AU2386800A - Dihydropyrimidines and uses thereof - Google Patents

Description

WO 00/37026 PCT/US99/30868 Dihydropyrimidines and Uses Thereof This application claims priority of U.S. Serial No. 5 09/221,668, filed December 23, 1998, and of U.S. Provisional Application No. 60/113,612, filed December 23, 1998, the contents of which are hereby incorporated by reference. Throughout this application, various references are referred 10 to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. 15 Background of the Invention The designation "alA" is the appellation recently approved by the IUPHAR Nomenclature Committee for the previously designated "ac" cloned subtype as outlined in the 1995 20 Receptor and Ion Channel Nomenclature Supplement (Watson and Girdlestone, 1995) . The designation alA is used throughout this application and the supporting tables and figures to refer to this receptor subtype. At the same time, the receptor formerly designated alA was renamed amD. The new 25 nomenclature is used throughout this application. Stable cell lines expressing these receptors are described herein; however, these cell lines were deposited with the American Type Culture Collection (ATCC) under the old nomenclature (infra). 30 Benign Prostatic Hyperplasia (BPH), also called Benign Prostatic Hypertrophy, is a progressive condition which is characterized by a nodular enlargement of prostatic tissue resulting in obstruction of the urethra. This results in 35 increased frequency of urination, nocturia, a poor urine stream and hesitancy or delay in starting the urine flow. Chronic consequences of BPH can include hypertrophy of bladder smooth muscle, a decompensated bladder and an WO 00/37026 PCT/US99/30868 2 increased incidence of urinary tract infection. The specific biochemical, histological and pharmacological properties of the prostate adenoma leading to the bladder outlet obstruction are not yet known. However, the development of 5 BPH is considered to be an inescapable phenomenon for the aging male population. BPH is observed in approximately 70% of males over the age of 70. Currently, in the United States, the method of choice for treating BPH is surgery (Lepor, H., Urol. Clinics North Amer., 17, 651 (1990)). Over 10 400,000 prostatectomies are performed annually (data from 1986). A medicinal alternative to surgery is clearly very desirable. The limitations of surgery for treating BPH include the morbidity rate of an operative procedure in elderly men, persistence or recurrence of obstructive and 15 irritative symptoms, as well as the significant cost of surgery. a-Adrenergic receptors (McGrath, et. al. Med. Res. Rev., 9, 407-533, 1989) are specific neuroreceptor proteins located 20 in the peripheral and central nervous systems on tissues and organs throughout the body. These receptors are important switches for controlling many physiological functions and, thus, represent important targets for drug development. In fact, many a-adrenergic drugs have been developed over the 25 past 40 years. Examples include clonidine, phenoxybenzamine and prazosin (treatment of hypertension) , naphazoline (nasal decongestant), and apraclonidine (treating glaucoma). a-Ad renergic drugs can be broken down into two distinct classes: agonists (clonidine and naphazoline are agonists) , which 30 mimic the receptor activation properties of the endogenous neurotransmitter norepinephrine, and antagonists (phenoxy benzamine and prazosin are antagonists), which act to block the effects of norepinephrine. Many of these drugs are effective but also produce unwanted side effects (for 35 example, clonidine produces dry mouth and sedation in addition to its antihypertensive effects). During the past 15 years a more precise understanding of WO 00/37026 PCT/US99/30868 3 a-adrenergic receptors and their drugs has evolved through increased scientific scrutiny. Prior to 1977, only one a-adrenergic receptor was known to exist. Between 1977 and 1988, it was accepted by the scientific community that at 5 least two a-adrenergic receptors (a1 and a 2 ) existed in the central and peripheral nervous systems. Since 1988, new techniques in molecular biology have led to the ident ification of at least six a-adrenergic receptors which exist throughout the central and peripheral nervous systems: alA 10 (new nomenclature) , aiB, alD (new nomenclature) , 2A, a2B and a2C (Bylund, D.B., FASEB J., 6, 832 (1992)). In many cases, it is not known precisely which physiological responses in the body are controlled by each of these receptors. In addition, current a-adrenergic drugs are not selective for 15 any particular a-adrenergic receptor. Many of these drugs produce untoward side effects which may be attributed to their poor a-adrenergic receptor selectivity. Since the mid 1970's, nonselective a-antagonists have been 20 prescribed to treat BPH. In 1976, M. Caine, et al. (Brit. J. Urol., 48, 255 (1976)), reported that the nonselective a-antagonist phenoxybenzamine was useful in relieving the symptoms of EPH. This drug may produce its effects by interacting with a-receptors located on the prostate. 25 However, this drug also produces significant side effects such as dizziness and asthenia which severely limit its use in treating patients on a chronic basis. More recently, the a-adrenergic antagonists prazosin and terazosin have also been found to be useful for treating BPH. However, these 30 drugs also produce untoward side effects. It has recently been discovered that the alA receptor is responsible for mediating the contraction of human prostate smooth muscle (Gluchowski, C. et. al., WO 94/10989, 1994; Forray, C. et. al., Mol. Pharmacol. 45, 703, 1994). This discovery 35 indicates that the a1A antagonists may be effective agents for the treatment of BPH with decreased side effects. Further studies have indicated that the aiA receptor may also WO 00/37026 PCT/US99/30868 4 be present in other lower urinary tract tissues, such as urethral smooth muscle (Ford et al. Br. J. Pharmacol., 114, 24P, (1995)). 5 This invention is directed to dihydropyrimidine compounds which are selective antagonists for cloned human alA receptors. This invention is also related to uses of these compounds for treating benign prostatic hyperplasia, and for the treatment of any disease where antagonism of the alA 10 receptor may be useful.

WO 00/37026 PCTIUS99/30868 5 Summary of the Invention This invention is directed to compounds having the structure: 5 F F 0 o 10 RHJ R3

R

2 N O H wherein R, is -OCH 3 or OH; 15 wherein R 2 is -CH 2 OH, -CH 2 0CH 3 , or -COOH; wherein R 1 and R 2 together form a 5-membered lactone ring; WO 00/37026 PCT/US99/30868 6 wherein R 3 is selected from the group consisting of - (CH 2 ) 3 0H, 5 -(H 2 C)--N

-(H

2 C) -- N \ -o 10 -(H 2 C) C) NO -(H 2C) -- N \/ -(H 2C) 3-N No OH N N 15 OH (H 2C) and OH 20 +I 20 --( H 2C) g N provided that when R 1 is OH, R 3 cannot be 25 -(H2C)-N\/ or a pharmaceutically acceptable salt thereof. 30 WO 00/37026 PCT/US99/30868 7 This invention is also directed to a compound having the structure: F F 5 0 o 0 N N OH 01 H N 0 H 10 This invention provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the compounds described herein and a pharmaceutically acceptable carrier. 15 This invention also provides a method of treating a subject suffering from benign prostatic hyperplasia which comprises administering to the subject an amount of any one of the compounds described herein effective to treat benign 20 prostatic hyperplasia. This invention provides a method of relaxing lower urinary tract tissue which comprises contacting the lower urinary tract tissue with an amount of any one of the compounds 25 described herein effective to relax lower urinary tract tissue. This invention provides a method of inhibiting contraction of prostatic tissue in a subject which comprises 30 administering an amount of any one of the compounds described herein effective to inhibit contraction of prostatic tissue. This invention provides a method of treating a disease which is susceptible to treatment by antagonism of the a,, receptor 35 which comprises administering to the subject an amount of any WO 00/37026 PCT/US99/30868 8 one of the compounds described herein effective to treat the disease. This invention provides a pharmaceutical composition 5 comprising a therapeutically effective amount of any one of the compounds described herein in combination with a therapeutically effective amount of finasteride and a pharmaceutically acceptable carrier. 10 This invention provides a process of making a pharmaceutical composition comprising combining a therapeutically effective amount of any one of the compounds described herein and a pharmaceutically acceptable carrier.

WO 00/37026 PCT/US99/30868 9 Brief Description of the Figures Figure 1A -1C: Figure 1A-1C show the structures of Compounds 1 to 9, described herein in the Examples.

WO 00/37026 PCT/US99/30868 10 Detailed Description of the Invention The present invention is directed to compounds having the following structure: 5 F F 0 0

~R

3

R

1 N N 10 1H N10 R2 N H wherein R, is -OCH 3 or OH; 15 wherein R 2 is -CH 2 OH, -CH 2

OCH

3 , or -COOH; wherein R 1 and R 2 together form a 5-membered lactone ring; WO 00/37026 PCT/US99/30868 11 wherein R 3 is selected from the group consisting of - (CH 2 ) 3 0H, 5 (H 2 C) f--NoK\ -(H 2 C) 5--N ND \N+ -O -- 0 10(H 2 C)-N+\ / No - No 10 -2 3NN/HO -(H 2 C) 5--N/ -(H 2 C) 3-N OHNA/I( 2 N1 15 OH -(H 2C) -- N and N OH 20 2C) I--N ; \/ N provided that when R 1 is OH, R 3 cannot be 25 -(H2C) 3-N -N1 or a pharmaceutically acceptable salt thereof. 30 The present invention also provides for the (+) and (-) enantiomers of all of the compounds described herein. The invention further provides for the cis and trans isomers 35 of all of the compounds described herein. It is noted that the terms "cis" and "trans" correspond to relative WO 00/37026 PCT/US99/30868 12 stereochemistry, as determined, for example by NOE (Nuclear Overhauser Effect) experiments. The compounds of the present invention may be present as 5 enantiomers, diasteriomers, isomers or two or more of the compounds may be present to form a racemic mixture. Furthermore, the compounds of the present invention are preferably at least 80% pure, more preferably 90% pure, and most preferably 95% pure. 10 In one embodiment the compound has the following structure: F F 15 0 o o0 N NR3 H N _0 H 20 In another embodiment of the present invention the compound is: 25 F F 0 o 30 0 N N OH N 0

H

WO 00/37026 PCTIUS99/30868 13 In a further embodiment of the present invention the compound is: F 5 F 0 o N N N H ON N 0 N o0 H 10 In yet a further embodiment of the present invention the compound is: 15 F F o o 20N N +N H 20 0 N 0 H In an embodiment of the present invention the compound is: 25 F F 0 0 -o _o N N +N H/ 30 N 0

H

WO 00/37026 PCT/US99/30868 14 In an embodiment of the present invention, R 3 is selected from the group consisting of 5N HO OH2C) 10 OH -(/H2C) and 15 OH -(H2 C) 3 20 In a further embodiment of the present invention the compound has the following structure: 25 F F 0 0 R N N N R2H 30 N-_

H

WO 00/37026 PCTIUS99/30868 15 In an embodiment of the present invention the compound is: F F 51 0 o N N N N H HO4 N 0 H 10 In yet another embodiment of the present invention the compound is: 15 F F 0 o 20 .N N N N HH N '0 H 0 In yet another embodiment of the present invention the 25 compound is: F F 0 o 30J

,

30NN N N 0 H N& \ N 0 H 35 WO 00/37026 PCTIUS99/30868 16 The present invention also provides a compound having the structure: F F 5 0 o 0 N N OH N 0 H 10 The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of any of the compounds described herein and a pharmaceutically acceptable carrier. In the subject invention, a "therapeutically 15 effective amount" is any amount of a compound which, when administered to a subject suffering from a disease against which the compounds are effective, causes reduction, remission, or regression of the disease. 20 In one embodiment, the therapeutically effective amount is an amount from about 0.01 mg per subject per day to about 500 mg per subject per day, preferably from about 0.1 mg per subject per day to about 60 mg per subject per day, more preferably from about 1 mg per subject per day to about 30 25 mg per subject per day. In the practice of this invention, the "pharmaceutically acceptable carrier" is any physiological carrier known to those of ordinary skill 30 in the art useful in formulating pharmaceutical compositions. In one embodiment the pharmaceutical carrier may be a liquid and the pharmaceutical composition would be in the form of 35 a solution. In another embodiment, the pharmaceutically acceptable carrier is a solid and the composition is in the WO 00/37026 PCT/US99/30868 17 form of a tablet or powder. In a further embodiment, the pharmaceutical carrier is a gel and the composition is in the form of a suppository or a cream. In yet a further embodiment, the compound may be formulated as a part of a 5 pharmaceutically acceptable transdermal patch. A solid carrier can include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, 10 binders or tablet-disintegrating agents; it can also be an encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary compression 15 properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active ingredient. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, 20 polyvinylpyrrolidine, low melting waxes and ion exchange resins. Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The 25 active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, 30 emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives 35 as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut WO 00/37026 PCTIUS99/30868 18 oil and arachis oil). For parenteral administration, the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral 5 administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellent. Liquid pharmaceutical compositions which are sterile 10 solutions or suspensions can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously. The compounds may be prepared as a sterile solid composition which may be dissolved or 15 suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium. Carriers are intended to include necessary and inert binders, suspending agents, lubricants, flavorants, sweeteners, preservatives, dyes and coatings. 20 The compound can be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents, for example, enough saline or glucose to make the solution isotonic, bile salts, acacia, gelatin, 25 sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The compound can be administered orally either in liquid or 30 solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, 35 emulsions, and suspensions. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular WO 00/37026 PCT/US99/30868 19 compound in use, the strength of the preparation, the mode of administration, and the advancement of the disease condition. Additional factors depending on the particular subject being treated will result in a need to adjust 5 dosages, including subject age, weight, gender, diet, and time of administration. In a further embodiment of the present invention, any one of the compounds described herein additionally does not cause 10 a fall in blood pressure at dosages effective to alleviate benign prostatic hyperplasia. This invention provides a method of treating a subject suffering from benign prostatic hyperplasia (BPH) which 15 comprises administering to the subject any one of the compounds described herein effective to treat BPH. The invention further provides that the compound additionally does not cause a fall in blood pressure at dosages effective to alleviate BPH. In one preferred embodiment the compound 20 effects treatment of BPH by relaxing lower urinary tract tissue and in particular where the lower urinary tract tissue is urethral smooth muscle. In the practice of this invention, the term "lower urinary 25 tract tissue" is used to include prostatic capsule, prostate urethra, bladderneck, urethral smooth muscle and prostatic smooth muscle. This invention also provides a method of treating a subject 30 suffering from benign prostatic hyperplasia, which comprises administering to the subject one of the compounds described herein in combination with a 5-alpha reductase inhibitor effective to treat benign prostatic hyperplasia. In one embodiment the 5-alpha reductase inhibitor is finasteride. 35 This invention further provides a method of relaxing lower urinary tract tissue which comprises contacting the lower urinary tract tissue with an amount of one of the compounds WO 00/37026 PCT/US99/30868 20 described herein effective to relax lower urinary tract tissue. In one embodiment the lower urinary tract tissue is urethral smooth muscle. In one preferred embodiment the compound additionally does not cause a fall in blood pressure 5 when it is effective to relax lower urinary tract tissue. The invention further provides for a method of inhibiting contraction of prostatic tissue, which comprises administering to the subject an amount of any of the 10 compounds described herein effective to inhibit contraction of prostatic tissue. In one preferred embodiment the prostatic tissue is prostatic smooth muscle and the compound additionally does not cause a fall in blood pressure. 15 This invention also provides a method of treating a disease which is susceptible to treatment by antagonism of the a1, receptor which comprises administering to the subject one of the compounds describe herein effective to treat the disease. 20 The invention provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the compounds described herein in combination with a therapeutically effective amount of finasteride and a pharmaceutically acceptable carrier. This invention also 25 provides for a pharmaceutical composition comprising any one of the compounds described herein present in an amount from about 0.01mg to about 500mg and the therapeutically effective amount of the finasteride is about 5mg. In one embodiment, the therapeutically effective amount of the compound is an 30 amount from about 0.1 mg to about 60 mg and the therapeutically effective amount of the finasteride is about 5 mg. In another embodiment, the therapeutically effective amount of the compound is an amount from about 1 mg to about 30 mg and the therapeutically effective amount of the 35 finasteride is about 5 mg. This invention provides a pharmaceutical composition made by WO 00/37026 PCT/US99/30868 21 combining a therapeutically effective amount of any of the compounds described herein and a pharmaceutically acceptable carrier. 5 This invention also provides a pharmaceutical composition made by combining a therapeutically effective amount of any of the compounds described herein with a therapeutically acceptable amount of finasteride and a pharmaceutically acceptable carrier. 10 Included in this invention are pharmaceutically acceptable salts and complexes of all of the compounds described herein. The salts include, but are not limited to, the following acids and bases: Inorganic acids which include hydrochloric 15 acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and boric acid; organic acids which include acetic acid, trifluoroacetic acid, formic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, maleic acid, citric acid, methanesulfonic acid, 20 trifluoromethanesulfonic acid, benzoic acid, glycolic acid, lactic acid, and mandelic acid; inorganic bases include ammonia and hydrazine; and organic bases which include methylamine, ethylamine, hydroxyethylamine, propylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, 25 ethylenediamine, hydroethylamine, morpholine, piperazine, and guanidine. This invention further provides for the hydrates and polymorphs of all of the compounds described herein. The present invention includes within its scope prodrugs of 30 the compounds of this inventions. In general, such prodrugs will be functional derivatives of the compounds of the invention which are readily convertible in vivo into the required compound. Thus, in the methods of treatment of the present invention, the term "administering" shall encompass 35 the treatment of the various conditions described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the WO 00/37026 PCT/US99/30868 22 specified compound in vivo after administration to the patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in Design of Prodrugs, ed. H. Bundgaard, 5 Elsevier, 1985. The present invention further includes metabolites of the compounds of the present invention. Metabolites include active species produced upon introduction of compounds of 10 this invention into the biological milieu. One skilled in the art will readily appreciate that appropriate biological assays will be used to determine the therapeutic potential of the claimed compounds for treating 15 the above noted disorders. This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and 20 results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter. 25 30 35 WO 00/37026 PCT/US99/30868 23 Experimental Details I. Synthesis of Compounds 1-9 5 A. Synthesis of Compound 1 (+)-1,2,3,6-Tetrahydro-1-(3-hydroxypropyl)carboxamido-5 methoxycarbonyl-4-methoxymethyl-6-(3,4-difluorophenyl)-2-oxo pyrimidine. 10 a) 5-Methoxycarbonyl-4-methoxymethyl-1,2,3,6-tetrahydro-2 oxo-6-(3,4-difluorophenyl)-pyrimidine. To a well-stirred mixture of methyl 4-methoxyacetoacetate (50 g, 0.351 mol), 3,4-difluorobenzaldehyde (51.39 g, 0.351 mmol), and urea (31.64 g, 0.527 mole) in THF (300 mL) at room 15 temperature were added copper(I) oxide (5.06 g, 0.035 mole) and acetic acid (2.05 mL) sequentially followed by dropwise addition of boron trifluoride diethyl etherate (56 mL, 0.456 mole). The mixture was stirred and refluxed for 8 h, whereupon TLC (1/1 EtOAc/hexanes) indicated completion of the 20 reaction. It was cooled and poured into a mixture of ice and sodium bicarbonate (100 g) and the resulting mixture was filtered through Celite. The Celite pad was washed with dichloromethane (400 mL). The organic layer was separated from the filtrate and the aqueous layer was extracted with 25 more dichloromethane (3 X 300 mL) . The combined organic extracts were dried (sodium sulfate) and the solvent evaporated. The crude product was purified by flash column on silica gel using 50% ethyl acetate in hexanes and then ethyl acetate as eluents to give the product as a pale yellow 30 foam, which on trituration with hexane became white powder (103.3 g, 94%) . 'H NMR (CDCl3) 6 3.476 (s, 3H), 3.651 (s, 3H), 4.653 (s, 2H), 5.39 (s, 1H), 6.60 (br s, 1H, NH), 7.00 7.20 (m, 3H), 7.72 (br s, 1H, NH). 35 b) (+) -5-Methoxycarbonyl-4-methoxymethyl-1,2,3, 6-tetrahydro 2-oxo-6- (3,4-difluorophenyl) -pyrimidine.

WO 00/37026 PCTIUS99/30868 24 The racemic intermediate 5-methoxycarbonyl-4-methoxymethyl 1,2,3,6-tetrahydro-2-oxo-6-(3,4-difluorophenyl)-pyrimidine was resolved by chiral HPLC [Chiralcel OD 20 X 250 mm #369 703-30604; 1 254 nm; hexanes/ethanol 90/10 ; 85 mg per 5 injection; retention time of the desired enantiomer: 16.94 min., the first enantiomer peak to elute] to give (+)-5 methoxycarbonyl-4-methoxymethyl-1, 2,3, 6-tetrahydro-2-oxo-6 (3,4-difluorophenyl)-pyrimidine (40-42 wt% isolation of the desired enantiomer from the racemate); [a]D + 83.8 (c 10 0.5, chloroform). c) (+) -5-Methoxycarbonyl-4-methoxymethyl-1,2,3, 6-tetrahydro 2-oxo-6-(3,4-difluorophenyl)-1-[(4-nitrophenyloxy) carbonyl]pyrimidine. 15 To a solution of (+)-5-methoxycarbonyl-4-methoxymethyl 1,2,3,6-tetrahydro-2-oxo-6-(3,4-difluorophenyl)-pyrimidine (1.98 g, 6.34 mmol) in anhydrous THF (20 mL) at -78 0 C under argon atmosphere, a solution of lithium hexamethyldisilazide in THF (lM, 18 mL, 18 mmol) was added over 2-3 min. and the 20 mixture was stirred for 10 min. This solution was added over 6 min. via a cannula to a stirred solution of 4-nitrophenyl chloroformate (4.47 g, 22.2 mmol) in THF (20 mL) at -78 OC. The stirring was continued for 10 min and the mixture was poured onto ice (50 g) and extracted with chloroform (2 X 50 25 mL) . The combined extracts were dried (sodium sulfate) and the solvent evaporated. The residue was purified by flash column chromatography using hexanes/ethyl acetate (4:1 to 3.5:1) as eluent. The product was obtained as a yellow syrup which on trituration with hexane became white powder (2.4 g, 30 79%). 'H NMR (CDCl 3 ) 6 3.522 (s, 3H), 3.744 (s, 3H), 4.65 4.80 (q, J = 16.5 Hz, 2H), 6.323 (s, 1H), 7.10-7.30 (m, 4H), 7.358 (d, J = 9 Hz, 2H), 8.273 (d, J = 9 Hz, 2H). d) (+)-1,2,3,6-Tetrahydro-1-(3-hydroxypropyl)carboxamido-5 35 methoxycarbonyl-4 -methoxymethyl-6- (3,4 -difluorophenyl) -2 -oxo pyrimidine.

WO 00/37026 PCTIUS99/30868 25 To a solution of (+)-1,2,3,6-tetrahydro-6-(3,4 difluorophenyl) -5-methoxycarbonyl-4-methoxymethyl-- [N- (4 nitrophenoxy) carbonyl] -2-oxopyrimidine (100 mg, 0.209 mmol) in dichloromethane (5 mL), 3-aminopropanol (0.2 mL, 2.62 5 mmol) was added at r.t. The resulting mixture was stirred for 2 hour and the crude product was purified through column chromatography on silica gel using EtOAc as the eluent (82 mg, 95%) . 'H-NMR (300 MHZ, CDCl 3 ) 6 1.67-1.75 (m, 2H) , 3.41-3.49 (m, 2H), 3.48 (s, 3H), 3.55-3.62 (m, 2H), 3.71 (s, 10 3H), 4.68 (s, 2H), 6.67 (s, 1H), 7.06-7.20 (m, 3H), 7.76 (s, 1H) , 8.92 (t, J=7 Hz, 1H) . B. Synthesis of Compound 2: (+)-1,2,3,6-Tetrahydro-l-{N-[4-(2-pyridyl)piperidin-1 15 yl]propyl}carboxamido-5-methoxycarbonyl-4-methoxymethyl-6 (3 ,4-difluorophenyl) -2-oxopyrimidine - pyridine-N-oxide hydrochloride. a) 4'-tert-Butoxycarbonyl-1',2',3',4',5',6' 20 hexahydro [2,4' ] bipyridinyl. To a solution of 1',2',3',4',5',6'-hexahydro[2,4']bipyridinyl (1.7 g, 10 mmol) in dichloromethane (100 mL) was added di tert-butyldicarbonate (2.2 g, 10 mmol) followed by saturated 25 aqueous sodium bicarbonate solution (100 mL). The mixture was stirred vigorously for 2 days, when the layers were separated and the aqueous layer extracted with dichloromethane (1 x 100 mL). The combined organic layers were dried with sodium sulfate, filtered and concentrated in 30 vacuo to provide crude 4'-tert-butoxycarbonyl 1,2',3',4',5',6'-hexahydro[2,4']bipyridinyl which was used as is in step b. b) 4'-tert-Butoxycarbonyl-2-oxo-1',2',3',4',5',6' 35 hexahydro [2,4' ] bipyridinyl.

WO 00/37026 PCT/US99/30868 26 A solution of 4'-tert-butoxycarbonyl-1',2',3',4',5',6' hexahydro[2,4']bipyridinyl (10 mmol) in dichloromethane (60 mL) was cooled to 0 C and was treated with 3 chloroperoxybenzoic acid (4.3 g, 12.5 mmol). The solution 5 was warmed to ambient temperature then stirred 18 h. The reaction was then treated with saturated aqueous sodium bicarbonate solution and the layers separated. The organic layer was dried with magnesium sulfate, filtered and concentrated in vacuo. The crude material was purified by 10 pressurized silica gel chromatography (1-3% methanol in dichloromethane) to afford 4'-tert-butoxycarbonyl-2-oxo 1',2',3',4',5',6'-hexahydro[2,4'1]bipyridinyl. FABMS (M + H) = 279 15 c) 2-oxo-1',2',3',4',5',6'-hexahydro[2,4']bipyridinyl. A solution of 4'-tert-butoxycarbonyl-2-oxo-1' ,2' ,3',4',5',6' hexahydro[2,4']bipyridinyl (2.1 g, 7.6 mmol) in ethyl acetate (150 mL) was cooled to -78 C. Hydrogen chloride gas was bubbled through the solution for 20 min and then the reaction 20 was warmed to ambient temperature. The solid that separated was collected by filtration and dried under vacuum to give the product as a hydrochloride salt. The free base was isolated by standard procedures to provide 2-oxo 1',2',3',4',5',6'-hexahydro[2,4']bipyridinyl. 25 d) N-tert-Butoxycarbonyl-3-(3',4',5',6'-tetrahydro-2-oxo 2'H- [2,4']bipyridinyl-1'-yl)propylamine To a solution of 2-oxo-1',2',3',4',5',6'-hexahydro[2,4'] 30 bipyridinyl (0.86 g, 4.8 mmol) in DMF (50 mL) was added 3 bromopropyl-tert-butoxycarbonylamine (1.4 g, 5.8 mmol) and cesium carbonate (1.2 g, 3.6 mmol) under argon. The solution was warmed to 50 0 C and stirred 2 days when the reaction was cooled to ambient temperature and the volatiles removed in 35 vacuo. The residue was taken up in dichloromethane (100 mL), washed with saturated sodium carbonate (1 x 50 mL), dried over magnesium sulfate, filtered, and concentrated in vacuo WO 00/37026 PCT/US99/30868 27 . The crude material was purified by pressurized silica gel chromatography (1-3% methanol in dichloromethane containing 0.5% NH 4 0H) to provide N-tert-butoxycarbonyl-3-(3',4',5',6' tetrahydro-2-oxo-2'H-[2,4']bipyridinyl-l'-yl)propylamine. 5 FABMS (M + H) = 336 e) 3-(3',4',5',6'-Tetrahydro-2-oxo-2'H-[2,4']bipyridinyl-1' yl) propylamine dihydrochloride. 10 A solution of N-tert-butoxycarbonyl-3-(3',4',5',6' tetrahydro-2-oxo-2'H-[2,4']bipyridinyl-l'-yl)propylamine (0.63 g, 1.9 mmol) in ethyl acetate (40 mL) was cooled to 78 C. Hydrogen chloride gas was bubbled through the solution for 10 min then the reaction was warmed to ambient 15 temperature. The solid that separated was collected by filtration and dried in vacuo to give the product. f) (+)-1,2,3,6-Tetrahydro-l-{N-[4-(2-pyridyl)piperidin-1 yl]propyl}carboxamido-5-methoxycarbonyl-4-methoxymethyl-6 20 (3,4-difluorophenyl)-2-oxopyrimidine - pyridine-N-oxide hydrochloride. A solution of (+) -5-methoxycarbonyl-4-methoxymethyl-1,2,3,6 tetrahydro-2-oxo-6-(3,4-difluorophenyl)-l-[(4 25 nitrophenyloxy)carbonyl]pyrimidine (200 mg, 0.42 mmol) (as described in Section I.A. (c)) and 3-(3',4',5',6'-Tetrahydro 2-oxo-2'H-[2,4']bipyridinyl-l'-yl)propylamine dihydrochloride (100 mg, 0.42) in THF (20 mL) was stirred at room temperature for 24 hours. Solvent was evaporated and 30 the residue was purified by flash chromatography on silica gel (dichloromethane:MeOH:2M ammonia in MeOH, 980:10:10 to 940:30:30 ) to give the desired product (252 mg, 98%). MH+ 574. 35 C. Synthesis of Compound 3: 4- (2 -Pyridyl) -1-{3 - [5-carboxymethyl-4-methoxymethyl-2-oxo- WO 00/37026 PCT/US99/30868 28 6(S)-(3,4-difluorphenyl)-1,2,3,6-tetrahydropyrimidin-l yl] carboxyaminopropyl}pyridinium chloride a) 1- (3-Aminopropyl) -4- (2-pyridyl)pyridinium bromide 5 hydrobromide A solution of 2,4'dipyridyl (820 g, 5.25 mol) and 3 bromopropylamine hydrobromide (1400 9, 6.39 mol) in DMF (5.OL) was heated to 95 0 C for 8 hours. The reaction mixture was cooled to room temperature and methyl tert-butyl ether 10 (3.7 L) was added over 3 hours. The slurry was stirred for 1 hour and filtered. The solid was washed with MTBE/DMF (1:1, 4.2L) and dried to afford 1-(3-aminopropyl)-4-(2 pyridyl)-pyridinium bromide hydrobromide as a tan solid. 15 b) 4- (2-Pyridyl)-l-{3- [5-carboxymethyl-4-methoxymethyl-2-oxo 6(S)-(3,4-difluorphenyl)-1,2,3,6-tetrahydropyrimidin-l yl] carboxyaminopropyl}pyridinium chloride Combined 1- (3-aminopropyl) -4- (2-pyridyl) -pyridinium bromide hydrobromide (227mg, O.60mmol), (+)-5-Methoxycarbonyl-4 20 methoxymethyl-1,2,3,6-tetrahydro-2-oxo-6- (3,4 difluorophenyl)-1-[(4-nitrophenyloxy)carbonyl]pyrimidine (287mg, 0.60mmol) (as described in Section I.A. (c)), and triethylamine (350mL, 2.51mmol) in 8mL of THF and 5mL of DMF. The yellow mixture was stirred at ambient temperature for 1 25 hour, then concentrated in vacuo. The resulting oil was purified by a Waters Delta Prep System using a reversed phase C18 cartridge column and a water : acetonitrile gradient containing 0.1% TFA. The product fractions were evaporated to dryness and the resulting residue was treated with HCl / 30 ethyl acetate. The mixture was evaporated to dryness and the residue was reconcentrated from ethyl ether, then triturated with ethyl ether and filtered to give the title compound. m.p.: 60-85 0 C (foam) NMR: consistent with structure 35 HPLC: 99% pure FAB MS: M+H @ m/e=552.2 WO 00/37026 PCT/US99/30868 29 Anal. cal'd for C 28

H

2 8 FPgT pl * HCl e 0.80 HP: C, 52.64; H, 4.83; N, 10.96. Found: C, 52.67; H, 4.68; N, 10.77. 5 D. Synthesis of Compound 4: (+)-4-(2-Pyridyl)-1-{3-[5-carboxymethyl-4-methoxymethyl-2 oxo-6-(3,4-difluorphenyl)-1,2,3,6-tetrahydropyrimidin-1 ylcarbonylamino] propyl}piperidine-1-oxide 10 a) 1- (3-Aminopropyl) -4- [2-pyridyl]pyridinium bromide hydrobromide. A solution of 2,4'-dipyridyl (25 g, 160 mmol) and 3 bromopropylamine hydrobromide (35 g, 160 mmol) in DMF (60 mL) was heated at 90-95 0 C for 10 h. After cooling to room 15 temperature, anhydrous ether (500 mL) was added to the mixture, the white solid that had formed was filtered, washed with Et 2 0 and dried to yield 60 g (100%) . 1H NMR (300 MHZ, DMSO-d) 5 2.35-2.44 (m, 2 H), 3.08-3.13 (m, 2 H), 4.76 4.81 (m, 2 H), 7.58 (dd, J = 4.8 Hz, J = 7.5 Hz, 1 H), 8.03 20 (dt, J = 1.8 Hz, J = 7.8 Hz, 1 H), 8.32 (d, J = 7.8 Hz, 1 H), 8.77-8.81 (m, 3 H), 9.12 (d, J = 6.3 Hz, 2 H). Anal. Calcd. for C,3H1 6 N3Br.HBr.0.5 H20: C, 40.65; H, 4.72; N, 10.94. Found: C, 40.83; H, 4.37; N, 11.05. 25 b) 3-(3',6'-Dihydro-2'-H-[2,4']bipyridinyl-1'-yl) propylamine. To a solution of 1- (3-aminopropyl) -4- [2-pyridyllpyridinium bromide hydrobromide (6 g, 16 mmol) in MeOH (150 mL) at 0-5 C was added NaBH 4 (2 g, 53 mmol) in small portions over a 30 period of 2 h. The reaction mixture was stirred overnight at room temperature and the solvent was evaporated. The residue was suspended in ether (200 mL) and treated with 50% NaOH solution (100 mL). The ether layer was separated and the aqueous layer was extracted with more ether (2 X 50 mL). 35 The combined ether extracts were dried over potassium carbonate and the solvent was removed to give 3-(3',6'- WO 00/37026 PCTIUS99/30868 30 dihydro-2'-H- [2,4']bipyridinyl-1'-yl)-propylamine (3.48 g) as an oil. It was used in the next step immediately without purification. 5 c) 3-Aminopropyl-4- (2-pyridyl)piperidine. To a solution of 3-(3',6'-dihydro-2'-H-[2,4']bipyridinyl-l' yl)-propylamine (3.48 g crude, 15.9 mmol) in MeOH (40 mL), was added 1.0 g of Pearlman's catalyst. The suspension was hydrogenated under 120 psi for 10 h after which the reaction 10 mixture was filtered through a pad of Celite and the solvent was removed. The residue was purified by column chromatography over silica gel (30 g) using

CH

2 Cl 2 /methanol /2M NH3 in MeOH (90:8:4 to 90:40:40) as eluent. The product was obtained as a pale yellow oil (3.21 g, 91%). 15 'H NMR 5 (CD 3 0D) 1.50-1.99 (m, 10 H) , 2.02-2.06 (m, 2 H) , 2.37-2.75 (m, 3 H), 3.02-3.06 (br m, 2 H), 7.05-7.09 (m, 4 H), 7.16 (dt, J = 0.9 Hz, J = 8.7 Hz, 1 H), 8.48 (dd, J = 0.9 Hz, J = 4.2 Hz, 1 H). 20 d) (+)-6- (3,4-Difluorophenyl)-1-{N- [4- (2-pyridyl)piperidin-1 ylJpropyl]}carboxamido-5-methoxycarbonyl-4-methoxymethyl-2 oxo 1,2,3,6-tetrahydropyrimidine. A solution of (+) -5-methoxycarbonyl-4-methoxymethyl-1,2,3,6 tetrahydro-2-oxo-6-(3,4-difluorophenyl)-l-[(4 25 nitrophenyloxy)carbonyl]pyrimidine (as described in Section I.A.(c)) (2.38 g, 5 mmol), 3-aminopropyl-4-(2 pyridyl)piperidine (1.206 g, 5.5 mmol) in THF (20 mL) was stirred at room temperature for 12 hours. Solvent was evaporated and the residue was redissolved in ethyl acetate 30 (100 mL) . It was washed with ice-cold 1N NaOH (4 X 50 mL), brine (2 X 50 mL) and dried over potassium carbonate. Solvent was evaporated at reduced pressure and the residue was purified by flash chromatography on silica gel (dichloromethane:MeOH:2M ammonia in MeOH, 980:10:10 to 35 940:30:30 ) to give 2.45 g (88%) of very pure product and 0.30 g (10%) of slightly impure fractions, as a foam. 'H NMR (CDCl 3 ) 5 1.60-2.00 (m, 6H), 2.05-2.15 (m, 2H), 2.38-2.43 (br WO 00/37026 PCTIUS99/30868 31 t, 2H), 2.65-2.80 (m, 1H), 3.05-3.06 (br d, 2H), 3.30-3.45 (m, 2H), 3.477 (s, 3H), 3.704 (s, 3H), 4.678 (s, 2H), 6.68 (s, 1H), 7.05-7.20 (m, 5H), 7.58-7.63 (dt, 1H), 7.702 (s, 1H, NH), 8.50-8.52 (dd, 1H), 8.875 (br t, 1H). 5 (e) (+)-4-(2-Pyridyl)-1-{3-[5-carboxymethyl-4-methoxymethyl 2-oxo-6-(3,4-difluorphenyl)-1,2,3,6-tetrahydropyrimidin-1 ylcarbonylamino] propyl}piperidine 1- oxide To a solution of (+)-6(S)-(3,4-difluorophenyl)-1-{3-[4-(2 10 pyridyl)piperidin-1-yl]propylaminolcarbonyl-5 methoxycarbonyl-4-methoxymethyl-2-oxo-1,2,3,6 tetrahydropyrimidine (205mg, 0.37mmol) in 4mL of methanol was added 50% hydrogen peroxide in water (75mL, 1.30mmol) and left stir 10 days. The reaction was quenched with the 15 addition of platinum black until oxygen evolution ceased. Filtered the reaction slurry over celite and evaporated the filtrate to dryness to yield a pale yellow oil. The resulting oil was purified by "flash" chromatography (94:6:0.6 of methylene chloride : methanol : ammonium hydroxide) . The 20 product fractions were evaporated to dryness, reconcentrated from ethyl ether several times to yield the title product as a yellow foam. m.p.: 89-115 0 C (foam) NMR: consistent with structure 25 HPLC: 100% pure FAB MS: M+H @ m/e=574 Anal. cal'd for C 28

H

3 3

F

2

N

5 0 6 * 0.60 H 2 0 e 0.10 Et 2 O: C, 57.63; H, 6.00; N, 11.83. Found: C, 57.64; H, 5.83; N, 11.80. 30 E. Synthesis of Compound 5: (+)-1,2,3,6-Tetrahydro-1-{N-[4-(2-pyridyl)-4 hydroxypiperidin-1-yl]propyl}carboxamido-5-methoxycarbonyl-4 methoxymethyl-6-(3,4-difluorophenyl)-2-oxopyrimidine 35 dihydrochloride WO 00/37026 PCT/US99/30868 32 a) N-Benzyl-4-hydroxy-4- (2-pyridyl)piperidine. To a stirred solution of 2-bromopyridine (10 mmol) in THF (120 mL) at -78 0 C under argon, 2.5 M n-butyl lithium solution in hexane (4.5 mL, 11.25 mmol) was injected over 5 5 min. After 25 min, 1-benzylpiperidone (10mmol) was added neat. After 30 min, the mixture was quenched by addition of aqueous NH 4 Cl solution (50 mL) . The mixture diluted with ethyl acetate (200mL), washed with brine (150mL), dried (Na 2

SO

4 ) . Solvent was evaporated and the residue was 10 purified by column chromatography (silica gel, EtOAc/Hexane from 20% to 60%) to give the desired product(1.78 g, 66.41 %). 'H-NMR was consistent with the structure. b) 4-Hydroxy-4- (2-pyridyl)piperidine. 15 A mixture of N-benzyl-4-hydroxy-4-(2-pyridyl)piperidine (1.2g, 4.48 mmol), 20% Pd-C (0.2 g) and EtOH (100 mL) was stirred at H2 (150 psi ) for 36 h. The catalyst was removed by filtration and washed with more ethanol. Solvent was evaporated to give pure 4-hydroxy-4-(2-pyridyl)piperidine 20 (0.75 g, 94 %). c) 3- [4-Hydroxy-4- (2-pyridyl)piperidin-1-yl]propylamine. A mixture of 4-hydroxy-4-(2-pyridyl)piperidine (0.730g, 4.1 mmol) , K 2

CO

3 (0.8 g) and KI (0.1 g) in Acetone (100mL) and 25 THF (20mL) was added by N-bromopropylphthalimide (4.5 mmol ) and the mixture was stirred for 14 h. The crude was purified by column chromatography (silica gel, CH 2 Cl 2 :MeOH:2M

NH

3 in MeOH from 90:4:1 to 75:0:25) to give the product to give the phthalimide-protected side chain (1.376 g, 92 %) as 30 a oil. To this, a solution of conc HCl and water (2:1, 25 mL) was added and the mixture was heated for 20 h. Then the reaction mixture was washed and extracted with ether (4 X 50 mL) to remove the phthalic acid formed. Water was evaporated from the aqueous layer to give a desired product as the HCl 35 salt (1.10 g, 100 %) . 'H-NMR was consistent with the product. This intermediate was treated with 6N NaOH and WO 00/37026 PCT/US99/30868 33 converted to the free base prior to use in the next step. d) (+)-1,2,3,6-Tetrahydro-1-{N-[4-(2-pyridyl)-4 hydroxypiperidin-1-ylJpropyl}carboxamido-5-methoxycarbonyl-4 5 methoxymethyl-6-(3,4-difluorophenyl)-2-oxopyrimidine dihydrochloride To a solution of (+)-5-Methoxycarbonyl-4-methoxymethyl 1,2,3,6-tetrahydro-2-oxo-6-(3,4-difluorophenyl)-1-[(4 nitrophenyloxy)carbonyl]pyrimidine (as described in Section 10 I.A.(c)) (201 mg, 0.421 mmol) in dichloromethane (5 mL) was added 3-[4-hydroxy-4-(2-pyridyl)piperidin-1-yl]propylamine (160 mg, 0.729 mmol) at r. t. The resulting mixture was stirred for 2 hours before concentrated to a residue. The residue was purified through column chromatography 15 (chloroform/MeOH/2 N ammonia in MeOH = 100:12:6) to afford desired product (212 mg, 88%) . [a]D = + 109 0 C = 0.24 in CHCl 3 ) . The compound was converted to HCl salt by dissolving in 1 N HCl in ether followed by concentrated to dryness. m. p. 135-138 0 C. 20 Anal. Calcd for C 2 8

H

33

F

2 N0 6 -2HCl (0.4CH 2 Cl 2 +1. 0H 2 0) C, 47.61; H, 5.60; N, 9.77. Found: C, 48.39; H, 5.45; N, 9.76. 'H-NMR (300 MHZ, CDCl 3 ) 5 1.57-1.68 (m, 2H), 1.75-1.82 (m, 2H), 2.02-2.16(m, 2H), 2.45-2.55 (m, 4H). 2.82-2.86 (m, 2H), 3.35-3.45 (m, 2H), 3.46 (s, 3H), 3.68 (s, 3H), 4.67 (s, 25 2H), 6.67 (s, 1H), 7.02-7.20 (m, 3H), 7.38 (d, J = 8 Hz, 1H), 7.66-7.79 (m, 2H), 8.48-8.50 (d, J = 5 Hz, 1H), 8.92(t, J = 7 Hz, 1H). F. Synthesis of Compound 6: 30 (+)-2-Oxo-3-{N-[4-(2-pyridyl)piperidin-1 yllpropyl}carboxamido-4-(3,4-difluorophenyl)-5 methoxycarbonyl -6 -carboxy-1,2,3,6- tetrahydropyrimidine a) Trimethylsilylethyl oxalate 35 Oxalic acid (13.64 g, 151.4 mmol), trimethylsilyl ethanol WO 00/37026 PCT/US99/30868 34 (43.4 ml, 303 mmole) and 0.5 ml of concentrated sulfuric acid were combined in 300 ml of benzene and heated to reflux using a Dean-Stark apparatus. After 18 hrs, the reaction mixture was cooled and diluted with 200 ml of ethyl acetate. The 5 reaction mixture was washed with 1N sodium hydroxide solution (2 X 100 ml), water (1 X 100 ml) and brine. Rotoevaporation of the dried (magnesium sulfate) organic phase yielded 9 g of the crude product as an oil. Chromatography on silica gel (chloroform elution) afforded 5.61 g of the title compound. 10 b) Methyl Trimethylsilylethyl oxaloacetate A solution of 25 ml of tetrahydrofuran containing 1.78 ml (22.38 mmole) of methyl acetate was cooled to -780 C under nitrogen and treated with 24.6 ml (24.62 mmole) of lithium 15 bis(trimethylsilyl)amide. The reaction mixture was stirred for 1 hr and cannulated into a solution of 25 ml of tetrahydrofuran containing trimethylsilylethyl oxalate at 780 C. After 3.5 hr, the reaction mixture was quenched with water and diluted with 150 ml of ethyl acetate. The organic 20 phase was separated and washed with 1 N hydrochloric acid and brine. Concentration of the dried (magnesium sulfate) extracts yielded and oil which was used without further purification in the next step. 25 c) Methyl 2-[(3,4-Difluorophenyl)methylene] trimethylsilylethyl oxaloacetate Methyl trimethylsilylethyl oxaloacetate (4.13 g, 16.78 mmole), 3,4-difluorobenzaldehyde (1.85 ml, 16.78 mmole) , piperidine (66 ml, 0.67 mmole), and acetic acid (201ml, 3.52 30 mmole) were mixed in 50 ml of benzene at ambient temperature. The reaction mixture was then refluxed for 2.5 hr in a Dean Stark apparatus. The reaction mixture was cooled, diluted with 100 ml of ethyl acetate, and washed with water and brine. The combined organic extracts were dried with 35 magnesium sulfate and concentrated to give 7 g of a free flowing yellow liquid which was used in the next step without further purification.

WO 00/37026 PCT/US99/30868 35 d) 2-Methoxy-4- (3,4-difluorophenyl) -5-methoxycarbonyl-6 trimethyl silylethylcarbonyl-1,6-dihydropyrimidine. A suspension of 3.7 g (10 mmole) of crude methyl 2-[(3,4 difluorophenyl)methylene]-trimethylsilylethyl oxaloacetate, 5 0-methyl isourea hemisulfate (2.23 g, 13 mmole), and sodium bicarbonate (3.27 g, 39 mmole) in 50 ml of dry N,N-dimethyl formamide was stirred at 550 C for 6 hr. The reaction mixture was cooled, filtered, and concentrated in vacuo. The residue was dissolved in ethyl acetate and the resulting 10 solution was washed with water. The organic phase was dried (sodium sulfate) and concentrated to afford approximately 3 g of the title compound in crude form. This material was used in the next step without further purification. 15 e) 4-(3,4-Difluorophenyl)-5-methoxycarbonyl-6 trimethylsilylethyl carbonyl-1, 2,3, 6-tetrahydropyrimidin-2 one. 2-Methoxy-4-(3,4-difluorophenyl)-5-methoxycarbonyl-6 trimethylsilylethyl carbonyl-1, 6-dihydropyrimidine (3 g, 7 20 mmole) in crude form was dissolved in 20 ml of tetrahydrofuran and treated with 2 ml of 12 N hydrochloric acid. The resulting reaction mixture was stirred at ambient temperature for 2.5 hr. The pH of the reaction mixture was adjusted to 7 with 2 N lithium hydroxide solution and the 25 reaction mixture was concentrated in vacuo to leave the aqueous phase. The aqueous layer was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried (magnesium sulfate) , and concentrated. The crude product was purified employing preparative centrifugal 30 thin layer chromatography (hexane-ethyl acetate elution) to yield 900 mg of the title compound. 'H NMR (CDCl3) d 0.06 (s, 9H), 1.07 (m, 2H), 3.63 (s, 3H), 4.36 (m, 2H,, 5.38 (d, 1H) 5.73 (br. s, 1H), 7.02-7.2 (m, 3H), 7.52 (br. s, 1H). 35 f) 2-oxo-3-[(4-nitrophenyloxy)carbonyl-4-(3,4 difluorophenyl)-5-methoxycarbonyl-6- WO 00/37026 PCT/US99/30868 36 trimethylsilylethylcarbonyl-1,2,3,6-tetrahydro pyrimidine. To a solution of 4-(3,4-difluorophenyl)-5-methoxycarbonyl-6 trimethylsilyl ethylcarbonyl-1,2,3,6-tetrahydropyrimidin-2 one (478 mg, 1.16 mmole) in 25 ml of dry tetrahydrofuran at 5 -78" C under nitrogen was added 0.7 ml (1.39 mmole) of lithium diisopropylamide. After addition was complete, the reaction mixture was stirred for 1 hr and cannulated into a solution of 15 ml of tetrahydrofuran containing 280 mg (1.39 mmole) of 4-nitrophenyl chloroformate) . The reaction was 10 stirred for 1 hr more, warmed to room temperature and partitioned between 100 ml of ethyl acetate and 50 ml of 1N sodium carbonate solution. The organic phase was washed once more with sodium carbonate and then with brine. The combined organic extracts were dried (sodium sulfate) and concentrated 15 to give a yellow semi-solid. The crude product was flash chromatographed on silica gel (chloroform-acetone elution, 9:1, v/v) to give the title compound as a pale yellow solid. g) (±)-2-Oxo-3-{N-[4-(2-pyridyl)piperidin-1 20 yllpropyl}carboxamido-4-(3,4-difluorophenyl)-5 methoxycarbonyl-6-trimethylsilylethylcarbonyl-1,2,3,6 tetrahydropyrimidine. 3-Aminopropyl-4-(2-pyridyl)piperidine ( 240 mg, 1.1 mmole) was combined with 2-oxo-3- [(4-nitrophenyloxy)carbonyll -4 25 (3,4-difluorophenyl)-5-methoxycarbonyl-6 trimethylsilylethylcarbonyl-1,2,3,6-tetrahydro pyrimidine (577 mg, 1.1 mmole) in 10 ml of dry tetrahydrofuran at ambient temperature. The resulting solution was stirred for 2 hr and then diluted with 50 ml of ethyl acetate. The 30 reaction mixture was then washed in succession with 1N hydrochloric acid, 10% sodium carbonate solution, 1N, hydrochloric acid, and brine. The combined organic extracts were dried (sodium sulfate) and concentrated to give 550 mg of crude product. This material was purified via preparative 35 centrifugal thin layer chromatography on silica gel (chloroform-methanol elution) to yield 228 mg of the title compound. 'H NMR (CDCl 3 ) d 0.06 (s, 9H), 1.07 (m, 2H), 1.78 WO 00/37026 PCTIUS99/30868 37 (m, 2H), 1.91 (m, 4H), 2.1 (m, 2H), 2.45 (m, 2H), 2.71 (m 1H), 3.05 (m, 2H), 3.31 (ddd, 1H), 3.42 (ddd, 1H), 3.72 (s, 3H) , 4.32 (m, 2H) , 6.60 (s, 1H) 7.05-7.35 (m, 6H) , 7.61 (ddd, 1H), 8.51 (d, 1H), 8.87 (dd, 1H). 5 h) (+)-2-Oxo-3-{N-[4-(2-pyridyl)piperidin-1 yl]propyl}carboxamido-4-(3,4-difluorophenyl)-5 methoxycarbonyl-6-trimethylsilylethylcarbonyl-1,2,3,6 10 tetrahydropyrimidine. Racemic 2-oxo-3-{N-[4-(2-pyridyl) piperidin-1 yl]propyl}carboxamido-4-(3,4-difluorophenyl)-5 methoxycarbonyl-6-trimethylsilylethylcarbonyl-1,2,3,6 tetrahydro pyrimidine was resolved by chiral HPLC [Chiralpak 15 AD, 250 X 20 mm; 1 315 nm; isocratic conditions 80% hexanes:20% 2-propanol containing 0.2% diethylamine; flow rate = 6.0 ml/min] . The first of the two enantiomers to elute was collected to give 110 mg of the title compound. Analytical chiral chromatography [Chiralpak AD, 250 X 4.6 mm, 20 1 280 nm; isocratic conditions 80% hexanes-20% 2-propanol containing 0.1% diethylamine; flow rate = 2.0 ml/min] showed the title compound to be 98.66% pure. i) (+)-2-Oxo-3-{N-[4-(2-pyridyl)piperidin-1 25 yl]propyl}carboxamido-4-(3,4-difluorophenyl)-5 methoxycarbonyl -6 -carboxy-1,2,3,6 - tetrahydropyrimidine (+)-2-Oxo-3-{N-[4-(2-pyridyl)piperidin-l yl]propyl}carboxamido-4-(3,4-di fluorophenyl)-5 methoxycarbonyl-6-trimethylsilylethylcarbonyl-1,2,3,6 30 tetrahydropyrimidine (110 mg, 0.16 mmole) was dissolved in 2 ml of dry tetrahydrofuran and treated with 33 mL (0.33 mmole) of 1M tetrabutyl ammonium fluoride solution in tetrahydrofuran. The reaction mixture was protected from moisture and heated to 600 for 2 hr. All volatiles were 35 removed under reduced pressure and the residual material was purified by preparative thin layer chromatography (ethyl WO 00/37026 PCT/US99/30868 38 acetate-pyridine-acetic acid-water elution, 90:10:1:1, v/v) to give the title compound as an off-white solid. HPLC: 95.5%. 'H NMR (DMSO-d 6 ) d 1.7-1.9 (m, 8H), 2.78 (m, 2H), 3.2-3.4 (m, 5H), 3.54 (s, 3H), 6.41 (s, 1H), 7.10 (m, 1H), 5 7.2 (m, 3H), 7.29 (d, 1H), 7.41 (dd, 1H), 7.71 (dd, 1H), 8.49 (d, 1H), 8.89 (dd, 1H), 9.91 (br. s, 1H). FAB MS: 558 (M' + H) , 580 (M* + Na) . G. Synthesis of Compound 7: 10 (+)-1,2,3,6-Tetrahydro-1-{N-[4-(2-pyridyl)piperidin-1 yl] propyl}carboxamido-5-methoxycarbonyl-4-hydroxymethyl-6 (3,4-difluorophenyl)-2-oxopyrimidine a) (+)-1,2,3,6-Tetrahydro-1-{N-[4-(2-pyridyl)piperidin-1 15 yl]propyl}carboxamido-5-methoxycarbonyl-4-hydroxymethyl-6 (3, 4-difluorophenyl) -2-oxopyrimidine To a solution of (+)-1,2,3,6-tetrahydro-1-{N-[4-(2 pyridyl)piperidin-1-yllpropyl}carboxamido-5-methoxycarbonyl 4-methoxymethyl-6- (3 ,4-difluorophenyl) -2-oxopyrimidine (250 20 mg, 0.448 mmol) (as described in Section I.D. (d) in dichloromethane (20 mL) at -78 0 C was added boron tribromide (1.0 M in dichloromethane, 1.20 mL, 1.20 mmol) dropwise. The cooling bath was removed and the reaction mixture was stirred while warmed gradually to r. t. for 4 hours before quenched 25 with MeOH (5 mL) and concentrated to a residue. The residue was purified through preparative thinlayer chromatography using chloroform/MeOH/2 N ammonia in MeOH = 100:4:1 as the eluent to afford desired product (15 mg, 6%) . MS: (M+H) 544. 1H-NMR (300 MHZ, CDCl 3 ) 5 1.79-1.98 (m, 6H), 2.05-2.12 (m, 30 2H), 2.45(t, J = 7 Hz, 2H), 2.68-2.76 (m, 1H), 3.04-3.08 (m, 2H), 3.25-3.42 (m, 2H), 3.68 (s, 3H), 4.95 (dd, J = 17 Hz, 23 Hz, 2H), 6.65 (s, 1H), 6.97-7.23 (m, 5H), 7.62 (t, J = 8 Hz, 1H), 8.48-8.50 (d, J = 5 Hz, 1H), 8.98(t, J = 7 Hz, 1H). 35 H. Synthesis of Compound 8 WO 00/37026 PCT/US99/30868 39 (+)-l-3-{[4-(3,4-Difluorophenyl)-2,5-dioxo-1,2,5,7 tetrahydro-4H-furo[3,4-d}-pyrimidine-3-carbonyl]amino) propyl-4- (2-pyridyl) -piperidine. 5 a) Methyl 2-{(3,4-difluorophenyl)methylene)-3-oxobutyrate. A mixture of 3,4-difluorobenzaldehyde (14.2 g, 0.1 mol), methyl acetoacetate (12.2 g, 0.105 mol), piperidine (0.430 g, 5 mmol), and acetic acid (0.30 g, 5 mmol) in benzene (150 mL) was stirred and refluxed with a Dean-Stark trap for 8 10 hours. Benzene was evaporated, the residue was dissolved in ethyl acetate (200 mL) and washed with brine (50 mL), saturated potassium bisulfate solution (50 mL), and saturated sodium bicarbonate solution in sequence. The ethyl acetate solution was dried (magnesium sulfate), solvent removed under 15 reduced pressure and the residue was purified by column chromatography (Si02, EtOAc/hexane, 10%-15%). The product, methyl 2-{ (3, 4-difluorophenyl)methylene} -3-oxobutyrate, was obtained as a yellow oil (0.98 g, 98.3%) and was used in the next step without any further characterization. 20 b) 6-(3,4-Difluorophenyl)-1,6-dihydro-2-methoxy-5 methoxycarbonyl-4-methylpyrimidine. A mixture of methyl 2-{ (3,4-difluorophenyl)methylene}-3 oxobutyrate (8.8 g, 36.6 mmol), 0-methylisourea hydrogen 25 sulfate (9.4 g, 55 mmol), and NaHCO 3 (12.3 g, 0.146 mol) in DMF (30 mL) was stirred and heated at 70 0 C for 16 hours. The mixture was cooled, diluted with EtOAc (300 mL) and washed with water (5 X 300 mL), brine (300 mL), and dried (MgSO 4 ). Solvent was evaporated and the crude product was purified by 30 flash column chromatography on silica gel using 10% through 20% EtOAc in hexane as the gradient eluent, to leave the product as an oil (3.82 g, 30.2%); 'H-NMR (CDCl 3 ) : 5 2.32,2.39 (2 s, 3 H), 3.58, 3.64 (2 s, 3 H), 3.72, 3.85 (2 s, 3 H), 5.55 ( s, 1H), 6.13-7.8 (m, 4 H). 35 c) 6-(3,4-Difluorophenyl)-1,6-dihydro-2-methoxy-5- WO 00/37026 PCTIUS99/30868 40 methoxycarbonyl-4-methyl-1-[(4-nitrophenyloxy) carbonyl]pyrimidine. To a solution of 6-(3,4-diifluorophenyl)1,6-dihydro-2 methoxy-5-methoxycarbonyl-4-methylpyrimidine (2.82 g, 9.52 5 mmol) and 4-dimethylaminopyridine (1.16 g, 9.52 mmol) in

CH

2 Cl 2 (50 mL), at 0-5 0 C, 4-nitrophenyl chloroformate (1.82 g, 9.04 mmol) was added and the mixture was allowed to warm to room temperature. After 12 hours solvent was evaporated and the residue was purified by flash column chromatography 10 (SiO 2 , EtOAc/hexane, 10-15%) to obtain the product as white crystals (3.72, 84.7%) ; m.p. 172-174 0 C; 1 H-NMR (CDCl 3 ) : 5 2.51 (s, 3 H), 3.72(s, 3 H), 3.97 (s, 3 H), 6.26 (s, 1H), 7.0-7.3 (m, 3 H) , 7.38 (d, J = 9.3 Hz, 2 H), 8.32 (d J = 9.3 Hz, 2 H). 15 d) (+)-6-(3,4-Difluorophenyl)-1,6-dihydro-2-oxo-5-methoxy carbonyl-4-bromomethyl-1-[(4-nitrophenyl oxy) carbonyl] pyrimidine. . To a well stirred solution of (+)-6-(3,4-difluorophenyl)-1,6 20 dihydro-2-methoxy-5-methoxycarbonyl-4-methyl-1-[(4 nitrophenyloxy)carbonyl]pyrimidine (1.5 mmol, 0.66 g) in 5 mL of chloroform was added a solution of bromine (1.5 mmol, 0.09 mL) in 3 mL of chloroform at 0 0 C and the solution was allowed to attain room temperature over 1.5 h. The solvent 25 was removed in vacuo and the residue was again dissolved in CHCl 3 (20 mL) and washed with brine. The organic layer was separated, dried over Na 2

SO

4 , filtered and the solvent was removed in vacuo to get 0.81 g of (+)-6-(3,4-difluorophenyl) 1,6-dihydro-2-oxo-5-methoxycarbonyl-4-bromomethyl-l-[(4 30 nitrophenyloxy)carbonyllpyrimidine as a yellow foam. It was used in the next step without any purification. 'H NMR 5 3.75 (s, 3 H), 4.67 (ABq, dA = 4.56, dB 4.78, J = 10.8 Hz, 2 H), 6.35 (s, 1 H), 7.09 - 7.19 (m, 4 H), 7.37 (d, J = 9.0 Hz, 2 H), 8.27 (d, J = 9.0 Hz, 2 H). 35 e) (+)-4-(3,4-Difluoro-phenyl)-2,5-dioxo-1,2,4,5,6,7- WO 00/37026 PCT/US99/30868 41 hexahydro-cyclopetapyrimidine-3-carboxylic acid-4 -nitrophenyl ester. (+)-6-(3,4-Difluorophenyl)-1,6-dihydro-2-oxo-5-methoxy c a r b o n y 1 - 4 - b r o m o m e t h y 1 - 1 - [ ( 4 5 nitrophenyloxy)carbonylIpyrimidine (1.5 mmol, 0.81 g) was heated in oil bath for 3 h (bath temperature 1300C) . The brown residue thus obtained was washed with CHCl 3 and (+)-4 (3,4-difluoro-phenyl)-2,5-dioxo-1,2,4,5,6,7-hexahydro cyclopenta pyrimidine-3-carboxylic acid-4-nitrophenyl ester 10 was obtained as a pale brown solid which was used in the next step without further purification (crude wt. 0.51 g) . 1H NMR (DMSO) 6 4.94 (br,s, 2 H), 6.08 (s, 1 H), 7.20 - 7.43 (m, 4 H), 8.35 (d, J = 10.2 Hz, 2 H). 15 f) (+)-1-3-{[4-(3,4-Difluorophenyl)-2,5-dioxo-1,2,5,7 tetrahydro-4H-furo[3,4-d]-pyrimidine-3-carbonyl]amino} propyl-4-(2-pyridyl)-piperidine A solution of (+)-4-(3,4-difluoro-phenyl)-2,5-dioxo 1,2,4,5,6,7-hexahydro-cyclopenta pyrimidine-3-carboxylic 20 acid-4-nitrophenyl ester ((0.33 mmol, 0.14 g) and 3-[4-(2 pyridyl)-piperidin-1-yl)propyl amine (0.33 mmol, 0.07 g) in 10 mL of anhydrous THF was stirred overnight at room temperature. The solvent was removed in vacuo and the residue was purified by column chromatography (CH 2 Cl 2 25 followed by 9:1 CH 2 Cl 2 /MeOH) to obtain (+)-1-3-{[4-(3,4 difluorophenyl) -2,5-dioxo-1,2,5,7-tetrahydro-4H-furo[3,4-d] pyrimidine-3-carbonyllamino}-propyl-4-(2-pyridyl)-piperidine as an oil. (0.16 g, 98%) . 1H NMR 5 1.77-1.98 (m, 6 H) , 2.20 2.25 (m, 2 H), 2.54 (t, J = 7.2 Hz, 2 H), 2.74-2.80 (m, 1 H), 30 3.11-3.18 (m, 2 H), 3.29-3.39 (m, 2 H), 4.86 (s, 2 H), 6.04 (br s, 1 H), 6.46 (s, 1 H), 7.07-7.26 (m, 5 H), 7.63 (dt, J = 7.8 Hz, J = 1.8 Hz, 1 H), 8.48 (d, J = 3.9 Hz), 9.22 (br t, J = 5.1 Hz, 1 H). It was characterized as a dihydrochloride salt(hygroscopic). M.P. 90-95 0 C; [a] = + 35 43. 4 (c = 0 . 25, acetone) ; Anal . Calcd. For C 2 6

H

29

N

5 0 4

F

2 Cl 2 . 4 .0 WO 00/37026 PCTIUS99/30868 42

H

2 0: C, 47.57; H, 5.68; N, 10.67. Found: C, 47.59; H, 5.32; N, 10.79. I. Synthesis of Compound 9: 5 3- (3-Carboxypropylcarbamoyl)-4(S)- (3,4-difluorophenyl) -2-oxo 1,2,3,4-tetrahydro-4H-furo[3,4-d]pyrimidine. a) (+) 5-Methoxycarbonyl-4-methoxymethyl-1, 2,3, 6-tetrahydro-2 oxo-6 (S) - (3,4-difluorophenyl) -1- [(2 10 ethoxycarbonylethylamino) - carbonyl] pyrimidine B-Alanine ethyl ester hydrochloride (87 mg, 0.57 mmol), (+) 5-methyoxycarbonyl-4-methoxymethyl-1, 2,3, 6-tetrahydro-2-oxo 6(S)-(3,4-difluorophenyl)-1-[(4-nitrophenyloxy) carbonylipyrimidine (as described in Section I.A.(c)) (227mg, 15 0.48mmol), and triethylamine (209mL, 1.50mmol) were combined in 5mL of dichloromethane. The yellow mixture was stirred at ambient temperature for 1 hour, then evaporated to dryness. The resulting oil was purified by "flash" chromatography (5.02, 98:2:0.2 of dichloromethane : methanol 20 : ammonium hydroxide) . The product fractions were evaporated to dryness to give the title compound as a clear oil. b) (+) -5-Methoxycarbonyl-4-methoxymethyl-1,2,3, 6-tetrahydro 2-oxo-6 (S) - (3,4-difluorophenyl) -1- [ (2-carboxyethylamino) 25 carbonyl]pyrimidine (+) -5-Methoxycarbonyl-4-methyoxymethyl-1, 2,3, 6-tetrahydro-2 oxo-6 (S) - (3,4-difluorophenyl) -l- [(2 ethoxycarbonylethylamino)carbonyl]pyrimidine (168mg, 0.37mmol) was dissolved in 5mL of methanol. A 2.OM solution 30 of aqueous sodium hydroxide (400mL, 0.80mmol) was added dropwise. The mixture was stirred at ambient temperature for 4.5 hours, then concentrated in vacuo. The resulting residue was partioned between ethyl acetate and 2.OM aqueous hydrochloric acid. The phases were separated and the aqueous 35 phase was extracted twice with ethyl acetate. The combined organic phases were then washed with brine. The organic WO 00/37026 PCT/US99/30868 43 phase was then dried over sodium sulfate, filtered, and the filtrate was then evaporated to dryness. The residue was reconcentrated from diethyl ether. The resulting white foamy solid was purified by "flash" chromatography (5.02, 98:2:0.2 5 of dichloromethane : methanol : glacial acetic acid). The product fractions were evaporated to dryness and the residue was reconcentrated from diethyl ether to give the title compound as a white foamy solid. m.p.: 52-550C 10 NMR: consistent with structure HPLC: 100% pure FAB MS: M+H @ m/e=428.0 Anal. cal'd for C 1 8 Hi 9

F

2

N

3 0 7 * 0.15H 2 0 e 0.10Et 2 O: C, 50.51; H, 4.68; N, 9.61. Found: C, 50.50; 15 H, 4.46; N, 9.51. c) 3- (2-Carboxyethylcarbamoyl) -4(S)-(3,4-difluorophenyl)-2,5 dioxo-1,2,3,4-tetrahydro-4H-furo[3,4-d]pyrimidine (+) -5--Methoxycarbonyl-4-methoxymethyl-1,2,3,6-tetrahydro-2 20 oxo - 6 ( S ) - ( 3 , 4 - d i f luor ophenyl ) - 1 - [ ( 2 c arboxye thyl amino) c arbonyl ] pyrimi dine (3 0 8mg, 0. 72mmol) was dissolved in 5mL of dichloromethane, and then cooled to -78'C in a dry ice/acetone bath while stirring under a nitrogen atmosphere. A 1.OM solution of boron tribromide in 25 dichloromethane (3.25mL, 3.3mmol) was then added. The mixture was stirred at -78 0 C for 45 minutes, then slowly warmed to ambient temperature over 1 hour. 15mL of sat'd sodium bicarbonate was added until a neutral pH was achieved. The mixture was stirred at ambient temperature for 10 30 minutes. Volatile components were removed by evaporation. The resulting aqueous solution was stirred at ambient temperature for 18 hours. Aqueous solution was then acidified with concentrated hydrochloric acid, precipitating a white solid which was filtered. The resulting white solid 35 was purified by preparative reverse phase HPLC using an H 2 0 : CH 3 CN gradient containing 0.1% TFA. The product fractions were evaporated to dryness to give the title compound as a WO 00/37026 PCT/US99/30868 44 white solid. NMR: consistent with structure HPLC: 96.2% pure FAB MS: M+H @ m/e=382.1 5 II. Synthesis of Other Possible Precursors Schemes 4A and 4B describe the synthesis of other possible precursors which may be utilized to prepare additional 10 compounds (Marquis, et al. J. Med. Chem. (1998) 41: 3563) III. Oral Composition As a specific embodiment of an oral composition of a compound 15 of this invention, 100 mg of one of the compounds described herein is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gel capsule. 20 IV. Pharmacological Profiles of the Comoounds in Cloned Human Adrenergic Receptors. Binding affinities were measured for selected compounds of 25 the invention at six cloned human ai and a 2 receptor subtypes, as well as at the L-type calcium channel. The protocols for these experiments are given below. 1. Protocol for the Determination of the Potency of al 30 Antagonists The activity of compounds at the different human receptors was determined in vitro using cultured cell lines that selectively express the receptor of interest. These cell lines were prepared by transfecting the cloned cDNA or cloned 35 genomic DNA or constructs .containing both genomic DNA and cDNA encoding the human a-adrenergic receptors as follows: WO 00/37026 PCT/US99/30868 45 ald Human Adrenergic Receptor: The entire ceding region of aid (1719 bp) , including 150 base pairs of 5' untranslated sequence (5' UT) and 300 bp of 3' untranslated sequence (3' UT) , was cloned into the BamHI and ClaI sites of the 5 polylinker-modified eukaryotic expression vector pCEXV-3, called EXJ.HR. The construct involved the ligation of partial overlapping human lymphocyte genomic and hippocampal cDNA clones: 5' sequence were contained on a 1.2 kb SmaI XhoI genomic fragment (the vector-derived BamHI site was used 10 for subcloning instead of the internal insert-derived SmaI site) and 3' sequences were contained on an 1.3 kb XhoI-ClaI cDNA fragment (the ClaI site was from the vector polylinker). Stable cell lines were obtained by cotransfection with the plasmid a1A/EXJ (expression vector containing the a, 15 receptor gene (old nomenclature)) and the plasmid pGCcos3neo (plasmid containing the aminoglycoside transferase gene) into LM(tk-) cells using calcium phosphate technique. The cells were grown, in a controlled environment (370C., 5% C0 2 ) , as monolayers in Dulbecco's modified Eagle's Medium (GIBCO, 20 Grand Island, NY) containing 25 mM glucose and supplemented with 10% bovine calf serum, 100 units/ml penicillin g, and 100Lg/ml streptomycin sulfate. Stable clones were then selected for resistance to the antibiotic G-418 (1mg/ml), and membranes were harvested and assayed for their ability to 25 bind [ 3 H]prazosin as described below (see "Radioligand Binding assays"). The cell line expressing the human aid receptor used herein was designated L-alA (old nomenclature) and was deposited 30 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The cell line expressing the human aid receptor, 35 was accorded ATCC Accession No. CRL 11138, and was deposited on September 25, 1992.

WO 00/37026 PCT/US99/30868 46 ailb Human Adrenergic Receptor: The entire coding region of alb (1563 bp), including 200 base pairs and 5' untranslated sequence (5' UT) and 600 bp of 3' untranslated sequence (3' UT), was cloned into the EcoRI site of pCEXV-3 eukaryotic 5 expression vector. The construct involved ligating the full length containing EcoRI brainstem cDNA fragment from X ZapII into the expression vector. Stable cell lines were selected as described above. The cell line used herein was designated L-alB and was deposited with the American Type Culture 10 Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The cell line L-alB was accorded ATCC Accession No. CR 11139, on September 29, 15 1992. a, Human Adrenergic Receptor: The entire coding region of aa (1401 bp) , including 400 base pairs of 5' untranslated sequence (5' UT) and 200 bp of 3' untranslated sequence (3' 20 UT), was cloned into the KpnI site of the polylinker-modified pCEXV-3-derived eukaryotic expression vector, EXJ.RH. The construct involved ligating three partial overlapping fragments: a 5' 0.6 kb HincII genomic clone, a central 1.8 EcoRI hippocampal cDNA clone, and a 3' 0.6Kb PstI genomic 25 clone. The hippocampal cDNA fragment overlaps with the 5' and 3' genomic clones so that the HincII and PstI sites at the 5' and 3' ends of the cDNA clone, respectively, were utilized for ligation. This full-length clone was cloned into the KpnI site of the expression vector, using the 5' and 30 3' KpnI sites of the fragment, derived from vector (i.e., pBluescript) and 3'-untranslated sequences, respectively. Stable cell lines were selected as described above. The stable cell line expressing the human a, receptor used herein was designated L-aic (old nomenclature) and was 35 deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International WO 00/37026 PCT/US99/30868 47 Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The cell line expressing the human aia receptor was accorded Accession No. CR 11140, on September 25, 1992. 5 Radioligand Binding Assays for a, receptors: Transfected cells from culture flasks were scraped into 5 ml of 5 mM Tris-HCl, 5 mM EDTA, pH 7.5, and lysed by sonication. The cell lysates were centrifuged at 1000 rpm for 5 min at 4 0 C, 10 and the supernatant was centrifuged at 30,000 x g for 20 min at 4 0 C. The pellet was suspended in 50 mM Tris-HCl, 1 mM MgCl 2 , and 0.1% ascorbic acid at pH 7.5. Binding of the ai antagonist [ 3 H]prazosin (0.5 nM, specific activity 76.2 Ci/mmol) to membrane preparations of LM(tk-) cells was done 15 in a final volume of 0.25 ml and incubated at 37 0 C for 20 min. Nonspecific binding was determined in the presence of 10 pM phentolamine. The reaction was stopped by filtration through GF/B filters using a cell harvester. Inhibition experiments, routinely consisting of 7 concentrations of the 20 tested compounds, were analyzed using a non-linear regression curve-fitting computer program to obtain Ki values. a 2 Human Adrenergic Receptors: To determine the potency of ai antagonists at the a2 receptors, LM(tk-) cell lines stably 25 transfected with the genes encoding the a2a, a2b, and a2c receptors were used. The cell line expressing the a2. receptor is designated L-a 2 A, and was deposited on November 6, 1992 under ATCC Accession No. CRL 11180. The cell line expressing the a2b receptor is designated L-NGC-a 2 B, and was 30 deposited on October 25, 1989 under ATCC Accession No. CRL10275. The cell line expressing the a2c receptor is designated L-a 2 c, and was deposited on November 6, 1992 under ATCC Accession No. CRL-11181. All the cell lines were deposited with the American Type Culture Collection, 12301 35 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes WO 00/37026 PCT/US99/30868 48 of Patent Procedure. Cell lysates were prepared as described above (see Radioligand Binding Assays), and suspended in 25 mM glycylglycine buffer (pH 7.6 at room temperature) . Equilibrium competition binding assay were performed using 5 [3H]rauwolscine (0.5nM), and nonspecific binding was determined by incubation with 10pM phentolamine. The bound radioligand was separated by filtration through GF/B filters using a cell harvester. 10 Determination of the Activity of a, Antagonists at Calcium Channels: The potency of ai antagonists at calcium channels may be determined in competition binding assays of [I3Hlnitrendipine to membrane fragments of rat cardiac muscle, essentially as 15 described by Glossman and Ferry (Methods in Enzymology 109:513-550, 1985) . Briefly, the tissue is minced and homogenized in 50 mM Tris-HCl (pH 7.4) containing 0.1 mM phenylmethylsulfonyl fluoride. The homogenates are centrifuged at 1000 g for 15 minutes, and the resulting 20 supernatant centrifuged at 45,000 g for 15 minutes. The 45,000 g pellet is suspended in buffer and centrifuged a second time. Aliquots of membrane protein are then incubated for 30 minutes at 370C in the presence of [3H]nitrendipine (1 nM), and nonspecific binding determined in the presence 25 of 10 gM nifedipine. The bound radioligand is separated by filtration through GF/B filters using a cell harvester. The compounds described above were assayed using cloned human alpha adrenergic receptors. The preferred compounds were 30 found to be selective a,, antagonists. The binding affinities of several compounds are illustrated in the following table. Binding affinities of selected compounds of the present 35 invention at cloned human ad, aib, aia, U2a' a2b, and a2c receptors (h = human).

WO 00/37026 PCT/US99/30868 49 Compound haia halb hald ha23 ha 2 b ha2c 1 260 >50,000 >50,000 40,000 26,000 >50,000 2 0.3 1,500 3,700 4,200 8,700 1,500 3 212 >2,000 >5,000 5,623 9,661 3,388 5 4 2.1 1,354 >5,000 22,000 17,000 8,000 6 23.5 >2,000 >5,000 >50,000 37,584 >50,000 7 0.8 530 1200 380 1200 260 8 3 1,600 1,970 1,500 970 1,500 9 >500 >2,000 >5,000 >50,000 >50,000 >50,000 10 WO 00/37026 PCT/US99/30868 50 00 0 0 0' 00 000 0 U 0 0 04 00 00 ++ +4

-

1 Q)) 0 0 0 + 0a 00 0 0 = 0H 4 WO 00/37026 PCTIUS99/30868 51 0 0 r-- q ro~ ~ / ro+ o 0 04 04449 z z ,4~ C) C)~ 0 ~ 0 0 0 Q) Q 0 0c0 + Z-I oz CN 0 z z ) 0 zN (N4 00 C U 00J

U))

WO 00/37026 PCT/US99/30868 52 0 OD0 / 0~ U2 o4 0 0 0 ) U) N4 z 0 00 WO 00/37026 PCT/US99/30868 53 0 z 00 ++ 4-0 En 4 00 0 w4 SU 04 WO 00/37026 PCT[US99/30868 54 0 0 0 Q) a o 0 zz A 4N m Cl 0 0 21 0 040) r~ CN ~ 0 zz C) C) WO 00/37026 PCTIUS99/30868 55 0 0 5"0 U0 00 0= u' 0 + ' 4 E-4 Q4- 0 0)H 41) U U u E-4 /U 0 ~ U) U 00 f14 . U = 04 0n b Uz~ 0 .H 0 0 rJ/ 0 ,C) U UU U wN rn rT4 - 0 -4 u' E-4 00U U-U0 0 WO 00/37026 PCTIUS99/30868 56 -Z N7C Q) C) 04 ) z o Q 0 7 7 0 + U

N

+~~O 0 z + Nx NO\< 0 0

NQI

WO 00/37026 PCT/US99/30868 57 0 0 + 0 0 U 41 r) oo o 0 4 0 0 4 0 0 OW O4 O 7O Ou WO 00/37026 PCTIUS99/30868 58 F F F F F O2N CH2CIa Et 3 N O 0 ON H0 N EtO

NH

2 Et0 O N -HCI H NaOH/ MeOH FF F F F F 0N F HO O 0

CH

2

CI

2 BBr 3 HO'K N IN O0 ON OH H O H O N H NaHCO 3 (aq) F I.F 0 0 00 HO) N N O N H Compound 9 Scheme 8: Synthesis of Compound 9

Claims (33)

1. A compound having the structure: 5 F F 0 o Ri N N 3 10 H R2 N H wherein R, is -OCH 3 or OH; 15 wherein R 2 is -CH 2 OH, -CH 2 0CH 3 , or -COOH; wherein R, and R 2 together form a 5-membered lactone ring; WO 00/37026 PCT/US99/30868 60 wherein R 3 is selected from the group consisting of - (CH 2 ) 3 0H, -(H2C) /-N -(H 2C)3-N -O 10 -(H 2 C) 3-N N 5(H2NC) 3--N (H2C) -N -(H2C)(H--N an 15OH N -2 N 10 OH (H( 2 Hc) 3-- and 20 OH 10 N provided that when R, is OH, R 3 cannot be 25 -(H2C)3 -N or a pharmaceutically acceptable salt thereof. 30

2. The (-) enantiomer of the compound of claim 1.

3. The (+) enantiomer of the compound of claim 1. WO 00/37026 PCTIUS99/30868 61

4. The compound of claim 1 having the structure: F F 5 0 0 0 N N R3 H N 0 H 10

5. The compound of claim 4 having the structure: F F 15 0 0 ON N OH H N 0 H 20

6. The compound of claim 4 having the structure: F F 25 0 0 ON N N N 0 H WO 00/37026 PCTIUS99/30868 62

7. The compound of claim 4 having the structure: F 5 F 0 0 N N +N1 o N N 0O 10 H

8. The compound of claim 4 having the structure: 15 F F 0 0 20 'K-0 20 N N +N H H N N 0_ H WO 00/37026 PCT/US99/30868 63

9. The compound of claim 4 wherein R3 is selected from the group consisting of Na 5 HO -(H2C) -NO 10 OH -(H 2 C)3-N 3 and 15 OH -(H2C) -N 20

10. The compound of claim 1 having the structure: F 25 F 0 o N N N R2 N 0 30 WO 00/37026 PCTIUS99/30868 64

11. The compound of claim 10 having the structure: F 5 F 0 o N N N HOHN N '0 10 0 H 15

12. The compound of claim 10 having the structure: F F 20 0 0 N N N N HH HO4 N _0 H 25

13. The compound of claim 10 having the structure: F F 30 0 o O N NN H 35 WO 00/37026 PCT/US99/30868 65

14. A compound having the structure: F F 5 0 o o N N OH N O H 10 15. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 1, 6, 8, 9, 12, 13 or 14 and a pharmaceutically acceptable carrier.

15

16. The pharmaceutical composition of claim 15, wherein the therapeutically effective amount is an amount from about 0.01 mg to about 500 mg.

17. The pharmaceutical composition of claim 16, wherein the 20 therapeutically effective amount is an amount from about 0.1 mg to about 60 mg.

18. The pharmaceutical composition of claim 17, wherein the therapeutically effective amount is an amount from 25 about 1 mg to about 30 mg.

19. The pharmaceutical composition of claim 15, wherein the carrier is a liquid and the composition is a solution. 30

20. The pharmaceutical composition of claim 15, wherein the carrier is a solid and the composition is a tablet.

21. The pharmaceutical composition of claim 15, wherein the carrier is a gel and the composition is a suppository. WO 00/37026 PCT/US99/30868 66

22. A method of treating a subject suffering from benign prostatic hyperplasia which comprises administering to the subject an amount of the compound of claim 1 or 14 effective to treat benign prostatic hyperplasia. 5

23. A method of treating a subject suffering from benign prostatic hyperplasia which comprises administering to the subject an amount of the compound of claim 1 or 14 in combination with a 5 alpha-reductase inhibitor 10 effective to treat benign prostatic hyperplasia.

24. The method of claim 23, wherein the 5-alpha reductase inhibitor is finasteride. 15

25. A method of relaxing lower urinary tract tissue which comprises administering to the subject an amount of the compound of claim 1 effective to relax lower urinary tract tissue. 20

26. The method of claim 25, wherein the lower urinary tract tissue is urethral smooth muscle.

27. A method of inhibiting contraction of prostatic tissue 25 in a subject which comprises administering an amount of a compound according to claim 1 or 14 effective to inhibit contraction of prostatic tissue.

28. A method of treating a disease which is susceptible to 30 treatment by antagonism of the a1A receptor which comprises administering to the subject an amount of the compound of claim 1 or 14 effective to treat the disease. 35

29. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 1 or 14 in combination with therapeutically effective amount of finasteride and a pharmaceutically WO 00/37026 PCT/US99/30868 67 acceptable carrier.

30. The pharmaceutical composition of claim 29, wherein the therapeutically effective amount of the compound is an 5 amount from about 0.01 mg to about 500 mg and the therapeutically effective amount of the finasteride is about 5 mg.

31. The pharmaceutical composition of claim 29, wherein the 10 therapeutically effective amount of the compound is an amount from about 0.1 mg to about 60 mg and the therapeutically effective amount of the finasteride is about 5 mg. 15

32. The pharmaceutical composition of claim 29, wherein the therapeutically effective amount of the compound is an amount from about 1 mg to about 30 mg and the therapeutically effective amount of the finasteride is about 5 mg. 20

33. A process for making a pharmaceutical composition which comprises combining a therapeutically effective amount of a compound of claim 1 or 14 and a pharmaceutically acceptable carrier.