AU747853B2 - Calcium receptor-active compounds - Google Patents

Description

S&F Ref: 376314D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

*j.

C

Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: NPS Pharmaceuticals, Inc.

Suite 240 420 Chipeta Way Salt Lake City Utah 84108-1256 United States of America Bradford C Van Wagenen, Scott T Moe, Manuel Balandrin, Eric G Delmar, Edward F Nemeth Spruson Ferguson St Martins Tower 31 Market Street Sydney NSW 2000 Calcium Receptor-active Compounds

C.

C

CC

C The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c

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.

Backaround of the Invention Certain cells in the body respond not only to chemical signals, but also to ions such as extracellular calcium ions (Ca 2 Changes in the concentration of *extracellular Ca^ (referred to herein as alter 10 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 Ca homeostasis in the blood and extracellular fluids.

15 PTH, by acting on bone and kidney cells, increases the level of Ca 2 in the blood. This increase in [Ca 2 then acts as a negative feedback signal, depressing PTH secretion. The reciprocal relationship between [Ca 2 and PTH secretion forms the essential mechanism maintaining 20 bodily Ca 2 homeostasis.

Extracellular Ca 2 acts directly on parathyroid cells to regulate PTH secretion. The existence of a parathyroid cell surface protein which detects changes in [Ca 2 has been confirmed. Brown et al., 366 Nature 574, 1993. In parathyroid cells, this protein acts as a receptor for extracellular Ca 2 ("the calcium receptor"), and detects changes in [Ca 2 and to initiate a functional cellular response, PTH secretion.

Extracellular Ca 2 can exert effects on different cell functions, reviewed in Nemeth et al., 11 Cell Calcium 319, 1990. The role of extracellular Ca in parafollicular (Ccells) 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 extracellular Ca 2 on bone osteoclasts is discussed by Zaidi, Bioscience Reports 493, 1990. In addition keratinocytes, juxtaglomerular cells, trophoblasts, pancreatic beta cells and fat/adipose cells all respond to increases in extracellular calcium which likely reflects activation of calcium receptors of these cells.

The ability of various compounds to mimic extracellular Ca 2 in vitro is discussed by Nemeth et al., 15 (spermine and spermidine) in "Calcium-Binding Proteins in Health and Disease," 1987, Academic Press, Inc., pp. 33-35; Brown et al., neomycin) 128 Endocrinology 3047, 1991; Chen et al., (diltiazem and its analog, TA-3090) 5 J. Bone and Mineral Res. 581, 1990; and Zaidi 20 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 25 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.

According to a first embodiment -of the present invention there is provided a process for producing a medicament comprising the step of combining a therapeutically effective amount of any one of the compounds selected from the group consisting of: N-(2-chlorophenylpropyl)- 1-(3-(2,2,2-trifluoroethoxy)phenyl)ethylamine -tri fluoromethoxy)propyl)- 1-(3 -methoxyphenyl)ethylamine (21 M); -(2-chlorophenyl)propyl)- 1 -propoxyphenyl)ethylamine (21 S); -(2-chlorophenyl)propyl)- 1-(3 -isopropoxyphenyl)ethylamine (21 T); -(2-chlorophenyl)propyl)- 1-(3 -isobutoxyphenyl)ethylamine (21 U); -(trifluoromethyl)phenyl)-2-butyl)- 1-(3 -methoxyphenyl)ethylamine (2 1Y); -(trifluoromethyl)phenyl)propyl)- 1-(1 -naphthyl)ethylamine (22J); -(trifluoromethoxy)phenyl)-2-butyl)-l1-(3 -methoxyphenyl)ethylamine (23A); -(trifluoromethoxy)phenyl)methyl)- 1 -naphthyl)ethylamine (23E); -methyl-4-methoxyphenyl)methyl)- 1-(2-(trifluoromethyl)phenyl)ethylamine (24B); -(3-(trifluoromethoxy)phenyl)propyl)- 1-(1 -naphthyl)ethylamine (24J); -difluorophenyl)propyl)- 1-(3-methoxyphenyl)ethylamine (24M); -methyl-4-methoxyphenyl)methyl)- 1-(3 -(ethylacetoxy)phenyl)ethylamine 20 (24V); -bromo-4-methoxyphenyl)methyl)- 1 -naphthyl)ethylamnine (24X); -chloro-4-ethoxyphenyl)methyl)- 1 -naphthyl)ethylamine (24Y); -tri fluoromethyl)phenyl)-2-butyl)- 1-(1 -naphthyl)ethylamine (25 C); -trifluoromethyl)phenyl)-2-butyl)- 1-(1 -naphthyl)ethylamine and -phenylprop-2-en- 1-yl)- 1-(3 -methoxyphenyl)ethylamine (25E); and a pharmnaceutically acceptable carrer.

According to a second embodiment of the present invention there is provided a process for producing a medicament comprising the step of combining a therapeutically effective amount of a compound having the formula: R9 Ar 3 N Ar 4 R- RD H 3 wherein Ar 3 is either naphthyl or phenyl optionally substituted with 1 to substituents each independently selected from the group consisting of halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH,

CH

2 OH, CONH 2 CN, acetoxy, benzyl, benzyloxy, dimethylbenzyl, NO 2

CHO,

CH

3 CH(OH), N(CH 3 2 acetyl, and ethylene dioxy; Ar 4 is either naphthyl or phenyl optionally substituted with 1 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, CH 2

OH,

10 CONH 2 CN, and acetoxy; provided that if Ar 3 and Ar 4 are both optionally substituted phenyl, then Ar 3 comprises at least one substituent and Ar 4 comprises at least one substituent, and if Ar 3 is 2-methoxyphenyl, then Ar 4 is not 3-methoxyphenyl;

R

8 is either hydrogen or phenyl; 15 R 9 is either hydrogen or methyl; and R10 is either hydrogen, methyl, or phenyl; a and a pharmaceutically acceptable carrier.

According to a third embodiment of the present invention there is provided a process for producing a medicament comprising the step of combining a therapeutically S 20 effective amount of a compound having the formula:

H

N Ar

R

1 1

R

12 wherein Ar 5 is either naphthyl or phenyl optionally substituted with 0 to substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH 2 0H, CONH 2 CN, acetoxy, benzyl, benzyloxy, a,adimethylbenzyl, NO 2 CHO, CH 3 CH(OH), acetyl, ethylene dioxy, and -CH=CH-phenyl; Ar 6 is phenyl substituted with 1 to 5 substituents each independently selected from ^the group consisting of acetyl, lower alkyl, halogen, lower alkoxy, lower thioalkyl, [R:\LIBVV]47036.doc:ais methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH 2 OH, CONII 2

CN,

carbomethoxy, OCH 2

C(O)C

2

H

5 and OCH 2 C(O)0C 2

H

5 and acetoxy, provided that at least one substituent is OCH 2 C(O)0C 2

H

5 RI 1 is hydrogen or methyl; and R 1 2 is hydrogen or methyl; provided that at least one of RI I and RI 2 is methyl; and a pharmaceutically acceptable camrier.

The present invention further provides for medicaments produced by the above defined processes.

R:\LIBVV147036.doc:ais Diseases or disorders which can be treated by modulating inorganic ion receptor activity include one or more of the following types: those characterized by abnormal inorganic ion homeostasis, preferably calcium homeostasis; 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; those characterized by an abnormal effect a different effect in kind or magnitude) of an intracellular or extracellular messenger which can itself be ameliorated by inorganic ion receptor activity, preferably calcium receptor activity; and other diseases or disorders in which modulation of inorganic ion receptor activity, preferably calcium receptor activity will exert a bene- Sficial 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, IP 3 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 Ca 2 concentration. For example, extracellular Ca 2 inhibits the secretion of parathyroid hormone from parathyroid 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- 4 stasis. Extracellular Ca 2 is 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: an abnormal increase or decrease in serum calcium; an abnormal increase or decrease in urinary excretion of calcium; an abnormal increase or decrease in bone calcium levels, for example, as assessed by bone mineral density measurements; an abnormal absorption of dietary calcium; 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 an abnormal change in the response 15 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.

20 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 25 example, in parathyroid cells, the calcium receptor is coupled to the G i protein which in turn inhibits cyclic AMP production. Defects in G i 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

H

N Ar 2 Arl 7 R

CH

3 where Ar, is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, 5 halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH 2 OH, CONH,, CN, acetoxy, N(CH3),, phenyl, phenoxy, benzyl, benzyloxy, a,adimethylbenzyl, NO,, CHO, CH3CH(OH), acetyl, ethylene dioxy; 10 Ar, 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.

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 stereochemistry. The CH 3 shown in Structure I is at a chiral center and provides an a-(R)-methyl structure. When R is

CH

3 the R shown in Structure I is also at chiral center which provides an (R)-methyl structure. Thus, when R is

CH

3 the Structure I compound has stereochemistry.

Inorganic ion receptor activities are those processes brought about as a result of inorganic ion receptor activation. Such processes include the production of molecules 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 evoke or potentiate) the effects of an inorganic ion at an inorganic ion receptor. Preferably, 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.

lonolytics are compounds which bind to an inorganic ion receptor and block inhibit or diminish) one or more activities caused by an inorganic ion at an inorganic ion receptor. Preferably, the compound affects one or •*eg more calcium receptor activities. Calcilytics are iono- 15 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 S..receptor site as the native inorganic ion ligand binds or 20 can bind at a different site 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 an 25 allosteric site) than extracellular calcium.

A measure of a compounds effectiveness can be determined by calculating the ECs, or IC,, for that compound.

The ECs is the concentration of a compound which causes a half maximal mimicking effect. The IC 0 is the concentration of compound which causes a half-maximal blocking effect. ECs, and IC 50 for compounds at a calcium receptor can be determined by assaying one or more of the activities of extracellular calcium at a calcium receptor.

Examples of assays for measuring EC 50 and ICs 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 incorporated by reference here) and below. Such assays include oocyte expression assays and measuring increases in intracellular calcium ion concentration ([Ca 2 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 preferably 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 •o 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 measured in vivo and the compound concentration is measured as the plasma concentration or extracellular fluid concentration and the measured effect is the production of 20 extracellular messengers such as plasma calcitonin, parathyroid hormone, or plasma calcium. For example, in Sa preferred embodiment, the compound selectively targets PTH secretion over calcitonin secretion.

Preferably, the compound is either a calcimimetic or 25 calcilytic having an EC, 5 or ICs, at a calcium receptor of less than or equal to 5 pM, and even more preferably less than or equal to 1 pM, 100 nmolar, 10 nmolar, or 1 nmolar using one of the assays described below. More preferably, the assay measures intracellular Ca 2 in HEK 293 cells transformed with nucleic acid expressing the human parathyroid calcium receptor and loaded with fura-2. Lower

EC

0 or IC 5 are advantageous since they allow lower concentrations of compounds to be used in vivo or in vitro. The discovery of compounds with low EC, 5 's and

IC

0 's enables the design and synthesis of additional compounds having similar or improved potency, effectiveness, and/or selectivity.

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

H

Ar 3 N Ar 4 RiO CH3 5 where Ar 3 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, CH 2 OH, CONH,, CN, acetoxy, benzyl, benzyloxy, a,e-dimethylbenzyl, NO,, CHO,

CH

3 CH(OH), N(CH 3 2 acetyl, ethylene dioxy.

Ar 4 is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of, lower alkyl, 15 halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CHOH, CONH,, CN, and acetoxy; R, is either hydrogen or phenyl;

R

9 is either hydrogen or methyl; and

R

1 i 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

H

Aro N Ar 6 R11 R 12 where Ar s 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, CH 2 OH, CONH,, CN, acetoxy, benzyl, benzyloxy, a,a-dimethylbenzyl, NO 2

CHO,

"CH

3 CH(OH), acetyl, ethylene dioxy, -CH=CH-phenyl; Ar 6 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, CH 2 OH, CONH,, CN, carbomethoxy, OCH 2

C(O)C

2

H

5 and acetoxy: 15 R, 1 is hydrogen or methyl; and

R

12 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 administration 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 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 preventing 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 administering to the patient a pharmaceutical composition conaining 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 receptormodulating 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 >ave a beneficial 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; 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 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 homeostasis include hyperparathyroidism, osteoporosis and other bone and mineral-related disorders, and the like (as described, 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 Ca 2 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 compound 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, gastrinsecreting 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 Ca 2 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 15 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.

"SThese examples are not intended in any way to limit the 25 invention.

Other features and advantages of the invention will be apparent from the following figures, detailed description 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 Ca 2 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 i. 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.

*S* e I. Calcium ReceDtors 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, 25 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. 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 subfornical 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 3 while osteoclasts respond to divalent cations such as calcium, but do not respond to Gd'. 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 20 and C-cells indicate that these receptors have a very similar amino acid structure. Nevertheless, calcimimetic compounds exhibit differential pharmacology and regulate Sdifferent activities at parathyroid cells and C-cells.

Thus, pharmacological properties of calcium receptors may 25 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 Ca" (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, calmodulin and the troponins.

Calcium receptors respond to changes in extracellular calcium levels. The exact changes depend on the particular 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 rapid (time to peak 5 seconds) and transient increase in [Ca 2 that is refractory to inhibition by 1 pM La 3 or 1 pM Gd 3 and is abolished by pretreatment with ionomycin (in the absence of extracellular Ca 2 The increase is not inhibited by dihydropyridines; The transient increase is abolished by pre- 15 treatment for 10 minutes with 10 mM sodium fluoride; 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 Pretreatment with pertussis toxin (100 ng/ml for 4 hours) does not affect the increase.

25 2. A rapid 30 seconds) increase in the formation of inositol-1,4,5-triphosphate or diacylglycerol. Pretreatment with pertussis toxin (100 ng/ml for 4 hours) does not affect this increase; 3. The inhibition of dopamine- and isoproterenolstimulated 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 Modulating 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 compounds can be identified by screening compounds which are Smodelled after a compound shown to have a particular activity a lead compound).

A preferred method of measuring calcium receptor activity is to measure changes in [Ca 2 Changes in [Ca 2 can be measured using different techniques such by using HEK 293 cells transduced with nucleic acid expressing 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 *oo 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 expressing a calcium receptor, such as a parathyroid cell, bone 25 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 resorption 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 secretion 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 25 concentration 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.

In another preferred embodiment, the compound has an

EC

5 or IC, 5 less than or equal to 5 pM at one or more, but not all cells chosen 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, glucagonsecreting 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. 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 compounds 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 different, effects on cells having different types of inorganic ion receptor morphology such as cells having normal inorganic ion receptors, a normal number of inor- 25 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 extracellular Ca 2 Similarly, calcilytics need not block all of the activities caused by extracellular calcium. Additionally, different calcimimetics and different calcilytics do not need to bind to the same site on the calcium receptor as does extracellular Ca 2 to exert their effects.

Inorganic modulating compounds need not effect inorganic receptor activity to the same extent or in exactly 19 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 affinity, 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:

H

N Ar 2 SArlq

-Y

R CH3 10 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, CONH,, CN, 15 acetoxy, N(CH 3 2 phenyl, phenoxy, benzyl, benzyloxy, at,adimethylbenzyl, NO CHO, CH3CH(OH) acetyl, ethylene dioxy, preferably each substituent is independently selected from the group consisting of, CH 3

CH

3 O, CH 3

CH

2

O,

methylene dioxy, Br, Cl, F, I, CF3, CHF,, CHF,

CF

3 CH20, CH 3 S, OH, CH 2 OH, CONH,, CN, NO 2

CH

3 CH,, propyl, isopropyl, butyl, isobutyl, t-butyl, and acetoxy. More preferably, Ar 1 is either a naphthyl or a phenyl having substituents each independently selected from the group consisting of isopropyl, CH 3 O, CH 3 S, CF30, I, Cl, F, CF 3 and CH 3 more preferably CF 3 O, I, Cl, F, and CF 3 Ar, 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, CH 2 OH, CONH 2

CN,

and acetoxy, preferably each substituent is independently selected from the group consisting of, CH 3

CH

3 O, CH 3

CH

2

O,

methylene dioxy, Br, Cl, F, I, CF3, CHF 2

CH

2 F, CF 3 0,

CF

3

CH

2 0, CH 3 S, OH, CH 2 OH, CONH,, CN, NO 2

CH

3

CH

2 propyl, isopropyl, butyl, isobutyl, t-butyl, and acetoxy. More preferably, Ar 2 is either a naphthyl or a phenyl having substituents each independently selected from the group consisting of isopropyl, CH 3 O, CH 3 S, CF30, I, Cl, F, CF 3 and CH 3 more preferably CF30, I, Cl, F, CH30, and CF 3 q is 0, 1, 2, or 3; and R is either H, or CH 3 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 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 25 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 is an oxygen joined to a lower haloalkyl.

a. Ar, and Ar 2 are Both Optionally Substituted Phenl s In a preferred embodiment both Ar 1 and Ar 2 are optionally substituted phenyls and the compound has following formula: H II Zm

N

R CH 3 where R is hydrogen or methyl m and n are each independently 0, 1, 2, 3, 4, or each X is independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower 5 thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH 2 OH, CONH 2 CN, acetoxy, N(CH 3 2 phenyl, phenoxy, benzyl, benzyloxy, a,a-dimethylbenzyl, NO 2

CHO,

CH

3 CH(OH), acetyl, ethylene dioxy. Preferably each X is independently selected from the group consisting of, CH 3 CH30, CH 3 CH20, methylene dioxy, Br, Cl, F, I, CF3, CHF,,

CH

2 F, CF 3 0, CF 3

CH

2 O, CH 3 S, OH, CH 2 OH, CONH 2 CN, NO 2 CH3CH 2 propyl, isopropyl, butyl, isobutyl, t-butyl, and acetoxy.

More preferably, each X is independently selected from the group consisting of isopropyl, CH 3 0, CH 3 S, CF 3 O, I, Cl, F, 15 CF3, and CH 3 more preferably CF 3 O, I, Cl, F, and CF 3 each Z is independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH 2 OH, CONH,, CN, and acetoxy. Preferably each Z is independently selected from the group consisting of,

CH

3

CH

3 O, CH 3

CH

2 O, methylene dioxy, Br, Cl, F, I, CF 3

CHF

2

CH

2 F, CF 3 0, CF 3 CH0O, CH 3 S, OH, CH 2 OH, CONH 2 CN, CH 3

CH

2 propyl, isopropyl, butyl, isobutyl, t-butyl, and acetoxy.

More preferably, each Z is independently selected from the group consisting of, isopropyl, CHO3, CH 3 S, CF 3 0, CF 3

I,

Cl, F, and CH 3 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: R CH 3 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 5 thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH 2 OH, CONH 2 CN, acetoxy, N(CH 3 phenyl, phenoxy, benzyl, benzyloxy, c,a-dimethylbenzyl, NO2, CHO,

CH

3 CH(OH), acetyl, ethylene dioxy, preferably each substituent is independently selected from the group consisting of, CH 3

CH

3 O, CH 3 CH20, methylene dioxy, Br, Cl, F, I, CF3,

CHF

2

CH

2 F 30, CF0 CFCH 2 O, CH 3 S, OH, CH 2 OH, CONH 2 CN, NO

CH

3 CH,, propyl, isopropyl, butyl, isobutyl, t-butyl, and acetoxy, more preferably, isopropyl, CH30, CH 3 S, CF30, CF 3 I, Cl, F, and CH3.

More preferably, the compound has the formula:

R

1 *ei *OC3 3 R CH 3 where R is either hydrogen or methyl; R, is either halogen or hydrogen, preferably R, is either F, or hydrogen;

R

2 is either hydrogen, halogen, lower alkyl, lower haloalkyl, or lower haloalkoxy, preferably, R 2 is either hydrogen, CF 3 CH3, OCF 3 or F, and

R

3 is either hydrogen, halogen, or alkoxy, preferably,

R

3 is either Cl, F, hydrogen, or methoxy, more preferably methoxy.

In alternative more preferred combinations; at least two of R 1

R

2 and R 3 is halogen, preferably F and R is hydrogen or CH 3 R is hydrogen or CH3, R 2 is either lower haloalkyl, or lower haloalkoxy, preferably OCF 3 or CF3, and RI and R 3 is hydrogen; and R is CH 3

R

3 is halogen, preferably Cl, R, is either halogen or hydrogen, preferably F or hydrogen, and R 2 is either hydrogen, lower alkyl, lower haloalkyl, or lower haloalkoxy, preferably, hydrogen, CF3, 15 CH 3

OCF

3 or F.

b. Ar, is NaDhthyl and a is 0 In another preferred embodiment, Ar 2 is naphthyl, q is 0, and the compound has the formula:

H

SAr 1

N

4* C where Ar 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, CH 2 OH, CONH,, CN, acetoxy, N(CH 3 phenyl, phenoxy, benzyl, benzyloxy, oa,adimethylbenzyl, NO CHO, CH 3 CH(OH), acetyl, ethylene dioxy, preferably each substituent is independently selected from the group consisting of, CH 3

CH

3 0, CH 3 CH0O, methylene dioxy, Br, Cl, F, I, CF3, CHF,, CH 2 F, CFO0,

CF

3 CH20, CH3S, OH, CH 2 OH, CONH,, CN, NO 2

CH

3

CH

2 propyl, isopropyl, butyl, isobutyl, t-butyl, and acetoxy. More preferably, Ar, is either a naphthyl or a phenyl having substituents each independently selected from the group consisting of isopropyl, CH 3 O, CH 3 S, CF3, CF30 I, Cl, F, and

CH

3 More preferably, Ar, is an optional substituted phenyl where the compound has the formula: R CH3

S

go C f 10 where Xi 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:

S*

CH3 where R is either CH 3 or hydrogen; R, is either lower alkyl, halogen, or alkoxy, preferably isopropyl, chlorine, or methoxy; and

R

5 is either hydrogen, lower alkyl, or halogen, preferably methyl, CH 3 Br, or Cl.

c. Ar, is NaDhthl and a is 2 In another preferred embodiment, Ar, is a substituted phenyl, Ar 2 is naphthyl, q is 2 and the compound has the formula: x

H

Ny R cl-b 0. where R is either hydrogen or CH 3 *:00 .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, CH, 2 OH, CONH 2 CN, acetoxy, N(CH 3 2 phenyl, S* phenoxy, benzyl,. benzyloxy, Q,a-dimethylbenzyl, NO 2

CHO,

CH

3 CH(OH), acetyl, ethylene dioxy, preferably each .substituent is independently selected from the group 15 consisting of, CH 3

CH

3 O, CH 3 CH20, methylene dioxy, Br, Cl, F, I, CF 3

CHF

2

CH

2 F, CF30, CF 3

CH

2 0, CH 3 S, OH, CH20H, CONH,, CN, NO,, CH 3 CH,, propyl, isopropyl, butyl, isobutyl, S. t-butyl, and acetoxy, more preferably, isopropyl, CH 3 0,

CH

3 S, CF 3 O, CF 3 I, C1, F, and CH 3 More preferably, the compound has the formula: where R, is either is either hydrogen, lower haloalkyl, or lower haloalkoxy, preferably hydrogen, OCF3 or CF 3 and R, is either halogen or hydrogen, preferably chlorine or hydrogen.

In other embodiments R, R 6 and R, are as described above (with the preferred substituents as described above), provided that when both R and R 6 are hydrogen, R, is not Cl; and R is CH 3 and R 6 and R, is as described above (with the preferred substituents as described above).

2. Structure II Compounds Structure II compounds have the formula:

H

H

SAr 3 N Ar 4 .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, CH 2 OH, CONH,, CN, acetoxy, benzyl, benzyloxy, a,a-dimethylbenzyl,

NO

2

CHO,

CH

3 CH(OH), N(CH 3 2 acetyl, ethylene dioxy, preferably

N(CH

3 2 lower alkoxy, or lower alkyl; Ar, 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, CHOH, CONH 2

CN,

and acetoxy, preferably lower alkoxy, more preferably methoxy; R. is either hydrogen or phenyl, preferably hydrogen;

R

9 is either hydrogen or methyl; and

R,

1 is either hydrogen, methyl, or phenyl, more preferably when R, 0 is methyl the chiral carbon it is attached to is the stereoisomer.

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

3. Structure III Compounds Structure III compounds have the formula:

H

N Ar 6 R11 R12 where Ar 5 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, CH 2 OH, CONH 2

CN,

acetoxy, benzyl, benzyloxy, a,a-dimethylbenzyl, NO 2

CHO,

15 CH 3 CH(OH), acetyl, ethylene dioxy, -CH=CH-phenyl, preferably, lower alkyl, phenoxy, -CH=CH-phenyl, dimethylbenzyl, methoxy, methylene, or ethylene; Ar 6 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, CH 2 OH, CONH 2 CN, carbomethoxy, OCH 2 C(O) C 2

H

5 and acetoxy, preferably methoxy, lower alkyl, phenyl, halogen, CF 3 CN, carbomethoxy or,

OCH

2

C(O)C

2

H

s R, is hydrogen or methyl, preferably when R 1 is methyl the carbon to which it is attached is an (R) stereoisomer; and

R,

2 is hydrogen or methyl, preferably when R 12 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 Ca 2 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 15 1 pM La 3 or 1 pM Gd 3 The increase in [Ca 2 i persists in the absence of extracellular Ca 2 but is abolished by pretreatment with ionomycin (in the absence of extracellular Ca 2 2. The compound potentiates increases in [Ca 2 elicited by submaximal concentrations of extracellular Ca 2 2 3. The increase in [Ca 2 elicited by extracellular Ca 2 is not inhibited by dihydropyridines; 4. The transient increase in [Ca 2 i caused by 25 the compound is abolished by pretreatment for 10 minutes with 10 mM sodium fluoride; The transient increase in [Ca 2 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 isoproterenol-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 [Ca 2 inositol-1,4,5-triphosphate, or diacylglycerol, nor decreases in PTH secretion; The compound elicits increases in Clcurrent 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 *15 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, 20 International Publication Number WO 94/18959.) Parallel definitions of compounds mimicking Ca 2 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 25 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 [Ca 2 occurring within thirty seconds; evokes a sustained increase (greater than thirty seconds) in [Ca 2 (preferably 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 [Ca 2 is preferably abolished by pretreatment of the cell for ten minutes with 10 mM sodium fluoride, or the transient increase is diminished by brief pretreatment (not more than ten minutes) of the cell with an activator of protein kinase C, preferably, phorbol myristate acetate (PMA), mezerein or indolactam V.

C. Calcilvtics The ability of a compound to block the activity of extracellular calcium at a calcium receptor can be determined using standard techniques based on the present disclosure. (See, also Nemeth et al., PCT/US93/01642, 15 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 Ca 2 to: a increase [Ca 2 mobilize intracellular Ca", increase the formation of inositol-1,4,5triphosphate, decrease dopamine- or isoproterenolstimulated cyclic AMP formation, and inhibit PTH secretion; The compound blocks increases in Cl- current in Xenopus oocytes injected with poly(A)'-mRNA from bovine or human parathyroid cells elicited by extracellular Ca 2 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 Ca 2 or a calcimimetic compound.

Parallel definitions of compounds blocking Ca 2 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 15 calcium receptor activity. Functional responses of cells regulated by calcium receptor are know in the art, including PTH secretion by parathyroid cells, calcitonin secretion by C-cells, and bone resorption by osteoclasts.

Such functional responses are associated with different diseases or disorders. For example, hyperparathyroidism results in elevated levels of PTH in the plasma.

Decreasing the plasma levels of PTH offers an effective means of treating hyperparathyroidism. Likewise, increasing plasma levels of calcitonin is associated with an 25 inhibition of bone resorption. Inhibiting bone resorption is an effective treatment for osteoporosis. Thus, modulation 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 resorption either directly an osteoclast ionomimetic compound) or indirectly by increasing endogenous calcitonin levels a C-cell calcimimetic) Alternatively, a calcilytic active on the parathyroid cell calcium receptor will increase circulating levels of parathyroid 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 hormone intermittent dosing with a parathyroid cell ionolytic) can increase bone mass in patients suffering .from osteoporosis.

15 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 20 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 ultimately producing a therapeutic effect. Calcium receptor- 25 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 parathyroid cells to negative feedback regulation by extracellular Ca 2 Thus, in tissue from patients with primary HPT, the "set-point" for extracellular Ca 2 is shifted to the right so that higher than normal concentrations of extracellular Ca 2 are required to depress PTH secretion.

Moreover, in primary HPT, even high concentrations of extracellular Ca 2 often depress PTH secretion only partially. In secondary (uremic) HPT, a similar increase in the set-point for extracellular Ca 2 is observed even though the degree to which Ca 2 suppresses PTH secretion is normal. The changes in PTH secretion are paralleled by changes in [Ca 2 the set-point for extracellular Ca 2 induced increases in [Ca 2 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 osteodystrophy in such patients.

15 Compounds that mimic the action of extracellular Ca 2 Sare beneficial in the long-term management of both primary and secondary HPT. Such compounds provide the added impetus required to suppress PTH secretion which the hypercalcemic condition alone cannot achieve and, thereby, help to 20 relieve the hypercalcemic condition. Compounds with greater efficacy than extracellular Ca 2 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.

25 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 proliferation 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 Ca 2 For example, calcitonin secretion from parafollicular cells in the thyroid (C-cells) is regulated by changes in the concentration of extracellular Ca Isolated osteoclasts respond to increases in the concentration of extracellular Ca 2 with corresponding increases in that arise partly from the mobilization of intracellular Ca 2 Increases in [Ca 2 in osteoclasts are associated with the inhibition of bone resorption.

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

Renin secretion from juxtaglomerular cells in the kidney, like PTH secretion, is depressed by increased concentrations of extracellular Ca". Extracellular Ca 2 causes the mobilization of intracellular Ca 2 in these cells. Other kidney cells respond to calcium as follows: elevated Ca 2 inhibits formation of 1,25(OH) 2 -vitamin D by proximal tubule cells, stimulates production of calcium- 15 binding protein in distal tubule cells, and inhibits tubular reabsorption of Ca 2 and Mg 2 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 20 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 25 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 Ca 2 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 Ca 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, depression, 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 aminoglycoside anti- 15 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 20 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 25 horses, dogs and cats.

1 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 localized administration. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA. Administration of ionomimetics 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 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 pharmacological use by altering the physical characteristic of the compound without preventing it from exerting its physio- 20 logical effect. Useful alterations in physical properties include lowering the melting point to facilitate transmucosal administration and increasing the solubility to facilitate administering higher concentrations of the drug.

25 Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, hydrochloride, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate. (See PCT/US92/03736, hereby incorporated 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, ethanesulfonic 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 appropriate 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 intravenously, intraperitoneal, subcutaneous, and intramuscular, orally, topically, or transmucosally.

For systemic administration, oral administration is preferred. Alternatively, injection may be used, e.g., 20 intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds of the invention are formulated in liquid solutions, preferably in physiologically 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 S* 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 transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays, for example, or using suppositories. For NW oral administration, the compounds can be formulated into conventional oral administration dosage forms such as capsules, tablet's, 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 Amole of the compound, preferably 0.1 nmole and 1 jmole depending on its EC.

0 or IC 50 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 15 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.

20 Example 1: Cloning of Human Parathyroid Calcium Receptor From a Human Parathyroid Gland Adenoma Tumor This example describes the cloning of a human parathyroid 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 hybridization 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 pBoPCaR1 insert was labeled by random-primed synthesis using 3 P]-dCTP to a specific activity of 1 x 109 cpm/Ag.

Library screening was performed at a hybridization stringency of 400 mM Na', 50% formamide at a temperature of 380C. 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 400C 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 pBoPCaR1 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.

25 Restriction enzyme mapping of the two different sized inserts indicate that they share regions of sequence similarity 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/Al RNA; 50 nl/oocyte) for functional analysis. Following W 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 concentrations 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 15 and pHuCaR 4.0. The nucleic acid sequence, and amino acid sequence, of the inserts are shown in SEQ. ID. Nos. 1 and S2.

Several differences were observed between the nucleic acid sequences of the two cDNA inserts. Sequence analyses 20 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 polyadenylation. In addition, sequence variation exists at the end of the inserts. These distinct sequences correspond 25 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 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, encodes glutamine (Gln) at aa#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 pharmacologically distinct.

Example 2: Selection of Stable Recombinant Cells Expressing 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 25 xanthine-guanine phosphoribosyltransferase (gpt) allowing stably transfected cells to overcome the blockade of the purine biosynthetic pathway imposed by addition of 2 Ag/ml aminopterin and 25 pg/ml mycophenolic acid. The second vector encodes a gene conferring resistance to the antibiotic hygromycin (used at 200 4g/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 bluntend 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 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 15 and 1 ml of buffer containing 1 AM fura2-AM, 1 mM CaCl 2 and 0.1% BSA and 1 mM CaC1 2 Measurements of fluorescence in response to calcium receptor agonists were performed at 37 0 C in a spectrofluorimeter using excitation and emission wavelengths of 340 and 510 nm, respectively. For signal 20 calibration, Fmax was determined after addition of ionomycin (40 AM) and the apparent Fmin was determined by addition of 0.3 M EGTA, 2.5 M Tris-HC1; pH 10. Robust increases in [Ca 2 were observed in response to the addition of the following calcium receptor agonists: Ca 2 25 (10 mM) Mg 2 (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 transfected 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: Usina 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 slauahtered calves (12-15 weeks old) at a local abattoir and transported to the laboratory in ice-cold parathyroid cell buffer (PCB) which contains NaC1, 126; KC1, 4; MgCl 2 1; Na-HEPES, 20; pH 7.4; glucose, 5.6, and variable amounts of CaCl 2 1.25 mM. Human parathyroid glands, were obtained from patients undergoing surgical removal of parathyroid tissue for primary or uremic hyperparathyroidism (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 20 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 25 cells were present as small clusters containing 5 to S* cells. Cellular viability, as indexed by exclusion of trypan blue or ethidium bromide, was routinely Although cells can be used for experimental purposes at this point, physiological responses suppressibility of PTH secretion and resting levels of [Ca 2 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 g/ml streptomycin, 100 U/ml penicillin, 5 pg/ml gentamicin and ITS*. ITS' is a premixed solution containing 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 cm 2 Falcon) and incubated overnight at 370C in a humid atmosphere of 5% CO 2 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 conditions 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 15 mM CaC1,-2% BSA-PCB containing 1 AM fura-2-acetoxymethylester and incubated at 37 0 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 0 C. The cells were subsequently washed twice with PCB containing 0.5 mM CaC 2 1 and 0.5% BSA and maintained at room temperature (about 20 0 Immediately before use, the cells were diluted five-fold with prewarmed 0.5 mM CaCl 2 S"PCB to obtain a final BSA concentration of The concentration of cells in the cuvette used for fluorescence S. 25 recording was 1-2 x 10 6 /ml.

The fluorescence of indicator-loaded cells was S"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 Ca'.

Fluorescence signals were calibrated using digitonin pg/ml, final) to obtain maximum fluorescence (Fax), 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 Sfirst 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 0 C for 2-3 minutes. The cell suspension was then removed, the cells pelleted, and the supernatant returned to a clean cuvette. The supernatant was then treated with digitonin and EGTA to estimate dye leakage, which is typically 10-15% of the total Ca 2 -dependent fluorescent signal. This estimate was subtracted from the apparent Fmi n Example 4: Using 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 15 293/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 AM 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 CaC12 and 1 mM MgC1 2 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: Measuring the Ability of Compounds to Modulate Calcium Receptor Activity The ability of different compounds to modulate calcium 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.l, l.b.2, and 2. Tables 1.a, l.b.l, l.b.2, and l.c summarizes the effects of compounds, at different concentrations, on calcium receptor activity assayed as a Ca..

described in Example 4 using HEK 293 cells transfected with nucleic acid encoding pHuPCaR4.0, which were loaded with fura-2).

Table 2, summarizes the results of different experiments where the EC 50 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 greater than 40% response at 3.3 ng/mL in HEK-293 cells expressing the human calcium receptor.

Compound activity Code at four concentrations (ng/mL) 3300 330 33 3.3 15 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 116 118 122 102 122 120 114 92 17E 116 110 110 92 24Z 138 138 135 14S 116 106 105 88 132 129 122 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 3.3 121 .115 ill 93 68 130 115 99 66 9R 108 101 64 12F 118 110 101 63 120 110 117 94 62 23Z 129 126 100 61 17M 115 99 59 16V 114 102 58 250 126 115 96 57 25J 119 123 105 56 *16L 146 138 98 56 12N 115 106 102 16T 97 88 107 107 95 17P 101 86 54 16Q 110 88 53 *23E 137 113 102 53 *17C 113 120 99 52 *25L 97 97 85 52 :20 8Z 101 97 52 17X 105 93 51 13R 132 98 51 170 112 96 51 23Q 122 114 98 51 16X 1l1 96 51 24V 127 98 71 130 115 94 17N 108 86 49 21V 122 116 99 48 24M 132 134 99 48 13U 108 79 47 Compound i activity Code at four concentrations (ng/mL) 3300 330 33 3.3 24P .140 138 110 46 17Y 109 94 79 46 lix 100 76 2SH 115 107 89 22J 99 71 9C 104 82 13S 102 87 103 100 84 44 13P 110 83 44 0*4010 8K 98 81 44 13N 114 88 43 1ON 106 97 77 43 12H 114 115 94 43 0 :4:4 25P 90 81 75 41 18A ill 88 14L -109 78 Table 1.b.l. Calcimimetic compounds which produce areater t-han 40l% response at 33 na/mL in H-EK-293 cells expressing 4.

4 4*e* 4* 4 20 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 0- activity Code at four concentrations (ng/mL) 3300 330 33 3 .3 17D 108 92. 38 17F ill 90 28 24C 116 113 87 32 K 124 107 86 13F 125 122 85 38 21F 109 85 36 21S 132 131 85 34 000. 1OF 96 84 27 0 014R 106 107 84 37 400 O 13G ill 128 82 29 toe:14Z 118 103 82 0 016N 122 159 82 8 8U 123 129 82 11 0 0 O23W 117 97 81 S.0 15 12G 139 139 81 113 80 32 118 100 79 13V 110 79 33 :*.14P 112 103 78 oboe* .0*20 6T 123 129 78 0 of 14Q 101 78 17L ill. 104 78 31 24 K 106 78 24U 106 106 78 25Q 116 95 77 8J 104 77 39 2311 121 114 77 28 21C=4U 134 114 76 17 97 85 76 28 16R 100 76 171 118 97 76 18 Compound activity Code at four concentrations (ng/mL) 3300 330 33 3 .3 24J 103 75 31 210 109 75 37 24G 1.09 94 75 22 151 ill 93 75 24 21D 104 75 17 117 95 74 24 lop 102 74 8 23M 113 97 74 26 14Y 109 73 17 17K 98 97 73 37 12E 117 121 73 23 17Z 99 73 37 *16W 102 73 4 *23K 106 107 72 24 25X 96 94 72 22 13W 109 71 12 23P 125 99 70 22 18B ill 96 69 26 *21Y 100 68 36 17W 92 67 13 23A 103 67 24 23G 127 93 67 13 13M 92 66 2 1U 104 104 66 18 21R 100 66 lOS/1OT 86 65 13 17R 98 65 13 13X 102 65 13 4N 100 65 13 21E 94 64 4 80 75 64 13 Compound Code activity at four concentrations (ng/mL) 3300 330 22Y 21G 24L 10W/10X 17B 23Y 11Y 114 88 105 99 98 92 87 103 33 3.3 64 28 63 18 62 62 8 61 9 61 19 61 16 61 106 Table l.b.2 Calcimimetic compounds which produce greater 10 than 40% 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 17P 17X 18C 23T 4V 8G 231 21M 240 3U 9A 12M 12B 99 102 137 98 130 101 105 87 74 93 84 102 102 114 89 82 86 110 Compound Code 21P 8T 1OL/1OM 241 14N 23R 23S 21T low/lox 13T 6R 201 24A 12D 6X 18T 21X 23J 10z 16Z 23N 16U 11D 23X 17A 22X 23U 9Z 16J 4P activity at four concentrations (ng/mL) 3300 330 33 3.3 92 56 13 85 55 13 99 55 4 109 84 55 11 89 55 104 86 54 13 97 53 3 133 112 53 3 81 53 4 90 53 6 94 52 7 87 52 12 122 85 52 9 128 109 52 84 52 99 74 52 14 119 101 51 2 102 61 51 29 96 51 88 51 9 96 50 2 85 50 4 96 50 4 94 49 1 88 49 7 80 48 8 86 48 87 483 74 48 4 92 76 47 31 94 73 46 8 81 46 8 Compound Code activity at four concentrations (ng/mL) 3300 330 33 3.3 230 111 79 46 13 13Q 95 46 4G 83 46 12Y 80 46 12L 88 45 23F 82 45 11W 81 44 2 8H 88 44 7 .0 25V 89 59 43 26 10 25W 95 69 42 8 10R 82 42 7 21N 124 98 42 4 8S 73 42 7 8X 75 40 19 S: 15 13E 123 94 40 2 Table 1.c. Calcimimetic compounds which produce areater 0o:eT1I f rn~rr LI rpc~~nccl 1( 1111 nr r\ r LU 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 3H 84 3L 81 28 160 129 81 21 2 80/8Q 124 80 14 0 Compound Code 14A 23L 1T 7W 4H 8D 4U 24E 16M 4M 2S 1 7V 2X 15 23D 4P 3M 16K 5D 4D 24B 24H 2Y 3V 2Q 14B 13Z 8A 24D %activity at four concentrations (ng/mL) 3300 330 33 3.3 98 78 10 7 107 77 37 9 76 76 77 37 73 21 72 94 71 35 6 130 68 11 4 68 34 67 29 91 66 27 -1 66 91 66 35 13 65 32 65 64 19 78 62 36 8 62 18 61 13 76 61 34 11 81 60 32 13 60 16 59 58 16 56 14 56 4 75 55 11 4 9.3 54 22 54 87 53 34 39 Compound Code %activity at four concentrations (ng/mL) 3300 330 33 3.3 1D 53 131 85 52 31 3B 52 8C 51 14H 112 49 s 7U 49 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 40 9 0 *TABLE 2 Arylalkylamine Calcimimetics from Figure 1 Active at the Parathyroid Cell Calcium Receptor In Vitro S 5 MM) ICompound Code IEC 50 ICompound Code FEC 5 1 (from Fig. 1) (MM) j (from Fig. 1) (AM) NPS R-467 2.0 liX 0.83 NPS R-568 0.60 ily 2.8 3U 0.64 12L 1.7 3V 1.8 12U 1.2 4A 1.4 12V 0.42 4B 2. 0 12W 3.2 000..*.

be..

4 C 2.0 12Y 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 i14Q 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. 4Y 1.3 16Q 0.04 4.4 16R 0.36 5B/5C 2.8 16T 0.04 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 8K 1.3 201 8R 2.6 20J 8S 1.7 20R 2.4 8T 1.8 20S 4.2 8u 0.44 21D 8X 0.76 21F 0.38 8Z 0.4 0 I21G 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 10F 0.89 25C 2 11D 1.8 25D 0.019 *0*0 0* 0*0*0 0

S.

0 5* 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 15 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-0, 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 triacetoxyborohydride 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 W 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 cyanoborohydride 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 HC1 to afford white solids.

The amines in these syntheses were purchased from Aldrich Chemical Co., Milwaukee, WI, or from Celgene 15 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 N- (2-Phenyl)propyl) (1-naphthyl)ethylamine 20 A mixture of 3-phenyl-l-propylamine (135 mg, 1 mmol), l'-acetonaphthone (170 mg, 1 mmol), and titanium (IV) isopropoxide (355 mg, 1.3 mmol) was stirred at room temperature for 1 hour. The reaction was treated with 1 M o 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 concentrated to a milky oil. A small portion of this material mg) was purified by HPLC (Phenomenex, 1.0 x 25 cm, pM silica) using a gradient of dichloromethane to methanol in dichloromethane containing 0.1% isopropylamine. This afforded the product (free base) as a single component by GC/E1-MS 10.48 min) m/z (rel. int.) 289 274 184 162 155 (100), 141 (18), 115 91 77(5).

59 O Example 7: Synthesis of Compound 8J N- (3-phenylpropyl) (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 OOC, and sodium nitrite (1.45 g, 21 mmol) dissolved in 3-5 mL of water was added over minutes while maintaining the temperature below 6 0

C.

Sodium thiomethoxide (1.75 g, 25 mmol) was dissolved in mL of water and cooled to 0°C. To this solution was added the diazonium salt over 10 minutes while maintaining the temperature below 10 0 C. The reaction was stirred for an additional hour while allowing the temperature to rise to ambient. The reaction mixture was partitioned between 15 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 *e 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 25 reaction was stirred overnight. The reaction was worked up by the addition of 4 mL of ether and 200 iL 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(R,=7.64 min) m/z (rel. int.)285 270(90) 180(17) 151(100) 136(32) 104(17) 91(54), 77(13).

W Example 8: Synthesis of Compound 8U N-3- (2-methoxyphenyl)-1-propyl-(R)-3-methoxy-amethylbenzylamine hydrochloride A mixture of (+)-3-methoxy-a-methylbenzylamine (3.02 g, 20 mmol), 2-methoxycinnamaldehyde (3.24 g, mmol), and titanium (IV) isopropoxide (8.53 g, 30 mmol, Eq.) was stirred 2 hours at room temperature and treated with 1 M (20 mL) ethanolic sodium cyanoborohydride. The reaction was stirred overnight (16 hours), diluted with diethylether, and treated with water (1.44 mL, 80 mmol, 4 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 15 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 HC1 in diethylether. The resulting precipitate (white solid) was collected, washed with diethylether, and air dried.

25 GC/E1-MS (Re 9.69 min) of this material (free base) showed a single component: m/z (rel. int.) 299 21), 284 (100) 164 (17) 150 135 (81) 121 (40) 102 91 77 (18) Example 9: Synthesis of Compound 9R (2-naphthyl)ethyl)-(R)-1 -naphthyl)ethylamine hydrochloride A mixture of (R)-(+)-1-(l-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 600C for 3 hours. Sodium cyanoborohydride (NaCNBH 3 (3.67 g, 58.4 mmol) was then added.

The reaction mixture was stirred at room temperature for 18 hours. Ether (1 L) and H 2 0 (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 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 yield), m.p.: softens 200-240 0 C (dec.).

Example 10: Synthesis of Compound 11X N- (4-Isopropylbenzyl) (1-naphthyl)ethylamine hydrochloride A mixture of (l-naphthyl)ethylamine (1.06 15 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 1000C for 5 min then allowed to stir at room temperature for 4 hours. Sodium cyanoborohydride (NaCNBH 3 (0.39 g, 6.2 mmol) was then added followed by EtOH (1 mL).

20 The reaction mixture was stirred at room temperature for 18 hours. Ether (100 mL) and H 2 0 (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 1% MeOH/ CHC1 3 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), 257- 259 0

C.

Example 11: Synthesis of Compound 12U N-3-(2-methylphenyl)-l-propyl-(R)-3-methoxy-omethylbenzylamine hydrochloride A solution of 2-methylcinnamonitrile (1.43 g, 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 minutes) with a 1 M solution of (R)-(+)-3-methoxy-amethylbenzylamine (1.51 g, 10 mmol) in dichloromethane 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 15 GC/E1-MS (R,=10.06 min) m/z (rel. int.) 281 17) 266 176 146 135 (73) 131 (100), 91 77 (13) The unsaturated intermediate in ethanol was hydrogenated (1 atm H 2 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/E1-MS 9.31 min) of this material (free base) showed a single component: m/z (rel. int.) 283 21), 268 (100), 164 25 148 135 (85) 121 (12) 105 91 77 (21) Example 12: Synthesis of Compound 12V N-3-(3-methylphenyl) propyl-(R)-3-methoxy-amethylbenzylamine hydrochloride The compound was prepared following the procedure described in Example 11, but using 2-methylcinnamonitrile.

The unsaturated intermediate was a single component by GC/EI-MS 10.21 min) m/z (rel. int.) 281 57), 266 146 135 131 (100), 115 102 (26), 91 77 Reduction of this material and hydrochloride formation using the procedure described Example 63 11 afforded the product. GC/EI-MS 9.18 min) of this material (free base) showed a single component; m/z (rel.

int.) 283 19) 268 (100) 164 148 135 121 105 91 77 (21).

Example 13: Synthesis of Compound 12Z N-3-(2-chlorophenyl) propyl- (l-naphthyl)ethylamine hydrochloride The compound was prepared following the procedures described in Example 11, but using 2-chlorohydrocinnamonitrile 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 methanol in dichloromethane). The hydrochloride was 15 prepared by treatment with 1 M HC1 in diethylether.

Example 14: Synthesis of Compound 14U (4-methoxyphenyl)ethyl naphthyl) ethylamine hydrochloride A mixture of (1-naphthyl)ethylamine (1.1 g, 20 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 0 C for 3 hours. Sodium cyanoborohydride (NaCNBH 3 (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 H 2 0 (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/CHC13). A portion of this material was HPLC chromatographed [Selectosil, 5 pM silica gel; 25 cm x 10.0 mm (Phenomenex, Torrance, CA), 4 mL per minute; UV 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 0 C(dec.).

Example 15: Synthesis of Compound 17M N- (3-chloro-4-methoxybenzyl)-(R)-1- (1-naphthyl)ethylamine hydrochloride A mixture of (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 0 C for 30 minutes then allowed to stir at room temperature for 3 hours.

Sodium cyanoborohydride (NaCNBH 3 (2.45 g, 39 mmol) was then added. The reaction mixture was stirred at room temperature for 18 hours. Ether (100 mL) and H 2 0 (2 mL) were added to the reaction mixture and the resulting 15 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 CH 2 Cl 2 The chromatographed material was then dissolved in hexane (500 mL), decolor- 20 ized with Norit® filtered (0.2 pM) and then ethereal HC1 was added to precipitate the product as a while solid (10.2 g, 56 yield), 241-242 0 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), K 2

CO

3 (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 H 2 0 (2 x 20 mL). The organic layer was dried (Na 2 SO,) and evaporated to yield 4.5 g, 82.4% yield. The ketone was used in the following reaction without further purification.

(R)-N-(1-(4-Methoxy-3-methylphenyl)ethyl) (1naphthyl)ethylamine hydrochloride [Compound 17P] A mixture of (R)-(+)-1-(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 0 C for 2 hours. Isopropanol (45 mL) was added and the reaction was then cooled to 10 0 C in an ice bath. Sodium triacetoxyborohydride (NaHB(O 2

CCH

3 3 (10.5 g, 49.5 mmol) was then added in portions over 15 minutes. The reaction mixture was then heated to 70 0 C for 18 hours. The mixture was S" cooled to room temperature and poured into ether (400 mL) The suspension was centrifuged, the supernatant was S collected and the pellet was washed with ether (400 mL).

•15 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 H 2 0 (2 x 50 mL) The oo e organic layer was dried (Na 2 S0 4 filtered and evaporated under vacuum. EtOH (abs.) was added to the wet residue which was then dried thoroughly on a rotary evaporator to Sprovide an oil. The mixture was then chromatographed on Ssilica gel (50 mm x 30 cm) [elution with MeOH:1% IPA:CHCl 3 to give 4.8 g of an oil].

The desired diastereomer was further purified by HPLC 25 chromatography [SUPELCOSIL'" PLC-Si, 18 iM silica gel; cm x 21.2 mm (Supelco, Inc., Bellefonte, PA), 7 mL per minute; UV det. 275 nM: 20% EtOAc-80% hexane (elution time 11.0 min)]. Injections (800 AL aliquots) of the mixture (100 mg/mL solution in eluent) provided 65 mg of the desired isomer. Multiple HPLC injections provided 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 0

C.

MW Example 17: Synthesis of Compound 17X 3-Chloro-4-methoxybenzaldehyde A mixture of 3-chloro-4-hydroxybenzaldehyde (25 g, 160 mmol), iodomethane (27.25 g, 192 mmol), K 2

CO

3 (granular, 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 (Na 2 SO,) 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) R,=0.24; GC R,=4.75 min; MS (EI) m/z 170(M) 172(M+2) e* 1-Methyl-(3'-chloro-4'-methoxybenzyl) alcohol A mixture of 3-chloro-4-methoxybenzaldehyde (13 g, 20 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. NH 4 Cl (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 (Na 2

SO

4 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 CHC1 2 to yield 11.3 g, 63% yield of an oil; TLC (CH 2 C1 2 Rf=0.25; GC R,=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 CH 2 Cl 2 (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 reaction without further purification. TLC (diethyl ether) GC R.=5.3 min; MS (EI) m/z 184 184(M+2)

G

est.

(1-Ethyl-4 -methoxy-3 -chlorophenyl) (1naphthyl ethyl) amine s. A mixture of 3'-chloro-4'-methoxyacetophenone (5.3 g, 29 mmol), (R)-(+)-l-(1-naphthyl)ethylamine (4.98 g, 29 mmol), titanium (IV) isopropoxide (10.2 g, 36 mmol), and isopropanol (20 mL) was heated to 100 0 C for 3 hours.

Sodium triacetoxyborohydride (NaB(0,CCH3) 3 12.29 g, 58 20 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) H 2 0 (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 (Na 2

SO

4 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%

CH

2 Cl 2 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 temperature 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 236-237%C (dec); TLC (MeOH-

CH

2 Cl 2 Rf=0.25; GC R,=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 15 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 re and 30 min at 100 0

C.

20 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 magnesium sulfate, filtered and concentrated to afford ethyl m-trifluoromethoxycinnamate as an oil; m/z (rel. int.) 260 19), 232 215 (100), 187 101 (28).

The ethyl ester.in ethanol (100 ml) was reduced under 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 16), 217 188 (100) 175 (28) 103 91 77 (23).

The saturated ethyl ester was hydrolyzed in a solution of ethanol-10 M sodium hydroxide for 16 hr at rt. After this time the solution was acidified and thp product extracted into diethyl ether. The ether solution was dried over anhydrous magnesium sulfate and concentrated to afford m-trifluoromethoxyhydrocinnamic acid as a solid; m/z (rel. int.) 234 46), 188 (100) 174 103 91 77 (17).

The acid was stirred in excess thionyl chloride for 4 hr at rt. The excess thionyl chloride was evaporated at reduced pressure (1000C) to afford m-trifluoromethoxyhydrocinnamyl 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 0 C and treated dropwise with a solution (13 ml of 3 M in tetrahydrofuran) of methylmagnesium bromide (39 mmol).

15 The reaction was stirred 4 hr at -78 0 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 68), 217 189 175 103 (28), 43 (100).

A solution of 4-(3-trifluoromethoxyphenyl)-2-butanone "25 (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 pM 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)-2butyl]-1-(3-methoxyphenyl)ethylamine, 22Z [m/z (rel. int.) 367 352 (20) 232 178 135 (100), 105 91 77 and (R,R)-N-[4-(3-trifluoromethoxyphenyl) -2-butyl] (3-methoxyphenyl)ethylamine, 23A; m/z (rel. int.) 367 352 232 178 135 (100), 105 91 77 (11).

Preparation of 22X and 22Y In a similar fashion an equal molar amount of 4-(3trifluoromethoxyphenyl)-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 15 chloroformafforded -N-[4-(3-trifluoromethoxyphenyl)- 2-butyl]-1-(1-naphthyl)ethylamine, 22X; m/z (rel. int.) 387 372 198 176 155 (100), 128 115 109 103 77 and (3trifluoromethoxyphenyl) -2-butyl] (1-naphthyl)ethylamine, 22Y; m/z (rel. int.) 387 372 198 176 (11) 155 (100) 128 115 109 103 77 Preparation of 4T In a similar fashion an equal molar amount of 4-(2chlorophenyl)-2-butanone, prepared from o-chlorobezaldehyde, (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-(2-chlorophenyl)-2-butyl]-1-(3-methoxyphenyl) ethylamine, 4T; m/z (rel. int.) 317 302 178 178 135 (100), 125 105 91 77 Preparation of 21Y In a similar fashion an equal molar amount of 4-(3trifluoromethylphenyl)-2-butanone, prepared from mtrifluoromethylbezaldehyde, (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]-l-(3-methoxyphenyl)ethylamine, 21Y [m/z (rel.

int.) 351 336 216 202 178 135 (100), 105 91 77 and trifluoromethylphenyl)-2-butyl]-1- (3-methoxyphenyl) ethylamine, 21X.

Preparation of 25C and In a similar fashion an equal molar amount of 4-(3trifluoromethylphenyl)-2-butanone, (R)-1-(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-(l-naphthyl)ethylamine, 25C [m/z (rel. int.) :371 (M 356 198 (15) 155 (100), 129 115 25 109 77 and (R,R)-N-[4-(3-trifluoromethylphenyl)-2-butyl]-1-(1-naphthyl)ethylamine, 25D; m/z (rel. int.) 371 (M 356 198 155 (100), 129 115 109 77 Preparation of 21D In a similar fashion an equal molar amount of 4phenyl-2-butanone (Aldrich Chemical (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 methanol in chloroform afforded (R,R)-N-(4-phenyl-2butyl)-1-(3-methoxyphenyl)ethylamine, 21D [m/z (rel. int.) 283 (M 268 178 135 (100), 105 (15) 91 77 and (S,R)-N-(4-phenyl-2-butyl)-1-(3methoxyphenyl)ethylamine, 21E.

Preparation of 21F In a similar fashion an equal molar amount of 4phenyl-2-butanone (Aldrich Chemical 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 methanol in chloroform afforded R) (4-phenyl-2butyl)-1-(1-naphthyl)ethylamine, 21F; m/z (rel. int.) 303 15 (M 288 198 155 (100), 129 115 91 77 (4) Preparation of 12Z A stirred solution of 2-chlorohydrocinnamonitrile (Aldrich Chemical Co., 1.66 g, 10 mmol) in dichloromethane 20 (100 ml) was cooled to -78 0 C and treated dropwise with diisobutylaluminum hydride (1.42 g, 10 mmol). The reaction was stirred 1 hr at rt, cooled to -78 OC and treated with a solution of 1-(l-naphthyl)ethylamine (1.71 g, mmol) in dichloromethane (25 ml). The reaction was transferred 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]-l-(1naphthyl)ethylamine, 12Z, as a clear oil; m/z (rel. int.) 323 2) 308 (63) 288 196 184 155 (100), 125 115 103 91 77 Preparation of 12B In a similar fashion, 4-methylcinnamonitrile was treated with diisobutyl aluminum hydride and the intermediate aluminum-imine complex treated with methoxyphenyl)ethylamine. The intermediate imine was treated with ethanolic sodium borohydride. Work-up and chromatography yielded (R)-N-[3-(4-methylphenyl)prop-2enyl]-1-(3-methoxyphenyl)ethylamine, 12B, as a clear, colorless oil; m/z (rel. int.) 281 266 176 146 135 131 (100), 115 105 (21), *91 77 (21).

o Preparation of 12C In a similar fashion, 2-methylcinnamonitrile was treated with diisobutyl aluminum hydride and the intermediate aluminum-imine complex treated with methoxyphenyl)ethylamine. The intermediate imine was treated with ethanolic sodium borohydride. Work-up and e 'chromatography yielded (R)-N-[3-(2-methylphenyl)prop-2enyl]-1-(3-methoxyphenyl)ethylamine, 12C, as a clear, colorless oil; m/z (rel. int.) 281 266 176 146 135 131 (100), 115 105 (19), 91 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 methoxyphenyl)ethylamine. The intermediate imine was treated with ethanolic sodium borohydride. Work-up and chromatography yielded (2,4,6-trimethylphenyl) prop-2-enyl]-1-(3-methoxyphenyl)ethylamine, 12D, as a clear, colorless oil; m/z (rel. int.) 309 294 174 159 (100), 135 129 105 (21), 91 77 (14).

Preparation of 12E In a similar fashion, 4-isopropylcinnamonitrile was treated with diisobutyl aluminum hydride and the intermediate aluminum-imine complex treated with methoxyphenyl)ethylamine. The intermediate imine was treated with ethanolic sodium borohydride. Work-up and chromatography yielded [3-(4-isopropylphenyl)prop-2enyl] (3-methoxyphenyl)ethylamine, 12E, as a clear, colorless oil; m/z (rel. int.) 309 294 174 15 159 135 117 (100), 105 91 77 (19).

Preparation of 12F In a similar fashion, 2,4-dimethylcinnamonitrile was treated with diisobutyl aluminum hydride and the inter- 20 mediate aluminum-imine complex treated with methoxyphenyl)ethylamine. The intermediate imine was treated with ethanolic sodium borohydride. Work-up and chromatography yielded (R)-N-[3-(2,4-dimethylphenyl)prop- S2-enyl]-1-(3-methoxyphenyl)ethylamine, 12F, as a clear, colorless oil; m/z (rel. int.) 295 294 174 160 145 (100), 135 117 105 91 77 (19).

Preparation of 12G In a similar fashion, 3-methylcinnamonitrile was treated with diisobutyl aluminum hydride and the intermediate aluminum-imine complex treated with methoxyphenyl)ethylamine. The intermediate imine was treated with ethanolic sodium borohydride. Work-up and chromatography yielded (R)-N-[3-(3-methylphenyl)prop-2enyl]-1-(3-methoxyphenyl)ethylamine, 12G, as a clear, colorless oil; m/z (rel. int.) 281 266 176 146 135 131 (100), 115 105 (19), 91 77 (18) Preparation of In a similar fashion, cinnamonitrile was treated with diisobutyl aluminum hydride and the intermediate aluminumimine complex treated with (R)-1-(3-methoxyphenyl)ethylamine. The intermediate imine was treated with ethanolic oe sodium borohydride. Work-up and chromatography yielded •eg (3-phenylprop-2-enyl) (3-methoxyphenyl)ethylamine, as a clear colorless oil; m/z (rel. int.) 267 3), 252 176 (17) 135 117 (100) 105 91 77 (33).

15 Preparation of In a similar fashion, e-methylcinnamonitrile was treated with diisobutyl aluminum hydride and the intermediate aluminum-imine complex treated with methoxyphenyl)ethylamine. The intermediate imine was 20 treated with ethanolic sodium borohydride. Work-up and chromatography yielded (R)-N-(2-methyl-3-phenylprop-2enyl)-1-(3-methoxyphenyl)ethylamine, 25G, as a clear, colorless oil; m/z (rel. int.) 281 266 190 146 135 131 (100), 115 105 (21), 91 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 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 0 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 x 300 ml) and brine. The organic layer was dried over anhydrous potassium carbonate and concentrated to yield 3chlorocinnamonitrile (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, 15 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 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 pM CR PTFE Acrodisc.

The ether wash was concentrated to an oil. Chromatography of this material (silica, preparative thin-layer chromatography) using 3% methanol-dichloromethane (containing 0.1% isopropylamine) afforded N-[3-(3-chlorophenyl)propyl]-l- (3-methoxyphenyl)ethylamine, 6X; m/z (rel. int.) 303 288 196 164 135 (100), 125 103 91 77 (29) Preparation of 6V An equal molar amount of 3-(4-chlorophenyl) propylamine (prepared in a similar fashion from 4chlorobenzaldehyde 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] (3-methoxyphenyl)ethylamine, 6V, as an oil; m/z (rel. int.) 303 288 (91) 196 164 (10) 135 (100) 125 103 91 77 (18) Preparation of In a similar fashion, an equal molar amount of 1-(1methoxyphenyl)ethylamine, 4- t-butylacetophenone and 1.25 10 molar equivalents titanium (IV) isopropoxide were mixed 4 hr at rt and the intermediate imine treated with an ethanolic sodium cyanoborohydride (5 ml of IM, 5 mmol) Workup and chromatography afforded (R)-N-[1-(4-t-butylphenyl) ethyl] -1-(1-naphthyl)ethylamine, 20A, as an oil; m/z (rel.

int.) 331 12) 316 161 155 (100) 131 127 (13) 115 (10) 105 91 77 (7) Preparation of 25H and 251 In a similar fashion, an equal molar amount of (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 e an ethanolic sodium cyanoborohydride (5 ml of 1 M, mmol). Work-up and chromatography afforded methyl-4-phenybut-3-enyl) (3-methoxyphenyl)ethylamine, 25H, as an oil; m/z (rel. int.) 283 268 178 135 (100), 105 91 77 (13) and (2-methyl-4-phenybut-3-enyl)-1-(3-methoxyphenyl) ethylamine, 251, as an oil; m/z (rel. int.) 283 268 178 (40) 135 (100), 105 91 77 (13) Preparation of 16L and 16M In a similar fashion, an equal molar amount of (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, mmol) Work-up and chromatography afforded methoxyphenyl)ethyl]-1-(3-methoxyphenyl)ethylamine, 16L, as an oil; m/z (rel. int.) 284 270 150 135 (100), 120 105 91 77 (23) and (S,R)-N-[1-(4-methoxyphenyl)ethyl]-1-(3-methoxyphenyl) ethylamine, 16M, as an oil; m/z (rel. int.) 284 1), 270 (53) 150 135 (100), 120 (11) 105 (33) 91 77 (23).

10 Preparation of 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 3methyl-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 20 and chromatography afforded N-(3-methyl-3-(4chlorophenyl)propyl]-1-(3-methoxyphenyl)ethylamine, as an oil; m/z (rel. int.) 317 12), 302 210 182 164 135 (100), 121 103 91 77 (28).

Preparation of 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 3methyl-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 17) 268 (71) 164 (13) 135 (100) 121 105 (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 3methyl-3-(2-chlorophenyl)propylamine. An equal molar 10 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-chlorophenyl)propyl]-1-(3-methoxyphenyl)ethylamine, 4Y, as an oil; m/z (rel. int.) 283 17) 268 164 (13) 135 S•(100), 121 105 91 77 (14) Preparation of 6T solution of NPS R-568 (30.3 g 100 mmol) in dichloromethane at -78 0 C was treated dropwise with borontribromide (50 g, 200 mmol). The reaction was stirred 1 Shr 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 HC1. The chloroform wash was dried over anhydrous magnesium sulfate and concentrated to afford chlorophenyl)propyl] -1-(3-hydroxyphenyl)ethylamine hydrochloride as a solid. A solution of sodium hydride (0.48 g, 20 mmol) in dimethylformamide was treated with [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 chloroformafforded (2-chlorophenyl)propyll (3ethoxyphenyl)ethylamine, 6T, as an oil; m/z (rel. int.) 316 302 (100) 282 (11) 196 178 149 121 103 91 77 (29).

Preparation of 6R NPS R-467 was used in a similar fashion to prepare (R)-N-(3-phenylpropyl)-1- (3-ethoxyphenyl)ethylamine, 6R, as an oil; m/z (rel. int.) 283 268 178 162 149 (100) 121 103 91 77 (29).

10 Preparation of 3U SoAn 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 20 decanted and concentrated to a milky oil. The oil was suspended in diethyl ether and filtered through a 0.45 AM CR PTFE Acrodisc. The diethyl ether filtrate was concentrated to afford N-(3,3-diphenylpropyl) -1-(l-naphthyl) ethylamine, 3U, as a clear, colorless oil; m/z (rel. int.) 365 17), 350 181 155 (100), 141 115 91 77 Preparation of 6F In a similar fashion equal molar amounts 1-(3methoxyphenyl)ethylamine (1.51 g, 10 mmol), 2'-acetonaphthone (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-(3methoxyphenyl)ethylamine, 6F, as a clear, colorless oil; m/z (rel. int.) 305 290 170 155 (100), 135 115 105 91 77 Preparation of 4G In a similar fashion equal molar amounts of phenylethylamine,, l'-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- [1-(1-naphthyl)ethyl)-1-phenylethylamine, 4G, as a clear, 10 colorless oil; m/z (rel. int.) 275 260 155 Poo.

(100), 127 105 77 (32).

Preparation of 4H In a similar fashion equal molar amounts of phenylethylamine, 2'-acetonaphthone and 1.25 equivalents 15 of titanium (IV) isopropoxide were mixed and the resulting intermediate imine was reduced with ethanolic sodium cyanoborohydride. Work-up and chromatography yielded N- (2-naphthyl)ethyl] -1-phenylethylamine, 4H, as a clear, colorless oil; m/z (rel. int.) 275 260 155 (100), 120 105 77 Preparation of 6E In a similar fashion equal molar amounts of 1-(3methoxyphenyl)ethylamine, 1'-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-l-(3-methoxyphenyl)ethylamine, 6E, as a clear, colorless oil; m/z (rel. int.) 305 290 170 155 (100), 135 115 105 91 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 0* 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 Dioxide Total 225 mg 1125 g 15 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 hydrochloride. 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 described, various modifications may be made, without departing from the spirit and scope of the present invention.

.0 0. 04 6 0 'S.04 of 6 S6 84 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NPS Pharmaceuticals, Inc.

(ii) TITLE OF INVENTION:

CALCIUM

COMPOUNDS

RECEPTOR-ACTIVE

(iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: Lyon Lyon STREET: First Interstate Center, Suite 4700 633 West Fifth Street World CITY: Lo! STATE: Ca COUNTRY:

US

ZIP: 90 COMPUTER READABLE

FORM:

MEDIUM TYPE:

COMPUTER:

OPERATING

SYSTEM:

SOFTWARE:

s Angeles lifornia

A

017 3.5" Diskette, 1.44 Mb storage IBM PC compatible

PC-DOS/MS-DOS

FastSeq (vi) CURRENT APPLICATION

DATA:

APPLICATION

NUMBER:

FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION

DATA:

Prior applications total, including application described below: 2 APPLICATION NUMBER: U.S. 08/353,784 FILING DATE: 8 December, 1994 APPLICATION NUMBER: PCT/US/94/12117 FILING DATE: 21 October, 1994 (viii) ATTORNEY/AGENT

INFORMATION:

NAME: Heber, Sheldon O.

REGISTRATION NUMBER: 38,179 REFERENCE/DOCKET NUMBER: 215/304 (ix) TELECOMMUNICATION INFORMATION:

TELEPHONE:

TELEFAX:

TELEX:

(213) 489-1600 (213) 955-0440 67-3510

S..

INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 5006 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 436..3699 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GCTGCTGTGG CCGGACCCGA AGGCGGGCGC CGGGAGCGCA

GCGAGCCAGA

AGGGAGCGGG

CTCATCCTCG

CCTCTGTGCG

CACAGGAGGC

GGACCACCCA

ATGGAGATTC

CAATCTGTAG

CTCCAAGGGA

CGCGCCTCTC

GCTGCGCGCA

TGGAGACCCA

AGGGAGCCCT

CTCTGCATGA

CATTACAAGT

AAACACCACG

ACATGTGTCC

GAAACTTCTG

CAAGACCGTG

GTCCTGAGAT

CGGCCGAGGG

GGCCGCGGCG

TGTGGCTTCC

CTGGATTGAG

TCTTCTATTA

CCACTGCAGG

GGAGCCTCCA

ACCTTGGCAT

CAGACCAGAG

GCCGGAGCTG

CAGAAGGCAT

AAAGACTCAA

GAAGGCAGAA

TTTTATTAAT

GAGTGAACTG

AACTCCTAGC

120 160 200 240 280 320 360 400 435 471 TGTCTCATCC CTTGCCCTGG AGAGACGGCA GAACC ATG GCA TTT TAT AGC TGC TGC TGG GTC CTC TTG GCA Met Ala Phe Tyr Ser Cys Cys Trp Val Leu Leu Ala 1 5 86

TAC

CTC ACC TGG CAC ACC TCT GCC GGG CCA GAC CAG 507 Leu Thr Trp His Thr Ser Ala Tyr Gly Pro Asp Gin

S.

CGA

Arg

CTC

Leu

CAA

Gin

ATC

Ile

GCT

Ala

CCA

Pro 85

ATA

Ile

GAA

Glu

GATI

Asp

GAC

Gi.

AC]

Th2 14!

CT(

Lei

GCC

Ala

TTT

Phe

GAT

Asp

AGG

Arg

ATG

Met

GCC

Ala

TTT

Phe

GCC

Ala 110

TCT

Ser

CAC

His

SGGC

Gly

GGG

aGly

CAA

Gln

CCT

Pro

CTC

Leu

TAT

Tyr

ATA

Ile 75

CTT

Leu

GAC

Asp

ACC

Thr

TTG

Leu

ATT

Ile 135

TC;

Sez

CTC

Le.

AAG

Lys

ATT

Ile

AAA

Lys

AAT

Asn

TTT

Phe

CTT

Leu

ACT

Thr 100

CTG

Leu

AAC

.Asn

*CCC

Pro

GGC

-Gly

TTC

1Phe 160

A.AG

Lys

CAT

H-is

TCA

Ser

TTC

Phe 65

GCC

Ala

CCC

Pro

TGC

Cys

AGT

Ser

CTI

Leu 125

TC'I

Sex

GTC

Val

TA(

Ty2

GGG

Giy 30

TTT

Phe

AGG

Arg

CGT

Arg

ATA

Ile

AAC

Asn 90

AAC

Asn

TTT

Phe

*GAT

Asp

ACG

-Thr

TCC

*Sex 150

ATI

lE GAC ATT Asp Ile GGA GTA Gly Val CCG GAG Pro Giu 55 GGG TTT Gly Phe GAG GAG Giu Giu TTG ACG Leu Thr ACC GTT Thr Val GTT GCT Val Ala 115 GAG TTC Glu Phe ATT GCT Ile Ala 140 ACG GCA Thr Ala CCC CAC Pro Gin.

ATC CTT Ile Leu GCA GCT Ala Ala TCT GTG Ser Val CGC TGG Arg Trp ATA AAC Ile Asn CTG GGA Leu Giy TCT AAG Ser Lys 105 CAA AAC Gin Asn TGC AAC Cys Asn 130 GTG GTG Val Vai GTG GCA Val Ala GTC AGT LVal Ser 165

GGG

Gly

~AA

Lys

GAA

Giu

TTA

Leu

AGC

Ser

TAC

Tyr

GCC

Ala

AAA

Lys

TGC

Cys

GGA

Gly

AAT

Asn 155

TAT

Tyr 3;GG Giy

GAT

Asp

TGT

Cys

CAG

Gin

AGC

Ser

AGG

Arg

TTG

Leu

ATT

Ile 120

TCA

Ser

GCA

Ala

CTG

Leu

GCC

Ala 543 579 615 651 687 723 759 795 831 867 903 939 TCC TCC AGC AGA CTC CTC AGC AAC AAG AAT CAA TTC 975 Ser Ser Ser Arg Leu Leu Ser Asn Lys Asn Gin 170 175 180 AAG TCT TTC CTC Lys Ser Phe Leu CGA ACC ATC Arg Thr Ile 185 CCC AAT Pro Asn GAT GAG CAC Asp Glu His 190 1011 CAG GCC ACT Gin Ala Thr 195 GCC ATG GCA GAC Ala Met Ala Asp

ATC

Ile 200 ATC GAG TAT TTC Ile Glu Tyr Phe 1047

CGC

Arg 205 TGG AAC TGG GTG Trp Asn Trp Val GGC ACA Gly Thr 210 ATT GCA Ile Ala GCT GAT GAC Ala Asp Asp 215 TTC CGA GAG Phe Arg Glu 1083 1119 GAC TAT GGG Asp Tyr Gly

CGG

Arg 220 CCG GGG ATT GAG Pro Gly Ile Glu

AAA

Lys 225 :.o GAA GCT Glu Ala 230 GAG GAA AGG Glu Glu Arg GAT ATC Asp Ile 235 TGC ATC GAC TTC Cys Ile Asp Phe

AGT

Ser 240 GAA CTC ATC TCC Glu Leu Ile Ser

CAG

Gin 245 TAC TCT GAT GAG Tyr Ser Asp Glu GAA GAG ATC Glu Glu Ile 250 1155 1191 1227 CAG CAT Gin His

GTG

Val 255 GTA GAG GTG ATT Val Glu Val Ile

CAA

Gin 260 AAT TCC ACG GCC Asn Ser Thr Ala

AAA

Lys 265 GTC ATC GTG GTT Val Ile Val Val TTC TCC Phe Ser 270 AGT GGC Ser Gly CCA GAT Pro Asp 275

CTT

Leu 1263 GAG CCC CTC Glu Pro Leu

ATC

Ile 280 AAG GAG ATT GTC Lys Glu Ile Val

CGG

Arg 285 CGC AAT ATC Arg Asn Ile 1299 ACG GGC Thr Gly 290 AAG ATC TGG Lys Ile Trp CTG GCC Leu Ala 295 AGC GAG GCC TGG Ser Glu Ala Trp

GCC

Ala 300 1335 AGC TCC.TCC CTG Ser Ser Ser Leu GTG GTT GGC GGC Val Val Gly Gly 315 GGG CAG ATC CCA Gly Gin Ile Pro 325 GTC CAT CCC AGG Val His Pro Arg 340

ATC

Ile 305 GCC ATG CCT Ala Met Pro CAG TAC TTC CAC Gin Tyr Phe His 310 GCT CTG AAG GCT Ala Leu Lys Ala ACC ATT GGA Thr Ile Gly GGC TTC CGG Gly Phe Arg 330

TTC

Phe 320 1371 1407 1443 1479 GAA TTC CTG AAG AAG Glu Phe Leu Lys Lys 335

AAG

Lys TCT GTC CAC Ser Val His AAT GGT TTT GCC Asn Gly Phe Ala 345 AAG GAG TTT TGG GAA GAA ACA Lys

CAA

Gin

TTT

Phe

AGC

Ser 385

GGG

Gly

ATA

Ile

TAC

Tyr

GAT

Asp

ACC

Thr 445

GCG

Ala Giu 350

GAA

Giu

CTG

Leu

AAC

As n

GAT

Asp

GAT

Asp 410

TTA

Leu

ATA

Ile

AAT

Asn

TGG

Trp Phe

GGT

Gly

AGA

Arg 375

AGC

Ser

GAG

Glu

TAC

Tyr

GCA

Ala

TAT

Tyr 435

GGC

Gly

CAG

Gin Trp

GCA

Ala

GGT

Gly

TCG

Ser

AAC

Asn 400

ACG

Thr

GTC

Val

ACC

Thr

TCC

Ser

GTC

Val 460 Giu

AAA

Lys 365

CAC

His

ACA

Thr

ATC

Ile

CAT

His

TAC

Tyr 425

TGC

Cys

TGT

Cys

CTG

Leu

ATG

Met

CTG

Leu 485

TCC

Ser Giu

GGA

Gly

CAA

Giu

GCC

Ala 390

AGC

Ser

TTA

Leu

TCC

Ser

TTA

Leu

GCA

Ala 450

AAG,

Lys

GGG

Gly

GTG

Val

CCA

Pro Thr 355

CCT

Pro

GAA

Giu

TTC

Phe

AGT

Ser

CGG

Arg 415

ATT

Ile

CCT

Pro

GAC

Asp

CAC

His

GAG

Glu 475

GGG

Gly

GAG

Giu TTT AAC Phe Asn TTA CCT Leu Pro AGT GGC Ser Gly 380 CGA CCC Arg Pro GTC GAG Val Giu 405 ATA TCC Ile Ser GCC CAC Ala His GGG AGA Gly Arg 440 ATC AAG Ile Lys CTA CGG Leu Arg 465 CAG GTG Gin Val MAC TAT Asn Tyr CAT GGC Asp Cly 500 TGC CAC Cys His GTG GAC Val Asp 370 GAC AGG Asp Arg CTC TGT Leu Cys 395 ACC CCT Thr Pro TAC AAT Tyr Asn GCC TTG Ala Leu 430 GGG CTC Gly Leu AAA GTT Lys Val 455 CAT CTA His Leu ACC TTT Thr Phe TCC ATC Ser Ile 490 TCC ATC Ser Ile

CTC

Leu 360

ACC

Thr

TTT

Phe

ACA

Thr

TAC

Tyr

GTG

Val 420

CAA

Gin

TTC

Phe

GAG

Giu

AAC

Asn

GAT

Asp 480

ATC

Ile

CTG

Val 1515 1551 1587 1623 1659 1695 1731 1767 1803 1839 1875 1911 1947 1983 TTT ACA AAC AAT Phe

GAG

Glu

AAC

Asn Thr 470

TGT

Cys

TGG

Trp Asn Asn GGT CAC Cly Asp CAC CTC His Leu 495 TTT AAG CAA GTC CGG TAT.TAC MAC GTC TAT GCC AAG Phe Lys Giu Val Gly 505 Tyr 510 Tyr Asn Val Tyr Ala Lys 515 AAG GGA GAA AGA CTC TTC ATC AAC GAG GAG AAA ATC Lys Gly Glu Arg Leu Phe Ile Asn

CTG

Leu

GTG

Val1

AGC

Ser

ATT

Ile 565

GAG

Glu

GCC

Ala

TCC

Ser

ATC

Ile

GCA

Ala 625

ACA

Thr

AAC

Asn

TCC

Ser

TCC

Ser TGG AGT Trp Ser 530 CTG TCT Leu Ser CGA GAC Arg Asp 555 GAG GGG Glu Gly TOT CCT Cys Pro AGT GCC Ser Ala 590 AAT GAG Asn Glu GAG TTT Giu Phe 615 CTC ACC Leu Thr GCC TTT Ala Phe ACA CCC Thr Pro 650 TAC CTC Tyr Leu AGC TCC Ser Ser 675 GGG TTC Gly Phe GTC CTC Val Leu 545 TGC CTG Cys Leu GAG CCC Glu Pro GAT GGG Asp Gly 580.

TGT AAC Cys Asn AAC CAC Asn His 605 CTG TCG Leu Ser CTC TTT' Leu Phe GTG CTG Val Leu 640 ATT GTC Ile Val CTC CTC Leu Leu 665 CTG TTC Leu Phe TCC AGG Ser Arg 535 CAG GTG Gin Val GCA GGG Ala Gly ACC TGC Thr Cys 570 GAG TAT Giu Tyr AAG TGC Lys Cys 595 ACC TCC Thr Ser TGG ACG Trp Thr GCC GTG Ala Val.

630 GGT GTG Gly Val.

AAG GCC Lys Ala 655 TTC TCC Phe Ser TTC ATC Phe Ile

GAG

Glu

CCC

Pro

ACC

Thr 560

TGC

Cys

AGT

Ser

CCA

Pro

TGC

Cys

GAG

Glu 620

CTG

Leu

TTT

Phe

ACC

Thr

CTG

Leu

GGG

Gly 680 Glu Glu Lys 525 CCA CTC ACC Pro Leu Thr TTC TCC AAC Phe Ser Asn 550 AGG AAA GGG Arg Lys Giy TTT GAG TGT Phe Giu Cys 575 GAT GAG ACA Asp Glu Thr 585 GAT GAC TTC Asp Asp Phe ATT GCC AAG Ile Ala Lys 610 CCC TTT GGG Pro Phe Gly GGC ATT TTC Gly Ile Phe 635 ATC AAG TTC Ile Lys Phe 645 AAC CGA GAG Asn Arg Glu CTC TGC TGC Leu Cys Cys 670 GAG CCC CAG Glu Pro Gin Ile

TTT

Phe 540

TGC

Cys

ATC

Ile

GTG

Val

GAT

Asp

TGG

Trp 600

GAG

Glu

ATC

Ile

CTG

Leu

CGC

Arg

CTC

Leu 660

TTC

Phe

GAC

Asp 2019 2055 2091 2127 2163 2199 2235 2271 2307 2343 2379 2415 2451 2487 TGG ACG Trp Thr 685 TGC CGC Cys Arg CTG CGC Leu Arg 690 CAG CCG GCC TTT GGC ATC Gin Pro Ala Phe Giy Ile 69S AGC TTC GTG Ser Phe Val CT c Leu 700 TGC ATC TCA TGC ATC CTG GTG AAA Cys Ile Ser Cys Ile Leu Val Lys 705 2523 2559 2595 2631 ACC AAC Thr Asn 710 CGT GTC CTC CTG Arg Val Leu Leu GTG TTT GAG GOC AAG Val Phe Giu Ala Lys 715

ATC

Ile 720 CCC ACC AGC TTC Pro Thr Ser Phe

CAC

His 725 CGC AAG TGG TGG GGG CTC AAC Arg Lys Trp Trp Gly Leu Asn 730 CTG CAG TTC Leu Gin Phe 735 CTG CTG GTT Leu Leu Val TTC CTC TGC ACC TTC ATG Phe Leu Cys Thr Phe Met 740 ATC TGG CTC TAC ACC GCG Ile Trp Leu Tyr Thr Ala 755

CAG

Gin 745 ATT GTC ATC TGT Ile Val Ile Cys

GTG

Val 750 CCC CCC TCA Pro Pro Ser

AGC

Ser 760 TAC CGC AAC CAG GAG CTG GAG GAT Tyr Arg Asn Gin Glu Leu Giu Asp 765 2667 2703 2739 2775 2811 2847 GAG ATC Giu Ile 770 ATC TTC ATC ACG Ile Phe Ile Thr TGC CAC GAG GGC TCC Cys His Giu Gly Ser 775

CTC

Leu 780 ATG GCC CTG GGC Met Ala Leu Gly

TTC

Phe 785 CTG ATC GGC TAC ACC TGC CTG Leu Ile Gly Tyr Thr Cys Leu 790

S

*5 CTG GCT GCC Leu Ala Ala 795 ATC TGC TTC Ile Cys Phe

CGG

Arg 805 AAG CTG CCG GAG Lys Leu Pro Giu

AAC

Asn 810 TTC TTT GCC TTC AAG, TCC Phe Phe Ala Phe Lys Ser 800 TTC AAT GAA GCC AAG, TTC Phe Asn Glu Ala Lys Phe 815 ATC TTC TTC ATC GTC TGG Ile Phe Phe Ile Val Trp 825 2883 ATC ACC TTC Ile Thr Phe

AGC

Ser 820 ATG CTC Met Leu CCA GCC Pro Ala 2919 2955 ATC TCC TTC ATT Ile Ser Phe Ile 830 TAT GCC AGC ACC TAT Tyr Ala Ser Thr Tyr 835

GGC

Gly 840 AAG TTT GTC TCT GCC GTA Lys Phe Val Ser Ala Vai 845 GAG GTG ATT GCC ATC CTG Glu Val Ile Ala Ile Leu 850 2991 GCA GCC Ala Ala AG C Ser 855 TTT GGC TTG CTG Phe Gly Leu Leu GCG TGC Ala Cys 860 TTC AAG Phe Lys

AAC

Asn 865 AAG ATC Lys Ile TAC ATC ATT Tyr Ile Ile 870 GAG GAG GTG Glu Glu Val 880

CTC

Leu ATC TTC TTC Ile Phe Phe CCA TCC CGC Pro Ser Arg 875 ACC GCA GCT Thr Ala Ala 3027 3063 3099 AAC ACC ATC Asn Thr Ile CGT TGC AGC Arg Cys Ser 885 CAC GCT His Ala 890 TTC AAG GTG GCT Phe Lys Val Ala

GCC

Ala 895 CGG GCC ACG CTG Arg Ala Thr Leu

CGC

Arg 900 3135 9 9999 CGC AGC AAC Arg Ser Asn GTC TCC CGC Val Ser Arg 905 AAG CGG TCC Lys Arg Ser GGA GGC Gly Gly

TCC

Ser 915 ACG GGA TCC ACC Thr Gly Ser Thr CCC TCC Pro Ser 920 AGC AGC CTT Ser Ser Leu 910 TCC TCC ATC Ser Ser Ile TTC CCA CGG Phe Pro Arg 935 CTG GCC CTA Leu Ala Leu 3171 3207

AGC

Ser 925 AGC AAG Ser Lys AGC AAC AGC Ser Asn Ser 930 CAG AAG CAG Gin Lys Gin 940 GAA GAC CCA Giu Asp Pro CAG CAG CCG Gin Gin Pro 945 3243 3279 CCC GAG AGG Pro Giu Arg ACC CAG Thr Gin 950 CAA GAG CAG CAG Gin Giu Gin Gin

CAG

Gin 955 CAG CCC CTG ACC Gin Pro Leu Thr CTC 3315 Leu 960 CCA CAG CAG Pro Gin Gin CAA CGA TCT Gin Arg Ser 965 CAG CAG CAG Gin Gin Gin AAG CAG Lys Gin

AAG

Lys 975 GTC ATC TTT GGC Val Ile Phe Giy AGC GGC Ser Gly 980 CCC AGA TGC Pro Arg Cys 970 ACG GTC ACC Thr Val Thr AAG, AAC GCC Lys Asn Ala 995 3351 3387

TTC

Phe 985 TCA CTG Ser Leu ATG CCC CAC Met Ala His GAG CCC CAG Giu Ala Gin 1010 AGC TTT CAT Ser Phe Asp 990 AGG AAT TCT Arg Asn Ser 1000 AAA AGC AGC Lys Ser Ser GAG CCT CAG Giu Pro Gin ACG CAC CAG AAC TCC CTG Thr His Gin Asn Ser Leu 1005 GAT ACG CTG ACC CGA CAC Asp Thr Leu Thr Arg His 1015 1020 3423 3459 3495 CAG CCA TTA CTC CCG CTG CAG TOC GGG GAA ACG GAC Gin Pro Leu Leu Pro Leu Gin Cys Gly Glu Thr Asp 1025 1030 TTA GAT CTG ACC GTC CAG GAA ACA GGT CTG CAA GGA Leu Asp Leu Thr Vai Gin Giu Thr Giy Leu Gin Giy 1035 .1040 CCT GTG GGT GGA GAC CAG CGG CCA GAG GTG GAG GAC Pro Val Gly Gly Asp Gin Arg Pro Giu Val Giu Asp 1045 1050 1055 CCT GAA GAG TTG TCC CCA GCA CTT GTA GTG TCC AGT Pro G1U Giu Leu Ser Pro Ala Leu Val Val Ser Ser 1060 1065 TCA CAG AGC TTT GTC ATC AGT GGT GGA GGC AGC ACT Ser Gin Ser Phe Vai Ile Ser Gly Gly Gly Ser Thr 1070 1075 1080 GTT ACA GAA AAC GTA GTG AAT TCA TAAAATGGAA 3531 3567 3603 3639 3675 3709 a.

a a. I a. *aa..

a a a.

a a. a a p Val Thr Giu Asn Val Val 1085 Asi *Ser

GGAGAAGACT

GGGTCCCAGG

TGAGGAAGAA

TTAGT CACAC

TCCCTTTAAA

TGGTTGCATT

TTTGCTGTTC

ATCCCACCCA

CTACCCTCCA

GCCACCACTA

CAGTCCTGCC

TGTTCACAAC

AAAACCATAT

TATGTAACAT

TGTCTGGTTC

GGGCTAGGGA

GATGAGGAAT

GGGATAATAG

CATCTTAAAT

ATTAAAAAAA

TGTCAAAGCA

ACCCACATCT

ACAGCTCAGA

GAGTGTGCA.G

GAGCTGAGAG

CAATGCCTTG

ATAAGGAGAA

GATATTTTGT

CCAGAAGGTT

TGTCCAGGAC

GAATG CAGAG

CGCCCCAGAC

ACACATCAAA

GACAGTGAAT

AGAAGAGCCT

TTGAGATCTC

AATGTCTCTT

GATGAAACTA

ACTGATGGGA

TCTGAAAGAC

ACAACAGACT

TGTATCTCCT

CTCC!TACCTG

TGCACCCCTC

ATGATACTGA

AGGTTTCTTG

TCCTTTCCTC

TGCCCCGAAT

TGACCCATGT

TGTGTTTCTG

CACGGTCAGA

CCTCTGTTCT

TGGCTTTAAA

CATCAAATTT

AGAATGTCAC

GAATTTTAAA

CCTATTTATG

CTGCTGCTAT

CTATACCATA

TGCCATGTTT

3749 3789 3829 3869 3909 3949 3989 4029 4069 4109 4149 4189 4229 4269 4309 4349 AGATTCCAGG ATCACAAGAA TCACCTLCAAA TTGTTAGGAA a..6.

'at, 004.

GGGACTGCAT AAACCAATGA TCCTATATGT AGCTTTATCC TAATAGTCCA TGGACAATAT GGTTTATATA AGGCAGTATT CCCACTATCC TCACTCCCAT CCCTTCAGGG ACTCAAGGGT TACCCCAAAG AATTCCTGAA GTACAGAGTA AGTTCTCAAT CTAGGGTAGG AAAGCGTGGT AAATGTCGGA GCTATGTTCC CTGCCGGTCA CCCAGGCTCT GGGTTCAAGC CATGGCTTCG TGTAGGCAGG GAACCTTAAC TCATCTTTAA AATAAGGATA GAGCTCTTAT GTGGATTAAA ACTTTAGCCT GGTACCTAGC ATATTAGTTA ATATTAT

GCTGTATCTG

TTAGGAAAAT

AAACTGAAAA

ATTGAGCTCT

AAGCTAAGCC

CCAGAAGTCC

GCCAGATCCA

TATTGGCCTG

TCCAAGAAAG

CTCCAGCAGT

GGAGCCAGAG

TCATTTGCAA

CTCTCTAA.GC

ATAAT CATT C

CGAGATAATG

ACACA-ATAAG

TAATTAATAT

GCTTCTGTTG

ATGT CAGTCT

ATTTCCCCAC

TTATGTGAGC

CTCCCATCTC

CCCTATCCCT

CTAATAGCTG

AT CCAC CCTC

GGTATTAATA

AGACAGACCG

GCTGAGTGAC

CACAGCTTCT

CTTCCCCTCA-.

TATATAAAGT

CATTCAATAA

4389 4429 4469 4509 4549 4589 4629 4669 4709 4749 4789 4829 4869 4909 4949 4989 5006 INFORMATION FOR SEQ ID NO: 2: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 3809 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 373. .3606 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CAACAGGCAC CTGGCTS3CAG CCAGGAAGGA CCGCACGCCC TTTCGCGCAG GAGAGTGGAA GGAGGG ACCGAGGTCT TGCGGCACAG GCAACG TGCAGAATGA AAGGCATCAC AGGAGG GGCTTCCAAA GACTCAAGGA CCACCC GATTGAGGAA GGCAGAAATG GAGATI TCTATTATTT TATTAATCAA TCTGTP CTGCAGGGAG TGAACTGCTC CAAGGC GCCTCCAAAC TCCTAGCTGT CTCATC GACGGCAGAA CC ATG GCA TTT TT Met Ala Phe T 1 GTC CTC TTG GCA CTC ACC TGG Val Leu Leu Ala Leu Thr Trp, 15 GGG CCA GAC CAG CGA GCC CAA Gly Pro Asp Gln Arg Ala Gln AGCT GTTTGCCAGC CTTG ACCTGAGTCT CCTC TGCATGATGT ACAT TACAAGTCTG CAAA CACCACGTCT LGACA TGTGTCCCCA AGAA ACTTCTGGGA CCTT GCCCTGGAGA ~T AGC TGC TGC TGG ~r Ser Cys Cys Trp CAC ACC TCT GCC TAC 120 160 200 240 280 320 360 396 o wes *904 0ge 0 06 432 His Thr Ser Ala Tyr 4.

ATC CTT GGG GGG Ile Leu Gly Gly GCA GCT AAA GAT Ala Ala Lys Asp TCT GTG GAA TGT Ser Val Glu Cys CGC TGG TTA CAG Arg Trp Leu Gln ATA AAC AGC AGC Ile Asn Ser Ser CTG GGA TAC AGG Leu Gly Tyr Arg CTC TTT CCT Leu Phe Pro CAA GAT CTC Gln Asp Leu 50 ATC AGG TAT Ile Arg Tyr GCT ATG ATA Ala Met Ile 75 CCA GCC CTT Pro Ala Leu AAG AAG GGG GAC ATT Lys Lys Gly Asp Ile ATT CAT.TTT GGA GTA Ile His Phe Gly Val AAA TCA AGG CCG GAG Lys Ser Arg Pro Glu AAT TTC CGT GGG TTT Asn Phe Arg Gly Phe TTT GCC ATA GAG GAG Phe Ala Ile Glu Glu 468 504 540 576 612

ACG

Thr CTT CCC AAC TTG Leu Pro Asn Leu 648 ATA TTT GAC ACT TGC AAC ACC GTT 684 Ile Phe Asp Thr Cys Asn Thr Val 100 TCT AAG GCC TTG GAA GCC ACC CTG AGT TTT GTT GCT Ser Lys Ala Leu Glu Ala Thr Leu Ser Phe Val Ala 105 110 115 720 CAA AAC AAA Gin Asn Lys ATT GAT TCT TTG AAC CTT Ile Asp Ser Leu Asn Leu 120 125 GAT GAG TTC Asp Glu Phe 756 TGC AAC Cys Asn 130 TGC TCA Cys Ser GAG CAC ATT CCC TCT ACG ATT GCT Glu His Ile Pro Ser Thr Ile Ala 135 140 792 GTG GTG GGA GCA Val Val Gly Ala ACT GGC Thr Gly 145 TCA GGC GTC Ser Gly Val TCC ACG GCA Ser Thr Ala 150 828 GTG GCA AAT Val Ala Asn 155 CTG CTG GGG CTC Leu Leu Gly Leu

TTC

Phe 160 TAC ATT CCC CAG Tyr Ile Pro Gin 864

GTC

Val 165 AGT TAT GCC Ser Tyr Ala TCC TCC Ser Ser 170 AGC AGA CTC Ser Arg Leu AAG AAT CAA Lys Asn Gin

TTC

Phe 180 AAG TCT TTC CTC Lys Ser Phe Leu

CGA

Arg 185 CTC AGC AAC Leu Ser Asn 175 ACC ATC CCC Thr Ile Pro GCA GAC ATC Ala Asp Ile 200 900 936 AAT GAT Asn Asp 190 GAG CAC CAG GCC Glu His Gin Ala

ACT

Thr 195 GCC ATG Ala Met 972 ATC GAG TAT TTC Ile Glu Tyr Phe GCA GCT GAT GAC Ala Ala Asp Asp 215

CGC

Arg 205 TGG AAC TGG Trp Asn Trp GTG GGC ACA ATT Val Gly Thr Ile 210 CCG GGG ATT GAG Pro Gly Ile Glu GAC TAT GGG Asp Tyr Gly

CGG

Arg 220 1008 1044 1080 1116

AAA

Lys 225 TTC CGA GAG GAA Phe Arg Glu Glu

GCT

Ala 230 GAG GAA AGG Glu Glu Arg ATC GAC TTC Ile Asp Phe GAG GAA GAG Glu Glu Glu 250

AGT

Ser 240 GAA CTC ATC TCC Glu Leu Ile Ser

CAG

Gin 245 ATC CAG CAT Ile Gin His

GTG

Val 255 GTA GAG Val Glu GAT ATC TGC Asp Ile Cys 235 TAC TCT GAT Tyr Ser Asp GTG ATT CAA Val Ile Gin 260 TTC TCC AGT Phe Ser Ser 270 GAG ATT GTC Glu Ile Val 1152 AAT TCC ACG GCC Asn Ser Thr Ala GGC CCA GAT CTT Gly Pro Asp Leu 275

AAA

Lys 265 GTC ATC GTG GTT Val Ile Val Val 1188 1224 GAG CCC CTC Glu Pro Leu ATC AAG Ile Lys 280 CGG CGC AAT ATC ACG GGC Arg Arg Asn 285 GAG GCC TGG Glu Ala Trp CAG TAC TTC Gin Tyr Phe 310 Ile Thr Gly 290 AAG ATC TGG CTG GCC AGC Lys Ile Trp Leu Ala Ser 295 1260 1296 GCC Ala 300 AGC TCC TCC CTG ATC Ser Ser Ser Leu Ile 305 GCC ATG CCT Ala Met Pro CAC GTG GTT His Val Val GGC GGC ACC ATT GGA TTC Gly Gly Thr Ile Gly Phe 315 320 1332 GCT CTG AAG GCT Ala Leu Lys Ala

GGG

Gly 325 CAG ATC CCA Gin Ile Pro GGC TTC CGG GAA Gly Phe Arg Glu 330 AAG TCT GTC CAC Lys Ser Val His 1368 1404 TTC CTG Phe Leu

AAG

Lys 335 AAG GTC CAT Lys Val His CCC AGG Pro Arg 340

AAT

Asn 345 GGT TTT GCC AAG Gly Phe Ala Lys

GAG

Glu 350 TTT TGG Phe Trp AAC TGC CAC Asn Cys His

CTC

Leu 360 CAA GAA GGT GCA Gin Giu Gly Ala CCT GTG Pro Vai 370 GAC ACC TTT Asp Thr Phe CTG AGA GGT Leu Arg Gly 375 AAC AGC TCG Asn Ser Ser GAA GAA ACA TTT Glu Glu Thr Phe 355 AAA GGA CCT TTA Lys Gly Pro Leu 365 CAC GAA GAA AGT His Giu Giu Ser 380 ACA GCC TTC CGA Thr Ala Phe Arg 390 ATC AGC AGT GTC Ile Ser Ser Val 1440 1476 1512 1548 1584 GGC GAC AGG TTT Gly Asp Arg Phe CCC CTC TGT ACA Pro Leu Cys Thr 395

AGC

Ser 385 GGG GAT GAG Gly Asp Glu

AAC

Asn 400

GAG

Glu 405 ACC CCT Thr Pro TAC ATA GAT Tyr Ile Asp 410 TAC ACG CAT Tyr Thr His GCA GTC TAC Ala Val Tyr 425 TTA CGG ATA Leu Arg Ile 415 TCC ATT GCC Ser Ile Ala 1620 TCC TAC AAT GTG TAC TTA Ser Tyr Asn Val Tyr Leu 420 1656 CAC GCC TTG CAA GAT ATA TAT His Ala Leu Gin Asp Ile Tyr 430 435 ACC TGC TTA CCT GGG Thr Cys Leu Pro Gly 440 1692 1728 AGA GGG CTC TTC ACC AAT GGC TCC TGT GCA GAC ATC 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 Lys Lys Val Glu Ala Trp Gin 9*

CGG

Arg 465

GTG

Val

TAT

Tyr

GGC

Gly

GTC

Val

GAG

Glu 525

GTG

Val

GGG

Gly

TGC

Cys

TAT

Tyr

TGC

Cys 585

TCC

Ser

ACG

Thr 455 CAT CTA His Leu ACC TTT Thr Phe TCC ATC Ser Ile 490 TCC ATC Ser Ile TAT GCC Tyr Ala 515 GAG AAA Glu Lys CCC TTC Pro Phe ACC AGG Thr Arg 550 TGC TTT Cys Phe AGT GAT Ser Asp 575 CCA GAT Pro Asp TGC ATT Cys Ile GAG CCC Glu Pro 610

AAC

Asn

GAT

Asp 480

ATC

Ile

GTG

Val

AAG

Lys

ATC

Ile

TCC

Ser 540

AAA

Lys

GAG

Glu

GAG

Glu

GAC

Asp

GCC

Ala 600

TTT

Phe TTT ACA AAC Phe Thr Asn 470 GAG TGT GGT Glu Cys Gly AAC TGG CAC Asn Trp His 495 TTT AAG GAA Phe Lys Glu 505 AAG GGA GAA Lys Gly Glu CTG TGG AGT Leu Trp Ser 530 AAC TGC AGC Asn Cys Ser GGG ATC ATT Gly Ile Ile 555 TGT GTG GAG Cys Val Glu 565 ACA GAT GCC Thr Asp Ala TTC TGG TCC Phe Trp Ser 590 AAG GAG ATC Lys Glu Ile GGG ATC GCA Gly Ile Ala 615 Val Leu Lys 460 AAT ATG GGG Asn Met Gly GAC CTG GTG Asp Leu Val 485 CTC TCC CCA Leu Ser Pro GTC GGG TAT Val Gly Tyr 510 AGA CTC TTC Arg Leu Phe 520 GGG TTC TCC Gly Phe Ser CGA GAC TGC Arg Asp Cys 545 GAG GGG GAG Glu Gly Glu TGT CCT GAT Cys Pro Asp 570 AGT GCC TGT Ser Ala Cys 580 AAT GAG AAC Asn Glu Asn GAG TTT CTG Glu Phe Leu 605 CTC ACC CTC Leu Thr Leu His Leu GAG CAG Glu Gin 475 GGG AAC Gly Asn GAG GAT Glu Asp 500 TAC AAC Tyr Asn ATC AAC Ile Asn AGG GAG Arg Glu 535 CTG GCA Leu Ala CCC ACC Pro Thr 560 GGG GAG Gly Glu AAC AAG Asn Lys CAC ACC His Thr 595 TCG TGG Ser Trp TTT GCC Phe Ala 620 1764 1800 1836 1872 1908 1944 1980 2016 2052 2088 2124 2160 2196 2232 GTG CTG GGC ATT TTC CTG Val Leu Gly GTG TTT ATC Val Phe Ile 635 Ile Phe Leu 625 AAG TTC CGC Lys Phe Arg ACA GCC TTT GTG CTG GGT Thr Ala Phe Val Leu Giy 630 AAC ACA CCC ATT GTC AAG 2268 2304 Asn Thr Pro '640 Ile Val Lys

GCC

Ala 645 ACC AAC CGA Thr Asn Arg GAG CTC Giu Leu 650 TCC TAC CTC CTC Ser Tyr Leu Leu CTC TTC Leu Phe 655 2340 TCC CTG CTC Ser Leu Leu

TGC

Cys 660 TGC TTC TCC Cys Phe Ser AGC TCC Ser Ser 665 CTG TTC TTC Leu Phe Phe 2376 S S 5* S S

S

5*

S

S

S.

ATC GGG Ile Gly 670 GAG CCC CAG GAC Giu Pro Gin Asp

TGG

Trp 675 ACG TGC CGC CTG Thr Cys Arg Leu

CGC

Arg 680 2412 2448 CAG CCG GCC TTT Gin Pro Aia Phe

GGC

Giy 685 ATC AGC TTC GTG Ile Ser Phe Vai

CTC

Leu 690 TGC ATC Cys Ile TCA TGC ATC Ser Cys Ile 695 CTG GTG AAA ACC Leu Vai Lys Thr AAC CGT Asn Arg 700 GTC CTC CTG Val Leu Leu 2484

GTG

Val 705 TTT GAG GCC AAG Phe Giu Ala Lys

ATC

Ile 710 CCC ACC AGC TTC Pro Thr Ser Phe

CAC

His 715

CGC

Arg 2520 AAG TGG TGG Lys Trp Trp

GGG

Giy 720 CTC AAC CTG CAG Leu Asn Leu Gin

TTC

Phe 725 CTG CTG GTT Leu Leu Vai 2556 2592 TTC CTC Phe Leu 730 TGC ACC TTC ATG Cys Thr Phe Met

CAG

Gin 735 ATT GTC ATC TGT Ile Val Ile Cys

GTG

Val 740 ATC TGG CTC TAC Ile Trp Leu Tyr

ACC

Thr 745 GCG CCC CCC Ala Pro Pro TCA AGC TAC CGC Ser Ser Tyr Arg 750 ATC TTC ATC ACG Ile Phe Ile Thr AAC CAG Asn Gin

GAG

Giu 755 CTG GAG GAT GAG Leu Giu Asp Giu

ATC

Ile 760 2628 2664 2700

TGC

Cys 765 CAC GAG GGC His Giu Giy TCC CTC ATG Ser Leu Met 770 GCC CTG GGC Ala Leu Gly TTC CTG Phe Leu 775 ATC GGC TAC ACC TGC CTG CTG Ile Gly Tyr Thr Cys Leu Leu 780 GCT GCC ATC TGC TTC Ala Ala Ile Cys Phe 785 2736 TTC TTT GCC TTC Phe Phe Ala Phe 790 TTC AAT GAA GCC Phe Asn Giu Ala AAG TCC CGG AAG CTG CCG GAG AAC Lys Ser Arg Lys Leu Pro Giu Asn 795 800

AAG

Lys 805 TTC ATC ACC TTC AGC ATG CTC Phe Ile Thr Phe Ser Met Leu 810 2772 2808 2844 ATC TTC Ile Phe TTC ATC Phe Ile 815 GTC TGG ATC TCC TTC ATT CCA GCC Val Trp Ile Ser Phe Ile Pro Ala 820 TAT GCC Tyr Ala 825 AGC ACC TAT Ser Thr Tyr GGC AAG TTT GTC TCT GCC GTA Gly Lys Phe Val Ser Ala Val 830 835 2880

S

GAG GTG Glu Val CTG GCG Leu Ala 850 ATT GCC Ile Ala 840 ATC CTG GCA GCC AGC TTT GGC TTG Ile Leu Ala Ala Ser Phe Gly Leu 845 2916 2952 TGC ATC TTC Cys Ile Phe TTC AAC AAG ATC TAC ATC ATT Phe Asn Lys Ile Tyr Ile Ile 855 860 CGC AAC ACC ATC GAG GAG GTG Arg Asn Thr Ile Giu Giu Val 870 CTC TTC AAG CCA Leu Phe Lys Pro

TCC

Ser 865 2988 CGT TGC Arg Cys AGC ACC Ser Thr 875

GCC

Ala 885 CGG GCC ACG Arg Ala Thr GCA GCT CAC GCT TTC.AAG GTG GCT Ala Ala His Ala Phe Lys Val Ala 880 CTG CGC CGC AGC AAC GTC TCC CGC Leu Arg Arg Ser Asn Val Ser Arg 890 895 AGC CTT GGA GGC TCC ACG GGA TCC Ser Leu Gly Gly Ser Thr Gly Ser 905 3024 AAG CGG TCC AGC Lys Arg Ser Ser 900 ACC CCC Thr Pro 910 TCC TCC TCC Ser Ser Ser ATC AGC AGC AAG AGC AAC AGC Ile Ser Ser Lys Ser Asn Ser 915 920 CAG CCC GAG AGG CAG AAG CAG Gin Pro Glu Arg Gin Lys Gin 930 3060 3096 3132 3168 3204 3240 GAA GAC CCA TTC Glu Asp Pro Phe

CCA

Pro 925 CAG CAG Gin Gin CCG CTG Pro Leu 935 GCC CTA ACC CAG CAA GAG CAG CAG Ala Leu Thr Gin Gin Glu Gin Gin 940 ACC CTC CCA CAG CAG CAA CGA TCT Thr Leu Pro Gin Gin Gin Arg Ser 950 955

CAG

Gin 945 CAG CCC CTG Gin Pro Leu CAG CAG CAG Gin Gin Gin GGC AGC GGC Gly Ser Gly 970 GAG CCT CAG Glu Pro Gin ACG CAC CAG Thr His Gin 995 GAT ACG CTG Asp Thr Leu 1005 CAG TGC GGG Gin Cys Gly GAA ACA GGT Glu Thr Gly 1030 CGG CCA GAG Arg Pro Glu CCC AGA Pro Arg 960 ACG GTC Thr Val AAG AAC Lys Asn 985 AAC TCC Asn Ser ACC CGA Thr Arg GAA ACG Glu Thr 1020 CTG CAA Leu Gin 100 TGC AAG CAG AAG GTC Cys Lys Gin Lys Val 965 ACC TTC TCA CTG AGC Thr Phe Ser Leu Ser 975 GCC ATG GCC CAC GGG Ala Met Ala His Gly 990 CTG GAG GCC CAG AAA Leu Giu Ala Gin Lys 1000 CAC CAG CCA TTA CTC His Gin Pro Leu Leu 1010 GAC TTA GAT CTG ACC Asp Leu Asp Leu Thr 1025 GGA CCT GTG GGT GGA Gly Pro Val Gly Gly 1035 ATC TTT Ile Phe TTT GAT Phe Asp 980 AAT TCT Asn Ser AGC AGC Ser Ser CCG CTG Pro Leu 1015 GTC CAG Vai Gin GAC CAG Asp Gin 1040 3276 3312 3348 3384 3420 3456 3492 3528 GTG GAG GAC CCT GAA GAG TTG TCC CCA Val Giu Asp Pro Giu Glu Leu Ser Pro 1045 1050 GCA CTT GTA GTG TCC AGT TCA CAG AGC TTT GTC ATC Ala Leu Vai Val Ser Ser Ser Gin Ser Phe Val Ile 1055 1060 AGT GGT GGA GGC AGC ACT GTT ACA GAA AAC GTA GTG Ser Gly Giy Giy Ser TIr Val Thr Giu Asn Val Val 1065 1070 1075 AAT TCA TAAAATGGAA GGAGAAGACT GGGCTAGGGA Asn Ser GAATGCAGAG AGGTTTCTTG GGGTCCCAGG GATGAGGAAT CGCCCCAGAC TCCTTTCCTC TGAGGAAGAA GGGATAATAG ACACATCAAA TGCCCCGAAT TTAGTCACAC CATCTTAAAT GACAGTGAAT TGACCCATGT TCCCTTTAAA AAAAAAAAAA AAAAAGCGGC

CGC

3564 3600 3636 3676 3716 3756 3796 3809

Claims (19)

1.A process for producing a medicament comprising the step of combining a therapeutically effective amount of the compound of any one of the compounds selected from the group consisting of: CN-(2-chlorophenylpropyl)- 1 2 -trifluoroethoxy)phenyl)ethylani ne);, 21 M -trifluoromethoxy)propyl)- 1-(3 -methoxyphenyl)ethylarnine)-I 21 S ((R)-N-(3-(2-chlorophenyl)propyl)-I 3 -propoxypheny)ethylamine),- 21 IT -chlorophenyl)propyl)- I -isopropoxyphenyl)ethylarnjne), 21 U ((R)-N-(3-(2-chlorophenyl)propyl)- 1 3 -isobutoxyphenyl)ethylamine); 21 Y (trifluoromethyl)ph eny1) -2-buty1) 1 -methox ypheny l)ethylIamn I n e) 0 22J -(trnfluoromethyl)phenyl)propyl)- 1-(1 -naphthyl)ethylarnine);, 3A -(trifluoromethoxy)phenyl)-2-butyL)- I 3 -methoxyphenyl)ethylarni ne); 23E -(trifluoromethoxy)phenyl)rnethyl)- 1 -naphthyl)ethyiarnine); :24B -methyl-4-methoxyphenyl)methyl) I 2 -(trifl uoromethyl)phenyl)ethy lam ine); 24J -(trifluoromethoxy)phenyl)propyly I -naphthyl)ethylamine); 24M S-difluorophenyl)propyl)- 1 -nethoxyphenyl)ethylamine); 24V -methyl-4-methoxyphenyl)methyl)- 1-(3 -(ethylIacetoxy)phenyl)ethyl amine); 24X -bromo-4-methoxyphenyl)methyl)- I -naphthyl)ethylamine); 24Y -chloro--4-ethoxyphenyl)methyl)- 1 -naphthyl)ethylarnine);- ((S,R)-N-(4-(3-trifluoromethyl)phenyl)>2-butyl) 1 -naphthyl)ethylamine); 2 5D -(3-trifluoromethyl)phenyl)-2-butyl). 1-(1 -naphthyl)ethylamrine); and 2 5E -(3-phenylprop-2--n- l-yl1)-l1 -methoxyphenylI)ethyl amine), and a pharmaceutically acceptable carrier.

2. A process for producing a medicamnent comprising the step of combining a therapeutically effective amount of a compound having the formula: LI 102 Ar 3 y N Ar 4 CHO whd-ein Arj3 is either naphthyl or phenyl optionally substituted with I to 5 substituents each independently selected froma the group consisting of halogen, lower alkoxy, lower thloalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH 2 OH, CONH, CN, acetoxy, benzyl, benzyloxy, dimethylbenzyl, NO 2 z, CHO, CH 3 CH(OH), N(CH- 3 2 acetyl, and ethylene dioxy; %000Ar 4 is either naphthyl or phenyl optionally substituted with 1 to 5 substituents each 0* C C....independently selected from the group consisting of lower alkyl, halogen, lower alkoxy, 0 lower thioalkyl, mnethiylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CI- 2 0H, CONH 2 CN, and acetoxy; C *provided that if Ar 3 and Ar 4 are both optionally substituted phenyl, then Ar 3 comprises at least one substituent and Ar 4 comprises at least one substituent, and if Ar 3 is 2- then tA4 isnot 3-ruethoxyphenyl; Rs is either hydrogen or phenyl; R 9 is either hydrogen or miethyl; and Rio is either hydrogen, methyl, or phenyl; C and a pharmnaceuticaly acceptable carrier.

3. A process for producing a mnedicament comprising the step of combining a therapeutically effective amount of a compound having the formula: wherein Ar 5 is either naphthyl or phenyl optionally substituted with 0 to 5 substituents each independently selected from the group consisting of lowxer alkyl, halogen, lowver alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH 2 OH, CONH 2 CN, acetoxy, beazyl, benzyloxy, crcL-dimethylbenzvl, NO 2 Cl-I, CH 3 C.H(OH), acetyl, ethylene dioxy, and -CH=CH-phenyl; Ar 6 is phenyl substituted with I 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, CH 2 OH, CONH 2 CN, carbomnethoxy, OCH 2 C(O)C 2 H 5 OCH 2 C(O)OC 2 H5 and acetoxy, provided that at least one substituent is OCH 2 C(O)OC 2 R, I0shdogno**ehl n R 1 2 is hydrogen or mnethyl;an :poided that at least one of R 1 1 and R 12 is methyl; and a pharmaceutically acceptable carrier.

4. The process of claimn 3, wherein Ar,6 is a substituted phenyl comprising a OCH 2 C(O)OC 2 Hs substituent in a rnet'a position.

The process of any of claims 1-4, wherein said medicament is for use in treating a patient having a diseae or disorder characterized by abnormal bone and mineral homeostasis.

6. The process of any of claims 1-4, wherein said medicament is for use in treating hyperparathyroidisni

7. The process of any of claims 1-4, wherein said medicament is for use in treating Paget's disease. 104

8. The process of any of claims 1-4, wherein said medicament is for use in treating osteoporosis.

9. The process of any of claims 1-4, wherein said medicament is for use in treating hypertension.

10. The process of any of claims 1-4, wherein said medicament is for use in treating renal osteodystrophy.

11. The process of any of claims 1-4, wherein said medicament is for use in treating a hypercalcemic disorder.

12. The process of any of claims 1-4, wherein said medicament is for use in o0 treating hypercalcemia of malignancy.

13. The process of any of claims 1-4, wherein said medicament is for use in decreasing parathyroid hormone.

14. The process of any of claims 1-4, wherein said medicament is for use in *o* decreasing serum ionized calcium.

15 15. A process for producing a medicament substantially as hereinbefore described with reference to any one of the examples. 9.

16. A medicament produced by the process of any one of the preceding claims.

17. A compound having the formula: R9 .R H wherein said compound is an inorganic ion receptor modulating compound; and wherein Ar3 is either naphthyl or phenyl optionally substituted with 1 to substituents each independently selected from the group consisting of halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CONH2, CN, acetoxy, benzyl, benzyloxy, dimethylbenzyl, NO2, CHO, S S CH3CH(OH), N(CH3)2, acetyl, and ethylene dioxy; wherein said compound is an inorganic ion receptor modulating compound; and wherein Ar 3 is either naphthyl or phenyl optionally substituted with 1 to 5 substituents each substituents each independently selected from the group consisting of lower alkyl, halogen, lower alkoxy, alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower haloalkoxy, OH, CH20H, CONH 2 CN,N, acetoxy, benzyl, benzyloxy, dimethylbenzyl, Nand acetoxy;CHO, CH 3 CH(OH), N(CH 3 2 acetyl, and ethylene dioxy; Ar 4 is either naphthyl or phenyl optionally substituted with 1 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, CH 2 OH, CONH 2 CN, and acetoxy; [R:\LIBVV)47001 .doc:ais 105 provided that if Ar 3 and Ar 4 are both optionally substituted phenyl, then Ar 3 comprises at least one substituent and Ar 4 comprises at least one substituent, and if Ar 3 is 2-methoxyphenyl, then Ar 4 is not 3-methoxyphenyl; R 8 is either hydrogen or phenyl; R 9 is either hydrogen or methyl; and RI0 is either hydrogen, methyl, or phenyl; or a pharmaceutically acceptable salt or complex thereof.

18. A compound having the formula: H Ar 5 N Ar 6 R 11 R 1 2 wherein said compound is an inorganic ion receptor modulating compound; and wherein Ar 5 is either naphthyl or phenyl optionally substituted with 0 to substituents each independently selected from the group consisting of, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylene dioxy, lower haloalkyl, lower S* i5 haloalkoxy, OH, CH 2 OH, CONH 2 CN, acetoxy, benzyl, benzyloxy, a,a- dimethylbenzyl, NO 2 CHO, CH 3 CH(OH), acetyl, ethylene dioxy, and -CH=CH-phenyl; Ar 6 is phenyl substituted with 1 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, CH 2 0H, CONH 2 CN, •0 20 carbomethoxy, OCH 2 C(O)C 2 H 5 and OCH 2 C(O)OC 2 H 5 and acetoxy, provided that at least one substituent is OCH 2 C(O)OC 2 H 5 R 1 1 is hydrogen or methyl; and R 12 is hydrogen or methyl; provided that at least one of R 1 1 and R 12 is methyl; or a pharmaceutically acceptable salt or complex thereof.

19. A compound having the formula H Ar 3 N Ar 4 R 8 Rio H 3 [R:\LIBVV]47001.doc:ais 106 H Ar 5 Y Ar 6 R 11 R 12 wherein said compound is an inorganic ion receptor modulating compound, substantially as hereinbefore described with reference to any one of Examples 5 to 19. Dated 15 March, 2002 NPS Pharmaceuticals, Inc Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON epee 0040 *0 0 [R \L1BVV47O0 .doc:ais