WO2006066070A2 - Prodrug constructs of pyrimidinone compounds as calcilytics - Google Patents

Abstract

Various calcilytic compounds and prodrugs of calcilytic compounds are disclosed. Calcilytic compounds are compounds capable of inhibiting calcium receptor activity. Methods for preparing these compounds, oral bioavailability of these compounds, pharmaceutical compositions containing these compounds and their use as calcium receptor antagonists are also disclosed.

Description

PRODRUG CONSTRUCTS OF PYRIMIDINONE COMPOUNDS AS CALCILYTICS

Technical Field

[0001] The present disclosure relates to substituted 3H-pyrimidin-4-ones, prodrugs of substituted 3f/-pyrimidin-4-ones able to inhibit calcium receptor activity, and to methods for preparing and the uses of such compounds. The compounds described herein are administered to patients to achieve a therapeutic effect.

Background

[0002] The present disclosure relates to novel calcilytic compounds and to novel prodrugs of calcilytic compounds, methods for preparing these compounds, oral bioavailability of these compounds, prolonged pharmacological effect of these compounds, pharmaceutical compositions containing these compounds and their uses as calcium receptor antagonists.

[0003] In mammals, extracellular Ca²⁺ is under rigid homeostatic control and regulates various processes such as blood clotting, nerve and muscle excitability, and proper bone formation. Extracellular Ca²⁺ inhibits the secretion of parathyroid hormone ("PTH") from parathyroid cells, inhibits bone resorption by osteoclasts, and stimulates secretion of calcitonin from C-cells. Calcium receptor proteins enable certain specialized cells to respond to changes in extracellular Ca²⁺ concentration.

[0004] PTH is believed to be the principal endocrine factor regulating Ca²⁺ homeostasis in the blood and extracellular fluids. PTH, by acting on bone and kidney cells, increases the level of Ca²⁺ in the blood. This increase in extracellular Ca²⁺ then acts as a negative feedback signal, depressing PTH secretion. The reciprocal relationship between extracellular Ca²⁺ and PTH secretion provides a mechanism for maintaining bodily Ca²⁺ homeostasis.

[0005] Extracellular Ca²⁺ acts directly on parathyroid cells to regulate PTH secretion. The existence of a parathyroid cell surface protein, which detects changes in extracellular Ca²⁺, has been confirmed [see Brown et al., Nature, 366, 574, (1993)]. In parathyroid cells, this protein, the calcium receptor, acts as a receptor for extracellular Ca²⁺, detects changes in the ion concentration of extracellular Ca²⁺ and initiates a functional cellular response, PTH secretion.

[0006] Extracellular Ca²⁺ influences various cell functions, as reviewed in Nemeth et al., Cell Calcium, 11 , 319 (1990). For example, extracellular Ca²⁺ plays a role in parafollicular (C-cells) and parathyroid cells [see Nemeth, Cell Calcium, 11 , 319 (1990)]. The role of extracellular Ca²⁺ on bone osteoclasts has also been studied [see Zaidi, Bioscience Reports, 10, 493 (1990)].

[0007] Various compounds are known to mimic the effects of extra-cellular Ca²⁺ on a calcium receptor molecule. Calcilytics are compounds able to inhibit calcium receptor activity, thereby causing a decrease in one or more calcium receptor activities evoked by extracellular Ca²⁺. Calcilytics are useful as lead molecules in the discovery, development, design, modification and/or construction of useful calcium modulators, which are active at Ca²⁺ receptors. Such calcilytics are useful in the treatment of various disease states characterized by abnormal levels of one or more components, e.g., polypeptides such as hormones, enzymes or growth factors, the expression and/or secretion of which is regulated or affected by activity at one or more Ca²⁺ receptors. Target diseases or disorders for calcilytic compounds include diseases involving abnormal bone and mineral homeostasis.

[0008] Abnormal calcium homeostasis is characterized by one or more of the following activities: an abnormal increase or decrease in serum calcium; an abnormal increase or decrease in urinary excretion of calcium; an abnormal increase or decrease in bone calcium levels (for example, as assessed by bone mineral density measurements); an abnormal absorption of dietary calcium; an abnormal increase or decrease in the production and/or release of messengers which affect serum calcium levels such as PTH and calcitonin; and an abnormal change in the response elicited by messengers which affect serum calcium levels.

[0009] Thus, calcium receptor antagonists offer a unique approach towards the pharmacotherapy of diseases associated with abnormal bone or mineral homeostasis, such as hypoparathyroidism, osteosarcoma, periodontal disease, fracture healing, osteoarthritis, rheumatoid arthritis, Paget's disease, humoral hypercalcemia associated with malignancy and fracture healing, and osteoporosis.

[0010] Abnormal calcium homeostasis is characterized by one or more of the following activities: an abnormal increase or decrease in serum calcium; an abnormal increase or decrease in urinary excretion of calcium; an abnormal increase or decrease in bone calcium levels (for example, as assessed by bone mineral density measurements); an abnormal absorption of dietary calcium; an abnormal increase or decrease in the production and/or release of messengers which affect serum calcium levels such as PTH and calcitonin; and an abnormal change in the response elicited by messengers which affect serum calcium levels.

[0011] Thus, calcium receptor antagonists offer a unique approach towards the pharmacotherapy of diseases associated with abnormal bone or mineral homeostasis, such as hypoparathyroidism, osteosarcoma, periodontal disease, fracture healing, osteoarthritis, rheumatoid arthritis, Paget's disease, humoral hypercalcemia associated with malignancy and fracture healing, and osteoporosis.

Brief Description of the Drawings

[0012] FIG. 1 is a graph, which depicts the effect of bolus i.v. injection of the compound of Example 4 on plasma PTH level in normal rats.

[0013] FIG. 2 is a graph, which depicts plasma levels of the compound of Example 4 after bolus i.v. injection in normal rats.

[0014] FIG. 3 is a graph, which depicts the effect of bolus i.v. injection of the compound of Example 1e on plasma PTH level in normal rats.

[0015] FIG. 4 is a graph, which depicts plasma levels of the compound of Example 1e after oral administration of the prodrug compound of Example 22 in normal fasted rats.

Detailed Description

[0016] The present disclosure features calcilytic compounds and prodrugs of calcilytic compounds. "Calcilytic compounds" refer to compounds able to inhibit calcium receptor activity. The ability of a compound to "inhibit calcium receptor activity" means that the compound causes a decrease in one or more calcium receptor activities evoked by extracellular Ca²⁺. "Prodrugs" refer to compounds of chemical modification of calcilytic compounds able to liberate calcilytic compounds upon in vivo enzymatic or hydrolytic attack.

[0017] The use of calcilytic compounds to inhibit calcium receptor activity and/or achieve a beneficial effect in a patient is described below. More specifically, the present application demonstrates the ability of calcilytic compounds to increase PTH secretion, thereby confirming that the parathyroid gland calcium receptor is a target site for these compounds. Also described below are techniques, which can be used to obtain additional calcilytic compounds and prodrugs of calcilytic compounds. [0018] Examples of the featured calcilytic compounds representing 2,3,5,6- substituted 3/-/-pyrimidin-4-ones are provided by the chemical formula depicted in Structure I and the accompanying description.

Structure I

[0019] wherein:

[0020] R¹ and R² are independently one of: H, halogen, CN, CF₃, lower alkyl, cycloalk, and aryl; or R¹ and R² are together -(CH₂)_n- and n is 5, 4, or 3; or R¹ and R² are together -CH(lower alkyl)(CH₂)_n ^" and n is 4, 3, or 2;

[0021] R³ is an aryl group, which may have 0 to 4 substituents in the aryl ring and each substituent is one of: halogen, CN, CF₃, OCF₃, lower alkyl, NO₂, NH₂, NH(lower alk), N(lower alk)₂, NH[S(O)₂lower alk], NH[S(O)₂alkyl aryl], NH[S(O)₂aryl],

NH[S(O)₂heterocycle], OS(O)₂NH₂, OS(O)₂N H(lower alkyl), OS(O)₂N(lower alkyl)₂, lower alkoxy, OH, OC(O)-lower alk, OC(O)-lower alkyl-NH₂, OC(O)-lower alkyl-NH(lower alk),

OC(O)-lower alkyl-N(lower alk)₂, OC(O)O-lower alk, OC(O)O-lower alkyl amino,

OC(O)O-lower alkyl-NH(lower alk), OC(O)O-lower alkyl-N(lower alk)₂, OC(O)NH-lower alk, OC(O)N(lower alk)₂, OC(O)heterocycle, O-lower alkyl-P(O)(OH)₂₎ O-lower alkyl-

P(O)OH(O-lower alkyl-OC(O)O-lower alkyl), O-lower alkyl-P(O)(O-lower alkyl-OC(O)O- lower alk)₂, OP(O)(OH)₂, OP(O)(O-lower alkyl-aryl)₂, OP(O)(O-lower alk)₂, OP(O)(O- lower alkyl-OC(O)O-lower alk)₂ and OP(O)(O-metal)₂;

[0022] R⁴ is one of H, lower alkyl, and a group of the formula -(CH₂)_n-R⁵ wherein n is

0, 1 , or 2, and R⁵ is an aryl group which may have 0 to 3 substituents on the aryl ring and each substituent is one of: halogen, CN, CF₃, OCF₃, lower alk, lower alkoxy, NH- lower alk, NH-alkyl aryl, N(lower alk)₂, OH, OC(O)-lower alk, OC(O)-lower alkyl amino, and OC(O)-lower alkyl-N(lower alk)₂; and

[0023] pharmaceutically acceptable salts, hydrates, tautomers, solvates and complexes thereof. [0024] In embodiments wherein R¹ and R² are independently selected, R¹ and R² may be one of: lower alkyl or cycloalkyl. In embodiments wherein R¹ and R² are together -(CH₂)_n-, n may be 5, 4, or 3. In embodiments wherein R¹ and R² are together -CH(lower alkyl)(CH₂)_n ^", n may be 3 or 2.

[0025] In embodiments wherein R³ is a phenyl group, the phenyl ring may have 0 to 3 substituents which are one of: halogen, NO₂, NH₂, NH[S(O)₂lower alk], NH[S(O)₂alkyl aryl], NH[S(O)₂heterocycle], lower alkoxy, OH, OC(O)-lower alk, OC(O)O-lower alk, O- lower alkyl-P(O)(OH)₂, O-lower alkyl-P(O)OH(O-lower alkyl-OC(O)O-lower alk), O-lower alkyl-P(O)(O-lower alkyl-OC(O)O-lower alk)₂, OP(O)(O-lower alkyl-OC(O)O-lower alk)₂, OP(O)(O-lower alkyl-aryl)₂, OC(O)NH-lower alkyl, OC(O)N(lower alkyl)₂, OC(O)heterocycle, OP(O)(O-lower alk)₂, OP(O)(OH)₂, OP(O)(O-metal)₂. Also, in other embodiments wherein R³ is a phenyl group, the phenyl ring may have 0 to 3 substituents which are one of: halogen, OH, NH₂, NH[S(O)₂lower alkyl], NH[S(O)₂heterocycle], OH, OC(O)-lower alkyl, OC(O)O-lower alkyl, OC(O)NH(lower alkyl), OC(O)N(lower alkyl)_2> OC(O)heterocycle, O-lower alkyl-P(O)OH(O-lower alkyl-OC(O)O-lower alkyl), OP(O)(O- lower alkyl)₂, OP(O)(O-lower alkyl-OC(O)O-lower alkyl)₂, OP(O)(OH)₂, and OP(O)(O- metal)₂.

[0026] In embodiments wherein R⁴ is a group of the formula -(CH₂)n-R⁵ _> n may be 1 or 2, and R⁵ may be an aryl group, the aryl ring may have O to 3 substituents which may be a halogen. More particularly, in embodiments wherein R⁴ is a group of the formula - (CH₂)_n-R⁵, n may be 2 and R⁵ may be an aryl group, having O to 3 substituents on the aryl ring which may be a halogen.

[0027] "AIk" refers to either alkyl or alkenyl. "Lower alk" refers to either lower alkyl or lower alkenyl.

[0028] "Alkenyl" refers to an optionally substituted hydrocarbon group containing at least one carbon-carbon double bond between the carbon atoms and containing 2-6 carbon atoms joined together. The alkenyl hydrocarbon group may be straight-chain. In some embodiments straight-chain alkenyl has 2 to 4 carbons.

[0029] "Alkyl" refers to an optionally substituted hydrocarbon group joined by single carbon-carbon bonds and having 1 to 6 carbon atoms joined together. The alkyl hydrocarbon group may be straight-chain or contain one or more branches. In some embodiments, branched- and straight-chain alkyl groups have 1 to 4 carbons, each of which may be optionally substituted. Alkyl substituents may be independently one of: lower alkyl, unsubstituted aryl, OH, NH₂, NH-lower alkyl, and N(lower alkyl)₂. In some embodiments, no more than two substituents are present. For example, alkyl may be a lower alkyl, which is unsubstituted branched- or straight-chain alkyl having 1 to 5 carbons. In other embodiments, alkyl may be a lower alkyl having 1 to 4 carbons.

[0030] "Cycloalk" refers to an optionally substituted cyclic alkyl or an optionally substituted non-aromatic cyclic alkenyl and includes monocyclic and multiple ring structures such as bicyclic and tricyclic. The cycloalkyl has 3 to 15 carbon atoms. In one embodiment, cycloalkyl has 3 to 5 carbon atoms. Optional substituents for cycloalk are independently selected from the group described above for alkyl and alkenyl. In one embodiment, no more than three substituents are present. In another embodiment, the cycloalk is unsubstituted. For example, the cylcoalk may be unsubstituted cyclic alkyl.

Examples of suitable cycloalkyl groups include cyclopropyl and cyclobutyl.

[0031] "Heterocycle" refers to an optionally substituted aromatic or non-aromatic ringed moieties. Heterocyclic moieties typically comprise one ring or two fused rings, where the ring(s) is 5 to 6-membered and typically contains 1 to 2 non-carbon atoms.

Non-carbon atoms for heterocycle are independently selected from nitrogen, oxygen and sulfur.

[0032] "Aryl" refers to an optionally substituted aromatic group with at least one ring having a conjugated or fused ring system. Aryl includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. In some embodiments, the aryl may be either optionally substituted phenyl or optionally substituted pyridyl.

[0033] "Alkoxy" refers to oxygen joined to an unsubstituted alkyl 1 to 4 carbon atoms in length. In one embodiment, the oxygen is joined to an unsubstituted alklyl 1 to 2 carbons in length. For example, the alkoxy may be methoxy.

[0034] "Metal" refers to the monovalent metal cation. For example, the metal may be sodium or potassium.

[0035] Exemplary embodiments include parent calcilytic compounds:

[0036] 3-[2-(3-fluoro-phenyl)-ethyl]-2-(2-hydroxy-phenyl)-5-isopropyl-6-methyl-3H- pyrimidin-4-one;

[0037] 3-[2-(3-fluoro-phenyl)-ethyl]-2-(3-fluoro-2-hydroxyphenyl)-5-isopropyl-6- methyl-3/-/-pyrimidin-4-one;

[0038] 3-[2-(3-fluoro-phenyl)-ethyl]-2-(2-hydroxy-phenyl)-5-isobutyl-6-methyl-3H- pyrimidin-4-one;

[0039] 5-(1 ,2-dimethylpropyl)-3-[2-(3-fluorophenyl)ethyl]-2-(2-hydroxyphenyl)-6- methyl-3H-pyrimidin-4-one;

[0040] 3-[2-(3-fluorophenyl)ethyl]-2-(2-hydroxyphenyl)-5-methyl-5,6,7,8-tetrahydro-

3AV-quinazolin-4-one; [0041] (5S)-3-[2-(3-fluorophenyl)ethyl]-2-(2-hydroxyphenyl)-5-methyl-5,6,7,8- tetrahexahydro-3/-/-quinazolin-4-one;

[0042] 2-(2-amino-phenyl)-3[2-(3-fluoro-phenyl)-ethyl]-5isobutyl-6-methyl-3H- pyrimidin-4-one;

[0043] and prodrugs of calcilytic compounds:

[0044] carbonic acid ethyl ester 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isopropyl-4-methyl-

6-0X0-1 ,6-dihydro-pyrimidin-2-yl}-phenyl ester;

[0045] carbonic acid ethyl ester 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-4-methyl~6- oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyl ester;

[0046] phosphoric acid mono-(2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isopropyl-4-methyl-6- oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyi) ester; and

[0047] phosphoric acid diethyl ester 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5 isopropyl-4- methyl-6-oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyl ester.

[0048] The calcilytic compounds of Structure I wherein R¹, R² and R⁴ are substituents other than hydrogen, and R³ is a phenyl group with 0 to 3 substituents which are one of: halogen, NO₂, lower alkoxy, can be prepared by Scheme I involving a method of reacting an appropriate 2-alk-3-aroylamino-alk-2-enoic acid methyl ester with an appropriate amine. A chemical synthesis for such compounds by Scheme I describes an approach to 2,3,5,6-substituted 3H-pyrimidin-4-ones which is an improvement in the art. This improvement is disclosed and claimed in International

Publications WO 2004/092120 (A2) and WO 2004/092121 (A2) titled "Pyrimidinone

Compounds as Calcilytics" and "Methods for Preparing 2,3,5,6-substituted 3H-pyrimidin-

4-ones", respectively.

[0049] More particularly, the compounds of Structure I, wherein R¹ is an alkyl group which contains more than one branch, R² is methyl, R⁴ is a substituent other than hydrogen, and R³ is an aryl group which may have substituents in the aryl ring, can be prepared by Scheme I provided below. Scheme I involves a method of reacting an appropriate 2-alkyl-3-aroylamino-alk-2-enoic acid methyl ester with a primary amine and trimethyl aluminum. The use of trimethyl aluminum during the last stage instead of previously used phenyl magnesium bromide (WO 2004/092120 A2, Example 13, Method

B) improves the yield of 2,3,5,6-substituted 3H-pyrimidin-4-ones of Structure I. Scheme I below provides a method for synthesizing 2,3,5,6-substituted 3H-pyrimidin-4-ones. Scheme I

[0050] The chemical synthesis involves a method of making 2-R¹-3-aroylamino-alk-2- enoic acid methyl ester of Structure Il by standard techniques, which includes acylation of an appropriate 3-amino-2-R¹-alk-2-enoic acid methyl ester of Structure III.

Structure Il Structure III

[0051] wherein:

[0052] R¹ and R² are independently one of: lower alkyl, cycloalk; or R¹ and R² are together -(CH₂)_n- and n is 5, 4, or 3;

[0053] R³ is an aryl group, which may have 1 to 4 substituents in the aryl ring and each substituent is one of: H₁ halogen, lower alkyl, lower alkoxy, NO₂.

[0054] When R¹ and R² are independently selected, R¹ and R² may, in some embodiments, be one of: lower alkyl and cycloalkyl. When R¹ and R² are together -

(CH₂)_n-. n may in some embodiments, be 4 or 3.

[0055] In embodiments wherein R³ is a phenyl group, the phenyl ring may have 0 to

3 substituents which are one of: halogen, lower alkoxy and NO₂.

[0056] The calcilytic compounds of Structure I wherein R¹, R² and R⁴ are substituents other than hydrogen, and R³ is a phenyl group with 0 to 3 substituents which are one of: halogen, and OH, and one of which is OH, can be prepared by demethylation of lower alkoxy in an appropriate 2,3,5,6-substituted 3H-pyrimidin-4-one of Structure I involving a method of reacting an appropriate 2-(lower alkoxy-aryl), 3,5,6- substituted 3H-pyrimidin-4-one with boron tribromide under conventional conditions, or with sodium cyanide under microwave irradiation as disclosed and claimed in

International Publication WO 2004/092120 (A2) (Example 13, Method B). [0057] The calcilytic compounds of Structure I wherein R¹, R² and R⁴ are substituents other than hydrogen, and R³ is a phenyl group with 0 to 3 substituents which are one of: halogen, OC(O)-lower alkyl, OC(O)O-lower alkyl, and one of which is OC(O)-lower alkyl or OC(O)O-lower alkyl, can be prepared by acylation of an appropriate 2,3,5,6-substituted 3/7-pyrimidin-4-one of Structure I involving a method of reacting an appropriate 2-(OH-aryl),3,5,6-substituted 3/7-pyrimidin-4-one with an appropriate carboxylic acid chloride or with an appropriate carbonic acid chloride in dry pyridine.

[0058] The calcilytic compounds of Structure I wherein R¹, R² and R⁴ are substituents other than hydrogen, and R³ is a phenyl group with 0 to 3 substituents which are one of: halogen, O-lower alkyl-P(O)(OH)₂, O-lower alkyl-P(O)OH(O-lower alkyl-OC(O)O-lower alkyl), OP(O)(O-lower alkyl-aryl)₂, OP(O)(O-lower alkyl)₂₎ OP(O)(OH)₂, OP(O)(O-metal)₂, and one of which is O-lower alkyl-P(O)(OH)₂, O-lower alkyl-P(O)OH(O-lower alkyl-OC(O)O-lower alkyl), OP(O)(O-lower alkyl-aryl)₂, OP(O)(O- lower alkyl)₂, OP(O)(OH)₂ or OP(O)(O-metal)₂, can be prepared by phosphorylation of an appropriate 2,3,5,6-substituted 3/-/-pyrimidin-4-one of Structure I involving a method of reacting an appropriate 2-(OH-aryl),3,5,6-substituted 3W-pyrimidin-4~one with an appropriate phosphite in the presence of dimethylaminopyridine, or involving the method of reduction of phosphoric dibenzyl ester of an appropriate 2,3,5,6-substituted 3H- pyrimidin-4-one of Structure I with H₂ on Pd/C, or involving a method of reacting phosphoric acid monoester of an appropriate 2,3,5,6-substituted 3H-pyrimidin-4-one of Structure I with sodium methoxide.

[0059] The calcilytic compounds of Structure I wherein R¹, R² and R⁴ are substituents other than hydrogen, and R³ is a phenyl group with 0 to 3 substituents which are one of: halogen, NH₂, and one of which is NH₂, can be prepared by reduction of the nitro group in an appropriate 2,3,5,6-substituted 3H-pyrimidin-4-one of Structure I involving a method of reacting an appropriate 2-(NO₂-aryl),3,5,6-substituted 3H- pyrimidin-4-one with H₂ on Pd/C.

[0060] The calcilytic compounds of Structure I wherein R¹, R² and R⁴ are substituents other than hydrogen, and R³ is a phenyl group with 0 to 3 substituents which are one of: halogen, NH[S(O)₂lower alkyl], NH[S(O)₂alkyl aryl], NH[S(0)₂heterocycle], and one of which is NH[S(O)₂lower alkyl], NH[S(O)₂alkyl aryl], NH[S(O)₂heterocycle], can be prepared by A/-sulfonylation of the amino group in an appropriate 2,3,5,6-substituted 3H-pyrimidin-4-one of Structure I involving a method of reacting an appropriate 2-(NH₂-aryl),3,5,6-substituted 3/-/-pyrimidin-4-one with an appropriate sulfonyl chloride.

[0061] In order to use a compound of Formula (I) or a pharmaceutically acceptable salt or complex thereof for the treatment of humans and other mammals, it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.

[0062] The calcilytic compounds can be administered by different routes including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical (transdermal), or transmucosal administration. For systemic administration, oral administration may be used. For oral administration, for example, the compounds can be formulated into conventional oral dosage forms such as capsules, tablets, and liquid preparations such as syrups, elixirs, and concentrated drops.

[0063] Alternatively, injection (parenteral administration) may be used, e.g., intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the calcilytic compounds are formulated in liquid solutions, such as in physiologically compatible buffers or solutions, such as saline solution, Hank's solution, or Ringer's solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms can also be produced.

[0064] Systemic administration can also be achieved by transmucosal or transdermal methods. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated may be used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration, for example, may be through nasal sprays, rectal suppositories, or vaginal suppositories.

[0065] For topical administration, the calcilytic compounds can be formulated into ointments, salves, gels, or creams, as is generally known in the art.

[0066] The amounts of various calcilytic compounds to be administered can be determined by standard procedures taking into account factors such as the compound

IC50, EC50, the biological half-life of the compound, the age, size and weight of the patient, and the disease or disorder associated with the patient. The significance of these and other factors to be considered are known to those of ordinary skill in the art. [0067] Amounts administered also depend on the routes of administration and the degree of oral bioavailability. For example, for compounds with low oral bioavailability, relatively higher doses may have to be administered. [0068] The composition may be in unit dosage form. For oral application, for example, a tablet or capsule may be administered, for nasal application, a metered aerosol dose may be administered, for transdermal application, a topical formulation or patch may be administered, and for transmucosal delivery, a buccal patch may be administered. In each case, dosing is such that the patient may administer a single dose.

[0069] Each dosage unit for oral administration contains suitably from 0.01 to 500 mg/Kg, such as from 0.1 to 50 mg/Kg, of a compound of Formula (I) or a pharmaceutically acceptable salt or complex thereof, calculated as the free base. The daily dosage for parenteral, nasal, oral inhalation, transmucosal or transdermal routes contains suitably from 0.01 mg to 100 mg/Kg, of a compound of Formula (I). A topical formulation contains suitably 0.01 to 5.0% of a compound of Formula (I). The active ingredient may be administered as a single dose or in multiple does, for example, from 2 to 6 times per day, sufficient to exhibit the desired activity, as is readily apparent to one skilled in the art.

[0070] As used herein, "treatment" of a disease includes, but is not limited to prevention, retardation and prophylaxis of the disease.

[0071] Diseases and disorders which might be treated or prevented, based upon the affected cells, include bone and mineral-related diseases or disorders; hypoparathyroidism; those of the central nervous system such as seizures, stroke, head trauma, spinal cord injury, hypoxia-induced nerve cell damage, such as occurs in cardiac arrest or neonatal distress, epilepsy, neurodegenerative diseases such as Alzheimer's disease, Huntington's disease and Parkinson's disease, dementia, muscle tension, depression, anxiety, panic disorder, obsessive-compulsive disorder, post-traumatic stress disorder, schizophrenia, neuroleptic malignant syndrome, and Tourette's syndrome; diseases involving excess water reabsorption by the kidney, such as syndrome of inappropriate ADH secretion (SIADH), cirrhosis, congestive heart failure, and nephrosis; hypertension; preventing and/or decreasing renal toxicity from cationic antibiotics (e.g., aminoglycoside antibiotics); gut motility disorders such as diarrhea and spastic colon; Gl ulcer diseases; Gl diseases with excessive calcium absorption such as sarcoidosis; autoimmune diseases and organ transplant rejection; squamous cell carcinoma; and pancreatitis.

[0072] In one embodiment, the present compounds are used to increase serum parathyroid hormone ("PTH") levels. Increasing serum PTH levels can be helpful in treating diseases such as hypoparathyroidism, osteosarcoma, periodontal disease, fracture, osteoarthritis, rheumatoid arthritis, Paget's disease, humoral hypercalcemia of malignancy, and osteoporosis.

[0073] In another embodiment, the present compounds are co-administered with an anti-resorptive agent. Such agents include, but are not limited to estrogen, 1 ,25-(OH)2- vitamin D3, calcitonin, selective estrogen receptor modulators, vitronectin receptor antagonists, V-H+-ATPase inhibitors, src SH2 antagonists, bisphosphonates and cathepsin K inhibitors.

[0074] Another aspect of the present disclosure includes a method of treating a patient comprising administering to the patient an amount of a present compound sufficient to increase the serum PTH level. By way of example, the method is carried out by administering an amount of the compound effective to cause an increase in duration and/or quantity of serum PTH level sufficient to have a therapeutic effect. [0075] In various embodiments, the compound administered to a patient causes an increase in serum PTH having a duration of up to one hour, about one to about twenty- four hours, about one to about twelve hours, about one to about six hours, about one to about five hours, about one to about four hours, about two to about five hours, about two to about four hours, or about three to about six hours.

[0076] In an alternative embodiment, the compound administered to a patient causes an increase in serum PTH having a duration of more than about twenty-four hours provided that it is co-administered with an anti resorptive agent.

[0077] In additional different embodiments, the compound administered to a patient causes an increase in serum PTH of up to two-fold, two- to five-fold, five- to ten-fold, and at least 10-fold, greater than peak serum PTH in the patient. The peak serum level is measured with respect to a patient not undergoing treatment.

[0078] Composition of Formula (I) and their pharmaceutically acceptable salts and/or complexes, which are active when given orally, can be formulated as syrups, tablets, capsules, and lozenges. A syrup formulation will generally consist of a suspension or solution of the compound or salt in a liquid carrier such as, for example, ethanol, peanut oil, olive oil, glycerine or water with a flavoring or coloring agent. Where the composition is in the form of a tablet, any pharmaceutical carrier routinely used for preparing solid formulations may be used. Examples of such carriers include magnesium stearate, terra alba, talc, gelatin, acacia, stearic acid, starch, lactose and sucrose. Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example using the aforementioned carriers in a hard gelatin capsule shell. Where the composition is in the form of a soft gelatin shell capsule, any pharmaceutical carrier routinely used for preparing dispersions or suspensions may be utilized. For example, aqueous gums, celluloses, silicates or oils may be used to form a soft gelatin capsule shell.

[0079] Typical parenteral compositions consist of a solution or suspension of a compound or salt in a sterile aqueous or non-aqueous carrier optionally containing parenterally acceptable oil, for example polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil.

[0080] Typical compositions for inhalation are in the form of a solution, suspension or emulsion that may be administered as a dry powder or in the form of an aerosol using a conventional propellant such as dichlorodifluoromethane or trichlorofluoromethane.

[0081] A typical suppository formulation comprises a compound of Formula (I) or a pharmaceutically acceptable salt or complex thereof which is active when administered in this, way, with a binding and/or lubricating agent, for example polymeric glycols, gelatins, cocoa-butter or other low-melting vegetable waxes or fats or their synthetic analogs.

[0082] Typical dermal and transdermal formulations comprise a conventional aqueous or non-aqueous vehicle, for example a cream, ointment, lotion or paste or are in the form of a medicated plaster, patch or membrane.

[0083] The composition may be in unit dosage form, for example a tablet, capsule or metered aerosol dose, so that the patient may administer a single dose.

[0084] No unacceptable toxological effects are expected when the compounds disclosed are administered in accordance with the methods described.

Examples

[0085] The following specific examples are included for illustrative purposes only and are not to be considered as limiting to this disclosure. The reagents and intermediates used in the following examples are either commercially available or can be prepared according to standard literature procedures by those skilled in the art of organic synthesis.

[0086] MPLC (Medium Pressure Liquid Chromatography) purifications for 95+% purity of the compounds were performed on a Biotage Horison HS1 Series MPLC equipped with a Silica Gel Cartridge Flash 65i (flow rate 65 mL/min). [0087] HPLC (High Pressure Liquid Chromatography) analyses for 95+% purity confirmation were performed on a Gilson Series HPLC equipped with an Agilent DAD 170 (diode array detector), and a Macherey-Nagel Nucleodur C18 (5μ, 4.6x150 mm) column.

[0088] MS (Mass spectrometry) analyses were performed on an Aqa single quad ThermoFinnigan spectrometer with an ESI (Electrospray Ionization) and were recorded in the positive-ion mode.

[0089] NMR (Nuclear Magnetic Resonance) spectroscopy was performed on a Bruker Advance 300 spectrometer and on a Varian Gemini 300 spectrometer. Proton and carbon spectra were recorded at 300 MHz and 75 MHz, respectively, in deuterochloroform (CDCI₃), methanol-^ (CH₃OH-^)₁ or dimethylsulfoxide-c/β (DMSO-αfe) solutions. NMR resonances are reported in δ (ppm) relative to tetramethylsilane (TMS) as internal standard with the following descriptors for the observed multiplicities: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), and m (multiplet). J_AB coupling constants are reported in Hz.

[0090] Solubility determination of the calcilytic compounds was performed on a Thermo Finnigan LCQ Deca XP with Finnigan Surveyor autosampler and accompanying Xcalibur software. The instrument parameters are general HPLC conditions; 300 μL/min flow rate, 5 μL injection, 5% mobile phase B up to 95% B. The column is 50 x 2.0 mm Synergi (Phenomenex). Prior to analysis, the detector was allowed to equilibrate with solvent flow for approximately one hour. During this time, a sequence table is created with the standard curve analyzed first, the samples immediately afterwards, with at least 3 injections per sample (giving a total of six replicates), followed by the standard curve to finish the analysis.

Example 1

Preparation of 3-f2-(3-fluoro-phenyl)-ethyll-2-(2-hvdroxy-phenyl)-5-isopropyl-6-methyl-

3/-/-pyrimidin-4-one

[0091] a). 2-Acetyl-3-m ethyl-butyric acid methyl ester

[0092] Sodium methoxide (7.06 g, 0.130 mol) was added portionwise to a solution of 2-acetyl-3-methyl-butyιϊc acid ethyl ester (15.0 g, 0.0871 mol) in dry MeOH (300 ml_). The reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and the residue was treated with EtOAc (200 ml_) and H₂O (200 mL). The organic residue was separated, dried over MgSO₄ and concentrated in vacuum to yield yellow oil (11.2g; 81.3% according to ¹H NMR and MS) that was used in the next step without purification.

[0093] ¹H NMR (CDCI₃): δ 3.65 (s, 3H), 3.13 (d, 1 H, J = 9.6), 2.37 (m, 1H), 2.16 (s, 3H), 0.88 (dd, 6H, J = 6.59, J = 11.11 ).

[0094] b). 3-Amino-2-isopropyl-but-2-enoic acid methyl ester.

[0095] 2-Methyl-3-oxo-butyric acid methyl ester of Example 1a (5.57 g, 0.035 mol) was dissolved in absolute ethanol (25 mL). After cooling the reaction mixture to -78°C, ammonia was bubbled to double the solution volume and the resulting mixture was stirred at room temperature in a sealed reaction vessel for 48 h. Excess ammonia and ethanol were removed under reduced pressure and the crude product (70% yield according to ¹H NMR and MS) was used in the next step without further purification. [0096] ¹H NMR (CDCI₃): δ 3.61 (s, 3H), 2.61 (m, 1 H), 1.91 (s, 3H), 1.06 (d, 6H, J = 6.97).

[0097] c). 2-lsopropyl-3-(2-methoxy-benzoylamino)-but-3-enoic acid methyl ester

[0098] 3-Amino-2-isopropyl-but-2-enoic acid methyl ester of Example 1b (4.86 g, 0.0309 mol) was dissolved in anhydrous THF (150 mL) and anhydrous pyridine (5.0 mL, 0.0617 mol) was added. Anisoyl chloride (4.34 mL, 0.0309 mol) was added dropwise, and the mixture was refluxed for 2 h. After cooling, water (150 ml_) was added and the organic layer was extracted with ethyl acetate (3 x 50 ml_). The combined organic extracts were washed with 1 N HCI (3 x 100 ml_), water (100 ml_), and brine (100 ml_), dried over sodium sulfate and concentrated on a rotary evaporator. The product was purified by column chromatography over silica gel (200-400 mesh) eluting with 3%

EtOAc/cyclohexanes to give 2-isopropyl-3-(2-methoxy-benzoylamino)-but-3-enoic acid methyl ester (1.53 g, 17%) as a white powder.

[0099] ¹H NMR (CDCI₃): δ 9.95 (s, 1 H), 8.21 (dd, 1 H, J = 9.66, 1.82), 7.5 - 7.42 (m,

1H), 7.12 - 7.03 ( m, 1H), 7.01 - 6.95 (m, 1H), 6.08 (s, 1H), 4.75 (s, 1H), 4.07 (s, 3H),

3.72 (s, 3H), 2.75 (d, 1 H, J = 10.96), 2.25 - 2.10 (m, 1 H), 1.02 - 0.90 (m, 6H).

[0100] HPLC (20-80% ACN/H₂O): > 95%

[0101] MS (ESI⁺) (+ 0.1% HCOOH): 292.1 [Ci₆H₂iNO₄+1]⁺ (m/z)

[0102] d). 3-[2-(3-Fluoro-phenyl)-ethyl]-5-isopropyl-2-(2-methoxy-phenyl)-6-methyl-

3H-pyrimidin-4-one

[0103] Trimethyl aluminum (2M solution in toluene, 0.0065 mol) was added to a solution of 3-fluoro-phenethyl amine (0.85 mL, 0.0065 mol) in anhydrous toluene (32 ml_). After stirring the mixture at 2O⁰C for 10 min, 2-isopropyl-3-(2-methoxy- benzoylamino)-but-3-enoic acid methyl ester of Example 1c (1.53 g, 0.0052 mol) was added. The mixture was refluxed for 10 hours, cooled and ethyl acetate (50 mL) was added followed by 1N HCI (50 mL). The organic layer was separated and the aqueous layer was extracted with EtOAc (3 x 50 mL). The combined organic extracts were washed with 1N HCI (3 x 100 mL), water (100 mL), and brine (100 mL). After drying over sodium sulfate and concentration on a rotary evaporator, the product was purified by column chromatography over silica gel (200-400 mesh) eluting with 10% EtOAc-hexanes to give 3-[2-(3-fluoro-phenyl)-ethyl]-5-isopropyl-2-(2-methoxy-phenyl)-6-methyl-3H- pyrimidin-4-one (0.756 g, 38 %) as a white solid.

[0104] ¹H NMR (CDCI₃): δ 7.51 - 7.42 (m, 1H), 7.18 - 7.08 (m, 1H), 7.07 - 7.05 (m, 1H)₁ 7.03 - 6.95 (m, 2H), 6.88 - 6.82 (m, 1H), 6.63 (d, 1 H, J = 7.63), 6.48 (dt, 1H, J = 9.75, 2.2), 4.35 - 4.22 (m, 1 H), 3.82 (s, 3H), 315 - 3.03 (m, 1 H), 3.25 - 3.10 (q, 1 H, J = 7.04), 2.90 - 2.70 ( m, 2H), 2.38 (s, 3H), 1.42 - 1.35 (m, 6H).

[0105] e). 3-[2-(3-Fluoro-phenyl)-ethyl]-2-(2-hydroxy-phenyl)-5-isopropyl-6-methyl- 3/-/-pyrimidin-4-one

[0106] Boron tribromide (1M in CH₂CI₂, 0.0057 mol) was added to a cold (-78⁰C) solution of 3-[2-(3-fluoro-phenyl)-ethyl]-5-isopropyl-2-(2-methoxy-phenyl)-6-methyl-3H- pyrimidin-4-one of Example 1d (0.73 g, 0.0019 mol) in CH₂CI₂ 25 ml_. The reaction mixture was stirred at room temperature overnight. Some CH₂CI₂ 100 mL was added followed by H₂O 100ml. The organic extracts were separated, washed with brine and dried over MgSO₄. After concentration on a rotary evaporator, the product was purified by column chromatography over silica gel (200-400 mesh) eluting with 20% EtOAc- hexanes to give 3-[2-(3-fluoro-phenyl)-ethyl]-2-(2-hydroxy-phenyl)-5-isopropyl-6-methyl-

3H-pyrimidin-4-one (0.702 g, 99%) as a white solid (m. p. 114-118°C).

[0107] ¹H NMR (CDCI₃) : δ 7.36 - 7.28 (m, 1 H), 7.25 - 7.23 ( m, 1 H), 7.18 - 7.11 (m,

1 H), 6.99 - 6.93 (m, 2H), 6.92 - 6.83 (m, 1H), 6.72 (d, 1 H, J = 7.59), 6.62 (d, 1 H, J

=9.78), 4.5 - 4.3 (m, 1H), 3.92 - 3.78 (m, 1 H), 2.60 - 2.38 (m, 2H), 2.32 (s, 3H), 2.10 -

1.92 (m, 1 H), 1.08 -0.9 (m, 6H).

[0108] HPLC (20-80% ACN/H₂O): > 97%.

[0109] MS (ESI⁺) (+ 0.1% HCOOH): 367.1 [C₂₂H₂₃FN₂O₂+1]⁺ (m/z).

Example 2

Preparation of 3-f2-(3-Fluorophenyl)-ethyll-5-isopropyl-2-(3-fluoro-2-methoxyphenyl)-6- methvl-3H-pyrimidin-4-one

[0110] Utilizing the procedures described in Example 1a-d except substituting 3- fluoro-2-methoxybenzoyl chloride for anisoyl chloride in step 1c, the title compound was prepared as yellow thick oil which was purified by MPLC (diethyl ether-hexanes, gradient 0-50%) to give the final product as a white solid (6.50 g, 81%).

¹H NMR (CDCI₃): δ 7.26-7.02 (m, 3H), 6.90-6.78 (m, 2H), 6.66 (d, 7.8 Hz, 1H), 6.60-6.50 (m, 1H), 4.30-4.18 (m, 1H), 3.91 (d, 2.4 Hz, 3H), 3.72-3.56 (m, 1H), 3.17 (m, 1H), 2.94- 2.74 (m, 2H), 2.37 (s, 3H), 1.44 -1.36 (m, 6H).

Example 3

Preparation of 3-f2-(3-Fluoro-phenyl)-ethyll-2-(3-fluoro-2-hvdroxyphenvπ-5-isopropyl-6- methvl-3H-pvrimidin-4-one

[0111] Utilizing the procedures described in Example 1e except substituting 3-[2-(3- fluorophenyl)-ethyl]-5-isopropyl-2-(3-fluoro-2-methoxyphenyl)-6-methyl-3H-pyrimidin-4- one for 3-[2-(3-fluoro-phenyl)-ethyl]-5-isopropyl-2-(2-methoxy-phenyl)-6-methyl-3H- pyrimidin-4-one, the title compound was prepared as a white solid.

[0112] ¹H NMR (CDCI₃): δ 7.18-7.00 (m, 2H), 6.92-6.82 (m, 3H), 6.70-6.62(m, 1H),

6.60-6.50 (m, 1H), 4.16-4.02 (m, 2H)₁ 3.08 (m, 1H), 2.94-2.80 (m, 2H), 2.25 (s, 3H), 1.37

(d, 7.2 Hz, 6H).

Example 4

Preparation of 3-r2-(3-Fluoro-phenyl)-ethyll-2-(2-hvdroxy-phenyl)-5-isobutyl-6-methyl-

3H-pvrimidin-4-one

[0113] Utilizing the procedures described in Example 1a-e except substituting 2- acetyl-4-methyl-pentanoic acid methyl ester for 2-acetyl-3-methyl-butyric acid methyl ester in step 1b, the title compound was prepared as off-white solid (1.59g, 83%).

[0114] ¹H NMR (CDCI₃): δ 7.40 (t, 1H, J = 1.02), 7.25 (d, 1 H, J = 1.49), 7.18 - 7.09

(m, 1 H)₁ 6.95 (d, 2H, J = 7.81), 6.92 - 6.82 (m, 1H), 6.71 (d, 1 H, J = 7.63), 6.61 (d, 1 H, J

= 7.0), 4.32 (m, 2H), 2.92 (t, 2H, J = 7.26), 2.45 (d, 2H, J = 7.24), 2.40 (s, 3H), 2.01 (m,

1 H), 0.98 (m, 6H).

[0115] HPLC (20-80% ACN/H₂O): > 98%.

[0116] MS (ESI⁺) (+ 0.1% HCOOH): 381.2 [C₂₃H₂₅FN₂O₂+^^* (m/z).

Example 5

Preparation of 2,2-dimethyl-propionic acid 2-{1-r2-(3-fluoro-phenyl)-ethyll-5- isobutyl-4-methyl-6-oxo-1,6-dihvdro-pyrimidin-2-yl}-phenvl ester

[0117] To a solution of 3-[2-(3-fluoro-phenyl)-ethyl]-2-(2-hydroxy-phenyl)-5-isobutyl- 6-methyl-3/-/-pyrimidin-4-one of Example 4 (0.3 g, 0.78 mmol) in dry pyridine (2.3 ml) was added pivaloyl chloride (0.14 ml, 1.5 mmol). The reaction mixture was stirred at 50⁰C under argon for 2 h and diluted with dichloromethane (50 ml). The organic layer was washed with water (50 ml) and concentrated under reduced pressure. The crude material was co-evaporated with toluene (3 x 50 ml) and purified by flash chromatography on silica gel, eluting with cyclohexane - ethyl acetate (9:1) to give 2,2- dimethyl-propionic acid 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-4-methyl-6-oxo-1 ,6- dihydro-pyrimidin-2-yl}-phenyl ester (0.25 g, 68%) as a colorless oil. [0118] ¹H NMR (DMSO-de): δ 7.60 - 7.58 (m, 1H), 7.35 - 7.25 (m, 4H)₁ 7.00 -6.99 (m, 1 H)₁ 6.64 - 6.57 (m, 2H), 4.11 - 4.06 (m, 1H), 3.57 - 3.53 (m, 1 H), 2.80 - 2.75 (m, 2H), 2.49 - 2.42 (m, 1 H), 2.32 - 2.25 (m 1H), 2.19 (s, 3H), 1.88 - 1.84 (m, 1H), 1.03 (s, 9H),

0.90 - 0.85 (m, 6H).

[0119] HPLC (isocratic 80% ACN/H₂O): > 95%.

[0120] MS (ESI⁺) (+ 0.1% HCOOH): 465.1 [C₂₈H₃₃FN₂O₃+! ]⁺ (m/z).

Example 6

Preparation of 3,3-dimethyl-butyric acid 2~f1-[2-(3-fluoro-phenyl)-ethvπ-5-isobutyl- 4-methyl-6-oxo-1 ,6-dihvdro-pyrimidin-2-yl}-phenyl ester

[0121] Utilizing the procedure described in Example 5 except substituting tert- butylacetyl chloride for pivaloyl chloride, the title compound was prepared as a colorless oil after purification by flash chromatography on silica gel eluting with cyclohexane - ethyl acetate (9:1).

[0122] ¹H NMR (DMSO-d₆): δ 7.59 - 7.58 (m, 1 H), 7.36 - 7.22 (m, 4H), 6.99 -6.98 (m,

1H), 6.66 - 6.58 (m, 2H), 4.11 - 4.01 (m, 1H), 3.57 - 3.52 (m, 1H), 2.79 - 2.77 (m, 2H),

2.49 - 2.31 (m, 2H), 2.27 (d, 2H, J = 2.2), 2.18 (s, 3H), 1.93 - 1.82 (m, 1 H), 0.91 - 0.87

(m, 6H), 0.81 (s, 9H).

[0123] HPLC (isocratic 90% ACN/H₂O): > 95%.

[0124] MS (ESI⁺) (+ 0.1% HCOOH): 479.0 [C₂₉H₃₅FN₂O₃+!]^"" (m/z).

Example 7

Preparation of isobutyric acid 2-(1-[2-(3-fluoro-phenyl)-ethvπ-5-isobutyl-4-methyl- 6-OXO-1 ,6-dihvdro-pyrimidin-2-yl)-phenyl ester

[0125] Utilizing the procedure described in Example 5 except substituting isobutyryl chloride for pivaloyl chloride, the title compound was prepared as a colorless oil after purification by flash chromatography on silica gel eluting with cyclohexane - ethyl acetate

(9:1).

[0126] ¹H NMR (DMSO-d₆): δ 7.59 - 7.55 (m, 1 H), 7.38 - 7.21 (m, 4H), 7.02 -6.99 (m,

1 H), 6.65 - 6.58 (m, 2H), 4.12 - 4.09 (m, 1 H), 3.61 - 3.57 (m, 1 H), 2.80 - 2.77 (m, 2H),

2.64 - 2.60 (m,1 H), 2.48 - 2.26 (m, 2H), 2.19 (s, 3H), 1.93 - 1.86 (m, 1 H), 0.96 - 0.85 (m,

12H).

[0127] HPLC (isocratic 90% ACN/H₂O): > 99%.

[0128] MS (ESI⁺) (+ 0.1 % HCOOH): 451.0 [C₂₇H₃₁ FN₂O₃+ 1]⁺ (m/z).

Example 8

Preparation of 2,2-dimethyl-butyric acid 2-{1-f2-(3-fluoro-phenyl)-ethvn-5-isobutyl- 4-methyl-6-oxo-1 ,6-dihydro-pyrimidin-2-vl}-phenyl ester

[0129] To a solution of butyric acid (0.015 g, 0.13 mmol) in chloroform (1 ml) was added dicyclohexyl-carbodiimide (0.027 g, 0.13 mmol) and dimethylaminopyridine (0.006 g, 0.052 mmol). The reaction mixture was stirred for one hour at room temperature, then 3-[2-(3-fluoro-phenyl)-ethyl]-2-(2-hydroxy-phenyl)-5-isobutyl-6-methyl-3H-pyrimidin-4- one of Example 4 (0.050 g, 0.13 mmol) was added and the mixture was stirred for one day before addition of water (5 ml). The aqueous layer was extracted with ethyl acetate (10 ml), the organic layers were combined, dried over magnesium sulphate and concentrated under reduced pressure. The resulting crude material was subjected to silica gel column chromatography with a stepwise gradient of ethyl acetate (20-50%) in cyclohexane to afford 2,2-dimethyl-butyric acid 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl- 4-methyl-6-oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyl ester (0.101 g, 41%) as a colorless oil. [0130] ¹H NMR (CDCI₃): δ 7.55 - 7.49 (m, 1 H), 7.30 - 7.09 (m, 4H), 6.90 -6.84 (m, 1H), 6.67 (d, 1H, J = 7.6), 6.59 - 6.54 (dt, 1H, J = 9.4, 2.0), 4.28 - 4.19 (m, 1 H), 3.72 - 3.62 (m, 1H), 2.90 - 2.82 (m, 2H), 2.61 - 2.54 (m, 1 H), 2.44 - 2.38 (m 1 H)₁ 2.32 (s, 3H), 2.03 - 1.96 (m, 1 H), 1.50 (q, 2H, J = 7.7), 1.08 (s, 6H), 1.00 - 0.95 (m, 6H), 0.77 (t, 3H, J

= 7.5).

[0131] HPLC (isocratic 90% ACN/H₂O): > 95%.

[0132] MS (ESI⁺) (+ 0.1 % HCOOH): 479.1 [C₂₅H₃₅FN₂O₃+1]⁺ (m/z).

Example 9

Preparation of (S)-2-methyl-butyric acid 2-(1-r2-(3-fluoro-phenvO-ethyri-5-isobutyl- 4-methyl-6-oxo-1 ,6-dihvdro-pyrimidin-2-yl)-phenyl ester

[0133] To a solution of (S)-(⁺)-2-methylbutyric acid (0.070 g, 0.68 mmol) in chloroform (3 ml) was added dicyclohexyl carbodiimide (0.141 g, 0.68 mmol) and dimethylaminopyridine (0.026 g, 0.21 mmol). The reaction mixture was stirred for 4 h at room temperature, and 3-[2-(3-fluoro-phenyl)-ethyl]-2-(2-hydroxy-phenyl)-5-isobutyl-6- methyl-3/-/-pyrimidin-4-one of Example 4 (0.2 g, 0.52 mmol) was added and the mixture was stirred overnight before addition of water (5 ml). The aqueous layer was extracted with ethyl acetate (10 ml), the organic layer was dried over magnesium sulphate and concentrated under reduced pressure. The resulting crude material was subjected to silica gel column chromatography using cyclohexane-ethyl acetate (8:2) as eluent to afford (S)-2-methyl-butyric acid 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-4-methyl-6- oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyl ester (0.056 g, 23%) as colorless oil. [0134] ¹H NMR (CDCI₃): δ 7.55 - 7.49 (m, 1 H)₁ 7.30 - 7.10 (m, 4H), 6.90 -6.84 (m, 1 H), 6.69 - 6.66 (m, 1H), 6.59 - 6.54 (m, 1H₁), 4.26 - 4.19 (m, 1H), 3.73 - 3.66 (m, 1H), 2.91 - 2.84 (m, 2H), 2.61 - 2.49 (m, 1H), 2.46 - 2.42 (m 2H), 2.32 (s, 3H), 2.03 - 1.96 (m, 1 H), 1.38 (m, 2H₁) 1.11 - 0.80 (m, 12H). [0135] HPLC (isocratic 90% ACN/H₂O): > 95%. [0136] MS (ESI⁺) (+ 0.1% HCOOH): 465.1 [C₂₈H₃₃FN₂O₃+1]⁺ (m/z). Example 10

Preparation of carbonic acid 2-{1-[2-(3-fluoro-phenyl)-ethvn-5-isobutyl-4-nnethyl-6- oxo-1 , 6-dihvdro-pyrimidin-2-yl)-phenyl ester isopropyl ester

[0137] To a solution of 3-[2-(3-fluoro-phenyl)-ethyl]-2-(2-hydroxy-phenyl)-5-isobutyl- 6-methyl-3H-pyrimidin-4-one of Example 4 (0.2 g, 0.52 mmol) in THF (4 ml) were added triethylamine (0.080 ml, 0.57 mmol) and isopropyl chloroformate (1.0 M solution in toluene) (0.58 ml, 0.57 mmol). The reaction mixture was stirred for 1.5 h at room temperature before addition of water (20 ml) followed by addition of ethyl acetate (20 ml). The organic layer was washed with brine (20 ml), dried over anhydrous sodium sulphate and concentrated under reduced pressure. The resulting crude material was purified by silica gel column chromatography using cyclohexane-ethyl acetate (8:2) as eluent to afford the carbonic acid 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-4-methyl-6- oxo-1,6-dihydro-pyrimidin-2-yl}-phenyl ester isopropyl ester (0.202 g, 82%). [0138] ¹H NMR (CDCI₃): δ 7.52 (m, 1 H), 7.35 - 7.27 (m, 2H), 7.18 - 7.09 (m, 2H), 6.85 (td, 1H, J = 2.2, 8.3), 6.85 (d, 1 H, J = 7.5), 6.56 (d, 1 H, J = 9.8), 4.84 (sept, 1 H, J= 6.2), 4.28 - 4.11 (m, 1 H), 3.83 - 3.68 (m, 1 H), 2.92 - 2.83 (m, 2H), 2.48 (d, 2H₁ J = 7.3), 2.31 (s, 3H), 2.13 - 1.98 (m, 1 H), 1.25 (br s, 6H), 0.98 (d, 6H, J = 6.6). [0139] HPLC (isocratic 90% ACN/H₂O): > 99%. [0140] MS (ESI⁺) (+ 0.1% HCOOH): 467.1 [C₂₇H₃₁ FN₂O₄+1]⁺ (m/z).

Example 11

Preparation of carbonic acid ethyl ester 2-(H2-(3-fluoro-phenyl)-e.thyl1-5-isobutyl- 4-methyl-6-oxo-1 ,6-dihvdro-pyrimidin-2-vl>-phenyl ester

[0141] Utilizing the procedure described in Example 10 except substituting ethyl chloroformate for isopropyl chloroformate, the title compound was prepared as a colorless oil after purification by flash chromatography on silica gel eluting with cyclohexane - ethyl acetate (85:15).

[0142] ¹H NMR (CDCI₃): δ 7.60 - 7.45 (m, 1H), 7.40 - 7.30 (m, 2H), 7.20 - 7.08 (m,

2H), 6.95 - 6.80 (m, 1 H), 6.75 - 6.65 (m, 1H), 6.62 - 6.52 (m, 1H), 4.33 - 4.15 (m, 3H),

3.85 - 3.65 (m, 1 H), 2.95 - 2.80 (m, 2H), 2.55 - 2.45 (m, 2H), 2.35 (s, 3H), 2.15 - 1.95 (m,

1 H), 1.3 (s, 3H), 1.05 - 0.91 (m, 6H).

[0143] HPLC (gradient 20-80% ACN/H₂O): > 98%.

[0144] MS (ESI⁺) (+ 0.1% HCOOH): 453.3 [C₂₆H₂₉FN₂(VI]⁺ (m/z).

Example 12

Preparation of carbonic acid ethyl ester 2-{1-[2-(3-fluoro-phenyl)-ethvn-5- isopropyl-4-methyl-6-oxo-1,6-dihvdro-pyrimidin-2-yiy-phenvl ester

[0145] Utilizing the procedure described in Example 10 except substituting 3-[2-(3- fluoro-phenyl)-ethyl]-2-(2-hydroxy-phenyl)-5-isopropyl-6-methyl-3/-/-pyrimidin-4-one for 3- [2-(3-fluoro-phenyl)-ethyl]-2-(2-hydroxy-phenyl)-5-isobutyl-6-methyl-3/-/-pyrimidin-4-one, the title compound was prepared was prepared as a colorless oil after purification by flash chromatography on silica gel eluting with cyclohexane - ethyl acetate (85:15). [0146] ¹H NMR (CDCI₃): δ 7.58 - 7.50 (m, 1 H), 7.38 - 7.28 (m, 2H), 7.20 - 7.11 (m , 2H), 6.92 - 6.83 (td, 1 H, J = 8.4, 2.4), 6.68 (d, 1 H, J = 7.6), 6.60 - 6.53 (d, 1 H, J = 9.7), 4.26 - 4.15 (q, 3H, J = 7.11), 3.80 - 3.65 (m, 1 H), 3.25 - 3.10 (m, 1 H), 2.92 - 2.82 (m,

2H), 2.35 (s, 3H), 1.48 (m, 6H), 1.25 (t, 3H, J = 7.14).

[0147] HPLC (gradient 20-80% ACN/H₂O): > 95%.

[0148] MS (ESI⁺) (+ 0.1 % HCOOH): 439.2 [C₂₉H₂₇N₂O₄+ 1]⁺ (m/z).

Example 13

Preparation of carbonic acid fe/t-butyl ester 2-{1-f2-(3-fluoro-phenvD-ethyl1-5- isobutyl-4-methyl-6-oxo-1 ,6-dihvdro-pyrimidin-2-yl}-phenvl ester

[0149] To a solution of 3-[2-(3-fluoro-phenyl)-ethyl]-2-(2-hydroxy-phenyl)-5-isobutyl- 6-methyl-3l-pyrimidin-4-one of Example 4 (0.1 g, 0.26 mmol) in dichloromethane (2 ml) were added dimethylaminopyridine (0.032 g, 0.26 mmol)) and di-tert-butyl dicarbonate (0.063 g, 0.29 mmol). The reaction mixture was stirred for 2 hours at room temperature before dilution with dichloromethane (20 mL). The organic layer was washed with water (20 mL), dried over anhydrous sodium sulphate and concentrated under reduced pressure. The resulting crude material was subjected to silica gel column chromatography with a stepwise gradient of ethyl acetate (20-30%) in cyclohexane to afford carbonic acid tert-butyl ester 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-4-methyl-6- oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyl ester (0.092 g, 73%).

[0150] ¹H NMR (CDCI₃): δ 7.55 - 7.49 (m, 1H), 7.33 - 7.28 (m, 2H), 7.18 - 7.09 (m, 2H), 6.86 (td, 1 H₁ J = 2.6, 8.2), 6.72 (d, 1 H, J = 7.5), 6.58 (d, 1 H, J = 9.4), 4.26 - 4.15 (m, 1 H), 3.77 - 3.67 (m, 1H), 2.91 - 2.85 (m, 2H), 2.51 - 2.47 (m, 2H), 2.32 (s, 3H), 2.07 - 1.96 (m, 1 H), 1.42 (s, 9H), 0.99 (d, 6H, J = 6.6). [0151] HPLC (isocratic 90% ACN/H₂O): > 95%. [0152] MS (ESI⁺) (+ 0.1% HCOOH): 481.1 [C₂₈H₃₃FN₂O₄+ 1]⁺ (m/z).

Example 14

Preparation of 3-r2-(3-Fluoro-phenyl)-ethyll-5-isobutyl-6-methyl-2-(2-nitro-phenvπ-3H- pyrimidin-4-one

[0153] Utilizing the procedures described in Example 1 a-e except substituting 2- nitrobenzoyl chloride for anisoyl chloride in step 1c, the title compound was obtained as a light yellow oil (2.21 g, 64%).

[0154] ¹H NMR (CDCI₃): δ 8.22 - 8.19 (m, 1 H), 7.66 - 7.62 (m, 2H), 7.14 - 7.02 (m,

2H), 6.88 (td, W, J₁ = 8.48, J₂ = 2.45), 6.69 (d, 1 H, J = 7.73), 6.54 (d, 1 H, J = 9.41 ), 4.22

- 4.09 (m, 1H), 3.52 - 3.35 (m, 1 H), 2.93 - 2.80 (m, 2H), 2.51 (d, 2H, J = 7.34), 2.29 (s,

3H), 2.08 - 1.91 (m, 1 H), 1.00 (d, 6H, J = 6.60).

[0155] MS (ESI⁺) (+ 0.1 % HCOOH): 410.2 [C₂₃H₂₄FN₃O₃+! ]⁺ (m/z).

Example 15

Preparation of 2-(2-amino-phenyl)-3f2-(3-fluoro-phenyl)-ethvn-5isobutyl-6-methyl-3H- pyrimidin-4-one

[0156] To a solution of 3-[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-6-methyl-2-(2-nitro- phenyl)-3H-pyrimidin-4-one (2.2 g, 0.00537 mol) of Example 13 in MeOH (50 mL) was added Pd/C 10% (0.220 g). The reaction mixture was stirred under H₂ atmosphere for 48 h, filtrated through a Celite® pad and concentrated in vacuum. The product was purified by column chromatography over silica gel (200-400 mesh) eluting with 15% EtOAc- cyclohexanes to give 2-(2-amino-phenyl)-3[2-(3-fluoro-phenyl)-ethyl]-5isobutyl-6-methyl- 3/-/-pyrimidin-4-one as a light yellow oil (1.3 g, 64%).

[0157] ¹H NMR (CDCI₃): δ 7.31 - 7.28 (m, 1 H), 7.17 - 7.09 (m, 1H), 7.05 (dd, 1 H, J, = 1.41 , J₂ = 7.63), 6.87 - 6.78 (m, 3H), 6.69 (d, 1H₁ J = 7.54), 6.56 (d, 1H₁ J = 9.23), 4.11 - 4.06 (m, 2H), 3.88 (broad s, 2H), 2.87 - 2.81 (m, 2H), 2.47 (d, 2H₁ J = 7.16), 2.34 (s, 3H), 2.09 - 1.95 (m, 1 H), 0.98 (d, 6H, J = 6.59) [0158] HPLC (20-80% ACN/H₂O): > 95%.

[0159] MS (ESI⁺) (+ 0.1% HCOOH): 380.1 [C₂₃H₂₆FN₃OH-I]⁺ (m/z).

Example 16

Preparation of /V-(2-{1-f2-(3-fluoro-phenyl)-ethvπ-5-isobutyl-4-methyl-6-oxo-1 ,6- dihvdropyrimidin-2-yll-phenyl)-methanesulfonamide

[0160] To a solution of 2-(2-amino-phenyl)-3[2-(3-fluoro-phenyl)-ethyl]-5isobutyl-6- methyl-3H-pyrimidin-4-one (0.25 g, 0.00066 mol) of Example 14 in dry pyridine (7 ml_) was added sulfonylchloride (0.056 ml_, 0.00073 mol). The reaction mixture was stirred at room temperature under argon for 2 days then concentrated under reduced pressure. The crude material was co-evaporated with toluene (3 x 50 ml) and was purified by column chromatography over silica gel (200-400 mesh) eluting with 25% EtOAc- cyclohexanes to give Λ/-(2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-4-methyl-6-oxo-1 ,6- dihydropyrimidin-2-yl}-phenyl)methanesulfonamide as a white powder (0.229 g, 76%). [0161] ¹H NMR (CDCI₃): δ 7.66 - 7.63 (m, 1 H), 7.54 - 7.48 (m, 2H)₁ 7.26 - 7.13 (m, 3H), 6.87 ( td, AH₁ J₁ = 8.47, J₂ = 2.45), 6.69 (d, 1 H, J = 7.53), 6.60 (d, 1H, J = 9.61), 4.10 - 4.05 (m, 2H), 3.05 (s, 3H), 2.91 - 2.82 (m, 2H), 2.48 (d, 2H, J = 7.16), 2.31 (s, 3H), 2.10 - 1.96 (m, 1H), 0.99 (d, 6H₁J = 6.59) [0162] HPLC (20-80% ACN/H₂O): > 95%. [0163] MS (ESI⁺) (+ 0.1% HCOOH): 458.0 [C₂₄H₂₈FN₃O₃-H]⁺ (m/z).

Example 17

Preparation of phosphoric acid dibenzyl ester 2-{1-[2-(3-fluoro-phenyl)-ethvn-5 isopropyl- 4-methyl-6-oxo-1 ^-dihydro-pyrimidin^-vD-phenvl ester

[0164] Carbon tetrachloride (0.278 ml_, 0.00287 mol), /-Pr₂EtN (0.200 mL, 0.00115 mol) and DMAP (0.007 g, 0.000057 mol) were added to a cold (-10⁰C) solution of 3-[2- (3-fluoro-phenyl)-ethyl]-2-(2-hydroxy-phenyl)-5-isopropyl-6-methyl-3H-pyrimidin-4-one of Example 1e in CH₃CN (3 mL) followed by addition of dibenzylphosphite (0.127 mL, 0.00057 mol). The reaction mixture was stirred at room temperature for 2 hours. KH₂PO₄ (0.5M, 20 mL) was added to the reaction mixture followed by extraction with EtOAc (50 mL). The organic extract was dried over MgSO₄ and concentrated in vacuum. The resulting crude material was subjected to a silica gel column chromatography using 20% EtOAc-cyclohexane as eluent to afford the title compound as an off-white solid (0.194 g, 57%).

[0165] HPLC (isocratic 80% ACN/H₂O): > 94%. [0166] MS (ESI⁺) (+ 0.1% HCOOH): 627.3 [C₃₆H₃₆FN₂O₅P+1]⁺ (m/z).

Example 18

Preparation of phosphoric acid dibenzyl ester 2-(1-f2-(3-fluoro-phenyl)-ethvπ-5-isobutyl-4 methyl-6-oxo-1 ,6-dihvdro-pyrimidin-2-vl|-phenvl ester

[0167] Utilizing the procedure described in Example 17 except substituting 3-[2-(3- fluoro-phenyl)-ethyl]-2-(2-hydroxy-phenyl)-5-isobutyl-6-methyl-3H-pyrimidin-4-one of Example 4 for 3-[2-(3-fluoro-phenyl)-ethyl]-2-(2-hydroxy-phenyl)-5-isopropyl-6-methyl- 3H-pyrimidin-4-one of Example 1e, the title compound was obtained as a beige solid (0.312 g, 62%) after a silica gel column chromatography using 20% EtOAc-cyclohexane. [0168] ¹H NMR (CDCI₃): δ 7.55 - 7.40 (m, 2H), 7.35 (s, 1 H), 7.30 - 7.25 (m, 5H), 7.24 - 7.10 (m, 6H), 7.03 (d, 1 H, J = 7.59), 6.88 (td, 1 H, J = 8.47, 2.49), 6.62 (d, 1 H, J = 7.61), 6.45 (dt, 1H, J = 9.45, 2.09), 5.0 - 4.85 (m, 4H), 4.35 - 4.22 (m, 1 H), 3.65 - 3.52

(m, 1 H), 2.9 - 2.75 (m, 2H), 2.52 - 2.35 ( m, 2H), 2.27 (s, 3H), 2.08 - 1.90 (m, 1 H), 1.00 -

0.85 (m, 6H).

[0169] HPLC (isocratic 80% ACN/H₂O): > 93%.

[0170] MS (ESI⁺) (+ 0.1 % HCOOH): 641.3 [C₃₇H₃₈FN₂O₅P+1]⁺ (m/z).

Example 19

Preparation of phosphoric acid mono-(2-{1-r2-(3-fluoro-phenyl)-ethvπ-5 isopropyl-4- methyl-6-oxo-1.6-dihydro-pyrimidin-2-yl)-phenyl) ester

[0171] To a solution of phosphoric acid dibenzyl ester 2-{1-[2-(3-fluoro-phenyl)-ethyl]- 5 isopropyl-4-methyl-6-oxo-1,6-dihydro-pyrimidin-2-yl}-phenyl ester of Example 17 (0.194 g, 0.00031 mol) in MeOH (5 mL) was added Pd/C 10% (0.194 g). The reaction mixture was stirred under H₂ atmosphere for 3 h, filtered off through a Celite^® pad and concentrated in vacuum to yield the title compound as a white solid (0.119 g, 86%) with m.p. 99-101⁰C.

[0172] ¹H NMR (DMSO-Qf₆): δ 7.70 - 7.60 (m, 1H), 7.50 - 7.35 (m, 1H), 7.30 - 7.15 (m, 1H)₁ 7.05 - 6.92 (m, 3H), 6.75 - 6.55 (m, 2H), 4.32 - 4.15 (m, 1H), 3.75 - 3.60 (m, 1 H), 3.22 - 3.0 (m, 1 H), 2.95 - 2.75 (m, 3H), 2.40 - 2.20 (s, 3H), 1.45 - 1.22 (m, 6H). [0173] HPLC (20-80% ACN/H₂O): > 95%. [0174] MS (ESI⁺) (+ 0.1% HCOOH): 447.1 [C₂₂H₂₄FN₂O₅P+^ (m/z).

Example 20

Preparation of phosphoric acid mono-(2-{1 -[2-(3-fluoro-phenyl)-ethyfl-5-isobutyl-4- methyl-6-oxo-1 ,6-dihvdro-pyrimidin-2-vl>-phenvl) ester

[0175] Utilizing the procedure described in Example 19 except substituting phosphoric acid dibenzyl ester 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-4 methyl-6-oxo-

1,6-dihydro-pyrimidin-2-yl}-phenyl ester of Example 18 for phosphoric acid dibenzyl ester

2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isopropyl-4-methyl-6-oxo-1 ,6-dihydro-pyrimidin-2-yl}- phenyl ester of Example 17, the title compound was obtained as a white solid (0.196 g,

87%).

[0176] ¹H NMR (CH₃OH-Of₄): δ 7.7 (s, 1H), 7.51 (s, 1H), 7.21 - 7.10 (m, 2H), 7.06 -

6.98 (m, 1H), 6.90 (t, 1 H, J = 7.57), 6.66 (d, 1 H, J = 7.31), 6.55 (d, 1 H, J = 10.17), 4.50 -

4.40 (m, 1 H), 4.92 - 4.80( m, 1 H), 2.90 (m, 2H), 2.60 - 2.40 (m, 2H), 2.32 (s, 3H), 2.10 -

1.93 (m, 1 H), 1.00 (m, 6H).

[0177] HPLC (20-80% ACN/H₂O): > 97%.

[0178] MS (ESI⁺) (+ 0.1 % HCOOH): 461.1 [C₂₃H₂₆FN₂O₅P+^ (m/z).

Example 21

Preparation of phosphoric acid mono-(2-{1-r2-(3-fluoro-phenyl)-ethyl1-5 isopropyl-4- methyl-6-oxo-1 ,6-dihvdro-pyrimidin-2-yl}-phenyl) ester disodium salt

[0179] Phosphoric acid mono-(2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-4~methyl-6- oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyl) ester of Example 19 (0.200 g, 0.00045 mol) was dissolved in 20 mL of anhydrous methanol. Sodium methoxide (0.048 g, 0.0009 mol) was added in one portion to this solution. The resulting mixture was stirred at room temperature for 2 h. Concentration under reduced pressure followed by recrystallization from water/acetone gave the disodium salt as a white powder (0.200 g, 91%). [0180] ¹H NMR (DMSO-dβ): δ 7.80 - 7.75 (m, 1 H), 7.15 -6.82 (m, 2H), 6.75 - 6.64 (m, 1H), 6.58 - 6.40 (m, 2H), 6.38 - 6.22 (m, 2H), 4.25 - 4.10 (m, 1 H), 3.60 - 3.40 (m, 1 H), 3.12 - 2.85 (m, 1 H), 2.55 - 2.45 (m, 2H), 2.30 (br s, 3H), 1.18 - 0.98 (m, 6H).

Example 22

Preparation of phosphoric acid diethyl ester 2-{1-r2-(3-fluoro-phenvO-ethvπ-5 isopropyl-4- methyl-6-oxo-1 ,6-dihvdro-pyrimidin-2-vl}-phenyl ester

[0181] Phosphoric acid mono-(2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isopropyll-4-methyl- 6-OXO-1 ,6-dihydro-pyrimidin-2-yl}-phenyl) ester disodium salt (0.100 g, 0.00020 mol) was dissolved in 20 mL of anhydrous DMF. Ethyl iodide (0.098 ml_, 0.0012 mol) was added in one portion to this solution. The resulting mixture was stirred and heated at 80 ⁰C for 4 h. Cooled, diluted with ethyl acetate (100 mL), washed with water, dried and concentrated. Column chromatography over silica gel using 50% EtOAc/Hexane afforded the title compound as a colorless oil, 0.08 g (78%)

[0182] ¹H NMR (CDCI₃): δ 7.51-7.34 (m, 2H), 7.22-6.92 (m, 3H), 6.86-6.74 (m, 1 H), 6.62-6.42 (m, 2H), 4.38-4.21 (m, 1H), 4.09-3.85 (m, 4H), 3.61-4.45 (m, 1 H)₁ 3.16-3.02 (m, 1 H), 2.90-2.67 (m, 2H), 2.29 (s, 3H), 1.38-1.05 (m, 12H).

Example 23

Preparation of 5-(1 ,2-dimethylpropyl)-3-r2-(3-fluorophenyl)ethvn-2-(2-hydroxyphenyl)-6- methvl-3A/-pvrimidin-4-one

[0183] a). Methyl 2-acetyl-4-methylpent-2-enoate

[0184] A mixture of methyl acetoacetate (38.84 g, 0.3 mol) and piperidine (0.3 ml_) was cooled down in ice bath, and a mixture of isobutyryl aldehyde (23.80 g, 0.33 mol) and ethanol (0.7 mL) was added in one portion. The mixture was kept at ~5 ⁰C for 48 h, diluted with diethyl ether (150 mL) and washed with water with a few drops of acetic acid (3 x 50 mL). Combined aqueous extracts were extracted with ether (50 mL). Combined ether solutions were washed with brine, dried with anhydrous magnesium sulfate and evaporated to give a pale yellow oil (48.6 g) which was distilled in vacuo. The fraction with boiling point at 50-70 °C/1 mm Hg was collected (17.1 g, 33.5%). GCMS indicated 90% pure mixture of E- and Z- isomers, which was used in the next step without further purification.

[0185] b). Methyl 2-acetyl-3,4-dimethylpentanoate

[0186] Lithium 2-thienylcyanocuprate (0.25M solution in THF, 200 mL, 50 mmol) was cooled to -78⁰C and methyllithium (1.6M solution in diethyl ether, 32 mL, 50 mmol) was added dropwise during 10 min. Cooling bath was removed, mixture allowed to warm to r.t. and cooled again to -78 ⁰C. Methyl 2-acetyl-4-methylpent-2-enoate Example 23a (7.74 Q₁ 45 mmol) was added dropwise during 5 min. and mixture was stirred for 1 h. Mixture was quenched with concentrated ammonium hydroxide (100 mL) mixed with saturated aqueous ammonium chloride solution (200 mL) and then diluted with ether (200 mL). After stirring for 1 h deep blue aqueous layer was separated and extracted with ether (2 x 100 mL). Combined organic extracts were washed twice with brine (100 mL), dried with anhydrous sodium sulfate. Solvent evaporation yielded a deep red oil (8.2 g). It was distilled in vacuo and fraction boiling 70-90 °C/1 mm Hg was collected (6.16 g, 73.5%). Purity by GCMS: 95%.

[0187] c). 5-(1 ,2-Dimethylpropyl)-3-[2-(3-fluorophenyl)ethyl]-2-(2-hydroxyphenyl)-6- methyl-3AV-pyrimidin-4-one

[0188] Utilizing the procedures described in Example 1b-e except substituting methyl 2-acetyl-3,4-dimethylpentanoate of Example 23b for 2-acetyl-3-methyl-butyric acid methyl ester of Example 1a in step 1b, the title compound was prepared as a white solid (4.49 g, 97%).

[0189] ¹H NMR (CDCI₃): δ 9.80 (bs, 1 H), 7.30-7.04 (m, 3H), 6.90-6.80 (m, 1H), 6.94- 6.76 (m, 3H), 6.72-6.62 (m, 1 H), 6.60-6.49 (m, 1 H), 4.28-4.02 (m, 2H), 2.96-2.76 (m, 2H), 2.62-2.24 (m, 2H), 2.29 (s, 3H), 1.32 (d, 6.9 Hz, 3H), 1.03 (d, 6.3 Hz, 3H), 0.78 (d, 6.6 Hz, 3H).

Example 24

Preparation of 3-r2-(3-fluorophenyl)ethvn-2-(2-hvdroxyphenyl)-5-methyl-5, 6,7,8- tetrahvdro-3H-quinazolin-4-one

[0190] a). Ethyl 6-oxocyclohex-1-enecarboxylate

[0191J To a solution of phenylselenenyl chloride (10.10 g, 52.5 mmol) in dichloromethane (800 ml_) cooled to 0 ⁰C, pyridine (4.35 g, 55.0 mmol) was added. In 15 min, ethyl 2-oxocyclohexanecarboxylate (8.51 g, 50 mmol) was added, and the mixture was stirred for 1 h. The resulting pale yellow solution was washed with 1 M HCI (2 x 100 ml_), 1M NaOH (2 x 100 ml_), and again with 1 M HCI (100 mL). The solution was cooled again in an ice bath and 30% hydrogen peroxide (5 x 3 mL) was added in 10 min intervals for each addition while the temperature was maintained below 5 ⁰C. The white suspension was washed with water (200 mL) and saturated aqueous solution of NaHCO₃ (2 x 100 mL), and then dried over anhydrous Mg₂SO₄. Evaporation of the solvent yielded a yellow oil (8.20 g) which was distilled in vacuo to give a fraction with boiling point at 85 °C/1mm Hg (4.94 g, 49.0%) and 97% purity (GC/MS).

[0192] b). Ethyl 2-methyl-6-oxocyclohexanecarboxylate

[0193] Lithium 2-thienylcyanocuprate (0.25M solution in THF, 127.7 mL, 32.0 mmol) was cooled to -78 ⁰C and methyllithium (1.6 M solution in diethyl ether, 20 mL, 32.0 mmol) was added dropwise during 10 min. The mixture was allowed to warm to room temperature and then cooled again to -78 ⁰C. Ethyl 6-oxocyclohex-1-enecarboxylate of Example 24a (4.88 g, 29.0 mmol) was added dropwise during 5 min, and mixture was stirred for 2 h. The mixture was quenched with concentrated ammonium hydroxide (15 mL) mixed with saturated aqueous ammonium chloride solution (300 mL) and then diluted with ether (200 mL). After stirring for 1 h, deep blue aqueous layer was separated and extracted with ether (3 x 100 mL). The combined organic extracts were washed twice with brine (100 mL) and dried over anhydrous Mg₂SO₄. The solvent evaporation yielded a deep red oil (5.0 g), which was distilled in vacuo to give a fraction with boiling point 60-75°C/1 mm Hg (3.31 g, 58.4%) and 94% purity (GC/MS).

[0194] c). 3-[2-(3-Fluorophenyl)ethyl]-2-(2-hydroxyphenyl)-5-methyl-5, 6,7,8- tetrahydro-3/-/-quinazolin-4-one

[0195] Utilizing the procedures described in Example 1b-e except substituting ethyl 2-methyl-6-oxocyclohexanecarboxylate of Example 24b for 2-acetyl-3-m ethyl-butyric acid methyl ester of Example 1a in step 1b, the title compound was prepared as a white solid (2.64 g, 88.3%).

[0196] ¹H NMR (CDCI₃): δ 9.80 (bs, 1 H), 7.26-7.08 (m, 3H), 6.92-6.77 (m, 3H), 6.70- 6.64 (m, 1 H), 6.58-6.51 (m, 1 H), 4.28-4.04 (m, 2H), 3.12-2.74 (m, 3H), 2.66-2.38 (m, 2H), 1.92-1.55 (m, 4H), 1.23 (d, 6.9 Hz, 3H).

Example 25

Preparation of (5>S)-3-[2-(3-fluorophenyl)ethvπ-2-(2-hvdroxyphenyl)-5-methyl-5,6.7,8- tetrahexahvdro-3/-/-quinazolin-4-one

[0197] Utilizing the procedures described in Example 1b-e except substituting methyl (2S)-2-methyl-6-oxocycloxenanecarboxylate for 2-acetyl-3-methyl-butyric acid methyl ester of Example 1a in step 1b, the title compound was prepared as a white solid (0.221 g, 76%) with 99% purity as determined by HPLC, and with 97.7% enantiomeric putity (ee) as determined by chiral HPLC by the following procedure: 20 μL of compound solution (1 mg/mL in 2% of ethanol in hexanes) was injected onto ChiralCel OJ-H (0.46 cm x 25 cm) using 2% ethanol in hexanes at a flow rate of 1 mL/min measuring UV absorbance at 254 nm and 280 nm for Rt(s)eπant. = 24.5 min. and Rtrøenant. = 28.7 min, respectively.

[0198] The biological activity and pharmacokinetic parameters of the compounds of Formula (I) were demonstrated by the following tests: uaicium Keceptor inhibitor Assay

[0199] Calcilytic activity was measured by determining the IC50 of the test compound for blocking increases of intracellular Ca²⁺ elicited by extracellular Ca²⁺ in HEK 293 4.0-7 cells stably expressing the human calcium receptor. HEK 293 4.0-7 cells were constructed as described by Rogers et al., J. Bone Miner. Res. 10 (Suppl. 1),

S483, (1995) (hereby incorporated by reference herein). Intracellular Ca²⁺ increases were elicited by increasing extracellular Ca²⁺ from 1.0 to 1.3 mM. Intracellular Ca²⁺ was measured using fluo-3, a fluorescent calcium indicator (Biotium). [0200] The procedure was as follows: Cells were maintained in DMEM with 10% FBS and 200 μg/ml hygromycin, under 5% CO2 at 37⁰C. At 24-hours prior to analysis, the cells were trypsinized and plated in the above medium at 120,000 cells/well in black sided, clear-bottom, collagen I coated, 96-well plates. Plates were centrifuged at 800 rpm for 2 minutes and incubated under 5% CO2 at 37⁰C overnight. The medium was then aspirated and 80 μL/well of 6 μM fluo-3 in assay buffer was added to the plate. Assay buffer contains 20 mM Na-Hepes, pH 7.4, 126 mM NaCI, 5 mM KCI, 1 mM MgCl2, 1 mM CaCl2, 1 mg/mL D-glucose and 1 mg/mL of bovine serum albumin (BSA; fraction V, ICN).

[0201] Cell-plates containing the fluo-3 solution were incubated in the dark, at room temperature, for 60 minutes. Following incubation plates were washed once, then refilled with 160 μL/well of assay buffer. Measurements of fluorescence were performed using the FLIPR system (Molecular Devices), with a laser setting of 0.8 W and a 0.4 second CCD camera shutter speed. A two-addition protocol was used with a 40-μL addition of buffer or test compound 95 seconds before the addition of extracellular Ca²⁺. The extracellular [Ca²⁺] is increased from 1.0 to 1.3 mM by adding 50 μL of 2.5 mM CaCl2 in assay buffer.

[0202] Calcilytic activity was determined by a compound's ability to block, in a concentration-dependent manner, increases in the concentration of intracellular Ca²⁺ elicited by increases in extracellular Ca²⁺. Fluorescence signals were measured as the peak height of the response and normalized to the response elicited by extracellular

Ca²⁺ in the absence of test compound. All compounds were tested at 8 concentrations in duplicate with the highest concentration being 30 μM. [0203] In general, those compounds having lower IC50 values in the Calcium Receptor Inhibitor Assay are desirable. Compounds useful typically have IC₅₀ values below 30 μM. Some desirable compounds are those having an IC50 of 10 μM or lower.

Other desirable compounds have an IC50 of 1 μM or lower. While yet other desirable compounds have an IC50 of 0.1 μM or lower.

General Protocol for Solubility Determination

[0204] The amount of sample to be weighed for the solubility determination is dependent upon the amount. Large amounts (>10 mg) will allow 2-3 mg to be weighed into 1mL buffer solution. Smaller amounts (1-5 mg) require a smaller amount to be weighed (around 1 mg) into a subsequently reduced amount of buffer solution ( 200-500 μL). If there is enough compound available, a duplicate is prepared. A blank is prepared along side the sample(s) and is to follow the sample(s) throughout the preparation procedure. Samples are weighed into 1.5 ml Axygen tubes.

[0205] The buffer solution is a 0.1 M sodium phosphate solution at a pH of 7.4. To prepare the phosphate buffer solution: weigh out 5.6 g of Dibasic Sodium Phosphate into

400 mL water for a final concentration of 0.1 M; weigh out 1.2 g of Monobasic Sodium

Phosphate into 100 mL water for a final concentration of 0.1 M; pour 81 mL of the dibasic phosphate into a beaker with a magnetic stirring bar; add approximately 25 mL of the monobasic phosphate solution incrementally until a pH of 7.4 is reached. This solution is then filtered through a 0.22-micron nylon filter by vacuum and refrigerated.

[0206] The preparation of Simulated Gastric Fluid (SGF) and Simulated Intestinal

Fluid SIF) is taken from the USP NF #25 and proceeds as follows:

[0207] SGF. Dissolve 2.0 g of sodium chloride and 3.2 g of purified pepsin in 7.0 mL of hydrochloric acid and sufficient water to make 1000 mL. This test solution has a pH of about 1.2.

[0208] SIF. Dissolve 6.8 g of monobasic potassium phosphate in 250 mL of water, mix, and add 77 ml of 0.2N sodium hydroxide and 500 mL of water. Add 10 g of pancreatin, mix, and adjust the resulting solution with 0.2N sodium hydroxide to a pH of

6.8.

[0209] Add the appropriate amount of buffer solution to the Axygen tubes containing the weighed sample. Seal the top of the tube with Paraffin paper. The phosphate buffer samples are placed on a shaker at low speed for 24 hours at ambient temperature. The gastric fluid samples are shaken for 2 hours at ambient temperature. The intestinal fluid samples are shaken for 4 hours at ambient temperature.

[0210] After the shaking is completed, the solid material is separated out by centrifuging once for five minutes at 6,000 Rpm's. The sample is then decanted into a new Axygen tube and centrifuged again for five minutes at 6,000 Rpm's. Follow this procedure for all replicates weighed, including any blanks. It is possible that after two spins the solution is not adequately cleared of solid material and is therefore placed in a Axygen tube with a built in filter and once again centrifuged until all the solution is forced through the filter.

[0211] A seven point standard curve is prepared from the compound and is dissolved into DMSO. The starting stock solution concentration is again dependent upon the amount of compound that is available to be weighed out. Unless otherwise noted, a 1 mM stock solution is prepared by weighing out the equivalent of the molecular weight of that particular compound into an appropriate amount of DMSO to yield a 1 mM final concentration. From this, seven serial dilutions are made to yield the following seven levels; 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, and 200 μM. The final volume of each level is 1mL. The dilutions for the curve are then: Level 7, 200 μl stock/800 μL DMSO; Level 6, 100 μL/900 μL; Level 5, 50 μL/950 μL; Level 4: 20 μL/980 μL. Using level 6 as stock: Level 3, 100 μL/900 μL; Level 2, 50 μL/950 μL; Level 1 , 20 μL/980 μL. [0212] Prior to analysis, both the samples and the seven concentrations for the curve are diluted 1 :1 with acetonitrile. For the case of the gastric fluid samples, due to the low pH, the samples are neutralized with a 0.2M phosphate buffer to a pH of 6.8 to protect the analytical instrument.

[0213] To determine the operating parameters of the instrument for analysis, one point from the curve is chosen and injected onto the instrument with the mass specter conducting a full scan and the PDA analyzing under the full range of wavelengths. From this, a molecular weight, an absorbance spectrum and the retention time of the compound can be determined.

General Protocol for Stressed Stability Determination

[0214] This protocol describes the stability of a compound after being incubated at 37⁰C. Initially, 1 mM stock solution of the compound is prepared in DMSO. Then, from the initial stock solution, a 10 μM solution of the compound is prepared in phosphate buffer solution (yielding a solution of one percent DMSO). The samples are then placed in an oven at 37°C. The phosphate buffer samples are incubated for 24 h, gastric samples for 2 h and intestinal samples incubated for 4 h. After incubation, the samples are analyzed against a fresh 10 μM solution prepared from the original stock solution. Again, for the gastric solution, both the incubated sample and the fresh stock are neutralized before analysis for reasons stated above. The two results are compared by dividing the area (or actual on-column amount) of the incubated sample by the area (or on-column amount) of the freshly prepared solution, and multiplying by 100 to give a result in percent stability. The instrument chromatogram is checked for any degradation products as well.

Caco-2 Assay Procedure

Solutions:

[0215] Basolateral Transport Buffer (pH 7.2 - 7.4): prepare HBSS, 25 mM Glucose,

20 mM Hepes solution; store at 4⁰C for up to 1 month; check / adjust pH on the day of use.

[0216] Apical Transport Buffer (pH 6.0): prepare HBSS, 25 mM Glucose, 6 mM MES

[(2-(A/-morpholino)ethanesulfonic acid] solution; store at 4⁰C for up to 1 month; check / adjust pH on the day of use.

[0217] Compound Stock Solution (1 mM; 100% DMSO): prepare 1 mM stock solution of each compound in 100% DMSO; store at 4⁰C up to 1 week.

[0218] Compound Dosing Solution (10 μM; 1 % DMSO final; 100 μM LY in Apical

Transport Buffer).

[0219] Lucifer Yellow (LY) Dosing Solution (100 mM): prepare 100 mM solution of LY in apical transport buffer, mix thoroughly; transfer 1000 mL aliquots into individual tubes; store at -2O⁰C wrapped in foil (light sensitive) for up to 1 month. On the day of use, thaw appropriate number of tubes; mix thoroughly prior to use.

Plate Preparation:

[0220] 12-well plate (0.500 mL per well; 5 wells per compound; 3.0 mL total): aliquot 0.030 mL (30 mL) of 1 mM stock solution into 5 mL vial; add 2.97 mL LY Dosing Solution; vortex thoroughly; prepare fresh on the day of use.

[0221] 24-well plate (0.100 mL per well; 5 wells per compound; 1.0 mL total): aliquot 0.01 mL (10 mL) of stock solution into 5 mL vial; add 0.990 mL apical transport buffer; vortex thoroughly; prepare fresh on the day of use. Method:

[0222] Prepare Cells. Assays can be run Day 21 - Day 26. Cells are plated at a density of 8X10⁴/well (12 well plate). Check/record TEER values in cell culture media. Also, record Day 21 date and passage number. Need sufficient wells to assay five replicates per compound. TEER values must be 300-600 Ωcm² (330-660Ω for 12 well plate; 1000-2000Ω for 24 well plate). Wash cells twice with apical transport buffer; for second wash, incubate cells for 10 min in CO₂ incubator.

[0223] Run Assay. Add basolateral transport buffer to each basolateral well (1.5 mL for 12-well plate; 0.6 ml_ for 24-well plate). Add Dosing Solution to each apical well in triplicate (0.5 ml_ for 12-well plate; 0.1 mL for 24-well plate). Incubate (with regular shaking) at 37⁰C in the CO₂ incubator for 1 hour.

[0224] Collect Samples. Transfer aliquot of dosing solution to appropriate sample tube (min. vol. is 100 μl_). Transfer basolateral well to appropriate sample tube (12-well = 1.5 mL). Transfer 0.2 mL aliquot into appropriate well in plate for LY analysis (i.e. single sample per well). Wash basolateral well with 1.3 mL ACN. Transfer to sample tube (same tube as above). Transfer apical well to appropriate sample tube (12-well = 0.5 mL). Wash apical well/cells with 0.5 mL ACN. Transfer solution and cells to sample tube (same tube as above)Store samples (11 per compound) at -2O⁰C until analysis. [0225] Test Monolayer Integrity with Lucifer Yellow. Prepare six point standard curve from LY dosing solution ranging from ~0.03 - 10 μM in basolateral transport buffer. (Recommended levels: 10, 3.0, 1.0, 0.3, 0.1 , 0.03 μM). Transfer 200 μL aliquots in duplicate to 96-well plate in first two columns (high (1^st (top) row) to low (6^th row). Transfer 200 μL aliquots of blank basolateral transport buffer to 96-well plate in duplicate for Background Blank into 7^th row. Read fluorescence at 485/530 (excitation/emission). Calculate P_app value for LY in each well (see separate calculation sheets). Acceptance criteria for LY P_app values: < 1.5E-06 cm/sec.

[0226] Analyze Samples. Prepare standard curve of each compound in duplicate. Final concentrations of standards are: 0.02, 0.04, 0.1 , 0.5, 2, 4, and 6 mM. Prepare standards in ACN and Basolateral Transport Buffer. Dilute dosing solution 4x prior to analysis - 50 mL dosing solution + 150 mL 1 :1 ACN:Transport Buffer. Final samples already diluted 2x by Tissue Culture during assay (1 :1 ACN:Transport Buffer). Dilute apical samples another 2x prior to analysis - 100 mL apical sample + 100 mL ACN:Transport Buffer. Analyze basolateral samples without diluting. Vortex all samples vigorously (including standards) and spin for 5 minutes at 10,000 rpm. Transfer aliquot of supernatant to injection vial for analysis by LC/MS. Acceptance criteria for standard curves are: r^ > 0.98; accuracy of calculated concentrations within +/- 20% of expected concentrations; a minimum of 3 levels with maximum number of points dropped < 25%; and lowest level of quantitation (mean peak area) > 3 x blank peak area. Calculate P_app value for each well (see separate calculation sheets). At least three wells per compound must be acceptable, with a %CV < 20%. Calculate Recovery value for each well. Recovery must be between 80% and 120%. Run high and low positive controls with each assay. Positive control mean P_app values must be within 2 ^* SD of historical accumulated means.

Clearance (CL) Assay Procedure

Solutions:

[0227] 0.5 M KPO₄ (pH 7.4) (final incubation concentration = 25 mM): 18.37 g potassium phosphate, monobasic; 63.58 g potassium phosphate, dibasic. Bring to 1000 mL with deionized H₂O. Stir to dissolve. Adjust pH. Store at room temperature for up to

1 year.

[0228] Cofactors (2OX stock solution) (final incubation concentrations = 1.3 mM

NADP+, 2.9 mM G6P, 3.3 mM MgCI₂): 200 mg NADP+; final incubation concentration =

1.3 mM; 200 mg glucose-6-phosphate disodium salt; final incubation concentration = 2.9 mM; 133 mg magnesium chloride hexahydrate; final incubation concentration = 3.3 mM.

Dissolve in about 9 mL deionized H₂O; adjust volume to 10 mL with deionized H₂O.

Store at -2O⁰C up to 6 months.

[0229] Glucose-6-Phosphate Dehydrogenase (G6PDH) (40 U/mL) (final incubation concentration = 0.4 U/mL): add appropriate amount of G6PDH solid to appropriate volume of 5 mM sodium citrate tribasic to achieve 40 units/mL of G6PDH. Stir to dissolve. For 5 mM sodium citrate tribasic, weigh 14.7 mg sodium citrate tribasic to 10 mL deionized H₂O. Store at -2O⁰C for up to 6 months.

[0230] Cofactor/Serial Dilution Buffer: 1.5 mL 0.5 M KPO₄, pH 7.4; 1.5 mL cofactors

(2OX stock); 0.3 mL G6PDH (40 U/mL); 11.7 mL deionized H₂O. Make fresh on the day of use.

[0231] Compound Incubation Solution (4 μM) (final incubation concentration = 1 μM).

Receive compounds as solids, usually varying in weight from 1-10 mg. Make stock solutions in ACN, usually at concentrations between 1-2.5 mM. Prepare 4 μM incubation solution from original compound stock. Dilute in deionized H₂O (final ACN incubation concentration is <1 %). Store at 4⁰C. [0232] Liver Microsomes (2 mg/mL) (final incubation concentration = 0.5 mg/mL). Prepare 2 mg/mL solution of human and rat liver microsomes in deionized H₂O from original 20 mg/mL microsomal stock solution. Store at -2O⁰C.

Method

[0233] Run Assay. Add 25 μL compound incubation solution (4 μM). Add 25 μL liver microsomes (2 mg/mL). Pre-warm compound and microsomes at 37⁰C for 10 min. Initiate reaction with addition of 50 μL Cofactor/Serial Dilution Buffer. Vortex and incubate samples at 37⁰C with caps off. Quench with 200 μL ACN at time points 0, 5, 15, 30, and 60 min. Include controls - samples without compound, samples without cofactor, samples without microsomes, and samples with boiled microsomes. [0234] Analyze Samples. Prepare standard curve of each compound in triplicate. Final concentrations of standards are: 0.01, 0.02, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.5 μM. Prepare standards in ACN and deionized H₂O. Add 50 μL of each standard to 50 μL Cofactor/Serial Dilution Buffer (no microsomes), and quench with 2-fold ACN (200 μL). Bracket each microsomal species with curves. For example, place the first curve at the beginning, then all human liver microsome samples, followed by the second curve. After the second curve, all rat liver microsome samples follow, and finally the third curve. After quenching, vortex all samples vigorously (including standards) and spin for 5 min at 13,000 rpm. Transfer aliquot of supernatant to injection vial for analysis by LC/MS. Acceptance criteria for standard curves are: r² > 0.98; accuracy of calculated concentrations within +/- 20% of expected concentrations; a minimum of 3 levels with maximum number of points dropped < 25%; and lowest level of quantitation (mean peak area) > 3 x blank peak area. Acceptance criteria for samples are: %CV of replicates < 20%. Calculate CL values for each compound in both human and rat liver microsomes.

[0235] Table 1. Solubility, stability and pharmacokinetic in vitro data for calcilytic compounds of Structure I.

No apparent clearance over 60 min.

In vivo effect of calcilvtic compounds of Structure I.

Bolus i.v. injection in normal rats.

[0236] 3-[2-(3-Fluoro-phenyl)-ethyl]-2-(2-hydroxy-phenyl)-5-isopropyl-6-methyl-3H- pyrimidin-4-one of Example 1e (1 or 3 μmol/kg) or vehicle, or 3-[2~(3-fluoro-phenyl)- ethyl]-2-(2-hydroxy-phenyl)-5-isobutyl-6-methyl-3H-pyrimidin-4-one of Example 4 (1 or 3 μmol/kg) or vehicle was administered by intravenous injection over about 15 seconds to normal conscious male Sprague-Dawley rats with chronic indwelling arterial and venous catheters. Arterial blood samples were collected at 30 min and immediately before, and at 1, 5, 10, and 30 min after the start of the injection for measurement of the levels of parathyroid hormone (PTH) and ionized calcium (Ca2⁺) in plasma. PTH was measured using a specific rat PTH (1-84) ELISA (Immutopics, San Clemente, CA). Injection of compound of Example 4 or 1e induced a rapid, but transient dose-related increase in plasma PTH levels that were maximal at 1 min after the injection. Plasma PTH levels had returned to pre-dose levels by 10 min after the injection (FIG. 1 for compound of Example 4, and FIG. 3 for compound of Example 1e). Plasma levels of compound of Example 4 were maximal at 1 min after injection and declined rapidly during the next 10-

30 min as shown in FlG. 2. There were no consistent changes in plasma Ca^⁺ levels during this experiment (not shown).

Oral administration in normal fasted rats.

[0237] Normal male Sprague-Dawley rats (275-300 g) were provided with unrestricted access to normal chow (Teklad 8640) and tap water and allowed to acclimate for at least 4 days. Each rat was anesthetized with isoflurane and a chronic blood sampling catheter was implanted in the abdominal aorta via the femoral artery. The rats were allowed to recover from surgery for at least 3 days. Each rat was weighed and prepared for study (n = 5/grόup). The oral group received phosphoric acid diethyl ester 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5 isopropyl-4-methyl-6-oxo-1 ,6-dihydro-pyrimidin-2- yl}-phenyl ester of Example 22 (50 μmol/kg) or vehicle, a 20% aqueous solution of 2- hydroxypropyl-β-cyclodextrin and 1-2% DMSO, pH ~ 3 by gavage (1 mL/200 g body weight). Blood samples (0.4 ml_) were collected at 0, 10, 20, 30, 60, and 120 min after dosing in the oral group. Plasma Ca²⁺ levels were measured immediately and plasma stored at -20⁰C for subsequent PTH assay and determination of exposure levels. PTH levels were measured using the rat PTH(I -84) ELISA (Immutopics). Compound plasma levels and drug solution concentration were determined by mass spectrometer at NPS. [0238] All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth.

[0239] Modifications and improvements of the embodiments specifically disclosed herein are within the scope of the following claims. Without further elaboration, it is believed that one skilled in the area can, using the preceding description, utilize the present disclosure to its fullest extent. Therefore the Examples herein are to be construed as merely illustrative and not a limitation of the scope of the present invention in any way. The embodiments disclosed in which an exclusive property or privilege is claimed are defined as follows.

[0240] It will be obvious to those having skill in the art that many changes may be made to the details of the above-described embodiments without departing from the underlying principles of the invention. The scope of the present invention should, therefore, be determined only by the following claims.

Claims

Claims

1. A compound having the chemical formula:

wherein:

R¹ and R² are independently chosen from: H, halogen, CN, CF₃, lower alkyl, cycloalk, and aryl; or R¹ and R² are together -(CH₂)_n- and n is 5, 4, or 3; or R¹ and R² are together -CH(lower alkyl)(CH₂)_n ^" and n is 4, 3, or 2;

R³ is an aryl group, which may have 0 to 4 substituents in the aryl ring and each substituent is chosen from: halogen, CN, CF₃, OCF₃, lower alkyl, NO₂, NH₂, NH(lower alk), N(lower alk)₂, NH[S(O)₂lower alk], NH[S(O)₂alkyl aryl], NH[S(O)₂aryl], NH[S(O)₂heterocycle], OS(O)₂NH₂, OS(O)₂NH(lower alkyl), OS(O)₂N(lower alkyl)₂, lower alkoxy, OH, OC(O)-lower alk, OC(O)-lower alkyl- NH₂, OC(O)-lower alkyl-NH(lower alk), OC(O)-lower alkyl-N(lower alk)₂, OC(O)O- lower alk, OC(O)O-lower alkyl amino, OC(O)O-lower a)kyl-NH(lower alk), OC(O)O-lower alkyl-N(lower alk)_2ϊ OC(O)NH-lower alk, OC(O)N(lower alk)₂₎ OC(O)heterocycle, O-lower alkyl-P(O)(OH)₂₎ O-lower alkyl-P(O)OH(O-lower alkyl- OC(O)O-lower alkyl), O-lower alky!-P(O)(O-lower alkyl-OC(O)O-lower alk)₂, OP(O)(OH)₂, OP(O)(O-lower alkyl-aryl)₂, OP(O)(O-lower alk)₂, OP(O)(O-lower alkyl-OC(O)O-lower alk)₂, and OP(O)(O-metal)₂;

R⁴ is chosen from: H, lower alkyl, and a group of the formula -(CH₂)_n-R⁵ wherein n is 0, 1 , or 2, and R⁵ is an aryl group which may have O to 3 substituents on the aryl ring and each substituent is chosen from: halogen, CN, CF₃, OCF₃, lower alk, lower alkoxy, NH-lower alk, NH-alkyl aryl, N(lower alk)₂, OH₁ OC(O)- lower alk, OC(O)-lower alkyl amino, and OC(O)-lower alkyl-N(lower alk)₂; and pharmaceutically acceptable salts, hydrates, tautomers, solvates and complexes thereof.

2. The compound according to claim 1 , wherein R¹ and R² are independently one of lower alkyl.

3. The compound according to claim 2, wherein said lower alkyl is chosen from: methyl, ethyl, isopropyl and isobutyl.

4. The compound according to claim 1, wherein R¹ is one of isopropyl and isobutyl.

5. The compound according to claim 1, wherein R² is methyl.

6. The compound according to claim 1 , where R¹ and R² are together - CH(lower alkyl)(CH₂)_n ^" and n is 4 or 3.

7. The compound according to claim 1, where R¹ and R² are together - CH(methyl)(CH₂)n^" and n is 4.

8. The compound according to claim 1, wherein R³ is phenyl optionally substituted with one of hydroxy, amino, OC(O)OEt, or OP(O)(OH)₂, hydroxy and fluorine, or methoxy and fluorine.

9. The compound according to claim 1 , wherein R⁴ further comprises the group -(CH₂),,- R⁵; wherein n is 1 or 2; and

R⁵ is phenyl optionally substituted with 1 or 2 halogens.

10. The compound according to claim 9, wherein n is 2 and said halogen is fluorine.

11. The compound according to claim 1, wherein the compound is chosen from:

3-[2-(3-fluoro-phenyl)-ethyl]-5-isopropyl-2-(2-methoxy-phenyl)-6-methyl-3H- pyrimidin-4-one;

3-[2-(3-fluoro-phenyl)-ethyl]-2-(2-hydroxy-phenyl)-5-isopropyl-6-methyl-3W- pyrimidin-4-one;

3-[2-(3-fluorophenyl)-ethyl]-5-isopropyl-2-(3-fluoro-2-methoxyphenyl)-6-methyl- 3H-pyrimidin-4-one; 3-[2-(3-fluoro-phenyl)-θthyl]-2-(2-hydroxy-phenyl)-5-isobutyl-6-mθthyl-3H- pyrimidin-4-one;

5-(1,2-dimethylpropyl)-3-[2-(3-fluorophenyl)ethyl]-2-(2-hydroxyphenyl)-6-methyl- 3W-pyrimidin-4-one;

3-[2-(3-fluorophenyl)ethyl]-2-(2-hydroxyphenyl)-5-methyl-5,6,7,8-tetrahydro-3H- quinazolin-4-one;

(5S)-3-[2-(3-fluorophenyl)ethyl]-2-(2-hydroxyphenyl)-5-methyl-5,6,7,8- tetrahexahydro-3/-/-quinazolin-4-one;

2,2-dimethyl-propionic acid 2-{1 -[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-4-methyl-6- oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyl ester;

3,3-dimethyl-butyric acid 2-{1 -[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-4-methyl-6- oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyl ester; isobutyric acid 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-4-methyl-6-oxo-1 ,6- dihydro-pyrimidin-2-yl}-phenyl ester;

2,2-dimethyl-butyric acid 2-{1 -[2-(3-fluoro-phenyl)-ethylJ-5-isobutyl-4-methyl-6- oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyl ester;

(S)-2-methyl-butyric acid 2-{1 -[2-(3-f luoro-phenyl)-ethyl]-5-isobutyl-4-methyl-6- oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyl ester; carbonic acid 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-4-methyl-6-oxo-1 ,6- dihydro-pyrimidin-2-yl}-phenyl ester isopropyl ester; carbonic acid ethyl ester 2-{1-[2-(3-fluoro-pheny()-ethyl]-5-isobutyl-4~methyl-6- oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyl ester; carbonic acid ethyl ester 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isopropyl-4-methyl-6- oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyl ester; carbonic acid ferf-butyl ester 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-4-methyl- 6-0X0-1 ,6-dihydro-pyrimidin-2-yl}-phenyl ester;

3-[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-6-methyl-2-(2-nitro-phenyl)-3/-/-pyrimidin-4- one;

2-(2-amino-phenyl)-3[2-(3-fluoro-phenyl)-ethyl]-5isobutyl-6-methyl-3H-pyrimidin- 4-one;

Λ/-(2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-4-methyl-6-oxo-1 ,6-dihydropyrimidin- 2-yl}-phenyl)-methanesulfonamide; phosphoric acid dibenzyl ester 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isopropyl-4- methyl-6-oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyl ester; phosphoric acid dibenzyl ester 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-4 methyl-6-oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyl ester; phosphoric acid mono-(2-{1-[2-(3-fluoro-phenyl)-ethyl]-5 isopropyl-4-methyl-6- oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyl) ester; phosphoric acid mono-(2-{1 -[2-(3-fluoro-phenyl)-ethyl]-5-isobutyl-4-methyl-6-oxo- 1 ,6-dihydro-pyrimidin-2-yl}-phenyl) ester; phosphoric acid mono-(2-{1 -[2-(3-fluoro-phenyl)-ethyl]-5-isopropyl-4-methyl-6- oxo-1 ,6-dihydro-pyrimidin-2-yl}-phenyl) ester disodium salt; and phosphoric acid diethyl ester 2-{1-[2-(3-fluoro-phenyl)-ethyl]-5 isopropyl-4-methyl- 6-0X0-1 ,6-dihydro-pyrimidin-2-yl}-phenyl ester.

12. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable diluent or excipient.

13. A method of treating a disease or disorder characterized by abnormal bone or mineral homeostasis which comprises the administration to a subject in need of treatment thereof an effective amount of a compound according to claim 1.

14. A method according to claim 13, wherein the bone or mineral disease or disorder is chosen from: osteosarcoma, periodontal disease, fracture healing, osteoathritis, rheumatoid arthritis, Paget's disease, humoral hypercalcemia malignancy, and osteoporosis.

15. A method according to claim 13, wherein the bone or mineral disease or disorder is osteoporosis.

16. A method of increasing serum parathyroid hormone levels in mammals, which comprises the administration to a subject in need of treatment thereof an effective amount of a compound according to claim 1.