HK1020041B - Calcium receptor-active compounds - Google Patents

Description

Calcium receptor active compounds

Technical Field

The present invention relates to the design, development, composition and use of compounds capable of modulating the activity of one or more inorganic ion receptors.

Background

Some cells in the body are not only sensitive to chemical signals, but also to ions (e.g., extracellular calcium (Ca)²⁺) Generates a response. Extracellular Ca²⁺Concentration of (referred to herein as [ Ca ]²⁺]) Changes in (b) alter the functional response of these cells. One such specialized cell is a parathyroid cell that secretes parathyroid hormone (PTH). PTH regulates Ca in blood and extracellular fluid²⁺The major endocrine factors of homeostasis.

PTH acts on bone and kidney cells to increase Ca in blood²⁺And (4) horizontal. This [ Ca ]²⁺]The enhancement action of (b) serves as a negative feedback signal to inhibit secretion of PTH. [ Ca ]²⁺]And PTH secretion, form and maintain Ca in vivo²⁺The primary mechanism of balance.

Extracellular Ca²⁺Directly acts on parathyroid cells to regulate PTH secretion. It has been demonstrated that there is a parathyroid cell surface protein which can detect [ Ca²⁺]And (4) changing. Brown et al, in the form of a tree,from However, the device is not suitable for use in a kitchen,366, 574, 1993. In parathyroid cells, the protein acts as extracellular Ca²⁺(ii) receptor of (Ca) ("calcium receptor") assay [ Ca²⁺]Changes and triggers a functional cellular response, i.e. PTH secretion.

Extracellular Ca²⁺Can act on different cell functions, for review, see Nemeth and the like,cellular calcium,11, 319, 1990. With respect to extracellular Ca in Parafollicular cells (C cells) and parathyroid cells²⁺For a discussion of the effects see Nemeth, cell calcium, 11, 323, 1990. These cells express similar Ca²⁺A receptor. Brown et alNature of nature366, 574, 1993; the chemical reaction of Mithal et al,bone and mineral research Study magazine9 supplement 1, page 282, 1994; the species Rogers et al,bone and mineral research complex Will (Chinese character)9 supplement 1, page 409, 1994; the number of the Garrett et al,journal of bone and mineral research"9 supple 1, page 409, 1994. About extracellular Ca²⁺For a discussion of the role of skeletal osteoclasts, see Zaidi,report on bioscience,10, 493, 1990. In addition, keratinocytes, juxtaglomerular cells, trophoblasts, pancreatic beta cells, and adipocytes all respond to changes in extracellular calcium, which may reflect activation of calcium receptors for these cells.

In vitro modelling of extracellular Ca with different compounds²⁺For a discussion of the ability of Nemeth et al (spermine and spermidine) calcium binding proteins in health and disease 1987 academic Press, pages 33-35; brown et al (e.g., neomycin)Endocrinology128, 3047, 1991; chen et al (diltiazem and its analogues TA-3090)Journal of bone and mineral research5, 581, 1990; and Zaidi et al (Verapamul),biochemical and biophysical research communiques167, 807, 1990. Nemeth et al, PCT/US93/01642, International publication No. WO94/18959, and Nemeth et al, PCT/US92/07152, International publication No. WO93/04373 describe various compounds capable of modulating the effect of inorganic ions on cells containing inorganic ion receptors.

References provided in the background of the invention are not considered prior art.

Summary of The Invention

Compounds that modulate one or more activities of an inorganic ion receptor and methods of treating diseases or disorders by modulating inorganic ion receptor activity are described. Preferred compounds mimic or block the effects of extracellular calcium on cell surface calcium receptors.

Diseases or disorders that can be treated by modulating inorganic ion receptor activity include one or more of the following types: (1) characterized by abnormal homeostasis in the inorganic plasma, preferably a disease or disorder of abnormal calcium homeostasis; (2) a disease or disorder characterized by an abnormal number of extracellular or intracellular messengers whose production is affected by the activity of an inorganic ion receptor, preferably a calcium ion receptor; (3) disorders characterized by abnormal effects of intracellular or extracellular messengers (e.g., one that differs in type or quantity), but which can be ameliorated by inorganic ion receptor activity, preferably calcium receptor activity; (4) other diseases or disorders in which modulation of inorganic ion receptor activity, preferably calcium receptor activity, may have a beneficial effect, such as a disease or disorder in which receptor activity triggers the production of one intracellular or extracellular messenger to compensate for an abnormal number of another messenger. Extracellular messengers whose secretion and/or action is affected by the regulation of inorganic ion receptor activity include inorganic ions, hormones, neurotransmitters, growth factors, chemokines and the like. Examples of intracellular messengers include cAMP, cGMP, IP₃And diglycerides.

Accordingly, the compounds of the present invention preferably modulate calcium receptor activity and are useful in the treatment of diseases or disorders that may be affected by modulation of one or more activities of the calcium receptor. Calcium receptor proteins target certain specialized cells to extracellular Ca²⁺The concentration produces a response. For example, extracellular Ca²⁺Inhibiting parathyroid hormone secretion by parathyroid cells, inhibiting bone resorption by osteoclasts, and stimulating calcitonin secretion by C cells.

In a preferred embodiment, the compounds are used to treat diseases or disorders characterized by abnormal bone and mineral homeostasis, more preferably abnormal calcium homeostasis. Extracellular Ca²⁺Is subject to strict homeostatic control andcontrol a variety of processes such as blood clotting, excitability of nerves and muscles, and normal bone formation. Abnormal calcium homeostasis is characterized by one or more of the following activities: (1) abnormal increase and decrease of serum calcium; (2) abnormal increase and decrease of calcium with urine output; (3) abnormal increase or decrease in bone calcium levels, as measured by, for example, bone mineral density; (4) abnormal absorption of calcium in food; (5) abnormal increase or decrease in production and/or release of messengers that affect serum calcium levels, such as parathyroid hormone and calcitonin; and (6) abnormal changes in response elicited by messengers that affect serum calcium levels. Abnormal increases and decreases in these various aspects of calcium homeostasis are associated with abnormal increases and decreases in calcium homeostasis present in the general population and are often associated with diseases or disorders.

Diseases or disorders characterized by abnormal calcium homeostasis may be caused by various cellular defects, such as a defect in calcium receptor activity, a defect in the number of calcium receptors, or a defect in intracellular proteins affected by calcium receptors. For example, in parathyroid cells, calcium receptors are associated with G_iProtein coupling, which in turn inhibits cyclic adenosine monophosphate production. G_iProtein defects may affect some ability to inhibit cyclic adenosine monophosphate production.

Accordingly, a first aspect of the present invention relates to compounds that modulate inorganic ion receptors, and pharmaceutically acceptable salts and complexes thereof, having the formula:

structure I

Wherein Ar is₁Is naphthyl or phenyl optionally substituted with 0 to 5 substituents, each substituent being independently selected from lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower haloalkyl, lower haloalkoxy, OH, CH₂OH、CONH₂CN, acetoxy, N (CH)₃)₂Phenyl, phenoxy, benzyl, benzyloxy, alpha-dimethylbenzyl、NO₂、CHO、CH₃Ch (oh), acetyl, ethylenedioxy;

Ar₂is naphthyl or phenyl optionally substituted with 0 to 5 substituents, each substituent being independently selected from lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower haloalkyl, lower haloalkoxy, OH, CH₂OH、CONH₂CN and acetoxy;

q is 0, 1, 2 or 3;

r is H or lower alkyl.

The compounds of the present invention have preferred stereochemistry. CH in Structure I₃Is located at a chiral center and provides an alpha- (R) -methyl structure. If R is CH₃R in structure I is also located at a chiral center and provides the (R) -methyl structure. Thus if R is CH₃Compounds of structure I have (R, R) stereochemistry.

Inorganic ion receptor activity is a process initiated by activation of an inorganic ion receptor. These processes include the production of molecules that can act as intracellular or extracellular messengers.

Compounds that modulate inorganic ion receptors include ion mimetics (ionomics), ion lysates (ionolytics), calcium mimetics, and calcium lysates. Ion mimetics bind to inorganic ion receptors and mimic (i.e., excite or enhance) the effect of inorganic ions on inorganic ion receptors. Preferably, the compound affects one or more calcium receptor activities. Calcimimetics are ionic mimetics that affect the activity of one or more calcium receptors and bind to calcium receptors.

Ion lysates are compounds that bind to an inorganic ion receptor and block (i.e., inhibit or attenuate) one or more activities of inorganic ions at the inorganic ion receptor. Preferably, the compound affects one or more calcium receptor activities. Calcium lysates are ionic lysates that block one or more extracellular calcium-triggered calcium receptor activities and bind to calcium receptors.

The ion mimetic and ion lysate can bind to the same receptor site that binds to the natural inorganic ion ligand or bind to another site (e.g., an allosteric site). For example, NPS R-467 binds to calcium receptors, resulting in calcium receptor activity, and therefore NPS R-467 is a calcimimetic. However, the site at which NPS R-467 binds to calcium receptors is distinct from the extracellular calcium binding site (i.e., the allosteric site).

Calculating the EC of the compound₅₀Or IC₅₀The effectiveness of the compound can be measured. EC (EC)₅₀Is the concentration of compound that produces half of the maximum mimicking effect. IC (integrated circuit)₅₀Is the concentration of compound that produces half of the maximum repressing effect. EC of Compounds at calcium receptor₅₀And IC₅₀Can be determined by detecting one or more activities of extracellular calcium at the calcium receptor. For measuring EC₅₀And IC₅₀Examples of such assays are described in Nemeth et al, PCT/US93/01642, International publication No. WO94/18959, and Nemeth et al, PCT/US92/07175, International publication No. WO93/04373, both of which are incorporated herein by reference, and infra. Such assays include egg cell expression assays and measurement of intracellular calcium ion concentration ([ Ca ] due to calcium receptor activity²⁺]_i) And (4) increasing. Preferably, such assays measure the release or inhibition of a particular hormone associated with calcium receptor activity.

Compounds that modulate inorganic ion receptors preferably selectively target inorganic ion receptor activity of specific cells. For example, selective targeting of calcium receptor activity is achieved if the effect of a given concentration of a compound on calcium receptor activity of one cell type is much greater than the effect on calcium receptor activity of another cell type. 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 plasma concentration or extracellular fluid concentration, and the measured effect is the production of an extracellular messenger such as plasma calcitonin, parathyroid hormone or plasma calcium. For example, in a preferred embodiment, the compound selectively targets PTH secretion rather than calcitonin secretion.

Preferably, the compound is a calcium mimetic or calcium lysate, administeredThe EC at the calcium receptor determined by the above assay₅₀Or IC₅₀Less than or equal to 5. mu.M, more preferably less than or equal to 1. mu.M, 100nM, 10nM or 1 nM. More preferably, the assay measures intracellular Ca in HEK293 cells transformed with a nucleic acid expressing the human parathyroid calcium receptor and loaded with (load) fura-2²⁺。EC₅₀Or IC₅₀This is advantageous because it allows for lower concentrations of the compound in vivo or in vitro. EC (EC)₅₀And IC₅₀The discovery of low compounds makes it possible to design and synthesize other compounds with similar or improved potency, effectiveness and/or selectivity.

In another aspect, the invention relates to compounds that modulate inorganic ion receptors, and pharmaceutically acceptable salts and complexes thereof, having the formula:

structure II

Wherein Ar is₃Is naphthyl or phenyl optionally substituted with 0 to 5 substituents, each substituent being independently selected from lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower haloalkyl, lower haloalkoxy, OH, CH₂OH、CONH₂CN, acetoxy, benzyl, benzyloxy, alpha-dimethylbenzyl, NO₂、CHO、CH₃CH(OH)、N(CH₃)₂Ethyl, ethylenedioxy;

Ar₄is naphthyl or phenyl optionally substituted with 0 to 5 substituents, each substituent being independently selected from lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower haloalkyl, lower haloalkoxy, OH, CH₂OH、CONH₂CN and acetoxy;

R₈is hydrogen or phenyl;

R₉is hydrogen or methyl; and

R₁₀is hydrogen, methyl or phenyl.

In another aspect, the invention relates to a compound that modulates an inorganic ion receptor having the formula:

structure III

Wherein Ar is₅Is naphthyl or phenyl optionally substituted with 0 to 5 substituents, each substituent being independently selected from lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower haloalkyl, lower haloalkoxy, OH, CH₂OH、CONH₂CN, acetoxyl group, benzyl group, benzyloxy group, alpha-dimethylbenzyl group, NO₂、CHO、CH₃CH (oh), acetyl, ethylenedioxy, -CH ═ CH-phenyl;

Ar₆is naphthyl or phenyl optionally substituted with 0 to 5 substituents, each substituent independently selected from acetyl, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower haloalkyl, lower haloalkoxy, OH, CH₂OH、CONH₂CN, carbomethoxy, OCH₂C(O)C₂H₅And an acetoxy group;

R₁₁is hydrogen or methyl; and

R₁₂is hydrogen or methyl.

Another aspect of the invention relates to a pharmaceutical composition consisting of a compound that modulates an inorganic ion receptor as described herein and a physiologically acceptable carrier. "pharmaceutical composition" means a composition in a form suitable for administration to a mammal, preferably a human. Preferably, the pharmaceutical composition contains a sufficient amount of a compound that modulates the calcium receptor, in a suitable pharmaceutical form, to be therapeutically effective in humans.

Considerations for appropriate forms of administration are known in the art and include toxicity, solubility, route of administration, and maintenance of activity. For example, a pharmaceutical composition injected into the blood should be soluble.

The pharmaceutical compositions may also be formulated as pharmaceutically acceptable salts (e.g., acid addition salts) and complexes thereof. The preparation of such salts facilitates their medicinal use by altering the physical properties of the compound without preventing it from exerting its physiological effects.

Another aspect of the invention relates to methods of treating a patient with a compound that modulates an inorganic ion receptor described herein to modulate inorganic ion receptor activity. The method involves administering to the patient a pharmaceutical composition containing a therapeutically effective amount of a compound that modulates an inorganic ion receptor. In a preferred embodiment, a disease or disorder is treated by administering to a patient a compound that modulates a calcium receptor in a therapeutically effective amount to modulate calcium receptor activity.

Compounds that modulate inorganic ion receptors and compositions containing such compounds are useful in treating patients. "patient" refers to a mammal in which modulation of an inorganic ion receptor has a beneficial effect. Patients in need of treatment by modulation of inorganic ion receptors can be identified using standard techniques known in the medical community.

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

Disorders characterized by abnormal homeostasis of calcium include hyperparathyroidism, osteoporosis, and other bone and mineral related disorders (described, for example, in standard medical texts such as Harrison's internal medicine principles). By mimicking or repressing extracellular Ca²⁺Modulating calcium receptors for one or more effects on calcium receptorsCompounds that directly or indirectly affect the levels of proteins or other compounds in a patient can treat these conditions.

"therapeutically effective amount" means that one or more symptoms of a disease or disorder in a patient are alleviated to some extent; or an amount of compound that partially or fully restores to normal one or more physiological or biochemical parameters associated or causative of the disease or disorder.

In a preferred embodiment, the patient has a disease or disorder characterized by abnormal levels of one or more calcium receptor-modulated components, and the compound is active at a cellular calcium receptor selected from the group consisting of; parathyroid cells, osteoclasts, proximal glomerulonephritis cells, proximal tubulointersnal renal cells, distal tubulointersal renal cells, central nervous system cells, peripheral nervous system cells, cells of the crude ascending limb of the Henle's loop and/or collecting vessel, keratinocytes of the epidermis, paracystic cells (C cells) in the thyroid gland, intestinal cells, platelets, vascular smooth muscle cells, atrial cells, gastrin-secreting cells, glucagon-secreting cells, mesangial cells, mammary cells, beta cells, adipocytes, immune cells, gastrointestinal cells, skin cells, adrenal cells, pituitary cells, hypothalamic cells, and cells of the posterior fornical (fundfornical) organ.

More preferably, the cell is selected from: parathyroid cells, central nervous system cells, peripheral nervous system cells, cells of the rough ascending branches of the kidney Henle's ring and/or collecting ducts, parathyroid vesicle cells (C cells) of the thyroid gland, intestinal tract cells, gastrointestinal tract cells, pituitary cells, hypothalamic cells, and cells of the posterior fornix organ.

In a preferred embodiment, the compound is a calcimimetic that acts on the calcium receptor of parathyroid cells and reduces the level of parathyroid hormone in the serum of a patient. More preferably, the level is reduced sufficiently to bring about plasma Ca²⁺The extent of the reduction. Most preferably, the parathyroid hormone level is reduced to that of a normal individual.

In another preferred embodiment, the compound is a calcium lysate that acts on the calcium receptor of parathyroid cells and elevates the level of parathyroid hormone in the serum of the patient. More preferably, the level is increased to a level sufficient to cause an increase in bone mineral density in the patient.

Patients in need of such treatment can be identified using standard medical techniques, such as blood or urine tests. For example by detecting defects in the production or secretion of proteins affected by changes in the concentration of inorganic ions. Or by detecting abnormal levels of inorganic ions or hormones that affect the balance within the inorganic plasma.

Various embodiments are used throughout this application. These examples are in no way intended to limit the invention.

Other features and advantages of the invention will be apparent from the following drawings, detailed description, examples and claims.

Brief Description of Drawings

FIGS. 1a-1r show the chemical structures of different compounds.

Physical data for the eutopic compounds described herein are presented in FIGS. 2-131.

Description of the preferred embodiments

The present invention relates to compounds capable of modulating the activity of one or more inorganic ion receptors, preferably the compounds mimic or block the effect of an extracellular ion, more preferably the extracellular ion is Ca, on cells bearing inorganic ion receptors²⁺This effect acts on cells containing calcium receptors. Publications relating to calcium activity, calcium receptors, and/or compounds that modulate calcium receptors include: brown et al, in the form of a tree,nature of nature366: 574, 1993; nemeth et al, PCT/US93/01642, International publication No. WO 94/18959; nemeth et al, PCT/US92/07175, International publication No. WO 93/04373; the short messages between the short messages and the Chen,journal of bone and mineral research， 9: 293 (1994); to be provided withAnd a jack, etc., and a rack, etc.,FEBS quick report(FEBS Lett.) 333: 132, (1993). These publications are not to be considered as

Prior art to the present invention.

I. Calcium receptors

Calcium receptors are present in different cell types and may have different functions in different cell types. The following medicinal effects of cells in response to calcium are consistent with the presence of calcium receptors: parathyroid cells, osteoclasts, proximal glomerulonephritis cells, proximal tubulointersnal renal cells, distal tubulointersal renal cells, central nervous system cells, peripheral nervous system cells, cells of the rough ascending limb of the Henle's loop and/or collecting vessel, keratinocytes of the epidermis, paracystic cells (C cells) of the thyroid gland, intestinal cells, platelets, vascular smooth muscle cells, atrial cells, gastrin-secreting cells, glucagon-secreting cells, mesangial cells, mammary cells, beta cells, adipocytes, immune cells, gastrointestinal cells, skin cells, adrenal cells, pituitary cells, hypothalamic cells, and cells of the posterior fornix organ. In addition, physical data confirm that calcium receptors are also present in parathyroid cells, central nervous system cells, peripheral nervous system cells, cells of the rough ascending limb of the kidney Henle ring and/or the collecting duct, the parathyroid vesicle cells (C cells) of the thyroid gland, intestinal tract cells, gastrointestinal tract cells, pituitary cells, hypothalamic cells, and cells of the posterior fornix organ.

The calcium receptors of these different cell types may differ. It is also possible that one cell contains more than one calcium receptor. Comparison of calcium receptor activity and amino acid sequence from different cell sources indicates the existence of different calcium receptor types. For example, calcium receptors can respond to a number of divalent and trivalent cations. Parathyroid calcium receptor responsive calcium and Gd³⁺Whereas osteoclasts respond to divalent cations such as calcium, but not Gd³⁺. Thus, the parathyroid calcium receptor is pharmacologically different from the osteoclast calcium receptor.

On the other hand, the nucleic acid sequences encoding the calcium receptors of parathyroid and C cells show that the amino acid structures of these receptors are similar. However, calcimimetics exhibit different pharmacological properties on parathyroid and C cells, modulating different activities. Thus, although calcium receptors may have similar or even identical structures. But their pharmacological properties may differ significantly depending on the type of cell or organ in which it is expressed.

Usually the calcium receptor is on extracellular Ca²⁺Low affinity (apparent K)_dTypically greater than 0.5 mM). Calcium receptors may include a free or bound effector mechanism, defined by Cooper, Bloom and Roth, Biochemical basis of neuropharmacology, Chapter four, and thus differ from intracellular calcium receptors such as calmodulin and troponin.

Calcium receptors respond to changes in extracellular calcium levels. The exact changes depend on the particular receptor and the cell line containing the receptor. For example, the in vitro effects of calcium on calcium receptors on parathyroid cells include:

1. intracellular calcium is increased. This increase is due to influx of extracellular calcium and/or recruitment of intracellular calcium. The characteristics of intracellular calcium increase include:

(a) rapid (time to peak < 5 seconds) and transient [ Ca [²⁺]_iIncrease, for 1. mu.M La³⁺Or 1 μ M Gd³⁺Inhibition of resistance, pretreatment with ionomycin (extracellular Ca)²⁺Absent) is eliminated;

(b) increase is not inhibited by dihydropyridine;

(c) the transient increase was eliminated by 10 min pretreatment with 10mM sodium fluoride;

(d) the temporary increase is reduced by pretreatment with an activator of Protein Kinase C (PKC), such as Phorbol Myristate Acetate (PMA), phorbol ester or (-) -indolactam V. The overall effect of protein kinase C is to shift the calcium concentration response curve to the right without affecting the maximum response; and

(e) pretreatment with pertussis toxin (100ng/ml greater than 4 hours) did not affect this increase.

2. The rapid (< 30 seconds) increase in inositol-1, 4, 5-trisphosphate or diglyceride formation. Pretreatment with pertussis toxin (100ng/ml > 4 hours) did not affect this increase;

3. inhibit dopamine and isoproterenol stimulated cyclic AMP formation. This effect was suppressed by pertussis toxin pretreatment (100ng/ml > 4 hours); and

4. inhibit PTH secretion. Pretreatment with pertussis toxin (100ng/ml > 4 hours) did not affect inhibition of PTH secretion.

The effect of calcium on other calcium receptors in different cells can be readily determined by techniques known in the art. These effects are similar in that an increase in intracellular calcium is observed in parathyroid cells. However, this effect is expected to vary in other respects, for example causing or inhibiting the release of a hormone other than parathyroid hormone.

Compounds for modulating inorganic ion receptors

Compounds that modulate inorganic ion receptors modulate the activity of one or more inorganic ion receptors. Preferred compounds that modulate calcium receptors are calcium mimetics and calcium lysates. Compounds that modulate inorganic ion receptors are identified by screening for compounds that have been modeled as compounds with specific activities (i.e., lead compounds).

A preferred method for measuring calcium receptor activity is measurement of [ Ca ]²⁺]₁A change in (c). HEK293 cells transduced with fura-2 and loaded using various techniques such as using nucleic acids expressing human parathyroid calcium receptor; and by measuring Cl in Xenopus ova injected with nucleic acids encoding calcium receptors^-Flow to measure [ Ca²⁺]_iA change in (c). (see Nemeth et al, PCT/US93/01642, International publication No. WO 94/18959.) for example, poly (A)⁺mRNA can be obtained from cells expressing a calcium receptor, such as parathyroid cells, osteoclasts, proximal glomerulonephritis cells, proximal tubular kidney cells, distal tubular kidney cells, cells of the rough ascending branch of the Henle ring and/or collecting vessel, epidermisKeratinocytes of the thyroid gland, accessory vesicle cells (C cells), intestinal tract cells, central nerve cells, peripheral nervous system cells, platelets, vascular smooth muscle cells, atrial cells, gastrin-secreting cells, glucagon-secreting cells, mesangial cells, mammary cells, beta cells, adipocytes, immune cells, and gastrointestinal tract cells. Preferably, the nucleic acid is from a parathyroid cell, a C cell or an osteoclast. More preferably, the nucleic acid encodes a calcium receptor and is located on a plasmid or vector.

In a preferred embodiment, the compound that modulates a calcium receptor is a calcimimetic that inhibits the bone resorption effect of osteoclasts in vivo; inhibiting the bone resorption by osteoclasts in vitro; activating calcitonin secretion from C cells in vitro or in vivo; inhibiting secretion of parathyroid hormone by parathyroid cells in vitro and reducing PTH secretion in vivo; increasing the in vivo calcitonin level; or inhibit bone resorption by osteoclasts in vitro and in vivo.

In another preferred embodiment the compound that modulates a calcium receptor is a calcium lysate, stimulates secretion of parathyroid hormone by parathyroid cells in vitro and increases parathyroid hormone levels in vivo.

Preferably, the compounds selectively target inorganic ion receptor activity, more preferably calcium receptor activity, in specific cells. "selectively" means that a given concentration of the compound has a greater effect on the activity of an inorganic ion receptor on one cell type than on another cell type. Preferably, the differential effect is 10 times or more. Preferably, the concentration refers to plasma concentration, and the effect measured is the production of an extracellular messenger such as plasma calcitonin, parathyroid hormone or plasma calcium. For example, in a preferred embodiment, the compound selectively targets PTH secretion rather than calcitonin secretion.

In another preferred embodiment, the EC of the compound in one or more, but not all, of the following cells₅₀And IC₅₀5 μ M or less: parathyroid cell, osteoclast, and julian glomerulusCells, proximal tubular kidney cells, distal tubular kidney cells, central nervous system cells, peripheral nervous system cells, cells of the Henle ring and/or rough ascending branches of the collecting vessel, keratinocytes of the epidermis, accessory vesicle cells (C cells) of the thyroid gland, intestinal tract cells, platelets, vascular smooth muscle cells, atrial cells, gastrin-secreting cells, glucagon-secreting cells, mesangial cells, mammary cells, beta cells, adipocytes, immune cells, gastrointestinal tract cells, skin cells, adrenal cells, pituitary cells, hypothalamic cells, and cells of the posterior fornix organ. More preferably, the cell is selected from the group consisting of parathyroid cells, central nervous system cells, peripheral nervous system cells, cells of the Henle ring and/or rough ascending branches of the collecting duct in the kidney, parathyroid vesicle cells (C cells) of the thyroid gland, intestinal tract cells, gastrointestinal tract cells, pituitary cells, hypothalamic cells, and cells of the posterior fornix organ. Physical data such as in situ hybridization and antibody staining have confirmed the presence of calcium receptors in this group of cells.

Preferably, the compound that modulates an inorganic ion receptor mimics or represses the action of a cell, extracellular ion, having an inorganic ion receptor, thereby having a therapeutic effect. Compounds that modulate inorganic ion receptors can have the same or different effects on cells having different types of inorganic ion receptor morphologies (e.g., cells having normal inorganic ion receptors, normal numbers of inorganic ion receptors, aberrant numbers of inorganic ion receptors).

Compounds that modulate calcium receptors preferably mimic or repress all effects of extracellular ions on cells having calcium receptors. However, calcium mimetics do not have extracellular Ca²⁺All biological activities of (a). Similarly, calcium lysates cannot block all the activity caused by extracellular calcium. In addition different calcium mimetics and different calcium lysates do not necessarily correspond to extracellular Ca²⁺Bind to the same site on the calcium receptor to function.

The inorganic modulatory compounds need not affect inorganic receptor activity to the same extent or in exactly the same form as the natural ligand. Calcium mimetics can affect calcium receptor activity to a different extent, for a different duration, bind to a different binding site or have a different affinity, for example, as compared to calcium acting on the calcium receptor.

A. Calcium mimetics (Calcimimetic)

1. Compounds of Structure I

Compounds of structure I (and pharmaceutically acceptable salts and complexes thereof) capable of modulating calcium receptor activity have the formula:

wherein Ar is₁Is naphthyl or phenyl optionally substituted with 0 to 5 substituents, each substituent being independently selected from lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower haloalkyl, lower haloalkoxy, OH, CH₂OH、CONH₂CN, acetoxy, N (CH)₃)₂Phenyl, phenoxy, benzyl, benzyloxy, alpha-dimethylbenzyl, NO₂、CHO、CH₃CH (OH), ethyl, ethylenedioxy, preferably each substituent is independently selected from CH₃、CH₃O、CH₃CH₂O, methylenedioxy, Br, Cl, F, Cl, CF₃、CHF₂、CH₂F、CF₃O、CF₃CH₂O、CH₃S、OH、CH₂OH、CONH₂、CN、NO₂、CH₃CH₂Propyl, isopropyl, butyl, isobutyl, tert-butyl and acetoxy. More preferably, Ar₁Is naphthyl or phenyl with 1-5 substituents. Each substituent is independently selected from: isopropyl group, CH₃O、CH₃S、CF₃O、I、Cl、F、CF₃、CH₃More preferably CF₃O、I、Cl、F、CF₃；

Ar₂Is naphthyl or phenyl optionally substituted with 0 to 5 substituents each independentlySelected from: lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower haloalkyl, lower haloalkoxy, OH, CH₂OH、CONH₂CN and acetoxy, preferably each substituent is independently selected from CH₃、CH₃O、CH₃CH₂O, methylenedioxy, Br, Cl, F, I, CF₃、CHF₂、CH₂F、CF₃O、CF₃CH₂O、CH₃S、OH、CH₂OH、CONH₂、CN、NO₂、CH₃CH₂Propyl, isopropyl, butyl, isobutyl, tert-butyl and acetoxy. More preferably, Ar₂Is naphthyl or phenyl having 1-5 substituents, each substituent independently selected from: isopropyl group, CH₃O、CH₃S、CF₃O、I、Cl、F、CF₃And CH₃More preferably CF₃O、I、Cl、F、CH₃O and CF₃。

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

R is H or CH₃。

"lower alkyl" refers to saturated hydrocarbons containing 1 to 4 carbons, preferably 1 to 3 carbon atoms, and may be straight or branched.

"lower alkoxy" means "O-lower alkyl". Wherein "O" is oxygen attached to a lower alkyl group.

"lower thioxo" means "S-lower alkyl". Wherein "S" is sulfur attached to a lower alkyl group.

"lower haloalkyl" refers to a lower alkyl substituted with at least one halogen. Preferably only the terminal carbons of the lower alkyl group are substituted by halogen and there are 1-3 halogens. More preferably, the lower haloalkyl group contains 1 carbon. Preferably, the halogen substituent is Cl or F.

"lower haloalkoxy" means "O-lower haloalkyl". Wherein "O" is oxygen attached to a lower haloalkyl.

a.Ar ₁ And Ar ₂ Are all optionally substituted phenyl

In a preferred embodiment, Ar₁And Ar₂Are each optionally substituted phenyl and the compound has the formula:

wherein R is hydrogen or a methyl group,

m and n are each independently 0, 1, 2, 3, 4 or 5;

each X is independently selected from: lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower haloalkyl, lower haloalkoxy, OH, CH₂OH、CONH₂CN, acetoxy, N (CH)₃)₂Phenyl, phenoxy, benzyl, benzyloxy, alpha-dimethylbenzyl, NO₂、CHO、CH₃CH (OH), ethyl, ethylenedioxy. Preferably each X is independently selected from CH₃、CH₃O、CH₃CH₂O, methylenedioxy, Br, Cl, F, I, CF₃、CHF₂、CH₂F、CF₃O、CF₃CH₂O、CH₃S、OH、CH₂OH、CONH₂、CN、NO₂、CH₃CH₂Propyl, isopropyl, butyl, isobutyl, tert-butyl and acetoxy. More preferably, each X is independently selected from isopropyl, CH₃O、CH₃S、CF₃O、I、Cl、F、CF₃And CH₃More preferred is CF₃O, I, Cl, F and CF₃；

Each Z is independently selected from: lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower haloalkyl, lower haloalkoxy, lower alkyl, lower haloalkoxy,OH、CH₂OH、CONH₂CN and acetoxy. Preferably each Z is independently selected from CH₃、CH₃O、CH₃CH₂O, methylenedioxy, Br, Cl, F, I, CF₃、CHF₂、CH₂F、CF₃O、CF₃CH₂O、CH₃S、OH、CH₂OH、CONH₂、CN、CH₃CH₂Propyl, isopropyl, butyl, isobutyl, tert-butyl and acetoxy. More preferably, each Z is independently selected from isopropyl, CH₃O、CH₃S、CF₃O、CF₃I, Cl, F and CH₃。

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

wherein R is hydrogen or methyl;

m is 0, 1, 2, 3, 4 or 5, preferably 1 or 2;

each X is independently selected from: lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower haloalkyl, lower haloalkoxy, OH, CH₂OH、CONH₂CN, acetoxy, N (CH)₃)₂Phenyl, phenoxy, benzyl, benzyloxy, alpha-dimethylbenzyl, NO₂、CHO、CH₃Ch (oh), ethyl, ethylenedioxy, preferably each substituent is independently selected from: CH (CH)₃、CH₃O、CH₃CH₂O, methylenedioxy, Br, Cl, F, I, CF₃、CHF₂、CH₂F、CF₃O、CF₃CH₂O、CH₃S、OH、CH₂OH、CONH₂、CN、NO₂、CH₃CH₂Propyl, isopropyl, butyl, isobutyl, tert-butyl and acetoxy. More preferred are isopropyl group,CH₃O、CH₃S、CF₃O、CF₃I, Cl, F and CH₃。

More preferably, the compound has the formula:

wherein R is hydrogen or methyl;

R₁is halogen or hydrogen, preferably, R₁Is F or hydrogen;

R₂is hydrogen, halogen, lower alkyl, lower haloalkyl, or lower haloalkoxy, preferably, R₂Is hydrogen, CF₃、CH₃、OCF₃Or F, and

R₃hydrogen, halogen or alkoxy of, preferably, R₃Is Cl, F, hydrogen or methoxy, more preferably methoxy.

In an alternative more preferred combination; r₁、R₂And R₃At least two of which are halogen, preferably F, and R is hydrogen or CH₃(ii) a R is hydrogen or CH₃，R₂Is lower haloalkyl or lower haloalkoxy, preferably OCF₃Or CF₃And R is₁And R₃Is hydrogen; r is CH₃，R₃Is halogen, preferably Cl, R₁Is halogen or hydrogen, preferably F or hydrogen, R₂Is hydrogen, lower alkyl, lower haloalkyl or lower haloalkoxy, preferably hydrogen, CF₃、CH₃、OCF₃Or F.

b.Ar ₂ Is naphthyl and q is 0

In another preferred embodiment, Ar₂Is naphthyl, q is 0, and the compound has the formula:

wherein Ar is₁Is naphthyl or phenyl optionally substituted with 0 to 5 substituents, each substituent independently selected from: lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower haloalkyl, lower haloalkoxy, OH, CH₂OH、CONH₂CN, acetoxy, N (CH)₃)₂Phenyl, phenoxy, benzyl, benzyloxy, alpha-dimethylbenzyl, NO₂、CHO、CH₃Ch (oh), ethyl, ethylenedioxy, preferably each substituent is independently selected from: CH (CH)₃、CH₃O、CH₃CH₂O, methylenedioxy, O,_Br、_Cl、_F、_I、CF₃、CHF₂、CH₂F、CF₃O、CH₃CH₂O、CH₃S、OH、CH₂OH、CONH₂、CN、NO₂、CH₃CH₂Propyl, isopropyl, butyl, isobutyl, tert-butyl and acetoxy. More preferably, Ar₁Is naphthyl or phenyl having 1-5 substituents, each substituent independently selected from: isopropyl group, CH₃O、CH₃S、CF₃、CF₃O, I, Cl, F and CH₃。

More preferably, Ar₁Is an optionally substituted phenyl group, the compound having the formula:

wherein X_nRepresents an optional substituent of the above optionally substituted phenyl group (the preferred substituents and number of substituents are as described above).

More preferably, the compound has the formula:

wherein R is CH₃Or hydrogen;

R₄is lower alkyl, halogen or alkoxy, preferably isopropyl, chloro or methoxy; and is

R₅Is hydrogen, lower alkyl or halogen is preferably methyl, CH₃Br or Cl.

c.Ar ₂ Is naphthyl and q is 2

In another preferred embodiment, Ar₁Is a substituted phenyl radical, Ar₂Is naphthyl, q is 2, and the compound has the formula:

wherein R is hydrogen or CH₃；

n is 0, 1, 2, 3, 4 or 5, preferably 1 or 2;

each X is independently selected from the group consisting of lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower haloalkyl, lower haloalkoxy, OH, CH₂OH、CONH₂CN, acetoxy, N (CH)₃)₂Phenyl, phenoxy, benzyl, benzyloxy, alpha-dimethylbenzyl, NO₂、CHO、CH₃Ch (oh), ethyl, ethylenedioxy, preferably each substituent is independently selected from: CH (CH)₃、CH₃O、CH₃CH₂O, methylenedioxy, Br, Cl, F, I, CF₃、CHF₂、CH₂F、CF₃O、CF₃CH₂O、CH₃S、OH、CH₂OH、CONH₂、CN、NO₂、CH₃CH₂Propyl, isopropyl, butyl, isobutyl, tert-butyl and acetoxyAnd (4) a base. More preferred are isopropyl group and CH group₃O、CH₃S、CF₃O、CF₃I, Cl, F and CH₃。

More preferably, the compound has the formula:

wherein R is₆Is hydrogen, lower haloalkyl or lower haloalkoxy, preferably hydrogen, OCF₃Or CF₃And is and

R₇is halogen or hydrogen, preferably chlorine or hydrogen.

In another embodiment R, R₆And R₇As described above (with the preferred substituents described above), provided that R and R₆Are each hydrogen, R₇Is not Cl; and R is CH₃，R₆And R₇As described above (with the preferred substituents described above).

2. Compounds of Structure II

The compound of structure II has the following formula:

wherein Ar is₃Is naphthyl or phenyl optionally substituted with 0 to 5 substituents, each substituent independently selected from: lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower haloalkyl, lower haloalkoxy, OH, CH₂OH、CONH₂CN, acetoxy, benzyl, benzyloxy, alpha-dimethylbenzyl, NO₂、CHO、CH₃CH(OH)、N(CH₃)₂Ethyl, ethylenedioxy, preferably N (CH)₃)₂Lower alkoxy or lower alkyl;

Ar₄is 0 toNaphthyl or phenyl optionally substituted with 5 substituents, each substituent independently selected from the group consisting of lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower haloalkyl, lower haloalkoxy, OH, CH₂OH、CONH₂CN and acetoxy, preferably lower alkoxy, more preferably methoxy;

R₈is hydrogen or phenyl, preferably hydrogen;

R₉is hydrogen or methyl;

R₁₀is hydrogen, methyl or phenyl, more preferably, when R is₁₀When methyl, the chiral carbon to which it is attached is the (R) stereoisomer.

Preferably, the α -methyl group in structure II is (R) - α -methyl.

3. Compounds of Structure III

The compound of structure III has the formula:

wherein Ar is₅Is naphthyl or phenyl optionally substituted with 0 to 5 substituents, each substituent independently selected from: lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower haloalkyl, lower haloalkoxy, OH, CH₂OH、CONH₂CN, acetoxyl group, benzyl group, benzyloxy group, alpha-dimethylbenzyl group, NO₂、CHO、CH₃CH (oh), ethyl, ethylenedioxy, -CH ═ CH-phenyl, preferably lower alkyl, phenoxy, -CH ═ CH-phenyl, dimethylbenzyl, methoxy, methylene or ethylene;

Ar₆is naphthyl or phenyl optionally substituted with 0 to 5 substituents, each substituent independently selected from ethyl, lower alkyl, halogen, lower alkoxy, lower thioalkyl, methylenedioxy, lower alkyl halideAlkyl, lower haloalkoxy, OH, CH₂OH、CONH₂CN, carbomethoxy, OCH₂C(O)C₂H₅And acetoxy, preferably methoxy, lower alkyl, phenyl, halogen, CF₃CN, carbomethoxy or OCH₂C(O)C₂H₅；

R₁₁Is hydrogen or methyl; preferably, when R is₁₁When methyl, the carbon to which it is attached is the (R) stereoisomer; and is

R₁₂Is hydrogen or methyl, preferably when R is₁₂When methyl, the carbon to which it is attached is the (R) stereoisomer.

4. Calcium mimetic Activity

Compounds which mimic Ca can be determined by methods known in the art²⁺The ability to have activity at calcium receptors is described in Nemeth et al, PCT/US93/01642, International publication No. WO 94/18959. For example, a calcimimetic has one or more, preferably all, of the following activities when tested on parathyroid glands in vitro:

1. this compound resulted in a rapid (time to peak < 5 seconds) transient increase in intracellular calcium concentration for 1. mu.M La³⁺Or 1 μ M Gd³⁺Is resistant to inhibition. [ Ca ]²⁺]_iIncrease of extracellular Ca²⁺Is not present but is pretreated with ionomycin (extracellular Ca)²⁺Absent) is eliminated;

2. the compound potentiates exposure to extracellular Ca at concentrations below the maximum²⁺Induced [ Ca²⁺]_iIncreasing;

3. extracellular Ca²⁺Induced [ Ca²⁺]_iIncrease is not inhibited by dihydropyridine;

4. caused by the compound [ Ca²⁺]_iThe transient increase was not eliminated by 10 min pretreatment with 10mM sodium fluoride;

5. caused by the compound [ Ca²⁺]_iThe temporary increase cannot be reduced by pretreatment with an activator of Protein Kinase C (PKC), such as phorbol tetradecyl ester (PMA), Eurasian balsam or (-) -indolilactam V. The overall effect of protein kinase C activator is to shift the calcium concentration response curve to the right, but not to affect the maximum response;

6. the compounds result in a rapid (< 30 seconds) increase in inositol-1, 4, 5-trisphosphate and/or diglyceride formation;

7. the compounds inhibit dopamine or isoproterenol stimulated cyclic AMP formation;

8. the compounds inhibit PTH secretion;

9. pretreatment with pertussis toxin (100ng/ml, > 4 hours) suppressed the inhibitory effect of this compound on cyclic AMP formation, but did not affect [ Ca²⁺]_iAn increase in inositol-1, 4, 5-triphosphate, or diglyceride and a decrease in PTH secretion;

10. the compound is injected with poly (A) of bovine or human parathyroid cell⁺Priming of Cl in mRNA-enriched Xenopus oocytes^-Increased flow, but no effect on xenopus oocytes injected with water or liver mRNA; and is

11. Similarly, using cloned calcium receptors from parathyroid cells, the compounds will elicit a response in xenopus eggs injected with a particular cDNA or mRNA encoding the receptor.

Different calcium activities can be measured using known techniques. (see Nemeth et al, PCT/US93/01642, International publication No. WO 94/18959). Simulated Ca²⁺A parallel definition of compounds active on calcium-responsive cells, preferably calcium receptors, is evident from the examples provided herein and also from Nemeth et al, PCT/US93/01642, International publication No. WO 94/18959.

Preferably, the compounds have one or more, more preferably all, of the following activities, as measured by the biological assays described herein or according to Nemeth et al, PCT/US93/01642, international publication number WO 94/18959: triggering a temporary increase in endogenous calcium, when sustainedLess than 30 seconds in between (preferably by recruitment of endogenous calcium); initiation of [ Ca ]²⁺]_iA rapid increase, occurring within 30 seconds; initiation of [ Ca ]²⁺]_i(ii) sustained increase (greater than 30 seconds) (preferably by exogenous calcium influx); initiating an increase in inositol-1, 4, 5-trisphosphate or diglyceride levels, preferably in less than 60 seconds; and inhibiting dopamine or isoproterenol stimulated cyclic AMP formation.

[Ca²⁺]_iThe transient increase in (A) is preferably abolished by a 10-mM sodium fluoride pretreatment of the cells for 10 minutes, or is reduced by a transient pretreatment of the cells with an activator of protein kinase C, preferably Phorbol Myristate (PMA), Eurasian balsam or (-) -indolactam V (not more than 10 minutes).

C. Calcium lysate

The ability of the compound to block extracellular calcium activity at calcium receptors can be determined using standard techniques disclosed in accordance with the present invention. (see also Nemeth et al, PCT/US93/01642, International publication No. WO 94/18959.) for example, when applied to parathyroid cells, compounds that block the extracellular calcium effect when tested on parathyroid cells in vitro have one or more, more preferably all, of the following characteristics:

1. the compound partially or completely blocks increased concentrations of extracellular Ca²⁺The following capabilities:

(a) increase [ Ca ]²⁺]_i，

(b) Recruitment of intracellular Ca²⁺，

(c) Increase the formation of inositol-1, 4, 5-triphosphate,

(d) reducing dopamine or isoproterenol stimulated cyclic AMP formation, and

(e) inhibition of PTH secretion;

2. the compounds block poly (A) injected bovine or human parathyroid cells⁺Extracellular Ca-dependent on Xenopus ova of mRNA²⁺Or Cl initiated by calcium mimetic compounds^-Increased flow, but absence of this Cl in Xenopus ova injected with water or liver mRNA^-A flow increase;

3. similarly, using cloned calcium receptors from parathyroid cells, the compounds would block extracellular Ca-bearing in Xenopus eggs injected with cDNA, mRNA or cRNA encoding the receptor²⁺Or calcium mimetic compounds.

A parallel definition of compounds that block the action of calcium on calcium-responsive cells, preferably calcium receptors, is evident in the examples provided herein as well as in Nemeth et al, PCT/US93/01642, International publication No. WO 94/18959.

Treatment of diseases or disorders

Diseases or disorders that can be treated by modulating calcium receptor activity are known in the art. For example, diseases or disorders that can be treated by modulating calcium receptor activity can be identified based on the functional response of cells that are modulated by calcium receptor activity. Functional responses of cells regulated by calcium receptors are known in the art and include PTH secretion by parathyroid cells, calcitonin secretion by C cells, and osteoclast resorption.

Such functional responses are associated with different diseases or disorders. For example, elevated plasma PTH levels caused by hyperparathyroidism. Lowering plasma PTH levels is effective in treating hyperparathyroidism. Similarly, elevated plasma calcitonin levels are associated with inhibition of reabsorption. The effect of inhibiting bone resorption can effectively treat osteoporosis. Thus, modulation of calcium receptor activity can be used to treat diseases such as hyperparathyroidism and osteoporosis, as well as paget's disease.

Compounds that modulate the activity of inorganic ion receptors, preferably calcium receptors, are useful in providing therapeutic benefit to patients suffering from a range of diseases or disorders. For example, osteoporosis is an age-related disease characterized by decreased bone mass and increased risk of fracture. The compounds may be used to block osteoclast resorption either directly (e.g., an osteoclast ion mimetic compound) or indirectly by increasing endogenous calcitonin levels (e.g., a C-cell calcium mimetic). Alternatively, calcium lysates active at the calcium receptor of parathyroid cells can elevate circulating levels of parathyroid hormone, stimulating bone formation. All three methods bring good curative effect to osteoporosis patients.

It is also known that intermittent low doses of PTH can cause anabolic effects of bone mass and corresponding bone remodeling. Thus compounds and dosage regimens that elicit a temporary increase in parathyroid hormone (e.g., intermittent administration with parathyroid cell ion lysate) can increase bone mass in osteoporotic patients.

Identification of other diseases or disorders can be carried out by identifying other cellular functional responses that are associated with a certain disease or disorder and are modulated by calcium receptor activity. Diseases or disorders that can be treated by modulation of other inorganic ion receptors can be identified in a similar manner.

The compounds of the present invention that modulate inorganic ion receptors may act on one inorganic ion receptor to produce one or more cellular effects that ultimately produce a therapeutic effect. The compounds of the invention that modulate calcium receptors are capable of acting on calcium receptors to produce one or more cellular effects that ultimately produce a therapeutic effect. The present invention allows the treatment of different diseases by targeting cells containing calcium receptors.

For example, primary hyperparathyroidism (HPT is characterized by abnormally elevated levels of hypercalcemia and circulating PTH. the deficiency associated with the major class of HPT is the parathyroid cell pair extracellular Ca²⁺The sensitivity of the negative feedback adjustment of (2) is reduced. Thus extracellular Ca is present in the tissues of primary HPT patients²⁺The "set point" of (A) shifts to the right, requiring the use of higher than normal concentrations of extracellular Ca²⁺To inhibit PTH secretion. And even high concentrations of extracellular Ca in primary HPT²⁺Often only partially inhibit PTH secretion. Ca-although in secondary (uremic) HPT²⁺Extent of inhibition of PTH secretion was normal but extracellular Ca was observed²⁺Set point ofAnd similarly becomes larger. Changes in PTH secretion and [ Ca²⁺]_iThe changes in (2) are in parallel: extracellular Ca-dependent cell line²⁺Induced [ Ca²⁺]_iThe increasing setpoint is shifted to the right and the magnitude of this increase is reduced.

Patients with secondary HPT may also suffer from renal osteodystrophy. Calcium mimetics appear to be useful in treating abnormal PTH secretion and osteodystrophy in these patients.

Mimicking extracellular Ca²⁺The active compounds are useful for long-term control of primary and secondary HPTs. These compounds provide additional stimulation required to inhibit PTH secretion but which hypercalcemia cannot be achieved independently and thus help alleviate hypercalcemia. Specific efficiency of extracellular Ca²⁺High compounds may overcome the non-suppressible component seen in PTH secretion, which is particularly troublesome in the primary form of HPT, caused by parathyroid tumors. Alternatively or additionally, these compounds inhibit PTH synthesis, whereas long-term hypercalcemia has been shown to inhibit the levels of the early PTH mRNA in parathyroid tissue of bovine and human adenomas. Long-term hypercalcemia also inhibits the proliferation of parathyroid cells in vitro, so that calcium mimetics can also effectively limit the proliferative character of parathyroid cells in secondary HPT.

Cells other than parathyroid cells can be directed against extracellular Ca²⁺A physiological change in concentration in response. Calcitonin secretion from e.g. parathyroid accessory vesicle cells (C cells) by extracellular Ca²⁺And adjusting concentration change.

Isolated osteoclast responsive extracellular Ca²⁺Increase in concentration of corresponding [ Ca ]²⁺]_iThe increase is partly from intracellular Ca²⁺Recruitment of (1). Osteoclast [ Ca ]²⁺]_iThe increase is associated with inhibition of bone resorption. Osteoblasts release basic phosphate directly upon calcium stimulation.

Secretion of renin by juxtaglomerular cells in the kidney is secreted by extracellular Ca as is secretion of PTH²⁺The increase in concentration is suppressed. Extracellular Ca²⁺Resulting in intracellular Ca in these cells²⁺Is recruited. The responses of other renal cells to calcium are as follows: ca²⁺Increasing inhibition of proximal tubular cells 1, 25(OH)₂Formation of vitamin D, stimulation of calcium binding protein production in distal tubular cells, inhibition of Ca²⁺And Mg²⁺Renal tubular reabsorption and angiotensin effects on the crude ascending branch of the Henle loop (MTAL), attenuate angiotensin effects on cortical collecting vessel cells and affect vascular smooth muscle cells in the glomerular vessels.

Calcium also promotes differentiation of intestinal goblet cells, breast cells and skin cells; inhibiting atrial natriuretic peptide secretion from the atrium; reducing cAMP aggregation in platelets; altering secretion of gastrin and glucagon; act on vascular smooth muscle cells to modify cellular secretion of vasoactive factors; and affect cells of the central and peripheral nervous systems.

Thus there is sufficient evidence to show Ca²⁺In addition to having a broad intracellular signaling role, it may also serve as an extracellular signal to regulate the response of certain specialized cells. The compounds of the invention are useful in the treatment of Ca disrupted in these cells²⁺Response to the associated disease or disorder.

Specific diseases or disorders that can be treated or prevented, depending on the affected cells, also include central nervous system diseases or disorders such as seizures, stroke, head trauma, spinal cord injury, hypoxia-induced nerve cell damage such as asystole or incipient bitter pain (neuropathic), epilepsy, degenerative neurological diseases such as senile dementia, Huntington's disease and Parkinson's disease, dementia, nervous tension, depression, anxiety, panic, obsessive-compulsive disorder, post-traumatic stress, schizophrenia, malignant neuroleptic syndrome, and Tourette's syndrome; diseases involving excessive reabsorption of water by the kidneys such as abnormal ADH secretion Syndrome (SIADH), cirrhosis, congestive heart failure and nephritis; hypertension, prevention and/or reduction of nephrotoxicity of cationic antibiotics (e.g., aminoglycoside antibiotics); digestive tract motility disorders such as diarrhea and large bowel spasm; gastrointestinal ulcers; gastrointestinal disorders such as sarcoidosis caused by excessive calcium absorption; and autoimmune diseases and organ transplant rejection.

Although the compounds of the invention which modulate calcium receptors are typically used in the treatment of humans, they may also be used to treat similar or identical diseases in other warm-blooded animals, such as other primates, livestock such as pigs, cattle, birds; and sports animals and pets such as horses, dogs and cats.

Administration of drugs

The various compounds described herein are useful for the treatment of various disorders by modulating inorganic ion receptor activity, particularly calcium receptor activity. The compounds of the invention may be formulated for administration in a variety of ways, such as systemic and local or localized administration. Techniques and formulations are generally described in Lemington's pharmaceutical sciences Mack publishing Inc., Easton, Bingzhou. Administration of ionic mimetics and ionic lysates is discussed in Nemeth et al, PCT/US93/01642, International publication No. WO 94/18959.

The appropriate dosage form depends in part on its use and the route of administration, e.g., oral, transdermal or injection. Whether the target cell is in a multicellular host or in culture, such dosage forms should allow the compound to reach the target cell. For example, a pharmaceutical compound or composition injected into the bloodstream should be soluble. Other factors are known in the art, including considerations of toxicity and dosage form to prevent the compound or composition from exerting its efficacy.

The compounds may also be formulated as pharmaceutically acceptable salts (e.g., acid addition salts) and complexes thereof. Pharmaceutically acceptable salts are salts that are non-toxic at the concentrations at which they are administered. The preparation of such salts can alter the physical properties of the compound without preventing it from performing its physiological function, thereby facilitating its pharmaceutical use. Useful changes in physical properties include lowering the melting point to facilitate transmucosal administration and increasing solubility to facilitate administration of high concentrations of the drug.

Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, hydrochloride, maleate, phosphate, sulfonamide, acetate, citrate, lactate, tartrate, mesylate, esylate, besylate, p-toluenesulfonate, cyclohexylsulfonamide and quinic acid salts. (see, e.g., PCT/US92/03736, incorporated herein by reference). Pharmaceutically acceptable salts can be derived 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 the compound is dissolved in a suitable solvent containing a suitable acid, such as water or a water-alcohol solution, and the solution is isolated by evaporation. In another embodiment the salt is prepared by reacting the free base with an acid in an organic solvent.

Carriers or excipients may also be used to facilitate administration of the compounds. Examples of carriers and excipients include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose or various types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents. The compositions or pharmaceutical compositions may be administered by different routes, including intravenous, intraperitoneal, subcutaneous and intramuscular, oral, topical or transmucosal administration.

The mode of systemic administration is preferably oral. Alternatively, injection may be performed, for example, intramuscularly, intravenously, intraperitoneally, and subcutaneously. For injection, the compounds of the invention are formulated in solution, preferably in a physiologically compatible buffer such as Hank's solution or ringer's solution. Alternatively, the compounds may be formulated in solid form for reconstitution or suspension prior to use. Can also be made into lyophilized form.

Systemic administration can also be transmucosal or transdermal, or oral. 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, cholates and fusidic acid derivatives for transmucosal administration. In addition detergents may be used to aid penetration. Transmucosal administration can be accomplished, for example, by nasal sprays or suppositories. For oral administration, the compounds may be formulated into conventional oral dosage forms such as capsules, tablets and liquid preparations.

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

The amount of each compound of the present invention to be administered is determined by standard procedures. Generally, a therapeutically effective amount is about 1 nanomolar to 1 micromolar of the compound, preferably 0.1 nanomolar and 1 micromolar, depending on its EC₅₀And IC₅₀And the age and size of the patient and the disease or disorder associated with the patient. Generally, for the treated animal, the dosage is between 0.1 and 50mg/kg, preferably 0.01 to 20mg/kg of the treated animal.

V. examples

The following examples are provided to illustrate various aspects and embodiments of the present invention. These examples are not intended to limit the present invention.

Example 1: cloning of human Parathyroid calcium receptor from human Parathyroid tumor

This example describes the cloning of human parathyroid calcium receptor from human parathyroid tumors using pBoPCaR1 as a hybridization probe (see Nemeth et al, PCT/US93/01642, International publication No. WO 94/18959). Nucleic acids encoding human parathyroid calcium receptor are identified by probe cross-hybridization under conditions of weak stringency.

Messenger RNA was prepared from human parathyroid adenoma removed from a 39 year old caucasian man diagnosed with primary hyperparathyroidism. Northern blot analysis of this mRNA using pBoPCaR1 as hybridization probe identified calcium receptor transcripts of about 5Kb and about 4 Kb. A cDNA library was constructed from the mRNA. Double-stranded cDNA larger than 3Kbp was selected on agarose gel according to size and ligated into the cloning vector lambda ZapII. 50 ten thousand primary recombinant phages were screened using the 5.2Kbp cDNA insert of pBoPCaR1 as hybridization probe. Use 2³²P]Random priming of dCTP Synthesis to tag the pBoPCaR1 insert to a specific Activity of 1X 10⁹cpm/μg。

The stringency of hybridization at which the library is screened is 400mM Na at 38 ℃⁺50% formaldehyde. The plaque lift filters (plaque lift filters) were hybridized for 20 hours with a probe concentration of 500,000 cmp/ml. Then washed with 1 XSSC 40 ℃ for 1 hour.

Approximately 250 positive clones were initially screened by hybridization with pBoPCaR 1. Of these, 7 were isolated as single clones hybridizing with the pBoPCaR1 probe by secondary and tertiary screening. These 7 clones were subjected to restriction enzyme mapping and Southern blot analysis. The 3 clones contained about 5Kbp of cDNA insert and appeared to correspond to a full length clone of 5Kb mDNA. 2 clones contained about 4Kbp cDNA insert and appeared to correspond to a full length clone of 4Kb mRNA.

Restriction enzyme mapping of two different sized inserts showed that they had similar sequence regions at the 5 'end, but different sequences at the 3' end. DNA sequence analysis showed that the small insert was probably due to alternative polyadenylation upstream of the polyadenylation site used for the large insert.

Representative cDNA inserts of two sizes were subcloned into the plasmid vector pBluescript 8K. After linearization, cRNA transcripts produced with T7 RNA polymerase were transcribed in vitro. The cRNA transcripts were injected into Xenopus ova (150 ng/. mu.l RNA; 50 nl/egg) for functional analysis. After 2-4 days of incubation, the egg cells were examined for the presence of functional calcium receptors. Calcium-activated Cl stimulated by addition of appropriate calcium receptor agonist^-Flow, both clonal types were detected to have functional calcium receptors. Known calcium receptor agonists, including NPS R-467 and NPS R-568 (see Nemeth et al, PCT/US93/01642, International publication No. WO94/18959), activate egg cell expression at a concentration of the receptor that is nearly the same as the effective concentration for activating the native parathyroid receptor. Thus both clones encode functional human parathyroid calcium receptor.

The inserts of each size were subcloned into pBluescript to prepare the plasmids pHuPCaR5.2 and pHuPCaR 4.0. The nucleic acid sequence and amino acid sequence of the insert sequence are represented by seq id NO: 1 and 2.

Several differences were observed in the nucleic acid sequences of the two cDNA inserts. Sequence analysis of the two cDNA inserts revealed at least two sequence differences in the 3' untranslated region, which may be due to alternative polyadenylation. Additionally, the 5' end of the insert sequence is sequence-different. These different sequences correspond to untranslated regions, possibly due to different transcription initiation and/or splicing.

Three additional sites of sequence differences were observed in the coding regions of the cDNA clones phupcrar 5.2 and phupcrar 4.0 (see seq. id. No.1 and 2), showing that these cDNA clones encode different proteins. Sequence analysis of the human CaR gene showed that 30 base pairs more abundant in the cDNA clone pHuPCaR5.2 than pHuPCaR4.0cDNA clone were derived from different mRNA splices. Splicing of different mRNAs predicts the insertion of 10 additional amino acids at positions #536 and #537 of the CaR polypeptide encoded by the pHuPCaR5.2 cDNA corresponding to the polypeptide encoded by the pHuPCaR4.0 cDNA. Furthermore pHuPCaR4.0 encodes glutamine (Gln) at amino acid #925 and glycine (Gly) at position 990, while pHuPCaR5.2 encodes arginine (Arg) at both equivalent positions. The human CaR gene encodes Gln and Arg at these positions, respectively. The differences between pHuPCaR4.0cDNA and human DNA appear to represent true sequence polymorphisms in the human population, whereas a single base change in pHuPCaR5.2 may reflect a mutation in its cloning. Both cDNAs encode functional calcium receptors, as measured by Cl^-Xenopus ova injected with cRNA prepared from these cDNA clones were all known to respond to extracellular calcium during conductance. However, it is also possible that the two receptors differ functionally and/or pharmacologically.

Example 2 selection of Stable recombinant cells expressing calcium receptors

A clonal cell line stably expressing two human and bovine calcium receptors was isolated. The calcium receptor cDNA was subcloned into two different commercially available expression vectors; pMSG (from Pharmacia) and Cep4B (from Invitrogen). The first vector contains a selectable marker gene for xanthine guanine phosphoribosyl transferase (gpt), and stably transformed cells cross the purine biosynthetic pathway barrier caused by the addition of 2. mu.g/ml aminopterin and 25. mu.g/ml mycophenolic acid. The second vector encodes the hygromycin (200. mu.g/ml) resistance gene. HuPCaR5.2 and HuPCaR4.0 cDNAs (SEQ ID NOS: 1 and 2, respectively) were removed from the parent pBluescript plasmid using Not I and Hind III restriction enzymes, and then ligated directly into Cep4B digested with Not I + Hind III or blunt-ended into SmaI-digested pMSG after treatment with Klenow fragment of DNA polymerase.

A pMSG subclone containing the hupcar5.2 insert as described above was transfected into CHO cells. The 20 resistant clones were selected and then characterized. As above, a sub-clone of Cep4B containing the hupcar5.2 insert was transfected into HEK293 cells. A stable clone bank was obtained by screening with hygromycin. Clones expressing the hupcar4.2 receptor isoform were prepared similarly.

Cells from the hygromycin-selected HEK293 cell bank were transfected with Cep4B containing the hupcar5.2 insert and plated onto collagen-coated AKlar patches in each well of a 12-well tissue culture plate. After 2-6 days the medium was removed and 1ml of 1. mu.Mfura 2-AM, 1mM CaCl, was added with balanced salt solution₂And 0.1% BSA, 1mM CaCl₂The buffer of (3) washes the cells. Fluorescence in response to calcium receptor agonists was measured at 37 ℃ with a fluorescence photometer, excitation and emission wavelengths of 340nm and 510nm, respectively. During signal calibration, ionic mycin (40. mu.M) was added to determine Fmax, 0.3M EGTA, 2.5M Tris HCl, pH10 was added to determine apparent Fmin. Animals were stimulated as the addition of the following calcium receptors: ca²⁺(10mM)，Mg²⁺(20mM) and NPS R-467, [ Ca ] observed²⁺]_iThe activity is increased. Control cells expressing a functional substance K receptor do not respond to these calcimimetic compounds.

Additional clonal isolates of HEK293 cells transfected with the phupcrar 4.0 sequence were obtained. Their response to calcium mimetics was purified by isolation as described above, except that these cells were tested in suspension.

Example 3: measurement of calcium receptor Activity Using Fura-2 loaded Parathyroid cells

This subsection describes methods for obtaining parathyroid cells from cattle and humans and for measuring calcium receptor activity using parathyroid cells.

Parathyroid glands were obtained from freshly slaughtered cattle (12-15 weeks old) from a nearby slaughterhouse, packaged in a container containing (mM): NaCl, 126; KCl, 4; MgCl₂1, 1; Na-HEPES, 20; pH 7.4; glucose, 5.6; and variable amounts of CaCl₂(e.g., 1.25mM) in ice cold Parathyroid Cell Buffer (PCB) to the laboratory. Human parathyroid glands were obtained from patients who received surgical removal of parathyroid tissue due to primary or vegetarian hyperparathyroidism (urea HPT), treated the same as bovine tissue.

The glands were freed of excess fat and connective tissue and cut into small squares of about 2-3mm length. Dissociated parathyroid cells were prepared by collagenase digestion and then purified by centrifugation in Percoll buffer. The resulting parathyroid cell preparation is substantially free of red blood cells, adipocytes, and capillary tissue; the test methods were phase contrast microscopy and sudan black B staining. Dissociated and purified parathyroid cells are small clusters (clusters) containing 5 to 20 cells. Cell viability, as indicated by exclusion of trypan blue or ethidium bromide, is typically 95%.

Although cells are available for experimentation at this time, the physiological response (e.g., inhibitory ability of PTH secretion and [ Ca ] should be determined after overnight culture of the cells²⁺]_iAt a resting level). The primary culture also has the advantage that the cells can be labelled with a radioisotope to isotopic equilibrium, which is essential for studies involving measurement of inositol phosphate metabolism.

After purification on a Percoll gradient, Ham's F12 Dulbecco's modified Eagle's medium (GIBCO) and ITS supplemented with 50. mu.g/ml streptomycin, 100U/ml penicillin, 5. mu.g/ml gentamicin 1: 1 were used⁺The mixture was washed several times with cells. ITS⁺Is a premixed solution containing insulin, transferrin, selenium, and Bovine Serum Albumin (BSA) -linolenic acid (collagenous research, Bedford, ma). The cells were then transferred to plastic flasks (75 or 150 cm)²(ii) a Falcon), 5% CO at 37 ℃₂Incubated overnight in humid air. Serum was not added to the overnight cultures because serum would attach cellsProliferate and dedifferentiate on plastic. Cells cultured under the above conditions can be conveniently removed from the flask after dehydration and have the same viability as freshly prepared cells.

The purified parathyroid cells were resuspended in 1.25mM CaCl containing 1. mu.M fura-2-acetoxymethyl ester_2-2% BSA-PSB, incubated at 37 ℃ for 20 minutes. The cells were then pelleted, resuspended in the same buffer lacking the ester, and incubated for an additional 15 minutes at 37 ℃. With a solution containing 0.5BSA and 0.5mM CaCl₂The PCB of (1) was washed twice and stored at room temperature (about 20 ℃ C.). Cells were pre-warmed with 0.5mM CaCl before use_2-The PCB was diluted 5-fold, and the final concentration of BSA obtained was 0.1%. Cell concentration in the cuvette for fluorescence recording was 1-2X 10⁶/ml。

Fluorescence excitation and emission wavelengths of indicator-loaded cells were measured at 340nm and 510nm, respectively, at 37 ℃ using a fluorescence photometer (biological Instrumentation Group, university of Bingzhou, Philadelphia, Bingzhou) equipped with a heat-resistant cup holder and a magnetic stirrer. The fluorescence shows Ca in the cell fluid²⁺And (4) horizontal. The fluorescence signal was normalized to the maximum fluorescence (Fmax) with digitonin (50. mu.g/ml, final concentration), to the minimum fluorescence (Fmin) with EGTA (10mM, pH8.3, final concentration), and to the dissociation constant at 224 nm. Dye leakage is temperature dependent, most often occurring in the first two minutes after heating the cells in the cuvette. After which the dye leakage increases very slowly. To correct for calibration for dye leakage, cells were placed in a cuvette and stirred for 2-3 minutes. The cell suspension was then removed, the cells were pelleted, and the supernatant was returned to a clean cuvette. Supernatant was treated with digitonin and EGTA to estimate total Ca leakage from the dye²⁺10-15% of the dependent fluorescence signal. This estimate is subtracted from the apparent Fimn.

Example 4: with Fura-2 loaded HEK 293/pHuPCaR 4.0

Cellular measurement of calcium receptor Activity

The subsection describes the measurement of calcium receptor Activity using Fura-2 loaded HEK 293/pHuPCaR 4.0 cellsA method. pHuPCaR4.0 transfected HEK293 cells in 5 u M fluoro-3/AM 20mM HEPES buffered Dulbecco's modified Eagle's medium room temperature 1 h incubation, loaded with fura-2. Then using a solution containing 1mM CaCl₂And 1mM MgCl₂20mM HEPES buffered Hank's balanced salt solution. The compound to be tested is then added to the cells and the fluorescence is measured (excitation and emission wavelengths 340 and 510nm, respectively).

Example 5: measuring the ability of a compound to modulate calcium receptor activity

Detection of [ Ca ] in Fura-2-loaded HEK293 cells transfected with a nucleic acid encoding pHuPCaR4.0 or Fura-2-loaded parathyroid cells²⁺]_iIncreased to test the ability of different compounds to modulate calcium receptor activity. The results of the different experiments are summarized in tables 1.a, 1.b.1, 1.b.2, 1.c and 2. Tables 1.a, 1.b.1, 1.b.2, 1.c summarize the effect of different concentrations of each compound on calcium receptor activity as determined according to the description of example 4 (i.e. HEK293 cells transfected with fura-2 loaded nucleic acid encoding phupcrar 4.0).

Table 2 summarizes the results of different experiments, in which EC₅₀Calculation was performed on parathyroid cells or HEK 293/pHuPCaR 4.0 loaded with fura-2. Cells were loaded with fura-2 and tested as described in example 2 (for parathyroid cells) or example 3 (for HEK 293/pHuPCaR 4.0 cells).

Table 1.a. production at 3.3ng/ml in HEK293 cells expressing human calcium receptor More than 40% of responding calcimimetic compounds

Percent Activity at 4 concentrations of Compound code

(ng/mL)

3300 330 33 3.3

Reference compound

R-568 95 69 24

17P 101 86 54

17X 105 93 51

24X 126 109 124 109

24Y 119 120 127 102

17J 116 118 122 102

25A 122 120 114 92

17E 116 110 110 92

24Z 138 138 135 90

14S 116 106 105 88

25E 132 129 122 85

17G 125 128 119 77

14T 126 125 117 77

17H 126 124 111 74

14O 119 119 102 74

25I 119 113 114 74

12J 131 130 113 68

Percent Activity at 4 concentrations of Compound code

(ng/mL)

3300 330 33 3.3

12I 115 111 93 68

25G 130 115 99 66

9R 108 101 64

12F 118 110 101 63

12O 110 117 94 62

23Z 129 126 100 61

17M 115 99 59

16V 114 102 58

25O 126 115 96 57

25J 119 123 105 56

16L 14 6 138 98 56

12N 115 106 102 55

16T 97 88 55

25U 107 107 95 55

17P 101 86 54

16Q 110 88 53

23E 137 113 102 53

17C 113 120 99 52

25L 97 97 85 52

8Z 101 97 52

17X 105 93 51

13R 132 98 51

17O 112 96 51

23Q 122 114 98 51

16X 11 96 51

24V 127 98 71 50

13O 115 94 50

17N 108 86 49

21V 122 116 99 48

24M 132 134 99 48

13U 108 79 47

Percent Activity at 4 concentrations of Compound code

(ng/mL)

3300 330 33 3.3

24P 140 138 110 46

17Y 109 94 79 46

11X 100 76 45

25H 115 107 89 45

22J 99 71 45

9C 104 82 45

13S 102 87 45

10Q 103 100 84 44

13P 110 83 44

8K 98 81 44

13N 114 88 43

10N 106 97 77 43

12H 114 115 94 43

25P 90 81 75 41

18A 111 88 40

14L 109 78 40

TABLE 1.b.1 production at 33ng/ml in HEK293 cells expressing human calcium receptors Calcimimetic compounds with more than 40% response

Percent Activity at 4 concentrations of Compound code

(ng/mL)

3300 330 33 3.3

Reference compound

R-568 95 69 24

17P 101 86 54

17X 105 93 51

12C 134 125 98 39

16I 121 117 96 36

Percent Activity at 4 concentrations of Compound code

(ng/mL)

3300 330 33 3.3

17D 108 91 38

17F 111 90 28

24C 116 113 87 32

25K 124 107 86 35

13F 125 122 85 38

21F 109 85 36

21S 132 131 85 34

10F 96 84 27

14R 106 107 84 37

13G 111 128 82 29

14Z 118 103 82 20

16N 122 159 82 8

8U 123 129 82 11

23W 117 97 81 25

12G 139 139 81 35

15G 113 80 32

25M 118 100 79 25

13V 110 79 33

14P 112 103 78 30

6T 123 129 78 15

14Q 101 78 35

17L 111 104 78 31

24K 106 78 30

24U 106 106 78 25

25Q 116 95 77 20

8J 104 77 39

23H 121 114 77 28

21C＝4U 134 114 76 17

25F 97 85 76 28

16R 100 76 25

17I 118 97 76 18

Percent Activity at 4 concentrations of Compound code

(ng/mL)

3300 330 33 3.3

24J 103 75 31

21O 109 75 37

24G 109 94 75 22

15I 111 93 75 24

21D 104 75 17

20Y 117 95 74 24

10P 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 111 96 69 26

21Y 100 68 36

17W 92 67 13

23A 103 67 24

23G 127 93 67 13

13M 92 66 15

21U 104 104 66 18

21R 100 66 15

10S/10T 86 65 13

17R 98 65 13

13X 102 65 13

4N 100 65 13

21E 94 64 4

15J 80 75 64 13

Percent Activity at 4 concentrations of Compound code

(ng/mL)

3300 330 33 3.3

22Y 114 64 28

21G 88 63 18

24L 105 62 10

10V 99 62 8

10W/10X 98 61 9

17B 92 61 19

23Y 106 87 61 16

11Y 103 61 20

Table 1.b.2 production at 330ng/ml in HEK293 cells expressing human calcium receptors More than 40% of responding calcimimetic compounds

Percent activity at 4 concentrations of compound No.: acids and amides

(ng/mL)

3300 330 33 3.3

Reference compound

R568 95 69 24

17P 101 86 54

17X 105 93 51

18C 99 87 60 18

23T 102 74 60 31

4V 93 59

8G 84 59 6

23I 102 58 3

21M 102 58 17

24O 137 114 58 8

3U 89 57

9A 82 56 6

12M 98 86 56 11

12B 130 110 56 4

Percent Activity at 4 concentrations of Compound code

(ng/mL)

3300 330 33 3.3

21P 92 56 13

8T 85 55 13

10L/10M 99 55 4

24I 109 84 55 11

14N 89 55 15

23R 104 86 54 13

23S 97 53 3

21T 133 112 53 3

10W/10X 81 53 4

13T 90 53 6

6R 94 52 7

20I 87 52 12

24A 122 85 52 9

12D 128 109 52 5

6X 84 52 10

18T 99 74 52 14

21X 119 101 51 2

23J 102 61 51 29

10Z 96 51 5

16Z 88 51 9

23N 96 50 2

16U 85 50 4

11D 96 50 4

23X 94 49 1

17A 88 49 7

20J 80 48 8

22X 86 48 10

23U 87 48 3

9Z 74 48 4

16J 92 76 47 31

25N 94 73 46 8

4P 81 46 8

Percent Activity at 4 concentrations of Compound code

(ng/mL)

3300 330 33 3.3

23O 111 79 46 13

13Q 95 46 5

4G 83 46

12Y 80 46 10

12L 88 45 10

23F 82 45 5

11W 81 44 2

8H 88 44 7

25V 89 59 43 26

25W 95 69 42 8

10R 82 42 7

21N 124 98 42 4

8S 73 42 7

8X 75 40 19

13E 123 94 40 2

Table 1.c. production at 3.3ng/ml in HEK293 cells expressing human calcium receptors Calcimimetic compounds with more than 40% response

Percent Activity at 4 concentrations of Compound code

(ng/mL)

3300 330 33 3.3

Reference compound

R5 68 95 69 24

17P 101 86 54

17X 105 93 51

7X 85

3H 84

3L 81 28

16O 129 81 21 2

8O/8Q 124 80 14 0

Percent Activity at 4 concentrations of Compound code

(ng/mL)

3300 330 33 3.3

14A 98 78 10 7

23L 107 77 37 9

1T 76

7W 76

4H 77 37

8D 75

5M 73 21

4U 72

24E 94 71 35 6

16M 130 68 11 4

4M 68 34

2S 67 29

17V 91 66 27 -1

2X 66 15

23D 91 66 35 13

4P 65 32

5B/5C 65 20

3M 64 19

16K 78 62 36 8

5D 62 18

4D 61 13

24B 76 61 34 11

24H 81 60 32 13

5L 60 16

2Y 59 10

5G 58 16

3V 56 14

2Q 56 4

14B 75 55 11 4

13Z 93 54 22 5

8A 54

24D 87 53 34 39

Percent Activity at 4 concentrations of Compound code

(ng/mL)

3300 330 33 3.3

1D 53

13I 85 52 3 1

3B 52 15

8C 51

14H 112 49 5 5

7U 49

5E 48 7

13H 88 48 36 12

13Y 106 47 2 4

4J 47 8

14I 80 45 11 7

4B 45 8

3D 45 4

3R 45 2

3A 41 7

14J 55 41 6 5

4I 40 9

TABLE 2Arylalkylamine calcium mimetics of FIG. 1 have in vitro activity on parathyroid cell calcium receptors (EC)₅₀≤5μM)

Compound code (from figure 1)	EC50(μM)	Compound code (from figure 1)	EC50(μM)
				NPSR-467	2.0	11X	0.83
NPSR-568	0.60	11Y	2.8
				3U	0.64	12L	1.7
3V	1.8	12U	1.2
				4A	1.4	12V	0.42
4B	2.0	12W	3.2

4C	2.0	12Y	2.0
				4D	4.4	12Z	0.11
4G	1.8	13Q	ca.0.8
				4H	≥3.0	13R	0.25
4J	2.2	13S	＜0.13
				4M	2.1	13U	0.19
4N	0.8	13X	＜0.75
				4P	1.6	14L	0.26
4R/6V	4.2	14Q	0.47
				4S	3.3	14U	0.13
4T/4U	1.6	14V	1.7
				4V	2.5	14Y	0.38
4W	2.3	15G	ca.0.5
				4Y	1.3	16Q	0.04
4Z/5A	4.4	16R	0.36
				5B/5C	2.8	16T	0.04
5W/5Y	3.6	16V	＜0.13
				6E	2.7	16W	0.59
6F(R，R-)	0.83	16X	0.10
				6R	3.4	17M	0.15
6T	2.9	17O	0.04
				6X	2.5	17P	0.04
7W	3.2	17R	0.39
				7X	1.1	17W	0.43
8D	2.5	17X	0.02
				8J	0.78	20F	＜1.0
8K	1.3	20I	＞1.0
				8R	2.6	20J	＞3.0
8S	1.7	20R	2.4
				8T	1.8	20S	4.2
8U	0.44	21D	3.0
				8X	0.76	21F	0.38
8Z	0.40	21G	1.1

9C	0.60	21O	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

Synthesis of Compounds of examples 6 to 17

The compounds described herein can be synthesized using standard techniques, as described, for example, in Nemeth et al, PCT/US93/01642, International publication No. WO 94/18959. The examples provided below describe representative syntheses of the compounds referred to herein.

The synthesis of compounds 9R, 14U and 17P is prepared by reductive amination of the commercially available aldehydes or ketones 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 25O were prepared in a similar manner.

It was found that the synthesis of these three compounds (9R, 14U and 16P) using sodium triacetoxyborohydride gave the desired diastereomer with greater diastereoselectivity than sodium cyanoborohydride. The enriched mixture is further purified to the individual diastereomers using normal phase HPLC or recrystallization from organic solvents.

Compounds 8J, 8U, 11X, 17M and 25Y are prepared by condensing a primary amine with an aldehyde or ketone in the presence of titanium (IV) isobutoxide. The resulting intermediate imine is reduced in situ (insitu) by the action of sodium cyanoborohydride, sodium borohydride or sodium triacetoxyborohydride. The intermediate, enamine, for the synthesis of compound 8U was catalytically reduced with palladium dihydroxide on carbon.

Compounds 12U, 12V and 12Z were prepared using a diisobutylaluminum hydride (DIBAL-H) mediated condensation of an amine with a nitrile. The obtained intermediate imine is reduced in situ by sodium cyanoborohydride or sodium borohydride. The intermediate alkenes (compounds 12U and 12V) were catalytically hydrogenated in EtOH with palladium on carbon. The compound was converted to its hydrochloride salt by the action of the free base with the volatile HCl to give a white solid.

The amines in these syntheses were purchased from Aldrich chemicals, Milwaukee, WI or Celgene, Warren, n.j. or synthesized using standard techniques. All other reagents were purchased from Aldrich chemicals.

Example 6: synthesis of Compound 25Y

N- (3- (2-phenyl) propyl) -1- (1-naphthyl) ethylamine

A mixture of 3-phenyl-1-propylamine (135mg, 1mmol), 1' -acetylnaphthalenone (170mg, 1mmol) and titanium (IV) isopropoxide (355mg, 1.3mmol) was stirred at room temperature for 1 hour. The reaction was treated with 1M sodium cyanoborohydride (1mL) 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, the ether layer removed and concentrated to a cream. A small portion of this material contained 0.1%. Isopropylamine was purified by gradient HPLC (Phenomenex, 1.0X 25cm, 5. mu.M silica) from dichloromethane to dichloromethane with 10% methanol. The product (free base) was a single component (R) as determined by GC/El-MS_t10.48 min) M/Z (relative intensity) 289 (M)⁺，11)，274(63)，184(5)，162(5)，155(100)，141(8)，115(8)，91(45)，77(5)。

Example 7: synthesis of Compound 8J

N- (3-phenylpropyl) -1- (3-thiomethylphenyl) ethylamine hydrochloride

3' -Aminoacetophenone (2.7g, 20mmol) was dissolved in 4mL concentrated HCl, 4g ice and 8mL water. The solution was cooled to 0 deg.C, sodium nitrite (1.45g, 21mmol) was dissolved in 3-5ml water and added to the solution over 5 minutes, maintaining the temperature below 6 deg.C. Sodium thiomethoxide (1.75g, 25mmol) was dissolved in 5mL of water and cooled to 0 ℃. The diazonium salt is added to the solution over 10 minutes maintaining the temperature below 10 ℃. The reaction was stirred for 1 hour and the temperature was raised to ambient. The reaction mixture synthesized ether and aqueous layers. The ether layer was separated, washed with sodium carbonate and sodium chloride and dried over sodium sulfate. The ether was evaporated to give 3' -thiomethyl acetophenone in 74% yield. The crude extract was purified by distillation under reduced pressure.

3-Phenylpropylamine (0.13g, 1mmol), 3' -thiomethyl acetophenone (0.17g, 1mmol) and titanium (IV) isopropoxide (0.36g, 1.25mol) were mixed and left for 4 hours. Ethanol (1mL) and sodium cyanoborohydride were added and the reaction was stirred overnight. The reaction was initiated by the addition of 4ml and 200. mu.L of water. The mixture was vortexed and then centrifuged to separate the solids. The ether layer was separated from the precipitate and the solvent was removed in vacuo. The oil was redissolved in dichloromethane and the compound was purified by preparative TLC on silica gel eluting with 3% methanol in dichloromethane to give the title compound as a pure oil: GC/EI-MS (R)_t7.64 min) M/Z (relative intensity) 285 (M)⁺，18)，270(90)，180(17)，151(100)，136(32)，104(17)，91(54)，77(13)。

Example 8: synthesis of Compound 8U

N-3- (2-methoxyphenyl) -1-propyl- (R) -3-methoxy-alpha-methylbenzylamine hydrochloride

A mixture of (R) - (+) -3-methoxy- α -methylbenzylamine (3.02g, 20mmol), 2-methoxycinnamaldehyde (3.24g, 20mmol) and titanium (IV) isopropoxide (8.53g, 30mmol, 1.5Eq) was stirred at room temperature for 2 hours and treated with 1M (20mL) sodium cyanoborohydride ethoxide. The reaction was stirred overnight (16 h), diluted with diethyl ether and treated with water (1.44mL, 80mmol, 4 Eq.). After mixing for 1 hour, the reaction mixture was centrifuged, the ether layer was removed, and concentrated to an oil. Dissolving the material in glacial acetic acid, and adding hydrogenThe palladium oxide was shaken and then hydrogenated in 60 lbs of hydrogen at room temperature. The catalyst was removed by filtration, and the resulting solution was concentrated to a thick oil. This material was redissolved in dichloromethane and neutralized with 1n naoh. The dichloromethane solution was separated from the aqueous phase, dried over anhydrous potassium carbonate and concentrated to an oil. This material was redissolved in ether and treated with 1M HCl in diethyl ether. The resulting precipitate was collected, washed with diethyl ether and dried in air. This material (GC/EI-MS (R) as the free base_t9.69 min) showed a single component: M/Z (relative intensity) 299(M +, 21), 284(100), 164(17), 150(8), 135(81), 121(40), 102(17), 91(43), 77 (18).

Example 9: synthesis of Compound 9R

(R) -N- (1- (2-naphthyl) ethyl) - (R) -1- (1-naphthyl) ethylamine hydrochloride

(R) - (+) -1- (1-naphthyl) ethylamine (10.0g, 58mmol), 2' -acetonaphthone (9.4g, 56mmol), titanium (IV) isopropoxide (20.7g, 730mmol) and EtOH (anhydrous) (100mL) were heated to 60 ℃ for 3 h. Then adding sodium cyanoborohydride (NaCNBH)₃) (3.67g, 58.4 mmol). The reaction mixture was stirred at room temperature for 18 hours. The ether (1L) and H₂O (10mL) was added to the reaction mixture and the resulting precipitate was removed by centrifugation. The supernatant was evaporated in vacuo and the crude product was recrystallized 4 times from hot hexane to yield 1.5g of pure (98 +%) diastereomer. The free base was dissolved in hexane, filtered and fuming HCl was added, precipitating the product as a white solid (1.1g, 6% yield), melting point: 200 ℃ and 240 ℃ softening (dec.).

Example 10: synthesis of Compound 11X

N- (4-isopropylphenyl) - (R) -1- (1-naphthyl) ethylamine hydrochloride

A mixture of (R) - (+) -1- (1-naphthyl) ethylamine (1.06g, 6.2mmol), 4-isopropylbenzaldehyde (0.92g, 6.2mmol) and titanium (IV) isopropoxide (2.2g, 7.7mmol) was heated to 100 ℃ for 5 minutes and then stirred at room temperature for 4 hours. Adding sodium cyanoborohydride (NaCNBH)₃) (0.39g, 6.2mmol) followed by EtOH (1 mL). The reaction mixture is stirred at room temperature 18And (4) hours. Ether (100mL) and H₂O (1mL) was added to the reaction mixture and the resulting precipitate was removed by centrifugation. The supernatant was evaporated in vacuo and the crude product was chromatographed on silica gel (50mm X30 cm column) (with 1% MeOH (CHCl)₃) Elution), the chromatographed material was redissolved in hexane, fuming HCl was added, and the product precipitated as a white solid (0.67g, 35% yield), melting point: 257 ℃ and 259 ℃.

Example 11: synthesis of Compound 12U

N-3- (2-methylphenyl) -1-propyl- (R) -3-methoxy-alpha-methylbenzylamine hydrochloride

A solution of 2-methylcinnamonitrile (143g, 10mmol) in dichloromethane (10mL) was cooled to 0 deg.C and treated dropwise (15 min) with 1M diisobutylaluminum hydride (10mL, dichloromethane). The reaction was stirred at 0 ℃ for 15 minutes and treated dropwise (15 minutes) with 1M (R) - (+) -3-methoxy-. alpha. -methylbenzylamine (1.51g, 10mmol) in dichloromethane (10 mL). The reaction was stirred at 0 ℃ for 1 hour and poured into a solution of sodium cyanoborohydride (1g, 16mmol) in ethanol (100 mL). The reaction mixture was stirred at room temperature for 48 hours. The reaction was diluted with ether and neutralized with 1N NaOH. The ether layer was removed, dried over anhydrous potassium carbonate, and concentrated to an oil. The material was chromatographed on silica with a gradient of 5% methanol in dichloromethane to dichloromethane. To give the unsaturated intermediate, GC/El-MS (R)_t10.06 minutes) was the single component. M/Z (relative intensity) 281(M +, 17), 266(59), 176(19), 146(65), 135(73), 131(100), 91(21), 77 (13).

The ethylated unsaturated intermediate was hydrogenated in the presence of palladium on charcoal at room temperature for 16 hours. The product of this reaction was treated with 1M HCl in diethyl ether and converted to the hydrochloride salt. GC/El-MS (R) of this material_t9.31 minutes) showed a single component: M/Z (relative intensity) 283(M +, 21), 268(100), 164(12), 148(8), 135(85), 121(12), 105(49), 91(23), 77 (21).

Example 12: synthesis of Compound 12V

N-3- (3-methylphenyl) -1-propyl- (R) -3-methoxy-alpha-methylbenzylamine hydrochloride

The compound was prepared according to the procedure of example 11, but using 2-methylcinnamonitrile. Unsaturated intermediates using GC/EI-MS (R)_t10.21 minutes) as a single component: M/Z (relative intensity) 281(M +, 57), 266(86), 146(98), 135(88), 131(100), 115(43), 102(26), 91(43), 77 (18). This material was reduced and the hydrochloride salt formed by the procedure of example 11 to afford the product. GC/EI-MS (R) of this substance (free base)_t9.18 minutes) are shown as single components: M/Z (relative intensity) 283(M +, 19), 268(100), 164(11), 148(8), 135(76), 121(16), 105(45), 91(23), 77 (21).

Example 13: synthesis of Compound 12Z

N-3- (2-chlorophenyl) -1-propyl- (R) -1- (1-naphthyl) ethylamine hydrochloride

The compound was prepared according to the method of example 11, but using 2-chlorohydrocinnamonitrile and (R) - (+) -1- (1-naphthyl) ethylamine in the order of 10 mmol. Chromatography over silica with a gradient of 5% methanol in dichloromethane afforded the product as a single component by TLC analysis (5% methanol in dichloromethane). The hydrochloride salt was prepared by treatment with 1M HCl in diethyl ether.

Example 14: synthesis of Compound 14U

(R) -N- (1- (4-methoxyphenyl) ethyl) - (R) -1- (1-naphthyl) ethylamine hydrochloride

A mixture of (R) - (+) -1- (1-naphthyl) ethylamine (1.1g, 6.2mmol), 4' -methoxyacetophenone (0.93g, 6.2mmol), titanium (IV) isopropoxide (2.2g, 7.7mmol) and EtOH (anhydrous) (1mL) was heated to 60 ℃ for 3 h. Then adding sodium cyanoborohydride (NaCNBH)₃) (0.39g, 6.2mmol) and the reaction mixture was stirred at room temperature for 18 h. Ether (200mL) and H₂O (2mL) was added to the reaction mixture and the resulting precipitate was removed by centrifugation. The supernatant was evaporated in vacuo and the crude product was chromatographed on silica gel (25 mm. times.25 cm column) (with 1% MeOH/CHCl)₃Elution). A portion of this material was chromatographed using HPLC [ selective, 5 μ M silica gel; 25cm × 10.0mm (Torrence, Calif.), 4mL per minute; UV detection 275 nM; 12% Ethyl acetate-88% Hexane (elution time 12.0 min)]. The HPLC purified diastereomer was redissolved in hexane and fuming HCl was added to precipitate the product as a white solid (20mg), melting point: 209 ℃ and 210 ℃ (dec.).

Example 15: preparation of Compound 17M

N- (3-chloro-4-methoxybenzyl) - (R) -1- (1-naphthyl) ethylamine hydrochloride

A mixture of (R) - (+) -1- (1-naphthyl) ethylamine (6.6g, 39mmol), 3 '-chloro-4' -methoxybenzaldehyde (16.6g, 39mmol), and titanium (IV) isopropoxide (13.8g, 48.8mmol) and EtOH (anhydrous) (30mL) was heated to 80 ℃ for 30 minutes and then stirred at room temperature for 3 hours. Then adding sodium cyanoborohydride (NaCNBH)₃) (2.45g, 39 mmol). The reaction mixture was stirred at room temperature for 18 hours. Ether (100mL) and H₂O (2mL) was added to the reaction mixture and the resulting precipitate was removed by centrifugation. The supernatant was evaporated in vacuo and the crude product was chromatographed on silica gel (50 mm. times.30 cm column) (using CH)₂Cl₂Elution). The chromatographed material was redissolved in hexane (500mL) and purified by Norit^@Decolorization was filtered (0.2 μ M) and then fuming HCl was added, precipitating the product as a white solid (10.2g, 56% yield), melting point: 241 ℃ and 242 ℃ (dec.).

Example 16: synthesis of Compound 17P

4-methoxy-3-methylacetophenone [17P precursor ]

4 '-hydroxy-3' -methylacetophenone (5.0g, 33.3mmol), methyl iodide (5.7g, 40.0mmol), K₂CO₃(granular, anhydrous) (23.0g, 167mmol) and acetone (250mL) were refluxed for 3 hours. The reaction mixture was cooled to room temperature, filtered to remove inorganic salts, evaporated in vacuo, the crude product was dissolved in ether (100mL) and washed with H₂O (2X 20 mL). The organic phase was dried (Na)₂SO₄) Post-evaporation gave 4.5g, 82.4% yield. The ketone is used in the following reaction without the need forAnd (5) further purifying.

(R) -N- (1- (4-methoxy-3-methylphenyl) ethyl) - (R) -1- (1-naphthyl) ethylamine hydrochloride [ Compound 17P ]

A mixture of (R) - (+) -1- (1-naphthyl) ethylamine (4.24g, 24.8mmol), 4 '-methoxy-3' -methylacetophenone (4.06g, 24.8mmol), titanium (IV) isopropoxide (8.8g, 30.9mmol) and EtOH (anhydrous) (1mL) was heated to 100 ℃ for 2 h. Isopropanol (45mL) was added and the reaction was cooled to 10 ℃ in an ice bath. Then adding sodium triacetoxyborohydride (NaHB (O) in several portions₂CCH₃)₃) (10.5g, 49.5mmol) for a total of 15 minutes. The reaction mixture was then heated to 70 ℃ for 18 hours. The mixture was cooled to room temperature and ether (400mL) was added. The suspension was centrifuged and the supernatant was collected and the precipitate was washed with ether (400 mL). The combined organic washes were evaporated under vacuum. The residue was redissolved in ether (400mL) with 1N NaOH (4X 50mL) and H₂O (2X 50 mL). The organic layer was dried (Na)₂SO₄) Filtered and evaporated in vacuo. EtOH (anhydrous) was added to the wet residue, which was then dried thoroughly on a rotary evaporator to give an oil. The mixture was chromatographed on silica gel (50 mm. times.30 cm) [ using (1% MeOH: 1% IPA: CHCl)₃Elution). Obtaining 48g of oil]。

The desired diastereomer was further purified by HPLC chromatography [ SUPELCOSIL^TMPLC-S: 18 μ M silica gel; 25cm × 21.2mm (Supelco, Inc. Bellefonte, Bingzhou), 7mL per minute; UV detection 275 nM; 20% EtOAc-80% hexane (elution time 9.5-11.0 min; 275nM UV detection; 20% EtOAc-80% hexane (elution time 9.5-11.0 min)]. Injection (800. mu.L aliquot) of this mixture (100 mg/mL solution in eluent) gave 65mg of the desired isomer. Multiple HPLC injections gave 1.0g of purified material. The HPLC chromatographed material was dissolved in hexane (50mL) and precipitated with fuming HCl to give the hydrochloride salt. The salt was collected on sintered glass and washed with hexane to give 1.0g of a white solid, melting point: 204 ℃ and 205 ℃.

Example 17: synthesis of Compound 17X

3-chloro-4-methoxybenzaldehyde

3-chloro-4-hydroxybenzaldehyde (25g, 160mmol), methyl iodide (27.25g, 192mmol), K₂CO₃A mixture of (granulated, anhydrous) (110.6g, 800mmol) and acetone (300mL) was refluxed for 3 hours. The reaction mixture was then cooled to room temperature. Diethyl ether (500mL) was added and the mixture was filtered over paper to remove inorganic salts. The filtrate was evaporated under reduced pressure, dissolved in diethyl ether (800mL) and washed with 0.1N NaOH (3X 100 mL). The organic layer was dried (Na)₂SO₄) Post-evaporation in vacuo gave 24g of crude product in 92% yield. The material was further purified by chromatography on silica gel (50mm X30 cm) (elution with hexane-EtOAc, 5: 1) to give 15.02g, 56% yield of a white solid: TLC (Hexane-EtOAc, 5: 1) R_f＝0.24；GCR_t4.75 minutes; MS (EI) M/Z170 (M)⁺) 172(M + 2). 1-methyl- (3 '-chloro-4' -methoxybenzyl) ethanol

A mixture of 3-chloro-4-methoxybenzaldehyde (13g, 76.5mmol), methyl magnesium chloride (52g, 153mmol) and THF (300mL) was refluxed for 3 hours. The reaction mixture was cooled to room temperature. Dropwise adding NH₄Diethyl ether (500mL) was added to the Cl (saturated solution, 6mL) and the mixture was filtered over paper to remove inorganic salts. The filtrate was evaporated under reduced pressure and the resulting solid was dissolved in diethyl ether (300mL) and washed with water (4X 25 mL). The organic layer was dried (Na)₂SO₄) Evaporation under vacuum gave 11.3g of crude product in 80% yield. The material was further chromatographed on silica gel (50 mm. times.30 cm) (using CH)₂CH₂Elution) to give 11.3g of oil in 63% yield; TLC (CH)₂Cl₂)R_f＝0.25；GC R_t5.30 minutes; MS (EI) M/Z186 (M)⁺)，188(M+2)。

3 '-chloro-4' -methoxyacetophenone

1-methyl- (3 '-chloro-4' -methoxybenzyl) ethanol (7.6g, 41mmol), pyridinium chlorochromate (PCC) (13.16g, 61.5mmol) and CH₂Cl₂The mixture (300mL) was stirred at room temperature for 2 hours. Diethyl ether (1000mL) was added and the resulting mixture was placed on a silica gel chromatography column (50 mm. times.30 cm) (eluted with diethyl ether) to give 7.3g, 97% yield of a crude solidAnd (3) obtaining a product in bulk. GC analysis of this material showed 99% purity and was used in the following reaction without further purification. TLC (diethyl ether) R_f＝1.0；GC R_t5.3 minutes; MS (EI) M/Z184 (M)⁺)，184(M+2)。

(R, R) -N- (1-ethyl-4 '-methoxy-3' -chlorophenyl) -1- (1-naphthylethyl) amine

A mixture of 3 '-chloro-4' -methoxyacetophenone (5.3g, 29mmol), (R) - (+) -1- (1-naphthyl) ethylamine (4.98g, 29mmol), titanium (IV) isopropoxide (10.2g, 36mmol) and isoamyl alcohol was heated to 100 ℃ for 3 hours. Sodium triacetoxyborohydride (NaB (O)₂CCH₃)₃(ii) a 12.29g, 58mmol) were added in portions for 10 minutes. The reaction mixture was heated to reflux for 30 minutes and then stirred at room temperature for 18 hours. The mixture was poured into diethyl ether (500 mL); addition of H₂O (2mL), the suspension was centrifuged to remove a tiny precipitate of the titanium salt. The supernatant was collected and the precipitate was washed with ether (500 mL). Mix organic layers and dry (Na)₂SO₄) Post-evaporation in vacuo gave 6.81g of crude product in 70% yield.

This material was chromatographed on silica gel (50 mm. times.30 cm) (3% MeOH-97% CH)₂Cl₂Elution) was further purified to give 2.01g of oil. The diastereoisomers were further purified by recrystallization. The free base (1.98g) was converted to its HCl salt with fuming HCl. The salt was dissolved in hot isopropanol (65mL) and the solution was filtered through paper. The filtrate was dried in vacuo and the resulting solid dissolved in isopropanol (30 mL). After 18 hours at room temperature, the crystalline solid was collected, washed with cold isopropanol (20mL) and dried to give 0.87g, 40% (from the free base) of the diastereomeric pure hydrochloride salt: melting point: 236-2371/2 ℃ (dec); TLC (MeOH-CH)₂Cl₂[99∶1])R_f＝0.25；GC R_t11.06 minutes; FTIR (KBr precipitate, 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: alternative synthetic schemes

Preparation of 22Z and 23A

To a stirred solution of sodium hydride (2.173g, 60% in oil, 54.325mmol) in dimethylformamide (100mL) was added dropwise triethyl phosphate (12.47g, 55.65mmol) and stirred at room temperature for 30 min. Then, a solution of m-trifluoromethoxybenzaldehyde (10.0g, 52.6mmol) in dimethylformamide (50ml) was added dropwise, and then the solution was stirred at room temperature for 30 minutes and at 100 ℃ for 30 minutes. The reaction was quenched by addition of water and transferred to another funnel with diethyl ether (500 mL). Washing the ether solution with saturated ammonium chloride (4 × 500ml), drying over anhydrous magnesium sulfate, filtering, and concentrating to obtain oily ethyl m-trifluoromethoxy cinnamate; M/Z (relative intensity) 260(M +, 19), 232(16), 215(100), 187(21), 101 (28).

Ethyl ester in ethanol (100ml) was reduced with a catalytic amount (10% by weight) of palladium hydroxide in 60 lbs of hydrogen. After reduction (2 hours at room temperature), the reaction was filtered and concentrated to give ethyl m-trifluoromethoxy hydrocinnamic acid as an oil: M/Z (relative intensity) 262(M +, 16), 217(7), 188(100), 175(28), 103(31), 91(18), 77 (23).

The saturated ethyl ester was hydrolyzed in ethanol-10M sodium hydroxide (1: 1) solution at room temperature for 16 hours. The solution was then acidified and the product was extracted into diethyl ether. The ether solution was dried over anhydrous magnesium phosphate and concentrated to give solid M-trifluoromethoxy hydrocinnamic acid M/Z (relative strength) 234(M +, 46), 188(100), (174), (65), (103), (27), (91), (12), 77 (17).

The acid was stirred in excess of mercapto chloride (thionyl chloride) at room temperature for 4 hours. Excess mercapto chloride was evaporated under reduced pressure (100 ℃) to give m-trifluoromethoxy-hydrocinnamyl chloride as an oil. The product was used without further purification.

A solution of M-trifluoromethoxy hydrocinnamoyl chloride (9.8g, 39mmol) in tetrahydrofuran was cooled to-78 deg.C and a solution of methylmagnesium bromide (39mmol) (3M 13mL in tetrahydrofuran) was added dropwise. The reaction was stirred at-78 ℃ for 4 hours, at room temperature for 8 hours, and then quenched with dilute HCl. The reaction mixture was extracted with diethyl ether. The ether was dried over anhydrous magnesium sulfate, filtered, and concentrated to an oil. Chromatography of the material on silica with a hexane to acetone gradient gave 4- (3-trifluoromethoxyphenyl) -2-butanone as an oil; M/Z (relative intensity) 232(M +, 68), 217(7), 189(59), 175(31), 103(28), 43 (100).

A solution of 4- (3-trifluoromethoxyphenyl) -2-butanone (2.32g, 10mmol), (R) -1- (3-methoxyphenyl) ethylamine (1.51g, 10mmol) and titanium (IV) isopropoxide (3.55g, 12.5mmol) was stirred at room temperature for 4 hours. The reaction mixture was treated with a solution of ethylated sodium cyanoborohydride (10mmol) (10ml, 1M) and stirred at room temperature for 16 h. The reaction was diluted with diethyl ether (50ml) and treated with water (0.72ml, 40 mmol). After thorough mixing, the solution was centrifuged and the ether layer was concentrated to dryness to give an oily solid. The solid was suspended in diethyl ether, filtered through 0.45 μ M CR PTFE Acrodisc, and concentrated to give a clear oil. Preparative thin layer chromatography was repeated using 5% methanol in chloroform to give the two diastereomers (S, R) -N- [4- (3-trifluoromethoxyphenyl) -2-butyl ] -1- (3-methoxyphenyl) ethylamine, 22Z [ M/Z (relative strength) 367(M +, 3), 352(20), 232(4), 178(47), 135(100), 105(14), 91(10), 77(11) and (R, R) -N- [4- (3-trifluoromethoxyphenyl) -2-butyl ] -1- (3-methoxyphenyl) ethylamine, 23A; M/Z (relative intensity) 367(M +, 3), 352(19), 232(7), 178(43), 135(100), 105(19), 91(10), 77 (11).

Preparation of 22X and 22Y

In a similar manner, equimolar amounts of 4- (3-trifluoromethoxyphenyl) -2-butanone, (R) -1- (1-naphthyl) ethylamine and 1.25 equivalents of titanium (IV) isopropoxide are combined and the intermediate imine is reduced with ethylated sodium cyanoborohydride. Separating and purifying (work-up) with 5% methanol in chloroform and performing repeated preparative thin layer chromatography to obtain (S, R) -N- [4- (3-trifluoromethoxyphenyl) -2-butyl ] -1- (1-naphthyl) ethylamine, 22X; M/Z (relative Strength) 387(M +, 3), 372(15), 198(15), 176(12), 155(100), 128(8), 115(6), 109(4), 103(5), 77(8) and (R, R) -N- [4- (3-1-trifluoromethoxyphenyl) -2-butyl ] -1- (1-naphthyl) ethylamine, 22Y; M/Z (relative intensity) 387(M +, 2), 372(12), 198(16), 176(11), 155(100), 128(8), 115(6), 109(4), 103(5), 77 (8).

Preparation of 4T

In a similar manner equimolar amounts of 4- (2-chlorophenyl) -2-butanone, prepared from O-chlorobenzaldehyde, (R) -1 (3-methoxyphenyl) ethylamine and 1.25 equivalents of titanium (IV) isopropoxide, were combined and the intermediate imine was reduced with ethylated sodium cyanoborohydride. Separating and purifying with 5% methanol in chloroform and performing repeated preparative thin layer chromatography to obtain (R, R) -N- [4- (2-chlorophenyl) -2-butyl ] -1- (3-methoxyphenyl) ethylamine, 4T; M/Z (relative intensity) 317(M +, 3), 302(16), 178(62), 135(100), 125(15), 105(10), 91(6), 77 (8).

Preparation of 21Y

In a similar manner equimolar amounts of 4- (3-trifluoromethylphenyl) -2-butanone, prepared from m-trifluoromethylbenzaldehyde, (R) -1- (3-methoxyphenyl) ethylamine and 1.25 equivalents of titanium (IV) isopropoxide were combined and the intermediate imine was reduced with sodium cyanoborohydride ethylated. Purification by separation with 5% methanol in chloroform and preparative thin layer chromatography on repeated column chromatography gave (R, R) -N- [4- (3-trichloromethylphenyl) -2-butyl ] -1- (3-methoxyphenyl) ethylamine, 21Y [ M/Z (relative intensity) 351(M +, 2), 336(18), 216(4), 202(3), 178(45), 135(100), 105(13), 91(9), 77(8) and (S, R) -N- [4- (3-trifluoromethylphenyl) -2-butyl ] -1- (3-methoxyphenyl) ethylamine, 21X.

Preparation of 25C and 25D

In a similar manner, equimolar amounts of 4- (3-trifluoromethylphenyl) -2-butanone, (R) -1- (1-naphthyl) ethylamine and 1.25 equivalents of titanium (IV) isopropoxide were combined and the intermediate imine was reduced with ethylated sodium cyanoborohydride. Separating and purifying with 5% methanol in chloroform and repeatedly performing preparative thin layer chromatography to obtain (S, R) -N- [4- (3-trichloromethylphenyl) -2-butyl]-1- (1-naphthyl) ethylamine, 25C [ M/Z (relative intensity) 37 (M)⁺，3)，356(16)，198(15)，155(100)，129(8)，115(5)，109(3)，77(2)]And (R, R) -N- [4- (3-trifluoromethylphenyl) -2-butyl]-1- (1-naphthyl) ethylamine, 25D; M/Z (relative intensity) 371 (M)⁺，3)，356(16)，198(15)，155(100)，129(8)，115(5)，109(3)，77(2)。

Preparation of 21D

In a similar manner, equimolar amounts of 4-phenyl-2-butanone (Aldrich chemical company), (R) -1- (3-methylchlorophenyl) ethylamine and 1.25 equivalents of titanium (IV) isopropoxide were combined and the intermediate imine was reduced with ethylated sodium cyanoborohydride. Separation and purification with 5% methanol in chloroform and repeated preparative thin layer chromatography to give (R, R) -N- (4-phenyl-2-butyl) -1- (3-methoxyphenyl) ethylamine, 21D [ M/Z (relative intensity) 283 (M)⁺，4)，268(13)，178(45)，135(100)，105(15)，91(43)，77(11)]And (S, R) -N- (4-phenyl-2-butyl) -1- (3-methoxyphenyl) ethylamine, 21E.

Preparation of 21F

In a similar manner, equimolar amounts of 4-phenyl-2-butanone (Aldrich chemical company), (R) -1- (1-naphthyl) ethylamine and 1.25 equivalents of titanium (IV) isopropoxide were combined and the intermediate imine was reduced with ethylated sodium cyanoborohydride. Separating and purifying with 5% methanol in chloroform and performing repeated preparative thin layer chromatography to obtain (R, R) -N- (4-phenyl-2-butyl) -1- (1-naphthyl) ethylamine, 21F; [ M/Z (relative intensity) 303 (M)⁺，6)，288(14)，198(22)，155(100)，129(8)，115(5)，91(19)，77(4)。

Preparation of 12Z

A stirred solution of 2-chlorohydrocinnamonitrile (Aldrich chemical, 1.66g, 10mmol) in dichloromethane was cooled to-78 deg.C and treated dropwise with diisobutylaluminum hydride (1.42g, 10 mmol). The reaction was stirred at room temperature for 1 h, cooled to-78 ℃ and treated with a solution of 1- (1-naphthyl) ethylamine (1.71g, 10mmol) in dichloromethane (25 ml). The reaction was transferred to an ice bath and stirred for 2 hours. The reaction was then poured directly into a stirred solution of sodium borohydride (50ml, 0.2M, 10mmol) in ethanol. The mixture was stirred at room temperature for 30 minutes and excess sodium borohydride was quenched with 10% HCl. The solution was made basic by addition of 10N NaOH and transferred to a separatory funnel and washed with diethyl ether (300 ml). The aqueous phase was removed and the remaining organic layer was washed with 1N NaOH (3X 100 mL). The organic layer was dried over anhydrous magnesium sulfate and concentrated to an oil. Chromatography of this material on silica gel with a chloroform to 10% methanol-chloroform gradient gave 2.34g (72% yield) of (R) -N- [3- (2-chlorophenyl) propyl) -1- (1-naphthyl) ethylamine, 12Z, as a clear oil; M/Z (relative intensity) 323(M +, 2), 308(63), 288(7), 196(5), 184(5), 155(100), 125(24), 115(8), 103(4), 91(3), 77 (7).

Preparation of 12B

In a similar manner 2-methylcinnamonitrile was treated with diisobutylaluminum hydride and the intermediate aluminum-imine complex was treated with (R) -1- (3-methoxyphenyl) ethylamine. The intermediate imine is treated with ethylated sodium borohydride. Separating, purifying and chromatographing to obtain (R) -N- [3- (2-methylphenyl) propyl-2-alkenyl) -1- (3-methoxyphenyl) ethylamine and 12B as clear colorless oil; M/Z (relative intensity) 281(M +, 6), 266(5), 176(27), 146(75), 135(63), 131(100), 115(25), 105(21), 91(21), 77 (21).

Preparation of 12C

In a similar manner 2-methylcinnamonitrile was treated with diisobutylaluminum hydride and the intermediate aluminum-imine complex was treated with (R) -1- (3-methoxyphenyl) ethylamine. The intermediate imine is treated with ethylated sodium borohydride. Separating, purifying and chromatographing to obtain (R) -N- [3- (2-methylphenyl) propyl-2-alkenyl) -1- (3-methoxyphenyl) ethylamine and 12C as clear colorless oil; M/Z (relative intensity) 281(M +, 4), 266(15), 176(18), 146(62), 135(58), 131(100), 115(23), 105(19), 91(38), 77 (17).

Preparation of 12D

In a similar manner, 2, 4, 6-trimethylcinnamonitrile was treated with diisobutylaluminum hydride and the intermediate aluminum-imine complex was treated with (R) -1- (3-methoxyphenyl) ethylamine. The intermediate imine is treated with ethylated sodium borohydride. Separating, purifying and electrophoresing to obtain clear colorless oily (R) -N- [3- (2, 4, 6-trimethylphenyl) prop-2-enyl) -1- (3-methoxyphenyl) ethylamine and 12D; M/Z (relative intensity) 309(M +, 8), 294(25), 174(82), 159(100), 135(52), 129(29), 105(21), 91(17), 77 (14).

Preparation of 12F

In a similar manner, 4-isopropylcinnamonitrile was treated with diisobutylaluminum hydride and the intermediate aluminum-imine complex was treated with (R) -1- (3-methoxyphenyl) ethylamine. The intermediate imine is treated with ethylated sodium borohydride. Separating, purifying and chromatographing to obtain (R) -N- [3- (4-isopropylphenyl) prop-2-alkenyl]-1- (3-methoxyphenyl) ethylamine, 12E; M/Z (relative intensity) 309 (M)⁺，9)，294(7)，174(98)，159(22)，135(80)，177(100)，105(35)，91(37)，77(19)。

Preparation of 12F

In a similar manner, 2, 4-dimethylcinnamonitrile was treated with diisobutylaluminum hydride and the intermediate aluminum-imine complex was treated with (R) -1- (3-methoxyphenyl) ethylamine. The intermediate imine is treated with ethylated sodium borohydride. Separating, purifying and chromatographing to obtain clear colorless oily (R) -N- [3-2, 4-dimethylphenyl) prop-2-enyl) -1- (3-methoxyphenyl) ethylamine and 12F; M/Z (relative intensity) 295 (M)⁺，8)，294(15)，174(29)，160(75)，145(100)，135(68)，117(21)，105(30)，91(26)，77(19)。

Preparation of 12G

In a similar manner 3-methylcinnamonitrile was treated with diisobutylaluminum hydride and the intermediate aluminum-imine complex was treated with (R) -1- (3-methoxyphenyl) ethylamine. The intermediate imine is treated with ethylated sodium borohydride. Separating, purifying and chromatographing to obtain (R) -N- [3- (3-methoxyphenyl) prop-2-alkenyl]-1- (3-methoxyphenyl) ethylamine, 12G; M/Z (relative intensity) 281 (M)⁺，5)，266(9)，176(24)，146(71)，135(62)，131(100)，115(23)，105(19)，91(41)，77(18)。

Preparation of 25E

In a similar manner, cinnamonitrile was treated with diisobutylaluminum hydride and the intermediate aluminum-imine complex was treated with (R) -1- (3-methoxyphenyl) ethylamine. The intermediate imine is treated with ethylated sodium borohydride. Separating, purifying and chromatographing to obtain clear colorless oily (R) -N- (3-phenylprop-2-enyl) -1- (3-methoxyphenyl) ethylamine, 25E; M/Z (relative intensity) 267 (M)⁺，3)，252(14)，176(17)，135(62)，117(100)，105(28)，91(56)，77(33)。

Preparation of 25G

In a similar manner α -methylcinnamonitrile was treated with diisobutylaluminum hydride and the intermediate aluminum-imine complex was treated with (R) -1- (3-methoxyphenyl) ethylamine. The intermediate imine is treated with ethylated sodium borohydride. Separating, purifying and chromatographing to obtain clear colorless oily (R) -N- (2-methyl-3-phenylprop-2-enyl) -1- (3-methoxyphenyl) ethylamine, 25G; M/Z (relative intensity) 281(M +, 5), 266(18), 190(12), 146(78), 135(82), 131(100), 115(21), 105(21), 91(62), 77 (19).

Preparation of 6X

A stirred solution of sodium hydride (1.8g, 75mmol) in dimethylformamide (150ml) was treated with a solution of diethylcyanomethyl phosphate (13.3g, 75mmol) in dimethylformamide (50 ml). The reaction was stirred at room temperature for 30 minutes. The reaction was then treated with 3-chlorobenzaldehyde (10.54g, 75mmol) and stirred at room temperature for 1 hour. Stirred at 60 ℃ for 30 minutes. The reaction was then quenched by the addition of water (200 ml). The reaction mixture was transferred to a separatory funnel with diethyl ether and the resulting organic phase was washed with water (5X 300ml) and salt solution. The organic layer was dried over anhydrous potassium carbonate and concentrated to give 3-chlorocinnamonitrile (11.06g) as a solid. The solid was dissolved in tetrahydrofuran (50ml) and treated with excess diborane and stirred at room temperature for 30 minutes. The reaction was poured into ice/10% HCl. The acidic aqueous phase was washed with diethyl ether (2X 200 ml). The aqueous phase was made alkaline with 10N NaOH and extracted with diethyl ether (200 ml). The ether extract was dried over anhydrous potassium carbonate and concentrated to give 3- (3-chlorophenyl) propylamine (0.6g, 354mmol) as an oil. 3- (3-chlorophenyl) propylamine (0.6g, 354mmol), 3' -methoxyacetophenone (0.53g, 354mmol) and 1.25 molar equivalents of titanium (IV) isopropoxide (1.26g, 443mmol) were stirred at room temperature for 4 hours and the intermediate imine was treated with ethylated sodium cyanoborohydride (5ml, 1M, 5 mmol). The reaction was stirred at room temperature for 16 h, diluted with diethyl ether (50ml) and treated with water (0.32ml, 17.7 mmol). After mixing well, the solution was centrifuged to concentrate the ether layer to a milky solid. This material was suspended in diethyl ether and filtered through 0.45. mu.M CR PTFE Acrodisc. The ether wash was concentrated to an oil. Chromatography of this material (silica, preparative thin layer chromatography) with 3% methanol-dichloromethane (containing 0.1% isopropylamine) gave N- [3- (3-chlorophenyl) propyl ] -1- (3-methoxyphenyl) ethylamine, 6X; M/Z (relative strength) 303(M +, 3), 288(40), 196(3), 164(8), 135(100), 126(46), 103(26), 91(29), 77 (29).

Preparation of 6V

Equimolar amounts of 3- (4-chlorophenyl) propylamine, prepared in a similar manner as above from 4-chlorobenzaldehyde, 3' -methoxyacetophenone and 1.25 molar equivalents of titanium (IV) isopropoxide, were mixed at room temperature for 4 hours and the intermediate imine was treated with sodium cyanoborohydride (5ml, 1M, 5mmol) ethylated, purified by isolation and chromatography to give N- [3- (4-chlorophenyl) propyl ] -1- (3-methoxyphenyl) ethylamine, 6V, M/Z (relative strength) 303(M +, 8), 288(91), 196(4), 164(10), 135(100), 125(61), 103(21), 91(21), 77(18) as an oil.

Preparation of 20A

In a similar manner, equimolar amounts of 1- (1-methoxyphenyl) propylamine, 4-tert-butylacetophenone and 1.25 molar equivalents of titanium (IV) isopropoxide were mixed at room temperature for 4 hours, and the intermediate imine was treated with ethylated sodium cyanoborohydride (5ml, 1M, 5 mmol). Purification by separation and chromatography gives (R) -N- [1- (4-tert-butylphenyl) ethyl ] -1- (1-naphthyl) ethylamine, 20A, M/Z (relative strength) 331(M +, 12), 316(29), 161(70), 155(100), 131(14), 127(31), 115(10), 105(6), 91(10), 77(10) in the form of an oil.

Preparation of 25H and 25I

In a similar manner equimolar amounts of (R) -1- (3-methoxyphenyl) ethylamine, trans-4-phenyl-3-buten-2-one and 1.25 molar equivalents of titanium (IV) isopropoxide were mixed at room temperature for 4 hours and the intermediate imine was treated with ethylated sodium cyanoborohydride (5ml, 1M, 5 mmol). Separating, purifying and chromatographing to obtain oily (R, R) -N- (2-methyl-4-phenyl J-3-alkenyl) -1- (3-methoxyphenyl) ethylamine, 25H; M/Z (relative strength) 283(M +, 4), 268(13), 178(40), 135(100), 105(15), 91(47), 77(13) and oily (S, R) -N- (2-methyl-4-phenyl J-3-alkenyl) -1- (3-methoxyphenyl) ethylamine, 25I; M/Z (relative intensity) 283(M +, 4), 268(13), 178(40), 135(100), 105(15), 91(47), 77 (13).

Preparation of 16L and 16M

In a similar manner equimolar amounts of (R) -1- (3-methoxyphenyl) -ethylamine, 3-methoxyacetophenone and 1.25 molar equivalents of titanium (IV) isopropoxide were mixed at room temperature for 4 hours and the intermediate imine was treated with ethanolated sodium cyanoborohydride (5ml, 1M, 5 mmol). Separating, purifying and chromatographing to obtain oily (R, R) -N- [1- (4-methoxyphenyl) ethyl]-1- (3-methoxyphenyl) ethylamine, 16L; M/Z (relative intensity) 284 (M)^-1,1),270(85),150(83),135(100),120(12),105(28),91(25),77(23). And oily (S, R) -N- [1- (4-methoxyphenyl) ethyl]-1- (3-methoxyphenyl) ethylamine, 16M; M/Z (relative intensity) 284(M-1, 1), 270(53), 150(98), 135(100), 120(11), 105(33), 91(25), 77 (23).

Preparation of 5R/5C

3-methyl-3- (4-chlorophenyl) cinnamonitrile was prepared in a similar manner using 4-chloroacetophenone. The nitrile was catalytically reduced (palladium hydroxide, acetic acid, 60 lbs. hydrogen for 2 hours) to produce 3-methyl-3- (4-chlorophenyl) propylamine. Equimolar amounts of the amine, 3' -methoxyacetophenone and 1.25 molar equivalents of titanium (IV) isopropoxide were mixed at room temperature for 4 hours and the intermediate imine was treated with ethylated sodium cyanoborohydride (5ml, 1M, 5 mmol). The oily N- (3-methyl-3- (4-chlorphenyl) propyl) -1- (3-methoxyphenyl) ethylamine, 5B/5C, M/Z (relative strength) 317(M +, 12), 302(74), 210(2), 182(4), 164(12), 135(100), 121(25), 103(40), 91(19), 77(28) are obtained by separation, purification and chromatography.

Preparation of 4Z/5A

3-methyl-3- (3-chlorophenyl) cinnamonitrile was prepared in a similar manner using 3-chloroacetophenone. The nitrile was catalytically reduced (palladium hydroxide, acetic acid, 60 lbs. hydrogen for 2 hours) to produce 3-methyl-3- (3-chlorophenyl) propylamine. Equimolar amounts of the amine, 3' -methoxyacetophenone and 1.25 molar equivalents of titanium (IV) isopropoxide were mixed at room temperature for 4 hours and the intermediate imine was treated with ethylated sodium cyanoborohydride (5ml, 1M, 5 mmol). Separating, purifying and chromatographing to obtain oily N- [ 3-methyl-3- (3-chlorphenyl) propyl ] -1- (3-methoxyphenyl) ethylamine, 4Z/5A; M/Z (relative intensity) 283(M +, 17), 268(71), 164(13), 135(100), 121(21), 105(27), 91(26), 77 (14).

Preparation of 4Y

3-methyl-3- (2-chlorophenyl) cinnamonitrile was prepared in a similar manner using 2-chloroacetophenone. The nitrile was catalytically reduced (palladium hydroxide, acetic acid, 60 lbs. hydrogen for 2 hours) to produce 3-methyl-3- (2-chlorophenyl) propylamine. Equimolar amounts of the amine, 3' -methoxyacetophenone and 1.25 molar equivalents of titanium (IV) isopropoxide were mixed at room temperature for 4 hours and the intermediate imine was treated with ethylated sodium cyanoborohydride (5ml, 1M, 5 mmol). Separating, purifying and chromatographing to obtain oily N- [ 3-methyl-3- (2-chlorphenyl) propyl]-1- (3-methoxyphenyl) ethylamine, 4Y; M/Z (relative intensity) 283 (M)⁺，17)，268(71)，164(13)，135(100)，121(21)，105(27)，91(26)，77(4)。

Preparation of 6T

A solution of NPS R-568(30.3g, 100mmol) in dichloromethane was treated dropwise with boron tribromide (50g, 200ml) at-78 ℃. The reaction was stirred at room temperature for 1 hour and poured onto ice. Hydrobromic acid was extracted from the aqueous phase with chloroform. The chloroform-soluble material was washed with 50% HCl (4X 100 ml). The chloroform washing solution was dried over anhydrous magnesium sulfate and concentrated to give (R) -N- [3- (2-chlorophenyl) propyl ] -1- (3-hydroxyphenyl) ethylamine hydrochloride as a solid. A solution of sodium hydride (0.48g, 20mmol) in dimethylformamide was treated with (R) -N- [3- (2-chlorophenyl) propyl ] -1- (3-hydroxyphenyl) ethylamine hydrochloride (3.25g, 10mmol) and the reaction was stirred at room temperature for 1 hour. The reaction was treated with iodoethane (1.17g, 11mmol) and stirred at room temperature for 16 h. Separating, purifying and chromatographing with 3% methanolic silica in chloroform to give (R) -N- [3- (2-chlorophenyl) propyl ] -1- (3-ethoxyphenyl) ethylamine, 6T as an oil; M/Z (relative intensity) 316(M +, 1), 302(100), 282(11), 196(5), 178(7), 149(74), 121(34), 103(25), 91(28), 77 (29).

Preparation of 6R

In a similar manner with NPS R-467 was prepared (R) -N- (3-phenylpropyl) -1- (3-ethoxyphenyl) ethylamine, 6R; M/Z (relative intensity) 283(M +, 10), 268(74), 178(11), 162(8), 149(100), 121(30), 103(16), 91(86), 77 (29).

Preparation of 3U

Equimolar amounts of 3, 3-diphenylpropylamine (2.11g, 10mmol), 1' -acetylnaphthalenone (1.70g, 10mmol) and 1.25 equivalents of titanium (IV) isopropoxide (3.55g, 12.5mmol) were stirred at room temperature for 4 hours, after which the reaction mixture was treated with a 1M solution of ethylated sodium cyanoborohydride (12.5ml, 12.5mmol) and stirred at room temperature for 16 hours. The reaction was diluted with diethyl ether (50ml) and treated with water (0.72ml, 40 mmol). Mixing, centrifuging to remove water from the ether layer, and concentrating to obtain milky oil. The oil was suspended in diethyl ether and filtered through 0.45. mu.M CR PTFE Acrodisc. The diethyl ether filtrate was concentrated to give N- (3, 3-diphenylpropyl) -1- (1-phenyl) ethylamine, 3U, as a clear colorless oil; M/Z (relative intensity) 365(M +, 17), 350(19), 181(23), 155(100), 141(25), 115(11), 91(13), 77 (6).

Preparation of 6F

Equimolar amounts of 1- (3-methoxyphenyl) ethylamine (1.51g, 10mmol), 2' -acetylnaphthalenone (1.70g, 10mmol) and 1.25 equivalents of titanium (IV) isopropoxide (3.55g, 12.5mmol) were treated in a similar manner as above. Separating and purifying to obtain clear colorless oily N- [1- (2-naphthyl) ethyl ] -1- (3-methoxyphenyl) ethylamine, 6F; M/Z (relative intensity) 305(M +, 1), 290(35), 170(49), 155(100), 135(55), 115(28), 105(10), 91(9), 77 (10).

Preparation of 4G

In a similar manner, equimolar amounts of (R) -1-phenylethylamine, 1' -acetylnaphthalenone and 1.25 equivalents of titanium (IV) isopropoxide were combined and the resulting intermediate imine was reduced with ethylated sodium cyanoborohydride. Separating, purifying and chromatographing to obtain clear colorless oily N- [1- (1-naphthyl) ethyl ] -1-phenylethylamine, 4G; M/Z (relative intensity) 275(M +, 16), 260(79), 155(100), 127(27), 105(70), 77 (32).

Preparation of 4H

In a similar manner, equimolar amounts of (R) -1-phenylethylamine, 2' -acetylnaphthalenone and 1.25 equivalents of titanium (IV) isopropoxide were combined and the resulting intermediate imine was reduced with ethylated sodium cyanoborohydride. Separating, purifying and chromatographing to obtain clear colorless oily N- [1- (2-naphthyl) ethyl ] -1-phenylethylamine, 4H; M/Z (relative intensity) 275(M +, 1), 260(61), 155(100), 120(36), 105(55), 77 (15).

Preparation of 6E

In a similar manner equimolar amounts of (1- (3-methoxyphenyl) ethylamine, 1' -acetylnaphthalenone and 1.25 equivalents of titanium (IV) isopropoxide were combined and the resulting intermediate imine was reduced with sodium cyanoborohydride ethylated, purified by separation and chromatography to give N-1- (1-naphthyl) ethyl-1- (3-methoxyphenyl) ethylamine, 6E, M/Z (relative strength) 305(M +, 10, 290(30), 170(43), 155(100), 135(69), 115(9), 105(15), 91(14), 77(18) as a clear colorless oil.

Example 19: pharmaceutical preparation

Pharmaceutical formulations suitable for administration to patients are given in table 3.

TABLE 3

Composition (I)	mg/capsule	g/typical 5000 Capsule batch
			NPS R-568	56.0	280.0
Pregelatinized starch NF	134.0	670.0
			Microcrystallized cellulose NF	34.0	170.0
Colloidal silicon dioxide	1.0	5.0
			Total amount of	225mg	1125g

Other examples of nps (r) -568 hydrochloride formulations and dosage forms include those suitable for sustained or extended release using standard techniques.

Appropriate dosages may also be determined using standard techniques. For example, in one set of experiments, an oral dose of 10-400mg of NPS (R) -568 hydrochloride showed pharmacological activity in human subjects. Significant levels of O-glucoside conjugates of 17Q (a major metabolite of NPS (R) -568) were observed in human plasma following oral administration of NPS (R) -568 hydrochloride. Thus, the 17Q O-glucoside conjugate may produce beneficial effects.

Other suitable dosage ranges for nps (r) -568 can be determined using standard techniques.

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

Other embodiments are within the scope of the following claims, and thus, while only a few embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the invention.

General data in sequence listing (1):

(i) the applicant: NPS Pharmaceuticals, Inc.

(ii) The invention name is as follows: calcium receptor active compounds

(iii) Sequence number: 2

(iv) The related address is as follows:

(A) the addressee: lyon & Lyon

(B) Street: first Interstate World

Center，Suite 4700

633 West Fifth Street

(C) City: los Angeles

(D) State: califomia (Califomia)

(E) The state is as follows: USA

(F) And E, postcode: 90017

(v) A computer-readable form:

(A) type of medium: 3.5 inch disk, 1.44Mb storage capacity

(B) A computer: IBM PC compatible

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: FastSeq

(vi) The current application information:

(A) application No.:

(B) submission date:

(C) and (4) classification:

(vii) prior application for information:

all prior applications include the following: 2

(A) Application No.: U.S.08/353,784

(B) Submission date: 12/8/1994

(A) Application No.: PCT/US/94/12117

(B) Submission date: day 21, 10 months in 1994

(viii) Lawyer/attorney profile:

(A) name: heber, Sheldon O.

(B) Registration number: 38,179

(C) Reference/registration number: 215/304

(ix) Remote communication data:

(A) telephone: (213)489-1600

(B) Electric transmission: (213)955-0440

(C) Telegraph: 67-3510(2) SEQ ID NO: 1, data:

(i) sequence characteristics:

(A) length: 5006 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: cDNA to mRNA

(ix) The characteristics are as follows:

(A) name/keyword CDS

(B) Position: 436 … 3699

(D) Other data:

(xi) Description of the sequence: SEQ ID NO: 1: GCTGCTGTGG CCGGACCCGA AGGCGGGCGC CGGGAGCGCA 40GCGAGCCAGA CGCGCCTCTC CAAGACCGTG ACCTTGGCAT 80 3980 80AGGGAGCGGG GCTGCGCGCA GTCCTGAGAT CAGACCAGAG 120CTCATCCTCG TGGAGACCCA CGGCCGAGGG GCCGGAGCTG 160CCTCTGTGCG AGGGAGCCCT GGCCGCGGCG CAGAAGGCAT 200CACAGGAGGC CTCTGCATGA TGTGGCTTCC AAAGACTCAA 240GGACCACCCA CATTACAAGT CTGGATTGAG GAAGGCAGAA 280ATGGAGATTC AAACACCACG TCTTCTATTA TTTTATTAAT 320CAATCTGTAG ACATGTGTCC CCACTGCAGG GAGTGAACTG 360CTCCAAGGGA GAAACTTCTG GGAGCCTCCA AACTCCTAGC 400TGTCTCATCC CTTGCCCTGG AGAGACGGCA GAACC 435ATG GCA TTT TAT AGC TGC TGC TGG GTC CTC TTG GCA 471Met Ala Phe Tyr Ser Cys Cys Trp Val Leu Leu Ala 1510 CTC ACC TGG CAC ACC TCT GCC TAC GGG CCA GAC CAG 507Leu Thr Trp His Thr Sar Ala Tyr Gly Pro Asp Gln

15 20CGA GCC CAA AAG AAG GGG GAC ATT ATC CTT GGG GGG 543Arg Ala Gln Lys Lys Gly Asp Ile Ile Leu Gly Gly25 30 35CTC TTT CCT ATT CAT TTT GGA GTA GCA GCT AAA GAT 579Leu Phe Pro Ile His Phe Gly Val Ala Ala Lys Asp

40 45CAA GAT CTC AAA TCA AGG CCG GAG TCT GTG GAA TGT 615Gln Asp Leu Lys Ser Arg Pro Glu Ser Val Glu Cys

50 55 60ATC AGG TAT AAT TTC CGT GGG TTT CGC TGG TTA CAG 651Ile Arg Tyr Asn Phe Arg Gly Phe Arg Trp Leu Gln

65 70GCT ATG ATA TTT GCC ATAGAG GAG ATA AAC AGC AGC 687Ala Met Ile Phe Ala Ile Glu Glu Ile Asn Ser Ser

75 80CCA GCC CTT CTT CCC AAC TTG ACG CTG GGA TAC AGG 723Pro Ala Leu Leu Pro Asn Leu Thr Leu Gly Tyr Arg85 90 95ATA TTT GAC ACT TGC AAC ACC GTT TCT AAG GCC TTG 759Ile Phe Asp Thr Cys Asn Thr Val Ser Lys Ala Leu

100 105GAA GCC ACC CTG AGT TTT GTT GCT CAA AAC AAA ATT 795Glu Ala Thr Leu Ser Phe Val Ala Gln Asn Lys Ile

110 115 120GAT TCT TTG AAC CTT GAT GAG TTC TGC AAC TGC TCA 831Asp Ser Leu Asn Leu Asp Glu Phe Cys Asn Cys Ser

125 130GAG CAC ATT CCC TCT ACG ATT GCT GTG GTG GGA GCA 867Glu His Ile Pro Ser Thr Ile Ala Val Val Gly Ala

135 140ACT GGC TCA GGC GTC TCC ACG GCA GTG GCA AAT CTG 903Thr Gly Ser Gly Val Ser Thr Ala Val Ala Asn Leu145 150 155CTG GGG CTC TTC TAC ATT CCC CAG GTC AGT TAT GCC 939Leu Gly Leu Phe Tyr Ile Pro Gln Val Ser Tyr Ala

160 165TCC TCC AGC AGA CTC CTC AGC AAC AAG AAT CAA TTC 975Ser Ser Ser Arg Leu Leu Ser Asn Lys Asn Gln Phe

170 175 180AAG TCT TTC CTC CGA ACC ATC CCC AAT GAT GAG CAC 1011Lys Ser Phe Leu Arg Thr Ile Pro Asn Asp Glu His

185 190CAG GCC ACT GCC ATG GCA GAC ATC ATC GAG TAT TTC 1047Gln Ala Thr Ala Met Ala Asp Ile Ile Glu Tyr Phe

195 200CGC TGG AAC TGG GTG GGC ACA ATT GCA GCT GAT GAC 1083Arg Trp Asn Trp Val Gly Thr Ile Ala Ala Asp Asp205 210 215GAC TAT GGG CGG CCG GGG ATT GAG AAA TTC CGA GAG 1119Asp Tyr Gly Arg Pro Gly Ile Glu Lys Phe Arg Glu

220 225GAA GCT GAG GAA AGG GAT ATC TGC ATC GAC TTC AGT 1155Glu Ala Glu Glu Arg Asp Ile Cys Ile Asp Phe Ser

230 235 240GAACTC ATC TCC CAG TAC TCT GAT GAG GAA GAG ATC 1191Glu Leu IleSer Gln Tyr Ser Asp Glu Glu Glu Ile

245 250CAG CAT GTG GTA GAG GTG ATT CAA AAT TCC ACG GCC 1227Gln His Val Val Glu Val Ile Gln Asn Ser Thr Ala

255 260AAA GTC ATC GTG GTT TTC TCC AGT GGC CCA GAT CTT 1263Lys Val IleVal Val Phe Ser Ser Gly Pro Asp Leu265 270 275GAG CCC CTC ATC AAG GAG ATT GTC CGG CGC AAT ATC 1299Glu Pro Leu Ile Lys Glu Ile Val Arg Arg Asn Ile

280 285ACG GGC AAG ATC TGG CTG GCC AGC GAG GCC TGG GCC 1335Thr Gly Lys Ile Trp Leu Ala Ser Glu Ala Trp Ala

290 295 300AGC TCC TCC CTG ATC GCC ATG CCT CAG TAC TTC CAC 1371Ser Ser Ser Leu Ile Ala Met Pro Gln Tyr Phe His

305 310GTG GTT GGC GGC ACC ATT GGA TTC GCT CTG AAG GCT 1407Val Val Gly Gly Thr Ile Gly Phe Ala Leu Lys Ala

315 320GGG CAG ATC CCA GGC TTC CGG GAA TTC CTG AAG AAG 1443Gly Gln Ile Pro Gly Phe Arg Glu Phe Leu Lys Lys325 330 335GTC CAT CCC AGG AAG TCT GTC CAC AAT GGT TTT GCC 1479Val His Pro Arg Lys Ser Val His Asn Gly Phe Ala

340 345AAG GAG TTT TGG GAA GAA ACA TTT AAC TGC CAC CTC 1515Lys Glu Phe Trp Glu Glu Thr Phe Asn Cys His Leu

350 355 360CAA GAA GGT GCA AAA GGA CCT TTA CCT GTG GAC ACC 1551Gln Glu Gly Ala Lys Gly Pro Leu Pro Val Asp Thr

365 370TTT CTG AGA GGT CAC GAA GAA AGT GGC GAC AGG TTT 1587Phe Leu Arg Gly His Glu Glu Ser Gly Asp Arg Phe

375 380AGC AAC AGC TCG ACA GCC TTC CGA CCC CTC TGT ACA 1623Ser Asn Ser Ser Thr Ala Phe Arg Pro Leu Cys Thr385 390 395GGG GAT GAG AAC ATC AGC AGT GTC GAG ACC CCT TAC 1659Gly Asp Glu Asn Ile Ser Ser Val Glu Thr Pro Tyr

400 405ATA GAT TAC ACG CAT TTA CGG ATA TCC TAC AAT GTG 1695Ile Asp Tyr Thr His Leu Arg Ile Ser Tyr Asn Val

410 415 420TAC TTA GCA GTC TAC TCC ATT GCC CAC GCC TTG CAA 1731Tyr Leu Ala Val Tyr Ser Ile Ala His Ala Leu Gln

425 430GAT ATA TAT ACC TGC TTA CCT GGG AGA GGG CTC TTC 1767Asp Ile Tyr Thr Cys Leu Pro Gly Arg Gly Leu Phe

435 440ACC AAT GGC TCC TGT GCA GAC ATC AAG AAA GTT GAG 1803Thr Asn Gly Ser Cys Ala Asp Ile Lys Lys Val Glu445 450 455GCG TGG CAG GTC CTG AAG CAC CTA CGG CAT CTA AAC 1839Ala Trp Gln Val Leu Lys His Leu Arg His Leu Asn

460 465TTT ACA AAC AAT ATG GGG GAG CAG GTG ACC TTT GAT 1875Phe Thr Asn Asn Met Gly Glu Gln Val Thr Phe Asp

470 475 480GAG TGT GGT GAC CTG GTG GGG AAC TAT TCC ATC ATC 1911Glu Cys Gly Asp Leu Val Gly Asn Tyr Ser Ile Ile

485 490AAC TGG CAC CTC TCC CCA GAG GAT GGC TCC ATC GTG 1947Asn Trp His Leu Ser Pro Glu Asp Gly Ser Ile Val

495 500TTT AAG GAA GTC GGG TAT TAC AAC GTC TAT GCC AAG 1983Phe Lys Glu Val Gly Tyr Tyr Asn Val Tyr Ala Lys505 510 515AAG GGA GAA AGA CTC TTC ATC AAC GAG GAG AAA ATC 2019Lys Gly Glu Arg Leu Phe Ile Asn Glu Glu Lys Ile

520 525CTG TGG AGT GGG TTC TCC AGG GAG CCA CTC ACC TTT 2055Leu Trp Ser Gly Phe Ser Arg Glu Pro Leu Thr Phe

530 535 540GTG CTG TCT GTC CTC CAG GTG CCC TTC TCC AAC TGC 2091Val Leu Ser Val Leu Gln Val Pro Phe Ser Asn Cys

545 550AGC CGA GAC TGC CTG GCA GGG ACC AGG AAA GGG ATC 2127Ser Arg Asp Cys Leu Ala Gly Thr Arg Lys Gly Ile

555 560ATT GAG GGG GAG CCC ACC TGC TGC TTT GAG TGT GTG 2163Ile Glu Gly Glu Pro Thr Cys Cys Phe Glu Cys Val565 570 575GAG TGT CCT GATGGG GAG TAT AGT GAT GAG ACA GAT 2199Glu Cys Pro Asp Gly Glu Tyr Ser Asp Glu Thr Asp

580 585GCC AGT GCC TGT AAC AAG TGC CCA GAT GAC TTC TGG 2235Ala Ser Ala Cys Asn Lys Cys Pro Asp Asp Phe Trp

590 595 600TCC AAT GAG AAC CAC ACC TCC TGC ATT GCC AAG GAG 2271Ser Asn Glu Asn His Thr Ser Cys Ile Ala Lys Glu

605 610ATC GAG TTT CTG TCG TGG ACG GAG CCC TTT GGG ATC 2307Ile Glu Phe Leu Ser Trp Thr Glu Pro Phe Gly Ile

615 620GCA CTC ACC CTC TTT GCC GTG CTG GGC ATT TTC CTG 2343Ala Leu Thr Leu Phe Ala Val Leu Gly Ile Phe Leu625 630 635ACA GCC TTT GTG CTG GGT GTG TTT ATC AAG TTC CGC 2379Thr Ala Phe Val Leu Gly Val Phe Ile Lys Phe Arg

640 645AAC ACA CCC ATT GTC AAG GCC ACC AAC CGA GAG CTC 2415Asn Thr Pro Ile Val Lys Ala Thr Asn Arg Glu Leu

650 655 660TCC TAC CTC CTC CTC TTC TCC CTG CTC TGC TGC TTC 2451Ser Tyr Leu Leu Leu Phe Ser Leu Leu Cys Cys Phe

665 670TCC AGC TCC CTG TTC TTC ATC GGG GAG CCC CAG GAC 2487Ser Ser Ser Leu Phe Phe Ile Gly Glu Pro Gln Asp

675 680TGG ACG TGC CGC CTG CGC CAG CCG GCC TTT GGC ATC 2523Trp Thr Cys Arg Leu Arg Gln Pro Ala Phe Gly Ile685 690 695AGC TTC GTG CTC TGC ATC TCA TGC ATC CTG GTG AAA 2559Ser Phe Val Leu Cys Ile Ser Cys Ile Leu Val Lys

700 705ACC AAC CGT GTC CTC CTG GTG TTT GAG GCC AAG ATC 2595Thr Asn Arg Val Leu Leu Val Phe Glu Ala Lys Ile

710 715 720CCC ACC AGC TTC CAC CGC AAG TGG TGG GGG CTC AAC 2631Pro Thr Ser Phe His Arg Lys Trp Trp Gly Leu Asn

725 730CTG CAG TTC CTG CTG GTT TTC CTC TGC ACC TTC ATG 2667Leu Gln Phe Leu Leu Val Phe Leu Cys Thr Phe Met

735 740CAG ATT GTC ATC TGT GTG ATC TGG CTC TAC ACC GCG 2703Gln Ile Val Ile Cys Val Ile Trp Leu Tyr Thr Ala745 750 755CCC CCC TCA AGC TAC CGC AAC CAG GAG CTG GAG GAT 2739Pro Pro Ser Ser Tyr Arg Asn Gln Glu Leu Glu Asp

760 765GAG ATC ATC TTC ATC ACG TGC CAC GAG GGC TCC CTC 2775Glu Ile Ile Phe Ile Thr Cys His Glu Gly Ser Leu

770 775 780ATG GCC CTG GGC TTC CTG ATC GGC TAC ACC TGC CTG 2811Met Ala Leu Gly Phe Leu Ile Gly Tyr Thr Cys Leu

785 790CTG GCT GCC ATC TGC TTC TTC TTT GCC TTC AAG TCC 2847Leu Ala Ala Ile Cys Phe Phe Phe Ala Phe Lys Ser

795 800CGG AAG CTG CCG GAG AAC TTC AAT GAA GCC AAG TTC 2883Arg Lys Leu Pro Glu Asn Phe Asn Glu Ala Lys Phe805 810 815ATC ACC TTC AGC ATG CTC ATC TTC TTC ATC GTC TGG 2919Ile Thr Phe Ser Met Leu Ile Phe Phe Ile Val Trp

820 825ATC TCC TTC ATT CCA GCC TAT GCC AGC ACC TAT GGC 2955Ile Ser Phe Ile Pro Ala Tyr Ala Ser Thr Tyr Gly

830 835 840AAG TTT GTC TCT GCC GTA GAG GTG ATT GCC ATC CTG 2991Lys Phe Val Ser Ala Val Glu Val Ile Ala Ile Leu

845 850GCA GCC AGC TTT GGC TTG CTG GCG TGC ATCTTC TTC 3027Ala Ala Ser Phe Gly Leu Leu Ala Cys Ile Phe Phe

855 860AAC AAG ATC TAC ATC ATT CTC TTC AAG CCA TCC CGC 3063Asn Lys Ile Tyr Ile Ile Leu Phe Lys Pro Ser Arg865 870 875AAC ACC ATC GAG GAG GTG CGT TGC AGC ACC GCA GCT 3099Asn Thr Ile Glu Glu Val Arg Cys Ser Thr Ala Ala

880 885CAC GCT TTC AAG GTG GCT GCC CGG GCC ACG CTG CGC 3135His Ala Phe Lys Val Ala Ala Arg Ala Thr Leu Arg

890 895 900CGC AGC AAC GTC TCC CGC AAG CGG TCC AGC AGC CTT 3171Arg Ser Asn Val Ser Arg Lys Arg Ser Ser Ser Leu

905 910GGA GGC TCC ACG GGA TCC ACC CCC TCC TCC TCC ATC 3207Gly Gly Ser Thr Gly Ser Thr Pro Ser Ser Ser Ile

915 920AGC AGC AAG AGC AAC AGC GAA GAC CCA TTC CCA CGG 3243Ser Ser Lys Ser Asn Ser Glu Asp Pro Phe Pro Arg925 930 935CCC GAG AGG CAG AAG CAG CAG CAG CCG CTG GCC CTA 3279Pro Glu Arg Gln Lys Gln Gln Gln Pro Leu Ala Leu

940 945ACC CAG CAA GAG CAG CAG CAG CAG CCC CTG ACC CTC 3315Thr Gln Gln Glu Gln Gln Gln Gln Pro LeuThr Leu

950 955 960CCA CAG CAG CAA CGA TCT CAG CAG CAG CCC AGA TGC 3351Pro Gln Gln Gln Arg Ser Gln Gln Gln Pro Arg Cys

965 970AAG CAG AAG GTC ATC TTT GGC AGC GGC ACG GTC ACC 3387Lys Gln Lys Val Ile Phe Gly Ser Gly Thr Val Thr

975 980TTC TCA CTG AGC TTT GAT GAG CCT CAG AAG AAC GCC 3423Phe Ser Leu Ser Phe Asp Glu Pro Gln Lys Asn Ala985 990 995ATG GCC CAC AGG AAT TCT ACG CAC CAG AAC TCC CTG 3459Met Ala His Arg Asn Ser Thr His Gln Asn Ser Leu

1000 1005GAG GCC CAG AAA AGC AGC GAT ACG CTG ACC CGA CAC 3495Glu Ala Gln Lys Ser Ser Asp Thr Leu Thr Arg His

1010 1015 1020CAG CCA TTA CTC CCG CTG CAG TGC GGG GAA ACG GAC 3531Gln Pro Leu Leu Pro Leu Gln Cys Gly Glu Thr Asp

1025 1030TTA GAT CTG ACC GTC CAG GAA ACA GGT CTG CAA GGA 3567Leu Asp Leu Thr Val Gln Glu Thr Gly Leu Gln Gly

1035 1040CCT GTG GGT GGA GAC CAG CGG CCA GAG GTG GAG GAC 3603Pro Val Gly Gly Asp Gln Arg Pro Glu Val Glu Asp1045 1050 1055CCT GAA GAG TTG TCC CCA GCA CTT GTA GTG TCC AGT 3639Pro Glu Glu Leu Ser Pro Ala Leu Val Val Ser Ser

1060 1065TCA CAG AGC TTT GTC ATC AGT GGT GGA GGC AGC ACT 3675Ser Gln Ser Phe Val Ile Ser Gly Gly Gly Ser Thr

1070 1075 1080GTT ACA GAAAAC GTA GTG AAT TCA TAAAATGGAA 3709Val Thr Glu Asn Val Val Asn Ser

1085GGAGAAGACT GGGCTAGGGA GAATGCAGAG AGGTTTCTTG 3749GGGTCCCAGG GATGAGGAAT CGCCCCAGAC TCCTTTCCTC 3789TGAGGAAGAA GGGATAATAG ACACATCAAA TGCCCCGAAT 3829TTAGTCACAC CATCTTAAAT GACAGTGAAT TGACCCATGT 3869TCCCTTTAAA ATTAAAAAAA AGAAGAGCCT TGTGTTTCTG 3909TGGTTGCATT TGTCAAAGCA TTGAGATCTC CACGGTCAGA 39 TTTGCTGTTC ACCCACATCT AATGTCTCTT CCTCTGTTCT 3989ATCCCACCCA ACAGCTCAGA GATGAAACTA TGGCTTTAAA 402 CTACCCTCCA GAGTGTGCAG ACTGATGGGA CATCAAATTT 4069GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC 41072 GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC 4149 GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC 413672 4229 GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC 4269 GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC 4309 GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC 4372 4349 GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC 4389 GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC 4472 4429 GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC 4469 GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC 4772 4509 GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC 4589 GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC 4672 4669 GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC 4709 GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC 4749 364772 364872 4829 GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC 4909 364949 GCCACCACTA GAGCTGAGAG TCTGAAAGAC AGAATGTCAC ATATTAGTTA ATATTAT 5006(2) SEQ ID NO: 2, data:

(i) sequence characteristics:

(A) length: 3809 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: cDNA to mRNA

(ix) The characteristics are as follows:

(A) name/keyword CDS

(B) Position: 373 … 3606

(D) Other data:

(xi) Description of the sequence: SEQ ID NO: 2: CAACAGGCAC CTGGCTGCAG CCAGGAAGGA CCGCACGCCC 40TTTCGCGCAG GAGAGTGGAA GGAGGGAGCT GTTTGCCAGC 80 3932 80ACCGAGGTCT TGCGGCACAG GCAACGCTTG ACCTGAGTCT 120TGCAGAATGA AAGGCATCAC AGGAGGCCTC TGCATGATGT 160GGCTTCCAAA GACTCAAGGA CCACCCACAT TACAAGTCTG 200GATTGAGGAA GGCAGAAATG GAGATTCAAA CACCACGTCT 240TCTATTATTT TATTAATCAA TCTGTAGACA TGTGTCCCCA 280CTGCAGGGAG TGAACTGCTC CAAGGGAGAA ACTTCTGGGA 320GCCTCCAAAC TCCTAGCTGT CTCATCCCTT GCCCTGGAGA 360GACGGCAGAA CC ATG GCA TTT TAT AGC TGC TGC TGG 396

Met Ala Phe Tyr Ser Cys Cys Trp

1 5GTC CTC TTG GCA CTC ACC TGG CAC ACC TCT GCC TAC 432Val Leu Leu Ala Leu Thr Trp His Thr Ser Ala Tyr

10 15 20GGG CCA GAC CAG CGA GCC CAA AAG AAG GGG GAC ATT 468Gly Pro Asp Gln Arg Ala Gln Lys Lys Gly Asp Ile

25 30ATC CTT GGG GGG CTC TTT CCT ATT CAT TTT GGA GTA 504Ile Leu Gly Gly Leu Phe Pro Ile His Phe Gly Val

35 40GCA GCT AAA GAT CAA GAT CTC AAA TCA AGG CCG GAG 540Ala Ala Lys Asp Gln Asp Leu Lys Ser Arg Pro Glu45 50 55TCT GTG GAA TGT ATC AGG TAT AAT TTC CGT GGG TTT 576Ser Val Glu Cys Ile Arg Tyr Asn Phe Arg Gly Phe

60 65CGC TGG TTA CAG GCT ATG ATA TTT GCC ATA GAG GAG 612Arg Trp Leu Gln Ala Met Ile Phe Ala Ile Glu Glu

70 75 80ATA AAC AGC AGC CCA GCC CTT CTT CCC AAC TTG ACG 648Ile Asn Ser Ser Pro Ala Leu Leu Pro Asn Leu Thr

85 90CTG GGA TAC AGG ATA TTT GAC ACT TGC AAC ACC GTT 684Leu Gly Tyr Arg Ile Phe Asp Thr Cys Asn Thr Val

95 100TCT AAG GCC TTG GAA GCC ACC CTG AGT TTT GTT GCT 720Ser Lys Ala Leu Glu Ala Thr Leu Ser Phe Val Ala105 110 115CAA AAC AAA ATT GAT TCT TTG AAC CTT GAT GAG TTC 756Gln Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe

120 125TGC AAC TGC TCA GAG CAC ATT CCC TCT ACG ATT GCT 792Cys Asn Cys Ser Glu His Ile Pro Ser Thr Ile Ala

130 135 140GTG GTG GGA GCA ACT GGC TCA GGC GTC TCC ACG GCA 828Val Val Gly Ala Thr Gly Ser Gly Val Ser Thr Ala

145 150GTG GCA AAT CTG CTG GGG CTC TTC TAC ATT CCC CAG 864Val Ala Asn Leu Leu Gly Leu Phe Tyr Ile Pro Gln

155 160GTC AGT TAT GCC TCC TCC AGC AGA CTC CTC AGC AAC 900Val Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn165 170 175AAG AAT CAA TTC AAG TCT TTC CTC CGA ACC ATC CCC 936Lys Asn Gln Phe Lys Ser Phe Leu Arg Thr Ile Pro

180 185AAT GAT GAG CAC CAG GCC ACT GCC ATG GCA GAC ATC 972Asn Asp Glu His Gln Ala Thr Ala Met Ala Asp Ile

190 195 200ATC GAG TAT TTC CGC TGG AAC TGG GTG GGC ACA ATT 1008Ile Glu Tyr Phe Arg Trp Asn Trp Val Gly Thr Ile

205 210GCA GCT GAT GAC GAC TAT GGG CGG CCG GGG ATT GAG 1044Ala Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Glu

215 220AAA TTC CGA GAG GAA GCT GAG GAA AGG GAT ATC TGC 1080Lys Phe Arg Glu Glu Ala Glu Glu Arg Asp Ile Cys225 230 235ATC GAC TTC AGT GAA CTC ATC TCC CAG TAC TCT GAT 1116Ile Asp Phe Ser Glu Leu Ile Ser Gln Tyr Ser Asp

240 245GAG GAA GAG ATC CAG CAT GTG GTA GAG GTG ATT CAA 1152Glu Glu Glu Ile Gln His Val Val Glu Val Ile Gln

250 255 260AAT TCC ACG GCC AAA GTC ATC GTG GTT TTC TCC AGT 1188Asn Ser Thr Ala Lys Val Ile Val Val Phe Ser Ser

265 270GGC CCA GAT CTT GAG CCC CTC ATC AAG GAG ATT GTC 1224Gly Pro Asp Leu Glu Pro Leu Ile Lys Glu Ile Val

275 280CGG CGC AAT ATC ACG GGC AAG ATC TGG CTG GCC AGC 1260Arg Arg Asn Ile Thr Gly Lys Ile Trp Leu Ala Ser285 290 295GAG GCC TGG GCC AGC TCC TCC CTG ATC GCC ATG CCT 1296Glu Ala Trp Ala Ser Ser Ser Leu Ile Ala Met Pro

300 305CAG TAC TTC CAC GTG GTT GGC GGC ACC ATT GGA TTC 1332Gln Tyr Phe His Val Val Gly Gly Thr Ile Gly Phe

310 315 320GCT CTG AAG GCT GGG CAG ATC CCA GGC TTC CGG GAA 1368Ala Leu Lys Ala Gly Gln Ile Pro Gly Phe Arg Glu

325 330TTC CTG AAG AAG GTC CAT CCC AGG AAG TCT GTC CAC 1404Phe Leu Lys Lys Val His Pro Arg Lys Ser Val His

335 340AAT GGT TTT GCC AAG GAG TTT TGG GAA GAA ACA TTT 1440Asn Gly Phe Ala Lys Glu Phe Trp Glu Glu Thr Phe345 350 355AAC TGC CAC CTC CAA GAA GGT GCA AAA GGA CCT TTA 1476Asn Cys His Leu Gln Glu Gly Ala Lys Gly Pro Leu

360 365CCT GTG GAC ACC TTT CTG AGA GGT CAC GAA GAA AGT 1512Pro Val Asp Thr Phe Leu Arg Gly His Glu Glu Ser

370 375 380GGC GAC AGG TTT AGC AAC AGC TCG ACA GCC TTC CGA 1548Gly Asp Arg Phe Ser Asn Ser Ser Thr Ala Phe Arg

385 390CCC CTC TGT ACA GGG GAT GAG AAC ATC AGC AGT GTC 1584Pro Leu Cys Thr Gly Asp Glu Asn Ile Sar Ser Val

395 400GAG ACC CCT TAC ATA GAT TAC ACG CAT TTA CGG ATA 1620Glu Thr Pro Tyr Ile Asp Tyr Thr His Leu Arg Ile405 410 415TCC TAC AAT GTG TAC TTA GCA GTC TAC TCC ATT GCC 1656Ser Tyr Asn Val Tyr Leu Ala Val Tyr Ser Ile Ala

420 425CAC GCC TTG CAA GAT ATA TAT ACC TGC TTA CCT GGG 1692His Ala Leu Gln Asp Ile Tyr Thr Cys Leu Pro Gly

430 435 440AGA GGG CTC TTC ACC AAT GGC TCC TGT GCA GAC ATC 1728Arg Gly Leu Phe Thr Asn Gly Sar Cys Ala Asp Ile

445 450AAG AAA GTT GAG GCG TGG CAG GTC CTG AAG CAC CTA 1764Lys Lys Val Glu Ala Trp Gln Val Leu Lys His Leu

455 460CGG CAT CTA AAC TTT ACA AAC AAT ATG GGG GAG CAG 1800Arg His Leu Asn Phe Thr Asn Asn Met Gly Glu Gln465 470 475GTG ACC TTT GAT GAG TGT GGT GAC CTG GTG GGG AAC 1836Val Thr Phe Asp Glu Cys Gly Asp Leu Val Gly Asn

480 485TAT TCC ATC ATC AAC TGG CAC CTC TCC CCA GAG GAT 1872Tyr Ser Ile Ile Asn Trp His Leu Ser Pro Glu Asp

490 495 500GGC TCC ATC GTG TTT AAG GAA GTC GGG TAT TAC AAC 1908Gly Ser Ile Val Phe Lys Glu Val Gly Tyr Tyr Asn

505 510GTC TAT GCC AAG AAG GGA GAA AGA CTC TTC ATC AAC 1944Val Tyr Ala Lys Lys Gly Glu Arg Leu Phe Ile Asn

515 520GAG GAG AAA ATC CTG TGG AGT GGG TTC TCC AGG GAG 1980Glu Glu Lys Ile Leu Trp Ser Gly Phe Ser Arg Glu525 530 535GTG CCC TTC TCC AAC TGC AGC CGA GAC TGC CTG GCA 2016Val Pro Phe Ser Asn Cys Ser Arg Asp Cys Leu Ala

540 545GGG ACC AGG AAA GGG ATC ATT GAG GGG GAG CCC ACC 2052Gly Thr Arg Lys Gly Ile Ile Glu Gly Glu Pro Thr

550 555 560TGC TGC TTT GAG TGT GTG GAG TGT CCT GAT GGG GAG 2088Cys Cys Phe Glu Cys Val Glu Cys Pro Asp Gly Glu

565 570TAT AGT GAT GAG ACA GAT GCC AGT GCC TGT AAC AAG 2124Tyr Ser Asp Glu Thr Asp Ala Ser Ala Cys Asn Lys

575 580TGC CCA GAT GAC TTC TGG TCC AAT GAG AAC CAC ACC 2160Cys Pro Asp Asp Phe Trp Ser Asn Glu Asn His Thr585 590 595TCC TGC ATT GCC AAG GAG ATC GAG TTT CTG TCG TGG 2196Ser Cys Ile Ala Lys Glu Ile Glu Phe Leu Ser Trp

600 605ACG GAG CCC TTT GGG ATC GCA CTC ACC CTC TTT GCC 2232Thr Glu Pro Phe Gly Ile Ala Leu Thr Leu Phe Ala

610 615 620GTG CTG GGC ATT TTC CTG ACA GCC TTT GTG CTG GGT 2268Val Leu Gly Ile Phe Leu Thr Ala Phe Val Leu Gly

625 630GTG TTT ATC AAG TTC CGC AAC ACA CCC ATT GTC AAG 2304Val Phe Ile Lys Phe Arg Asn Thr Pro Ile Val Lys

635 640GCC ACC AAC CGA GAG CTC TCC TAC CTC CTC CTC TTC 2340Ala Thr Asn Arg Glu Leu Ser Tyr Leu Leu Leu Phe645 650 655TCC CTG CTC TGC TGC TTC TCC AGC TCC CTG TTC TTC 2376Ser Leu Leu Cys Cys Phe Ser Ser Ser Leu Phe Phe

660 665ATC GGG GAG CCC CAG GAC TGG ACG TGC CGC CTG CGC 2412Ile Gly Glu Pro Gln Asp Trp Thr Cys Arg Leu Arg

670 675 680CAG CCG GCC TTT GGC ATC AGC TTC GTG CTC TGC ATC 2448Gln Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile

685 690TCA TGC ATC CTG GTG AAA ACC AAC CGT GTC CTC CTG 2484Ser Cys Ile Leu Val Lys Thr Asn Arg Val Leu Leu

695 700GTG TTT GAG GCC AAG ATC CCC ACC AGC TTC CAC CGC 2520Val Phe Glu Ala Lys Ile Pro Thr Ser Phe His Arg705 710 715AAG TGG TGG GGG CTC AAC CTG CAG TTC CTG CTG GTT 2556Lys Trp Trp Gly Leu Asn Leu Gln Phe Leu Leu Val

720 725TTC CTC TGC ACC TTC ATG CAG ATT GTC ATC TGT GTG 2592Phe Leu Cys Thr Phe Met Gln Ile Val Ile Cys Val

730 735 740ATC TGG CTC TAC ACC GCG CCC CCC TCA AGC TAC CGC 2628Ile Trp Leu Tyr Thr Ala Pro Pro Ser Ser Tyr Arg

745 750AAC CAG GAG CTG GAG GAT GAG ATC ATC TTC ATC ACG 2664Asn Gln Glu Leu Glu Asp Glu Ile Ile Phe Ile Thr

755 760TGC CAC GAG GGC TCC CTC ATG GCC CTG GGC TTC CTG 2700Cys His Glu Gly Ser Leu Met Ala Leu Gly Phe Leu765 770 775ATC GGC TAC ACC TGC CTG CTG GCT GCC ATC TGC TTC 2736Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys Phe

780 785TTC TTT GCC TTC AAG TCC CGG AAG CTG CCG GAG AAC 2772Phe Phe Ala Phe Lys Ser Arg Lys Leu Pro Glu Asn

790 795 800TTC AAT GAA GCC AAG TTC ATC ACC TTC AGC ATG CTC 2808Phe Asn Glu Ala Lys Phe Ile Thr Phe Ser Met Leu

805 810ATC TTC TTC ATC GTC TGG ATC TCC TTC ATT CCA GCC 2844Ile Phe Phe Ile Val Trp Ile Ser Phe Ile Pro Ala

815 820TAT GCC AGC ACC TAT GGC AAG TTT GTC TCT GCC GTA 2880Tyr Ala Ser Thr Tyr Gly Lys Phe Val Ser Ala Val825 830 835GAG GTG ATT GCC ATC CTG GCA GCC AGC TTT GGC TTG 2916Glu Val Ile Ala Ile Leu Ala Ala Ser Phe Gly Leu

840 845CTG GCG TGC ATC TTC TTC AAC AAG ATC TAC ATC ATT 2952Leu Ala Cys Ile Phe Phe Asn Lys Ile Tyr Ile Ile

850 855 860CTC TTC AAG CCA TCC CGC AAC ACC ATC GAG GAG GTG 2988Leu Phe Lys Pro Ser Arg Asn Thr Ile Glu Glu Val

865 870CGT TGC AGC ACC GCA GCT CAC GCT TTC AAG GTG GCT 3024Arg Cys Ser Thr Ala Ala His Ala Phe Lys Val Ala

875 880GCC CGG GCC ACG CTG CGC CGC AGC AAC GTC TCC CGC 3060Ala Arg Ala Thr Leu Arg Arg Ser Asn Val Ser Arg885 890 895AAG CGG TCC AGC AGC CTT GGA GGC TCC ACG GGA TCC 3096Lys Arg Ser Ser Ser Leu Gly Gly Ser Thr Gly Ser

900 905ACC CCC TCC TCC TCC ATC AGC AGC AAG AGC AAC AGC 3132Thr Pro Ser Ser Ser Ile Ser Ser Lys Ser Asn Ser

910 915 920GAA GAC CCA TTC CCA CAG CCC GAG AGG CAG AAG CAG 3168Glu Asp Pro Phe Pro Gln Pro Glu Arg Gln Lys Gln

925 930CAG CAG CCG CTG GCC CTA ACC CAG CAA GAG CAG CAG 3204Gln Gln Pro Leu Ala Leu Thr Gln Gln Glu Gln Gln

935 940CAG CAG CCC CTG ACC CTC CCA CAG CAG CAA CGA TCT 3240Gln Gln Pro Leu Thr Leu Pro Gln Gln Gln Arg Ser945 950 955CAG CAG CAG CCC AGA TGC AAG CAG AAG GTC ATC TTT 3276Gln Gln Gln Pro Arg Cys Lys Gln Lys Val Ile Phe

960 965GGC AGC GGC ACG GTC ACC TTC TCA CTG AGC TTT GAT 3312Gly Ser Gly Thr Val Thr Phe Ser Leu Ser Phe Asp

970 975 980GAG CCT CAG AAG AAC GCC ATG GCC CAC GGG AAT TCT 3348Glu Pro Gln Lys Asn Ala Met Ala His Gly Asn Ser

985 990ACG CAC CAG AAC TCC CTG GAG GCC CAG AAA AGC AGC 3384Thr His Gln Asn Ser Leu Glu Ala Gln Lys Ser Ser

995 1000GAT ACG CTG ACC CGA CAC CAG CCA TTA CTC CCG CTG 3420Asp Thr Leu Thr Arg His Gln Pro Leu Leu Pro Leu1005 1010 1015CAG TGC GGG GAA ACG GAC TTA GAT CTG ACC GTC CAG 3456Gln Cys Gly Glu Thr Asp Leu Asp Leu Thr Val Gln

1020 1025GAA ACA GGT CTG CAA GGA CCT GTG GGT GGA GAC CAG 3492Glu Thr Gly Leu Gln Gly Pro Val Gly Gly Asp Gln

1030 1035 -1040CGG CCA GAG GTG GAG GAC CCT GAA GAG TTG TCC CCA 3528Arg Pro Glu Val Glu Asp Pro Glu Glu Leu Ser Pro

1045 1050GCA CTT GTA GTG TCC AGT TCA CAG AGC TTT GTC ATC 3564Ala Leu Val Val Ser Ser Ser Gln Ser Phe Val Ile

1055 1060AGT GGT GGA GGC AGC ACT GTT ACA GAA AAC GTA GTG 3600Ser Gly Gly Gly Ser Thr Val Thr Glu Asn Val Val1065 1070 1075AAT TCA TAAAATGGAA GGAGAAGACT GGGCTAGGGA 3636Asn SerGAATGCAGAG AGGTTTCTTG GGGTCCCAGG GATGAGGAAT 3676CGCCCCAGAC TCCTTTCCTC TGAGGAAGAA GGGATAATAG 3716ACACATCAAA TGCCCCGAAT TTAGTCACAC CATCTTAAAT 3756GACAGTGAAT TGACCCATGT TCCCTTTAAA AAAAAAAAAA 3796AAAAAGCGGC CGC 3809

Claims (12)

1. The compound N-3- (3-trifluoromethoxyphenyl) -1-propyl- (R) -1- (3-methoxyphenyl) ethylamine, or a pharmaceutically acceptable salt thereof.

2. The compound N-3- (3-trifluoromethylphenyl) -1-propyl- (R) -1- (1-naphthyl) ethylamine, or a pharmaceutically acceptable salt thereof.

3. The compound (R, R) -N- [4- (3-trifluoromethylphenyl) -2-butyl ] -1- (1-naphthyl) ethylamine, or a pharmaceutically acceptable salt thereof.

4. The compound (R, R) -N- [4- (3-trifluoromethylphenyl) -2-butyl ] -1- (3-methoxyphenyl) ethylamine, or a pharmaceutically acceptable salt thereof.

5. A pharmaceutical composition for treating a patient suffering from a disorder characterized by one or all of the following characteristics:

(1) abnormal homeostasis of calcium; and

(2) an abnormal amount of extracellular or intracellular messengers affected by calcium receptor activity, said composition comprising an effective amount of a compound according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

6. The composition of claim 5, wherein the disease is selected from the group consisting of: primary and secondary hyperparathyroidism, paget's disease, malignant hypercalcemia, osteoporosis, hypertension, and renal osteodystrophy.

7. The composition of claim 6, wherein the composition is a composition for treating a patient with primary and secondary hyperparathyroidism.

8. The composition of claim 6, wherein the composition is a composition for treating a patient with Paget's disease.

9. The composition of claim 6, wherein the composition is a composition for treating a patient suffering from hypercalcemia of malignancy.

10. The composition of claim 6, wherein the composition is a composition for treating a patient suffering from osteoporosis.

11. The composition of claim 6, wherein the composition is a composition for treating a patient suffering from hypertension.

12. The composition of claim 6, wherein the composition is a composition for treating a patient suffering from renal osteodystrophy.