1728 Arg Gly Leu Phe Thr Asn Gly Ser Cys Ala Asp Ile 445 450 AAG AAA GTT GAG GCG TGG CAG GTC CTG AAG CAC CTA 1764 Lys Lys Val Glu Ala Trp Gln Val Leu Lys His Leu 455 460 CGG CAT CTA AAC TTT ACA AAC AAT ATG GGG GAG CAG 1800 Arg His Leu Asn Phe Thr Asn Asn Met Gly Glu Gln 465 470 475 GTG ACC TTT GAT GAG TGT GGT GAC CTG GTG GGG AAC 1836 Val Thr Phe Asp Glu Cys Gly Asp Leu Val Gly Asn 480 485 TAT TCC ATC ATC AAC TGG CAC CTC TCC CCA GAG GAT 1872 Tyr Ser Ile Ile Asn Trp His Leu Ser Pro Glu Asp 490 495 500 GGC TCC ATC GTG TTT AAG GAA GTC GGG TAT TAC AAC 1908 Gly Ser Ile Val Phe Lys Glu Val Gly Tyr Tyr Asn 505 510 GTC TAT GCC AAG AAG GGA GAA AGA CTC TTC ATC AAC 1944 Val Tyr Ala Lys Lys Gly Glu Arg Leu Phe Ile Asn 515 520 GAG GAG AAA ATC CTG TGG AGT GGG TTC TCC AGG GAG 1980 Glu Glu Lys Ile Leu Trp Ser Gly Phe Ser Arg Glu 525 530 535 GTG CCC TTC TCC AAC TGC AGC CGA GAC TGC CTG GCA 2016 Val Pro Phe Ser Asn Cys Ser Arg Asp Cys Leu Ala 540 545 GGG ACC AGG AAA GGG ATC ATT GAG GGG GAG CCC ACC 2052 Gly Thr Arg Lys Gly Ile Ile Glu Gly Glu Pro Thr 550 555 560 TGC TGC TTT GAG TGT GTG GAG TGT CCT GAT GGG GAG 2088 Cys Cys Phe Glu Cys Val Glu Cys Pro Asp Gly Glu 565 570 TAT AGT GAT GAG ACA GAT GCC AGT GCC TGT AAC AAG 2124 Tyr Ser Asp Glu Thr Asp Ala Ser Ala Cys Asn Lys 575 580 TGC CCA GAT GAC TTC TGG TCC AAT GAG AAC CAC ACC 2160 Cys Pro Asp Asp Phe Trp Ser Asn Glu Asn His Thr 585 590 595 TCC TGC ATT GCC AAG GAG ATC GAG TTT CTG TCG TGG 2196 Ser Cys Ile Ala Lys Glu Ile Glu Phe Leu Ser Trp 600 605 ACG GAG CCC TTT GGG ATC GCA CTC ACC CTC TTT GCC 2232 Thr Glu Pro Phe Gly Ile Ala Leu Thr Leu Phe Ala 610 615 620 GTG CTG GGC ATT TTC CTG ACA GCC TTT GTG CTG GGT 2268 Val Leu Gly Ile Phe Leu Thr Ala Phe Val Leu Gly 625 630 GTG TTT ATC AAG TTC CGC AAC ACA CCC ATT GTC AAG 2304 Val Phe Ile Lys Phe Arg Asn Thr Pro Ile Val Lys 635 640 GCC ACC AAC CGA GAG CTC TCC TAC CTC CTC CTC TTC 2340 Ala Thr Asn Arg Glu Leu Ser Tyr Leu Leu Leu Phe 645 650 655 TCC CTG CTC TGC TGC TTC TCC AGC TCC CTG TTC TTC 2376 Ser Leu Leu Cys Cys Phe Ser Ser Ser Leu Phe Phe 660 665 ATC GGG GAG CCC CAG GAC TGG ACG TGC CGC CTG CGC 2412 Ile Gly Glu Pro Gln Asp Trp Thr Cys Arg Leu Arg 670 675 680 CAG CCG GCC TTT GGC ATC AGC TTC GTG CTC TGC ATC 2448 Gln Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile 685 690 TCA TGC ATC CTG GTG AAA ACC AAC CGT GTC CTC CTG 2484 Ser Cys Ile Leu Val Lys Thr Asn Arg Val Leu Leu 695 700 GTG TTT GAG GCC AAG ATC CCC ACC AGC TTC CAC CGC 2520 Val Phe Glu Ala Lys Ile Pro Thr Ser Phe His Arg 705 710 715 AAG TGG TGG GGG CTC AAC CTG CAG TTC CTG CTG GTT 2556 Lys Trp Trp Gly Leu Asn Leu Gln Phe Leu Leu Val 720 725 TTC CTC TGC ACC TTC ATG CAG ATT GTC ATC TGT GTG 2592 Phe Leu Cys Thr Phe Met Gln Ile Val Ile Cys Val 730 735 740 ATC TGG CTC TAC ACC GCG CCC CCC TCA AGC TAC CGC 2628 Ile Trp Leu Tyr Thr Ala Pro Pro Ser Ser Tyr Arg 745 750 AAC CAG GAG CTG GAG GAT GAG ATC ATC TTC ATC ACG 2664 Asn Gln Glu Leu Glu Asp Glu Ile Ile Phe Ile Thr 755 760 TGC CAC GAG GGC TCC CTC ATG GCC CTG GGC TTC CTG 2700 Cys His Glu Gly Ser Leu Met Ala Leu Gly Phe Leu 765 770 775 ATC GGC TAC ACC TGC CTG CTG GCT GCC ATC TGC TTC 2736 Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys Phe 780 785 TTC TTT GCC TTC AAG TCC CGG AAG CTG CCG GAG AAC 2772 Phe Phe Ala Phe Lys Ser Arg Lys Leu Pro Glu Asn 790 795 800 TTC AAT GAA GCC AAG TTC ATC ACC TTC AGC ATG CTC 2808 Phe Asn Glu Ala Lys Phe Ile Thr Phe Ser Met Leu 805 810 ATC TTC TTC ATC GTC TGG ATC TCC TTC ATT CCA GCC 2844 Ile Phe Phe Ile Val Trp Ile Ser Phe Ile Pro Ala 815 820 TAT GCC AGC ACC TAT GGC AAG TTT GTC TCT GCC GTA 2880 Tyr Ala Ser Thr Tyr Gly Lys Phe Val Ser Ala Val 825 830 835 GAG GTG ATT GCC ATC CTG GCA GCC AGC TTT GGC TTG 2916 Glu Val Ile Ala Ile Leu Ala Ala Ser Phe Gly Leu 840 845 CTG GCG TGC ATC TTC TTC AAC AAG ATC TAC ATC ATT 2952 Leu Ala Cys Ile Phe Phe Asn Lys Ile Tyr Ile Ile 850 855 860 CTC TTC AAG CCA TCC CGC AAC ACC ATC GAG GAG GTG 2988 Leu Phe Lys Pro Ser Arg Asn Thr Ile Glu Glu Val 865 870 CGT TGC AGC ACC GCA GCT CAC GCT TTC. AAG GTG GCT 3024 Arg Cys Ser Thr Ala Ala His Ala Phe Lys Val Ala 875 880 GCC CGG GCC ACG CTG CGC CGC AGC AAC GTC TCC CGC 3060 Ala Arg Ala Thr Leu Arg Arg Ser Asn Val Ser Arg 885 890 895 AAG CGG TCC AGC AGC CTT GGA GGC TCC ACG GGA TCC 3096 Lys Arg Ser Ser Ser Leu Gly Gly Ser Thr Gly Ser 900 905 ACC CCC TCC TCC TCC ATC AGC AGC AAG AGC AAC AGC 3132 Thr Pro Ser Ser Ser Ile Ser Ser Lys Ser Asn Ser 910 915 920 GAA GAC CCA TTC CCA CAG CCC GAG AGG CAG AAG CAG 3168 Glu Asp Pro Phe Pro Gln Pro Glu Arg Gln Lys Gln 925 930 CAG CAG CCG CTG GCC CTA ACC CAG CAA GAG CAG CAG 3204 Gln Gln Pro Leu Ala Leu Thr Gln Gln Glu Gln Gln 935 940 CAG CAG CCC CTG ACC CTC CCA CAG CAG CAA CGA TCT 3240 Gln Gln Pro Leu Thr Leu Pro Gln Gln Gln Arg Ser 945 950 955 CAG CAG CAG CCC AGA TGC AAG CAG AAG GTC ATC TTT 3276 Gln Gln Gln Pro Arg Cys Lys Gln Lys Val Ile Phe 960 965 GGC AGC GGC ACG GTC ACC TTC TCA CTG AGC TTT GAT 3312 Gly Ser Gly Thr Val Thr Phe Ser Leu Ser Phe Asp 970 975 980 GAG CCT CAG AAG AAC GCC ATG GCC CAC GGG AAT TCT 3348 Glu Pro Gln Lys Asn Ala Met Ala His Gly Asn Ser 985 990 ACG CAC CAG AAC TCC CTG GAG GCC CAG AAA AGC AGC 3384 Thr His Gln Asn Ser Leu Glu Ala Gln Lys Ser Ser 995 1000 GAT ACG CTG ACC CGA CAC CAG CCA TTA CTC CCG CTG 3420 Asp Thr Leu Thr Arg His Gln Pro Leu Leu Pro Leu 1005 1010 1015 CAG TGC GGG GAA ACG GAC TTA GAT CTG ACC GTC CAG 3456 Gln Cys Gly Glu Thr Asp Leu Asp Leu Thr Val Gln 1020 1025 GAA ACA GGT CTG CAA GGA CCT GTG GGT GGA GAC CAG 3492 Glu Thr Gly Leu Gln Gly Pro Val Gly Gly Asp Gln 1030 1035 1040 CGG CCA GAG GTG GAG GAC CCT GAA GAG TTG TCC CCA 3528 Arg Pro Glu Val Glu Asp Pro Glu Glu Leu Ser Pro 1045 1050 GCA CTT GTA GTG TCC AGT TCA CAG AGC TTT GTC ATC 3564 Ala Leu Val Val Ser Ser Ser Gln Ser Phe Val Ile 1055 1060 AGT GGT GGA GGC AGC ACT GTT ACA GAA AAC GTA GTG 3600 Ser Gly Gly Gly Ser Thr Val Thr Glu Asn Val Val 1065 1070 1075 AAT TCA TAAAATGGAA GGAGAAGACT GGGCTAGGGA 3636 Asn Ser GAATGCAGAG AGGTTTCTTG GGGTCCCAGG GATGAGGAAT 3676 CGCCCCAGAC TCCTTTCCTC TGAGGAAGAA GGGATAATAG 3716 ACACATCAAA TGCCCCGAAT TTAGTCACAC CATCTTAAAT 3756 GACAGTGAAT TGACCCATGT TCCCTTTAAA AAAAAAAAAA 3796 AAAAAGCGGC CGC 3809