AU610591B2 - Phosphorus-containing hmg-coa reductase inhibitors, new intermediates and method - Google Patents

Description

USSN 53,281

COMMONWEALTH OF AUSTRALIA Patent.; Act

DECARAIONFOR A PATENT APPLICATION 'Y rritsTaucnotits mInit "Conviitions" In support of the convent ion application made by itapplicablis (b E. R. SQUIBB SONS, Insert FULL name(s) INC., a corporation duly organized and existing of applicant(s) under the laws of the State of Delaware, United States of America, having its offices at Lawrenceville-Princeton Road, Princeton, New Insert "of addition"~ Jersey, United States of America, if applicable (hereinafter called "applicant for a pa,:nt for an (it) Insert IITLE of invention entitled (d) invention PHO SPHORUS- CONTAINING HMG-CoA REDUCTASE INHIBITORS, NEW INTERMEDIATES AND METHOD Wi Insert FULL niae(s) AND addrit~es) of I Nicholas P. Malatestinic of Princdeton, New Jersey, United States declaranits) (Set lseadnoteiv) of America,

do solemnly and sincerely declare as follows:

1. 1 am authorized to make this declaration on behalf of the applicant(s),

JOInsert FULL name(s) 2. DONALD STEVEN KARANEWSKY; SCOTT ADAMS BILLER; and ANDXddre~s(cs))of tt actual inventor(,) ERIC MICHAEL GORDON; 8 Charred Oak Lane, East Windsor, New Jersey, United States of America; 136 Nancy Lane, Ewing, New Jersey, United States of America; and 126 Laning Avenue, Pennington, New Jersey, United States of America

W: R~ecite how, appli- *is/are the actual inventor(s) of the invention ind the facts upon which the applicant cant(%)derivels) title from actual is entitled to make the application are as follows: inventor(v) See headnoiee) The said E. R. SQUIBB SONS, INC., is the assignee of the c3aid DONALD STEVEN KARANEWSKY; SCOTT ADAMS BILLER; and ERIC MICHAEL GORDON by virtue of assignments dated: July 9, 1987 arnd Nce''roler 113, 1987 *14

Insert country. 3, The basic application(s) for patent or similar protection on which the application is halscd ,filmns date,and for the/.or EACH .is/are identified by country, filing date, and basic applicant(s) as follows: tasic application

In: United States of America on: May 22, 1987

By: DONALD STEVEN KARANEWSKY; SCOTT ADAMS BILLER; and ERIC MICHAEL GORDON

In: United States of America on: October 19, 1987 By: DONALD STEVEN KARANEWSKY; SCOTT ADAMS BILLER; and ERIC MICHAEL GORDON 4. The basic application(s) referred to in paragraph 3 hereof wts'wec the first appllction(sd made in a Convention country in respect of the invention the subject of the applicitton. Ili) Invert PI ACE of 'ining Insert DATE of Declared at WhPrin."eton, New Jersey, U.S.A., signing

Dated (ll April 19 88 Im) Sionatureli) of ujeclarsnlli) 'nii SQUIBB SONS, INC. 1404st His litealoyalimin or ollier -itrc required

To: The Commisiioner of Patents Nicholas P. MaJ~atestinic (SEAL) Asisan Se---a

PHILLIPS ORMONDE FITZPATRICK Patent Decliration Patent and Trade Mark Attorneys No CCSamiIInf or othizr witnesi requorrL Melbourne. AuistraliaI

(12) PATENT ABRIDGMENT (11) Document No. AU-B-1 6508/88 (19) AUSTRALIAN PATENT OFFICE (10) Acceptance No. 610591

(54) Title PHOSPHORUS-CONTAINING HMG-COA REDUCTASE INHIBITORS, NEW !NTERMEDIATES AND METHOD International Patent Classification(s) (51)4 C07F 009/40 A61 K031/66 C07F 009/32 C07F 009/36 Ct)7c 009/44 (21) Application No. 16508/88 (22) Application Date 20.05.38 Priority Data (31) Number (32) Date (33) Country 053281 22.05.87 US UNITED STATES OF AMERICA 109,G'80 19.10.87 UNITED STATES OF AMERICA (43) Publication Date :24.11 .88 (44) Publication Date of Accepted Application;. 23.05. )i (71) Applicant(s) E.R. SQUIBB AND SONS, ING;. (72) Inventor(s) DONALD STEVEN KARA,-EWSKY; SCOTT ADAMS BILLER; ERIC MICHAEL GORDON (74) Attorney or Agent PHILLIPS ORMONDE FITZPATRICK, 367 Colins Street, MELBOURNE VIC 3000 (56) Prior Art Documents FR 2596393 (57) Claim

1. A compound useful in inhibiting the enzyme 3 -hydroxy-3 -methylglutaryl -coenzyme A redu~rtase having the moiety

0

X OH (173 2 )n~

wherein X is or n is 1 or 2 and Z is a hydrophobic anchor. 3, The compound as~ defined, in claim I having the structure zAxm~n: bdouwjq!61 apqn j1. 25 L.4 zAXAfisdd,-?Nw1AHCINiC)8V *Id 01. 11111 1.25 J.6 111.4

7? (11) AU-B- 16508/88 610591

0 I- 29C2- C0 2 RX X OH

(FE2 )n z including salts thereof, wherein R is OH, lower alkoxy or lowex alkylian R sHo oe ly~ X is or -NH-; n is 1 or 2 Z is a hydrophobic anchor which is

R2a

(R6a)

wherein the dotted lines represent optional double bonds, wherein R1, R 2, R2 a and R2bmay be the same or different and are H, h'alogen, lower alkyl, haloalkyl, phenyl, substituted phenyl or ORY in which Ry is H, alkanoyl, benzoyl, phenyl, halophenyl, phenyl-lovn alkyls lower alkyl, cinnamyl, haloalkyl, allyl, cycloalkyl-lower alkyl., adamantyl-lower alkyl or substituted phenyl-lower alkyl.; wherein Z is

6012q/1 2 0 MlAY 1288 Welbouxne- NEHUNDHEIJ ULLAS

AITAVID~ M4AIL OFPICF

-L

A (11) AU-B-I1 6508/88 610591

(R6

Rand R5 are the same or different and are H, lower alkyl or OH,. one or R5and R61being present when the carbon to whict-" it is attached is linked to a double bond; 0 6 11 Ris alkyl-C-or arylCH2 R 6 a is lower alkyl, hydroxy, oxo or halogen, and q is 0, 1, 2 or 3, wherein Rx is hydrogen or lower alkyl in free acid form or in the foxm, of a physiologically- hydrolysable and -acceptable ester in salt form.

7. A hypocholesterolemic or hypoliipemic composition comprising a compound as de: ined in Clai 'm 3 and a pharmaceutically acceptable carrier thetlefor.

'n)t E.R. SQUIBB SONS, INC. zet mf or othe .1 44ep quwed

To: The Commisysioner of Patent.,, Nicholas P. Malatestinic (SEAL) Assistant Secretary I

PHILLIPS ORMONDE FITZPATRICK Patent Declaration Patent and Trade Mark Attorneys No legalization or other witness reqvirE4 Melbourne. Australia -4.

Patenits Act o o 5 9 1 9 COMPIEM' SPECIFICAIN (ORIGINAL)

Class Int. Class Application Numnber: Lodged:

Complete Specification Lodged: Accepted: Published: Priority

,C-Rp-lated Art: at t

4t APPLICANT'S REFERENCE: R-53,281*1 lad

Name(s) of Applicant(s): E.R. Squibb and Sons Inc.,

t~dress(es) of Applicant(s): Lawrenceville-Princeton Road, Princeton, New Jersey, UNITED) STATES OF AMERICA. ,,Address for Service is: I tI

PHILLIPS ORMOM1E FITZPATRICK 4 Patent and Trade mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Complete Specification for the invention entitled:

PH0PH=RS-C1XthTN]ING HW-Cah 7JSE ]U\MIBITORS, NEW INTERMEDIATES AND

our Ref 92770 POF Code: 8448/43804 I

The following statement is a full description of this invention, including the best method of performing it known to applicant(s):

600 3q1 LI

I I

i t

PHOSPHORUS-CONTAINING HMG-CoA REDUCTASE INHIBITORS, NEW INTERMEDIATES AND METHOD

5 _This Appli atinn iq a rCnntnahtn-in-nart- of application Serial No. filed October 19, 1987 whichis--acontinuation-in-part of application Serial.No-053,281, filed May v22, 9

The present invention relates to new phosphorus-containing compounds which inhibit the activity of 3-hydroxy-3-methylglutaryl.-coenzyme A reductase and thus is useful in inhibiting cholesterol biosynthesis, to hypocholesterolemic compositions containing such compounds, to new ,o intermediates formed in the preparation of such compounds and to a method of using such compounds for such purposes.

F. M. Singer et al., Proc. Soc. Exper. Biol. Med., 102, 370 (1959) and F. H. Hulcher, Arch. Biochem. Biophys:, 146, 422 (1971) disclose that certain mevalonate derivatives inhibit the r biosynthesis of cholesterol. SEndo et al in U. S. Patents Nos. 4,049,495, 4,137,322 and 3,983,140 disclose a fermentation

HX8b -2-

product which is active in the inhibition of cholesterol biosynthesis. This product is called compactin and was repo~rted by Brown et al., Chem. Soc. Perkin 1165 (1976)) to have a complex mevalonolactone structure. GB 1,586,152 discloses a group of synthetic compounds of the formula

CH

R100 9$R.

R2 E

R R9

R5 3

R 49 *in whict E represents a direct bond, a C 1 3

~.alkylene bridge or a vinylene bridge and the various R's represent a.variety of substituents. 20 The activity reported in the U.K. patent is less than 1% that of compactin. U. Patent No. 4,375,475 to Willard et al discloses hypocholesterolemic and hypolipemic compounds having the structure

H

6- 2 R

RI hjY 3

HX8b -3-

wherein A is H or methyl; E is a direct bond, -CH2 '-CH2 -CH2 -CH2 -CH2 -CH2 or -CH=CH-; R1 R 2 and R3 are each selected from H, halogen, C-_ 4 alkyl, C1 4 haloalkyl, phenyl, phenyl substituted by halogen, Cl14 alkoxy, C2_ 8 alkanoyloxy, C1-4 alkyl, or C1 -4 haloalkyl, and OR4 in which R4 is H, C2- 8 alkanoyi, benzoyl, phenyl, halophenyl, phenyl C1-3 alkyl, C19 alkyl, cinnamyl, C1 -4 haloalkyl, allyl, cycloalkyl-C 1 3 -alkyl, adamantyl-C 1 3 -alkyl, or substituted phenyl SCl_ 3 -alkyl in each of which the substituents are selected from halogen, C1 -4 alkoxy, C1 4 alkyl, or C1 -4 haloalkyl; and the corresponding dihydroxy acids resulting from the hydrolytic opening of the lactone ring, and the pharmaceutically acceptable salts of said acids, and the C1 -3 alkyl and .phenyl, dimethylamino or acetylamino substituted *04 C_3-alkyl esters of the dihydroxy acids; all of the compounds being the enantiomers having a 4 R 20 configuration in the tetrahydropyran moiety of the trans racemate shown in the above formula. WO 84/02131 (PCT/EP83/00308) (based on U. S. application Serial No. 443,668, filed November 22, 1982, and U. S. application Serial No. 548,850, filed November 4, 1983), filed in the name of Sandoz AG discloses heterocyclic analogs of mevalono lactone and derivatives thereof having the Sstructure

R R 4

6

Ro

i -o

HX8b -4-

wherein one of R and Ris and the

other is primary* or secondary C 1 6 alkyl, C 3 6 cycloalkyl or phenyl-(CH 2m-, wherein R 4 is hydrogen, C 1 4 al'kyl, C 1 4 alkoxy, (except t-butoxy), trifluoromethyl, fluoro, chioro, phenoxy or benzyloxy, R 5 is hydrogen, C -31-..koy 3 alkyl, C 1 akxy trifluoromethyl, fluoro, chioro, phenoxy or

beny a.yis hydrogen, C 1 2 alkyl, C 1 2 lo

fluoro or chioro, and mis 1, 2 or 3, a .with the provisos that both Rand 5 Ra 5aa must be hydrogen weR4ishydrogen, R 5a mutbe a.hydrogen when R shydrogen, not more than one'of R4* and R5is trifluoromethyl, not more than one of 20 R4and R 5 is phenoxy and not more than one ofR4 and Ris benzyloxy, s 2s hydrogen, C 1 alkyl, 3-6 *cycloalkyl, C1 4 alkoxy (except t-butoxy), trifluoromethyl, fluoro, chioro, phenoxy or benzyloxy, R3 is hydrogen, C 1 alkyl, C1 3 alkoxy, 3 trif luoromethyl, fluoro, chioro, phenoxy or benzyloxy, with the provisos that R 3 must be hydrogen when R2is hydrogen, not more than one of R2and R 3 is trifluoromethyl, not more than one of R2 and R3 isphenoxy, and not more than one of R2 and R3 is benzyloxy.

3I

HX8b

K i ~CH2 n~or -CH=CH- (n0O, 1, 2 or 3), R6 4 312 1 Z is -CH-CH -C-CH C HII 1 2 1 2 -OHI OH OH wherein Ris hydrogen or C 3 alkyl in free acid form or in the form of a physiologically- hydrolysable and -acceptable ester or a 6 lactone 04 thereof or in salt form. 0As 10 GB 2162-179-A discloses naphthyl analogues 0:of mevalolactone useful as cholesterol biosynthesis inhibitors having the structure Me Me

R

wherein R1 l-3C alkyl; Z is a gp. of formula Z1 orZ 2

0.AH OH -CHCH CHCH COOR7 OH OH0

0

(Z1 (Z2

R7 H, a hydrolysable, ester gp. or a cation.

HX8b, -6-

European Patent No. 164-698-A discloses preparation of lactones useful as anti-hyper- cholesterolemic agents by treating an amide with an organic suiphony]. halide R 5 so 2 X, then removing 'the protecting group Pr.

0 R5 0X 99 3 R4 2 9 ~9

9499 9 9999

9.9 99 949

99 o 9-49

999*9 9 9 wherein X halo; Pr a c arb inol1-protecting group; o R= H or CH3 99 R3 R4 H, 1-3C alkyl or phenyl-(1-3C 99

99 9 alkyl), the phenyl, being optionally substituted by 9 99 1-3C alkyl, 1-3C alkoxy or halo; .99 9 994 9 R a group of formula or 0 9* .9 9 9

*9 9 99 49 R CH 3

L.

-7,n

HX8b -7-

I I Q R-C- or R6 -CH I

CH3 R H or OH; R H or CH 3 a, b, c and d optional double bonds; R7 phenyl or benzyloxy, the ring in each case being optionally substituted by 1-3C alkyl or halo; R8 R9 1-3C alkyl.or halo; 5 1-3C alkyl, phenyl or mono- or SR 00 di-(1-3C alkyl)phenyl. Anderson, Paul Leroy, Ger. Offen. DE 3,525,256 discloses naphthyl analogs of mevalonolactones of the structure 0 0

M .°Me

H OH H Z -CHCH2 CHCH 2 CO2 R7 ISC=CN A A 0 0 oH' OH OH 1 Q 4040 T a Q1

S* 1 7 wherein R is alkyl, Z Q, Q R H, or a hydrolyzable ester group useful as inhibitors of cholesterol biosynthesis and in treatment of Satherosclerosis. WO 8402-903 (based on U.S. application Serial No. 460,600, filed January 24, 1983) filed in the name of Sandoz AG discloses mevalono-lactone analogues useful as hypolipoproteinaemic agents having the structure

Ak,

HX8b -8-

wherein the two groups Ro together form a radical of formula

8 7 6 CC C C or'-C

R R* 2 R3

wherein Ri J hydrogen, C 1 4 alkyl, C1 alkoxy, (except t-butoxy), trifluoromethyl, 4 .9.,'fluoro, chioro, phenoxy or benzyloxy, R 3. is hydrogen, C 1 3 alkyl, C 1 3 alkoxy, trifluoromethyl, fluoro, chioro, phenoxy or 20 benzyloxy, with the provisos that not more than

one of Rand R3 is phenoxy, and not more than one 0040#4of R2and R3 is benzyloxy, Ris hydrogen,'C 1-1 1 alkyl, fluoro, Ca~loro or benzyloxy, Ris hydrogen, C1 4 alkyl, C 1 4 alkoxy, (except t-butoxy), trifluoromethyl, fluoro, chioro, phenoxy or benzyloxy, hydrogen, C 1 3 alkyl, C1 3 alkoxy, trifluoromethyl, fluoro, chioro, phenoxy or benzyloxy, R 5a is hydrogen, C 1 2 t4 C1-C 1 2 alkoxy, fluoto or chioroo and with the provis.os that not

HX~b

more than one of R4and R5 is trifluoromethyl, not more than one of R4and R5is pheiioxy and not more than one of R4and R5is benzyloxy, H /(CH 2 )q x is -(CH2 )n-1 /C C\ (CH2 q H

wherein n is 0, 1, 2 or 3 and both g's are 0 or one is 0 and the other is 1, Z is

4 3 2.

#444-CH-CH 1 C-CH -COOH II OH OH wherein R6 is hydrogen or C 3 alkyl, with the general proviso that X-anthR 4 bern phenyl group are ortho to each other; in free acid form or in the formn of a physiologically-hydrolysable and acceptable ester or a 6 lactone thereof or in salt form.

Eur~pean patent application 127,848-A (Merck 11 4 1& Co, Inc.) discloses derivatives of thia-w-aryl-alkanoic acids having the structural formula: HO 0 Ox n E

H~

wherein Z is:

R6 R

R3

V ot n is 0, 1 or 2; E is -CH 2 -CH2 -CH 2 -t -CH 2 -CH 2 -CH 2 -1 -CH=CH-CH-;o -CH -CH=CH-; R 1 R2 and R3 are, hydrogen, chioro, I 4 '.bromo, fluoro, C1 -alkyl, phenyl, substituted phenyl or OR7 in which R7 is, hydrogen, 7 7

C2 8 alkanoyl, benzoyl, phenyl, substituted phenyl, C1 9 alkyl, cinnamyl, C1 4 haloalkyl, allyl, cycloalkyl-C 1 3 alkyll adamantyl-C 3 alkyl, or

R R- and R are hydrogen,, chioro, bromo, 20 fluoro or~ C 1 3 alkyl; and X is, hydrogen, C 3 alkyl, a cation derived from an alkali metal or is ammonium. .4 4Those compounds have antihypercholesterolrntic p activity by virtue of their ability to inhibit 3-h4-ydroxy-3-methylglutaryl-coenzyme A (UI4G-CoA) reductase and antifungal activity. French patent applicatio2n. 2,596,393 A filed on April 1986 (Sanofi SA) discloses 3-carboxy-2-hydroxy-propane-phosphonic acid derivatives including salts thereof which ar*e useful as hypolipaemic agents and have tae formula:

-3.1-HX8b

R2 0/OR3 ROOC-CH 2 -C-CH 2 QH OR4

wherein Rand 2=H, lower alkyl or optionally substituted aralkyl; R3 and R4 H, lower alkyl or optionally substituted aryl or aralkyl. These comounds ares disclosed as giving greater reductions in cholesterol, triglyceride and phospholipid levels than meglutol. European patent application 142,146-A (Merck Inc) discloses mevinolin-like compounds of 15 the struotural formula:

HO 0 t

~'0

*'0

z wherein: Ris, hydrogen or C akl Es CH 2 CH 2 1 -CH=CH-, or -(CH2 r- n Zis

HX8b -12-

R Ax

whereinh X is or -NR9 wherein R9 is hydrogen or C1- 3 alkyl; 4, R7 is C2 8 alkyl; and 4 4 4 4. Ris hydrogen or CH3 *4~4 4 4~ 4.

4, 4 4 044 4 4, 4 4 444@

4 R0 4$44*4 t

4. 4 S 4 44

4 4* 4 4 .4 wherein R10 10 R11 and R 2 are independently, e.g., 4 44 hydrogen, halogen or C1 4 alkyl.; 444 4

3)

4.4$ I 4.44

44 4 (R1 4 )n S 4*

In1 accordance with the present invention, there is provided phosphorus-containing compounds

L~.

HX8b

HX8b -13-

which inhibit the enzyme 3-hydroxy-3-methylglutaryl- coenzyme A reductase (HMG-CoA Reductase) and thus are useful as hypocholesterolemic agents and include the following moiety

0 SI -P-CH -CH-CH -CO- 1 2 a 2 X OH (CF, )n 2

wherein X is or n is 1 or 2 and Z is a "hydrophobic anchor". The term hydrophobic anchor as employed 15 herein refers to a lipophilic group which wheh linked to the HMG-like upper side chain of the molecule by the appropriate linker binds to a hydrophobic pocket of the enzyme not utilized in binding the substrate UMG CoA, resulting in 20 enhanced potency relative to compounds where Z=H. In preferred eibodiments, the compounds of the invention have the formula I 9 9

O H II I R-P-CH -C-CH-COR x

0 X OH

SCH2 n z

including salts thereof, wherein R is OH, lower alkoxy or lower alkyl; Rx is H or lower alkyl;

A|

L I

47 HX8b -14-

X is or -NH-; n is 1 or 2; Z is a hydrophobic anchor; and including pharmaceutically acceptable salts thereof. The terms "salt" and "salts" refer to basic salts formed with inorganic and organic bases. Such salts include ammonium salts, al:ali metal salts like, lithium, sodium and potassium salts (which are preferred), alkaline earth metal salts like the calcium and magnesium salts, salts with organic bases, such as amine like salts, e.g., *i 9 9 dicyclohexylamine salt, benzathine, N-methyl-D- 9999

r*9 4 glucamine, hydrabamine salts, salts with amino 94 944 44

9 15 acids like arginine, lysine and the like. The 99~ 99 4 nontoxic, pharmaceutically acceptable salts are 9 preferred, although other salts are also useful, in isolating or purifying the product. Examples of hydrophobic anchors which may be 9o o 99 4 20 included in accordance with the present invention 9 *4 a include, but are not limited to 9 94

9 ia *r

9* 44 0 9 a

4 4 i a R2a

L t

HX8b -l

.lkyl

(R a

wherein the dotted lines represen,\. optional double bonds, for example, .4 4 4 44

4q 4 4 *44*

44 44 4 .44 9

44- 9 t 4444

I a4 t 4 R

R 44 4 4 4 44

4 9 49

94 4 I I 4 44 1- alkyl alkyl 44 9 444 4

6a a 44.4 (R 4 444..

44 4 44 *4

'i

-16-

.alkyl alkyl

'6a ,R C

p .4 p alkyl 44

*4, p 4~*4

~4 0 4 p (R6 o 44 p4 p

.4440k p

whren 1 R2 2 a an 2 b, R, R wherin R ad R may be the same or p. 4 4 20 different and are each independently selected from p

9 0 *4

44 p H, halogen, l.ower alkyl, haloalkyl, phenyl, substituted phenyl or ORY wherein Ry is H, *44 4 4 S alkanoyl, benzoyi, phenyl, halophenyl, phenyl-lower alkyl, lower alkyl, cinna~myl, 25 haJ.oalkyl, allyl, cycloalkyl-lower alkyl, 4, 4.

44£ 0 adamantyl-lower alkyl or substituted phenyl-lower 4 04 4 45 5 alkyl. Where Z is

Pt

HX8b -17-

6 .R

~-alky.

(R6

.R5and R51are the same or different and 4t I .4 a are H, lower alkyl or OH; 4 44

&40q 0 0 4 6 I, 4.4,4 R6 is lower alkyl-C such as CH -CH2 7C 4 4. 4 4 4 CH3 R a or arylCH 2 4444*9 R 6 a.is lower' alkyl, hydroxy, oxo or halogen; 4

q is 0, 1, 2 or 3, and R7is H or lower alkyl; a 4, I Thus, the compounds of formula I encompass 4.

4 4 I. 0 IA R-P-CH QHC -CO RX I 2 2 2 #4 444 4 4 0 OH and IO2 2) 4*44 4 4 444, 4. 4 4 44 0 *4 1' IB R-P-CH -gHC 1 2 -C 2 -CC 2 R X NH OH

z The term "lower alkyl" or "alkyl." as employed herein alone or as part of another group includes

r- I1

HX8b contining 1 to 12 carbons in the normal chain, preferably 1 to 7 carbons, such as methyl, ethyl, *propyl, isopropyl, butyl,. t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethyl- pentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, the various branched chain isomers thereof, and the like as well as such groups including a halo-substituent, such as F, Br, Cl or I or CF3 ;jan alkoxy substituent, an aryl

td substituent, an alkyl-aryl substituent, a haloaryl substituent, a cycloalkyl substituent, an alkyl- cycloalkyl substituent, hydroxy, and alkylamino substituent, an alkanoylamino substituent, an aryl- carbonylamino substituent, a nitro substituent, a cyano substituent, a thiol substituent or an alkylthio substituent. ~I The term "cycloalkyl" as employed herein alone or as part of another group includes I saturated cyclic hydrocarbon groups containing 3 to 12 carbons, preferably 3 to 8 carbons, which include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl and cyclododecyl, any of which groups may be substi- tuted with 1 or 2 halogens, l'or 2 lower alkyl groups, 1 or 2 lower alkoxy groups, 1 or 2 hydroxy groups, 1 or 2 alkylamino groups, 1 or 2 alkanoyl- amino groups, 1 or 2 arylcarbonylamino groups, 1 or 2 amino groups, 1 or 2 nitro groups, 1 or 2 cyano groups, 1 or 2 thiol groups, and/or 1 or 2 alkyl- thio groups. The term "aryl" or. "Art" as employed herein refers to monocyclic or bicyclic aromatic groups portion, such as phenyl, naphthyl, substituted

9

HX8b -19- -19- I,

containing from 6 to 10 carbons in the ring

phenyl or substituted naphthyl wherein the the substituent on either the phenyl or naphthyl may may be 1, 2 or 3 lower alkyl groups, halogens (Cl, Br or 1, 2 or 3 lower alkoxy groups, 1, 2 or 3 hydroxy groups, 1, 2 or 3 phenyl groups, 1, 2 or 3 alkanoyloxy group, 1, 2 or 3 benzoyloxy groups, 1, 2 or 3 haloalkyl contpreferably groups, containing 1, 2 or 3 halophenyl 3 substituents. portion, 1, groups, such 2 oras 3phen"aralkyl, naphthy,1, 2 allyl groups, substituted or 3 cyclo- alkylalkyl groups, 1, 2 or 3 adamantylalkyl groups, be aryl-lower 1, 2 or2 3 alkylamino 3 lower alkys used herein groups, alone 1, 2 or o asr part 3 alkanoyl- amino or F, groups, 1, refers 2 oup 2 or lower 3 to arylcarbonylamino groups, alkoxy 2 groups, or 3as 1, 2 or 3 amino hydroxycussed groups, 1, 2 or an above having 2 or 3 nitro 3 phenyl ar, groups, substituent, or 1, such2 3 15 22 or 33 cyano group, alkanoyloxy groups, 1, groups, 2 2or 1, 2 or3 3 thiol groups, benzoyloxygroups, groups,and/or and/or 2 or 1, 3 haloalky 22 or groups, 33 alkylthio 2 or the groups with aryl group 3 halophenyl,or preferably containing 3 substituents. as part 25 anotherr 3 allyl of 2 or 3 cyclo- group includes any, alkylower alkyl groupsalkyl, 2r or adamantylalky groupsnked 3aky

S, 20 of another 2 The group refers alkythiooups term "lower to lower alkyl3 groups ora2 lkanoylas discussed above having "arythiaminogroups, 3 an aryl 2 oaralkylthio substituent, as employed such herein as aloneoups, or30 part benzyl. as of ano ther includes group any of the, The term "lower alkoxy", "alkoxy", or *I "aryloxy" "aryloxy" or "aralkoxy" as employed herein alone or or above loweralkyl, alkyl, aralkyl or aryl groups as part of another group includes any of the above "aryl-lowerd to asulfurky used herein alone or as part atom.s lower alkyl, alkyl; aralkyl or aryl groups linked to an oxygen atom. atom. The term "lower alkylthio", "alkylthio",

"arylthio" or "aralkylthio" as employed herein alone or as part of another group includes any of the above lower alkyi, alkyl, aralkyl or aryl groups f 'linked to a sulfur atom.

HX8b

The term "lower alkylamino", "alkylamino", "arylamino", "arylalkylamino" as employed herein alone or as part of another group includes any of the above lower alkyl, alkyl, aryl or arylalkyl groups linked to a nitrogen atom. The term "alkanoyl" as used herein as part of another group refers to lower alkyl linked to a carbonyl group. The term "halogen" or "halo" as used herein refers to chlorine, bromine, fluorine, iodine and CF3 with chlorine or fluorine being preferred. Preferred are those compounds of formula I. 'which have the following structure *#f 4

15 II R 0 CH H CH S 2\ P C CO R /X CH OH 20 z

wherein R is OH, OLi; R is Li or H; X is 0 or NH; and Z is R 1R wherein R is phenyl which includes an alkyl 0. and/or halo substitutent

R 2a

or R1 is benzyloxy which includes a halo substituent; 2 R2 and R aa are the same and are halogen or lower alkyl; i

A

HX8b -21-

Z may also preferably be

wherein.R and R2are as defined immediately above with respect to the compound of formula II, or

R6 N".0

99 99

99 9 z is 9.99 9 9 9,99

99

wherein R is1 H, 9 5 CH3 or OH and R6 is 99 999 9 15 9 99 99 9 9999

999999 9

CH 3 or(substituted)phenyalmethyl *9 99 9 9 99 20 wherein R' is H or CH 3 1 99 69 9 *9 9 The compounds of formula I of the invention 99 may be prepared according to the following 4 9 999 9 reaction sequence and description thereof.

9999 9

*9 9 9 99 9 99 a *a Ca. a *9.Q. ta a a a C a a a a a a. a a. a. a a a a. a a a a a C a a a a a -a a a C a C 8 a a a -a a a a a a a a a a a

C H 16 5 R -P(Oalkyl) 2 0 0si-C (Ci 3 Ra P-CH -CH-CH -CO a1Icy1 1 2t- 2 2 I-CH 2 -C-C112 -C0 2 alkyl Arbuzov Reaction Oalkyl 0Oi-C (CH 3 3 H (Ra lower alkyl or A lower alkoxy) C6H5 C6

IV 0 1. (al 3 3 -SiBr, CH12 C1 2 R -P-CH -CU-CU -COaly IV 2. if2 12= 2 2 aly

Phosphorus ester Si-C(C11 3 3 cleavage /UH CH 6 5 6

VA. R a=lower alkyl where Ra was lower alkyl VB. R 0l=H. where Rawas lower alkoxy

L

.Pae a. 44 a a. a a. a -e 4. a a a a a a a a a a a at a a a a a a 4. a a a

VB RbOH-DCC 0 (R a0OH) (Rb lower alkyl)" Rb9-P-Cl 2 Cll-CH CO alkyl

Pyridine OH 0 (Esterification) *Ai-C(C11 3 3

c6H5 c6

cI~ tr

V

-j

I

a a a~a a a 4 a a a S S a, a a a a a a

a9-H2O acfi alkyl

VI (COI) 2 CH2 C12 (Acid Cl Formation) or Z-(CH 2 n-XH (coupling reaction) VA. x 0 (C 2 If5 )33 N, D14AP (a 1 :5i-C (al3

VII

(Rc lower alkyl or lower alkoxy)

1) (n-C 4 119 4 NF, C11 3 C0011, TfiF (silyl 0 VII ether cleavage) R-P-CH -CH-CH -Co RX 2) OH ,dioxane (Hlydrolysis) 1 2- 2 2 I OH (CII I2

I

HX8b

As seen in the above reaction sequ-ence,, compounds of Formula I may be prepared by subjecting iodide A to an Arbuzov reaction by heating iodide A

C16H O-Si-C(CH3 6H A I-OH 2-C.-CH2C2aly H

and phosphonite/phosphite III

III RaP(Oalkyl)2 15 o 4a .wherein Ra is lower alkyi or lower alkoxy, employing standard Arbuzov conditions and procedures to form phosphinate/phosphonate IV

0 44 IV aP-CH CH-C 2 -CO alkyl Oalkyl g.si-C(CO 3 3

Phosphinate/phosphonate IV is a novel compound and as such is a part of the present .:invention. Phosphinate/phosphonate IV is then subjected to a phosphorus ester cleavage by treating a solution of compound IV in an inert or-ganic solvent, such as methylene chloride, sequentially with bis(trimethylsilyl)trifluoroacetamide (BSTFA) and trimethylsilyl bromide, under an inert atmosphere

HX8b -26-

such as argon to form the phosphinic acid VA where R a in IV is lower alkyl, that is,

0 VA lower allkyl- -CH 2 -9H-CH 2 -CO2 al kyl OH 0 ~Si-C( CH 3 C6 H C6 H

or phosphonic acid VB (wher-'n R ain IV is lower alkoxy), that is

00

HO-T'-CH2 -gH-CH 2 -C02 alkyl OH 0

4~ Cf 5 C6 H

ft Compounds VA and VB are novel intermediates and as such as part of the present invention. Where phosphonic acid VB is'obtaiined, it is esterified by treating VB in dry pyridine with 44t alcdhol

VC RbOH (where Rbis lower alkyl)

2 and dicyclohexyl carbodiimide and the resulting reaction mixture is stirred under an inert atmosphere, such as argon, to form phosphonic mono alkyl ester VI i laurr~--Rlur~a~r~-*rarnsns~- rr

HX8b -27-

0 b 11 vI R O-P-CH -CH-CH 2 CO2 alkyl -OH O Si-C(CH3) C6 H 5 C6 H

Ester VI or phosphinic acid VA is then dissolved in an inert organic solvent, such as, methylene chloride, benzene or tetrahydrofuran (THF) and treated with trimethylsilyldiethylamine and stirred under an inert atmosphere such as argon; the mixture is evaporated and then dissolved in methylene chloride (or other appropriate inert organic solvent). The resulting solution is cooled S 15 to a temperature within the range of from about 0 C to about 25 0 C, treated with oxalyl chloride and then evaporated to give crude phosphorochloridate. The phosphonochlsridate is dissolved in inert organic solvent such as methylene chloride, benzene, pyridine or THF; the solution is cooled to a temperature within the range of from about -200C oa to about 0 C and treated with *a

B Z-(CH2 )n-XH

,employing a molac ratio of VI or VA:B of within the range of from about 0.5:1 to about 3:1 and preferably from about 1:1 to about 2:1, followed by triethylami'e and catalytic 4-dimethylaminopyridine (DMAP) to form adduct VII

4.

,Kt. -28- H~

0 VII RC IIP-CU2 -H-CH -C0 alkyl

H2 )n i-C(CU 3 3 Z C H5 CH

wherein Rc is lower alkyl or lower alkoxy.

Compound VII is subjected to silyl ether cleavage by treating a VII in an inert organic solvent such as tetrahydrofuran, with glacial acetic acid and tetrabutylammonium fluoride to form ester VIII

I RCc- -CH -QC2-C Rx 2

X OH

1 n aaa

9 0* (RX= alkyl)

The ester VIII may then be hydrolyzed to the corresponding alkali metal salt or acid, that 4*4*,is, where Rx is alkali metal or H by treatment with a *strong base such as lithium hydroxide in the presence of dioxane, tetrahydrirfuran or other inert organic solvent, under an inert atmosphere such as argon, at 25*C, employing a molar ratio of base:ester VIII of within th'e range of from about 1:1 to alout 1.1:1 to form the corresponding alkali metal salt

At

HX8b -29-

0 VilA -PCH CH-CH2 -CO 2 alkali metal X OH (CH) z

wherein R is lower alkyl or lower alkoxy.

Compound VIIIA may then be treated with strong acid such as HCl to form the corresponding acid VIIIB

4 OH

4(Uri

Th este IIwhriRislerakx mayI bcovrtedCto-theCHcorsodigH-akl mea salt by tratn eseOIIihsrogbs

abu 4: tofr II

200 VIII Thealest VII -C whria slwralikoxya 244-H2 may~~ becnetdt h OHsodin iakl 4 (CH tetn 2)eanatby strVI ih togbs at5060C mpoin amoarrai o bseese

HX8b

The di-alkali metal salt VIIIC may be converted to the corresponding acid wherein R is OH by treatment with strong acid such as HCl to form VIIID

0 11 VII ID HO-P-CH -QH-C 2 OH X OH (CH) 12 n

The iodide starting material A may be prepared starting with the bromide C

OH C Br-CH 2 -H-HCO alkyl

20 (prepared employing procedures as described in Tetrahedron Lett. 26, 2951 (1985)) o which is dissolved in solution in dimethylformamide (DMF) with imidazole and 4-dimethylamino pyridine and the resulting solution is treated with t-butyldiphenyl silyl chloride under an inert atmosphere such as argon to form the silyl ether D

C5 C6 H

cSi-C(CH3 D Br-CH 2 -CH-CH2 CO2 alkyl

I

i ~aarr~ r h

HX8b -31-

A solution of silyl ether D in an inert organic solvent such as methyl ethyl ketone or DMF is treated with sodium iodide under an inert atmosphere such as argon, to form iodide A. The starting compound B

Z-(CH 2)n-XH

may be prepared as described below depending upon the definition of Z and X. Thus, compounds of formula B wherein Z is

a

a a. RR 1 a 15 a. a

R2a *r

.6 U a. 20 and X is 0, that is, compounds of the structure a.l a 0 U *4

Ir 9 1i*4

2a R (CH )n-OH *u ae a aJ ha a a

may be prepared by treating aldehyde E

i

1HX8b -32-

2a (CH 2 )n-1 CHO R

with a reducing agent such as lithium aluminum hydride or sodium borohydride. Compounds of formula B where Z is

4. 9 49 44A 9 4099

4 4. 4 R2 a 99, 9

99 4 44,..

94*99 9 9

and X is N, that is compounds of the structure 94 9 9 4 &4

44 9 9 9 44

44 94 9

R2 a 444 9 CH 2 nNH2

4944 4 'we. 44 4 9 9 *4 may be -ed by oxidizing the aldehyde E by treating E iii solution with acetone with, for example, Jones reagent to form the acid F

HX8b -33-

R1 F R2 a 0 (CH )n- 1 -COOH

2

which in suspension with methylene chloride is treated with oxalyl chloride to form the corresponding acid chloride which is dissolved in an inert organic solvent such as tetrahydrofuran, and treated with a mixture of concentrated ammonium hydroxide in tetrahydrofuran to form an amide of *the structure

,co R1 *G 2a II R 0 (CH2 )-1-C-NH2

20 20 4 2 elk R

Amide G is then reduced to the corresponding amide B by treating G with a reducing agent such as lithium aluminum hydride. Starting compounds of formula B wherein Z 4 is

HX8b -34-

6 a) q alkyl

and X is 0 or -that is, compounds of the structure

.4 4 4 4.

4

4 *4 4 15 HI .4 (CH2 n-X 4 9 .4.4 4 6 a)q alkyl *4e ft. ft where X is 0 are disclosed by C. H. Heathcock et al, 20 J. Org. Chem. 50, 1190 (1985). Compounds of 44 *4 .4. 9 formula H' were X is NH may be prepared by the 4 4S 4 reductive aMination of

.4 4 9 4 6 CHO I I alkyl 4*ft ft 4 4 .4 ft. ft ft ft 4 4~

(prepared as disclosed by C. H. Heathcock et al, supra) p

HX8b

by treating J with ammonium acetate and sodium cyanoborohydride in the presence of an alcohol solvent such as methanol. Starting compound of formula B wherein Z is

Ra

99 99 9 9,

994. 9 .999 and X is 0, that is, compounds of the structure 99 9 9 999 0

9 99 99 9 9999 9 9999,9 9 9 Ra

9 99 '9 9 99 9 (CH2 n-0H 9 99 99 9 9 99

9 99 99 4 999 9

are disclosed in WO 8402-903-A and GB 2,162,1.79A 9,. 9 9449 9 both filed in the name of Sandoz, 9 99 99 9 Starting compounds of formula B wherein Z 9 99 is

HX8b -36-

Ra

and X is NH, that is, compounds of the structure

94 99 9 9 99 Ra .444 9 9 9499 9 99 9 944 4 9 (CH 2 -H *4 99 4 444.

949946 I

94 99 9 20 may be prepared by the reductive amination of the 4 44

99 9 49 9 aldehyde 0 94

99 9* 4 994 4

R2 a 0 9444 999, I Io~o+(CH 2 CHO *9 4 44 4 94

by treating 2 wi th amnmonium acetate and sodium cyanoborohydride in the presence of an alcohol solvent such as methanol.

r i, I i_ i

HX8b -37-

The compounds of the invention may be prepared as Lacemic mixtures and may later be resolved to obtain the S-isomer which is preferred. However, the compounds of the invention may be prepared directly in the forrn of their S-isomers as described herein and in th e working examples set out hereinafter. The compounds of the invention are inhibitors of 3-hydroxy-3-methyl-glutaryl coe, lzyme A (HMG-CoA) reductase and thus are useful in inhibiting cholesterol biosynthesis as demonstrated by the following tests. 4.

1) Rat Hepatic HMG-CoA Reductase 4, 15 Rat hepatic HMG-CoA reductase activity is c measured using a modification of the method U -described by Edwards (Edwards, et al., i* 4r4. *e J. Lipid Res. 20:40, 1979). Rat hepatic microsomes are used as a source of enzyme, and the S .4 20 enzyme activity is determined by measuring the 14 44# *4 4 conversion of the 14 C-HMG-CoA substrate to 4 4' 14 C-mevalonic acid. *C 4

a. Preparation of Microsomes Livers are removed from 2-4 444,* V: cholestyramine-fed, decapitated, Sprague Dawley 4, rats, and homogenized in phosphate buffer A *a (potassium phosphate, 0.04 M, pH 7.2; KCl, 0.-05 M; sucrose, 0.1 M; EDTA, 0.03 M; :protinin, 500 KI units/ml). The homogenate is spun at 16,000 x g for 15 minutes at-40C. The supernatant is removed and recentrifuged under the same conditions a second time. The second 16,000 x g supernatant is

BX8b

spun at 100,000 x g for 70 minutes at V0 C. Pelleted microsomes are resuspended in a minimum volume, of buffer A (3-5 ml per' liver), and homogenized in a glass/glass homogenizer, Dithiothreitol is added (10 mM), and the preparation is aliquoted, quick frozen in acetone/dry ice, and stored at -80 0 C. The specific activity of the first microsomal preparation was 0.68 nmole mevalonic acid/mg protein/minute.

44 b. Enzyme Assay 4 4 4 3 4, The reductase is assayed in 0.25 ml which 4~4, 4 .4'.4 contains the following components at the indicated 4 4* 44 @44 4 4 15 final concentrations; 44 44 4 4-444

S~334*4 4 0.04 M Potassium phosphate, pH 4 4 0.05 M Y'Cl 0.10 M Sucrose 4 4; 1 44 0.03 M EDTA 43 44 S 0.01 M Dithiothreitol 4 *4 mM NaCl 4 44 4$4 4 4 1% Dimethylsulf oxide 50-200 pg Microsomal protein 100 pM 14 C-CDL]HMG-CoA (0.05 pCi, 44 4 444 30-60 mCi/mmole) 4 3' 4 2.7 mM NADPH (nicotinamide adenine 4 dinucleotide phosphate)

Reaction mixtures are incubated at 37 0 C. Under conditions described, enzyme activity increases linearly up to 300 pg iuicrosomal protein per reaction mixture, and is linear with respect to a

.'i

r" i~ I j

HX8b -39-

incubation time up to 30 minutes. The standard incubation time chosen for drug studies is minutes, which results in 12-15% conversion of HMG-CoA substrate to the mevalonic acid product.

[DL-]HMG-CoA substrate is used at 100 pM, twice the concentration needed to'saturate the enzyme under the conditions described, NADPH is used in excess at a level 2.7 times t..e concentration required to achieve maximum enzyme velocity.

Standardized assays for the evaluation of S99 inhibitors are conducted according to the *9 4 following procedure. Microsomal enzyme is incubated in the presence of NADPH at 370 C for *9O

49

4* .9 15 minutes. DMSO vehicle with or without test 9 I

44 4 compound is added, and the mixture further 4 494 tS incubated for 15 minutes at 370 C. The enzyme assay is initiated by adding 14C-HMG-CoA *9 44t substrate. After 20 minutes incubation at 37°C the reaction is stopped by the addition of 25 pl of 33A KOH. H-mevalonic acid (0.05 pCi) is 9 4 added, and the reaction mixture allowed to stand 49

at rtum temperature for 30 minutes. Fifty pl HCl is added to lactonize the mevalonic acid, 94 4 Bromophenol blue is added as a pH indicator to monitor an adequate drop in pH. Lactonization is 9* 4 allowed to proceed for 30 minutes at room temperature. Reaction mixtures are centrifuged for 15 minutes at 2800 rpm, The supernatants are layered onto 2 grams AG 1-X0 anion exchange resin (Biorad, formate form) poured in 0.7 cm (id) glass columns, and eluted with 2.0 ml H20. The first ml is discarded, and the next 1.5 ml is

i I

V A :i -silll~lC1~I

HX8b

collected and counted for both tritium and carbon 14 in 10.0 ml Opti-fluor scintillation fluid. Results are calculated as nmoles mevalonic acid produced per 20 minutes, and are corrected to 100% recovery of tritium. Drug effects are expressed. as I50 values (concentration of drug producing inhibition of enzyme activity) derived from composite dose response data with the confidence interval indicated. Conversion of drugs in lactone form to their sodium salts is accomplished by solubilizing the lactone in DMSO, adding a 10-fold molar excess 0i of NaOH, and allowing the mixture to stand at room *0 temperature for 15 minutes. The mixture is then partially neutralized (pH 7.5-8.0) using 1N HC1, 0 I; and diluted into the enzyme reaction mixture. *000

2) Cholesterol Synthesis in Freshly Isolated Rat Hepatocytes *0r 20 Compounds which demonstrate activity as 0i 0 inhibitors of HMG-CoA reductase are evaluated for their ability to inhibit 14C-acetate incorporation into cholcsterol in freshly isolated rat hepatocyte ii suspensions using methods originally described by 0000:

0i 4 i 25 Capuzzi et al. (Capuzzi, D.M. and Margolis, S., Lipids, 6:602, 1971). r* t

a. Isolation of Rat Hepatocytes Sprague Dawley rats (180-220 grams) are anesthetized with Nembutol (50 mg/kg). The abdomen is opened and the first branch of the portal vein is tied closed. Heparia (100-200 units) is injected directly into the abdominal

N

An

HX8b -41-

vena, cava. A single closing suture is placed on the distal section of the portal vein, and the portal vein is canulated between the suture and the first branching vein. The liver is perfused at a rate of 20 ml/minute with prewarred (37 0 C), oxygenated buffer A (HBSS without calcium or magnesium containing 0.5 mM EDTA) after severing the vena cava to allow drainage of the effluent. The liver is additionally perfused with 200 ml of prewarmed buffer (HBSS containing 0.05% bacterial collagenase). Following perfusion with buffer B; the liver is excised and decapsulated in ml Waymouth's medium allowing free cells to *4 disperse into the medium. Hepatocytes are isolated by low speed centrifugation for minutes 44*4 at 50xg at room temperature. Pelleted hepatocytes I are washed once in Waymouth's medium, counted and assayed for viability by trypan blue exclusion. 14 These hepatocyte enriched cell suspensions routinely show 70-90% viability.

4 *6 b. 1 4 C-Acetate Incorporation into Cholesterol 4 0

Hepatocytes are resuspended at 5x10 6 cells *44 4 25 per 2.0 ml in incubation medium (IM) [0.02 M Tris-HCl (pF 0.1 M KC1, 3.3 mM sodium 4*64 citrate, 6.7 mM nicotinamide, 0.23 mM NADP, 1.7 mM 44 4 4 4 glucose-6-phosphate]. Test compounds are routinely dissolved in DMSO or DMSO:H20 and added to the IM. Final DMSO concentration in the IM is and has no significant effect on cholesterol synthesis.

a i ~J

.cc HX8b -42-

Incubation is initiated by adding 14 C-acetate (58 mCi/mmol, 2 pCi/ml), and placing the cell suspensions (2.0 ml) in 35 mm tissue culture dishes, at 370 C for 2.0 hours. Following incubation, cell suspensions are transferred to glass centrifuge tubes and spun at 50xg for 3 minutes at room temperature. Cell pellets are resuspended and lysed in 1.0 ml H 2 0, and placed in an ice bath. Lipids are extracted essentially as described by Bligh, E. G. and W. J. Dyer, Can. J. Biochem. and'Physiol., 37:911, 1959. The lower *9 organic phase is removed and dried under a stream 0 of nitrogen, and the residue resuspended in (100 15 pl) chloroform:methanol The total sample is spotted on silica gel (LK6D) thin-layer plates and developed in hexane:ethyl ether:acetic acid 99 9 (75:25:1). Plates are scanned and counted using a BioScan automated scanning system. Radiolabel in 20 the cholesterol peak (RF 0.28) is determined and 99 expressed at total counts per peak and as a percent of the label in the total lipid extract. 9 0

9 B Cholesterol peaks in control cultures routinely 999 i9 contain 800-1000 cpm, and are 9-20% of the label present in the total lipid extract; results *99 compatable w.,th Capuzzi, et al., indicating 9% of 9 CC o: ii i+ extracted label in cholesterol. S ee, o Drug effects inhibition of cholesterol synthesis) are determined by cop,,aring of label in cholesterol for control and drug treated cultures, Dose response curves are constructed from composite data from two or more studies, and

HX8b -43-

*results are expressed as 150 values with a confidence interval.

3) Cholesterol Synthesis in Human Skin Fibroblasts Compound selectivity favoring greater inhibitory activity in hepatic tissue would be an attribute for a cholesterol synthesis inhibitor. Therefore, in addition to evaluating cholesterol synthesis inhibitors in hepatocytes, these compounds are also tested for their activity as inhibitors of .cholesterol synthesis in cultured fibroblasts. Q

a. Human Skin Fibroblast Cultures 15 Human skin fibroblasts (passage 7-27) are grown in Eagles' minimal essential medium (EM) e. containing 10% fetal calf serum. For each experiment, stock cultures are trypsonized to disperse the cell monolayer, counted, and plated 20 in 35 mm tissue culture wells (5x10 ml). Cultures are incubated for 18 hours at 37°C in 5% C02 /95% humidified room air. Cholesterol So. biosynthetic enzymes are induced by removing the serum containing medium, washing the cell monolayers, and adding 1.0 ml of EM containing 1.0% fatty acid free bovine serum albumin, and incubating the cultures an additional 24 hours.

b. 14C-Acetate Incorporation into Cholesterol Induced fibroblast cultures are washed with EMEM 1 00 (Earle's minimal essential medium). Test compounds are dissolved in DMSO or DMSO:EM (1:3)

I

S ii'

HX8b -44-

(final DMSO concentration in cell cultures added to the cultures, and the cultures preincubated for 30 minutes at 3'/C in 5% CO2/95% humidified room air: Following preincubation with drugs, [1- 1 4 C]Na acetate (2.0 pCi/ml, 58 mCi/mmole) is added, and the cultures reincubated for 4 hours. After incubation, the culture medium is removed, and the cell monolayer (200 pg cell protein per culture) is scraped into 1.0 ml of H20. Lipids in the lysed cell suspension are extracted into chloroform:methanol as described for hepatocyte suspensions. The organic phase is dried under 5945 nitrogen, and the residue resuspended in chloroform:methanol (100 pl), and the total 15 saple spotted on silica gel (LK6D) thin-layer S. plates, and analyzed as described for hepatocytes. Inhibition of cholesterol synthesis is S"determined by comparing the percent of label in the cholesterol peak from control and drug-treated 20 cultures. Results are expressed as 150 values, S and are derived from composite dose response curves from two or more experiments. A 95% confidence interval for the 10 value is also calculated from the composite dose response curves. A further aspect of the present invention is a pharmaceutical composition consisting of at least one of the compounds of formula I in Sassociation with a pharmaceutical vehicle or diluent. The pharmaceutical composition can be formulated employing conventional solid or liquid vehicles of diluents and pharmaceutical additives of a type appropriate to the mode of desired administration. The compounds can be administered i r

HX8b

by an oral route, for example, in the form of tablets, capsules, granules or powders, or they can be administered by a parenteral route in the form of injectable preparations, such dosage forms containing from 1 to 2000 mg of active compound per dosage, for use in the treatment. The dose to be administered depends on the unitary dose, the symptoms, and the age and the body weight of the patient. The compounds of formula I may be administered in a similar manner as known compounds suggested for use in inhibiting cholesterol biosynthesis, such as lovastatin, in mammalian species such as humans, dogs, cats and 0:o 15 the like. Thus, the compounds of the invention may 0 be administered in an amount from about 4 to 2000 mg in a single dose or in the form of individual doses from 1 to 4 times per day, preferably 4 to 200 mg in divided dosages of 1 to 100 mg, suitably S 20 0.5 to 50 mg 2 to 4 times daily or in sustained Srelease form.

4 0

0

I

HX8b -46-

The following working Examples represent preferred embodiments of the present invention. Unless otherwise indicated, all temperatures are expressed in degrees Centigrade. Flash chromatography was performed on either Merck 60 or Whatmann LPS-I silica gel. Reverse phase chromatography was performed on CHP-20 MCI gel resin supplied by Mitsubishi, Ltd.

Example 1 (S)-4-[[[4'-Fluoro-3,3',5-trimethyl[1,l'-biphenyl]- 2-yl]methoxy]methoxyphosphinyl]-3-hydroxy-butanoic So acid, monolithium salt A. N-(2,4-Dimethylbenzylidene )benzeneamine so 15 Ref. Merck U. S. Patent No. 4,375,475, pg. 39. A solution of freshly distilled 2,4-dimethylbenzaldehyde (Aldrich, 6.97 ml, mmole) and distilled aniline (Aldrich, 4.56 ml, mmole) in dry toluene (80.0 ml) was refluxed for 61 20 3.0 hours under argon in a flask equipped with a Dean-Stark U apparatus. The mixture was cooled, then evaporated in vacuo to a yellow oil. The crude oil was purified by Kugelrohr distillation mm Hg, 160-1800 C) to give 8.172 g of desired title benzeneimine as a light yellow oil 9, which crystallized on standing to a low melting o* solid. TLC Hex-acetone, RF=0.67 and 0.77 erc pg 39 (geometric isomers),ef. U.V. U. 15 PtentNo.4,37,47, and 12.

HX8b -47-

B. B. N

H3 C Pd 0 OCCH 3 0 CH3 2

Ref. Merck U.S. Patent No. 4,375,475, pg. 39. A mixture of Part A benzeneimine (6.0 g, 28.7 mmol) in glacial HOAc (144 .ml) was treated with palladium (II) acetate (6.44 g, 28.7 mmole) and the clear, red homogeneous solution refluxed 15 under argon for one hour. The resulting turbid .9t. mixture was filtered warm through a packed bed of Celite into 900 ml of H2 0. Precipitated orange solid was collected by filtration and dried in vacuo at 65°C over P2 05 for 16.0 hours to give o 20 10.627 g of desired title palladium complex as an orange solid with m.p. 194°-1960 C. (Literature m.p. of a recrystallized .9 analytical sample 203 0 -205 0 C).

C. 4'-Fluoro-3,3',5-trimethyl[1,1'- "biphenyl -2-carboxaldehyde I Bromo[4-fluoro-3-methylphenyl]- magnesium Ref. Merck U. S. Patent No. 4,375,475, pp. 37 and 38. The title Part C(l) Grignard reagent was prepared by adding 5-bromo-2-fluorotoluene (22.5 g, 60.9 mmole, Fairfield Chemical Co.) dropwise i i j

.1L.I

HX8b -48-

at a rate sufficient to maintain the reaction at reflux to stirred magnesium turnings (1.35 g, 55.4 mmole, 8.0 eq.) in dry Et20 (70.0 ml). The reaction was initiated in an ultrasound device. After bromide addition was complete, the mixture was stirred for one hour under argon at room temperature, refluxed for 15 minutes and then allowed to cool to room temperature.

4'-Fluoro-3,3',5-trimethyl[l,l'- biphenyl -2-carboxaldehyde 9 In a second flask, a mixture of the Part B 99* dipalladium complex (3.0 g, 6.92 mmole) and 9 triphenylphosphine (14.52 g, 55.4 mmole, 8.0 eq.) 99 9 15 in dry benzene (100 ml) was stirred at room 99 9 ft 9 temperature under argon for 30 minutes. Freshly prepared and filtered (glass wool plug) Part C (1) 999

Grignard reagent was then added in one portion by means of a cannula to this solution and the 99 20 mixture was stirred for 1.5 hours at room 99 .9* temperature under argon. 6.0 N HC1 (35 ml) was 99 added, the mixture stirred an additional hour at *9

994 9 room temperature, -then filtered through packed Celite bed). The filtrate was extracted with Oi ii •o i~ 25 Et20 (250 ml), the extract washed with brine (2 x i 9 100 ml), dried over anhydrous MgSO 4 and evaporated 99 in vacuo to give 13.35 g of a viscous orange oil 9 99* which crystallized on standing. The crude orange solid was purified by flash chromatography on silica gel (700 g) eluting with hexane, followed by (95:5) hexane-Et 2 0. Product fractions were evaporated to give 1.507 g of desired title aldehyde as a light yellow solid with m.p.

I.

HX8b -49-

720 -750 C) (Literature reports m.p. 730-74'C). TLC: (95:5) Hex-Et20, Rf 0.40, U.V. and PMA.

D.4'-Fluoro-3,3',5-trimethyl[l,l'- biphenyl]-2-methanol A cooled ice bath) solution of dry (15.0 ml) was treated with LiAlH 4 (259 mg, 6.82 mmole, 0.55 eq.) and the gray suspension treated dropwise over 15 minutes with a solution of the Part C aldehyde (3.0 g, 12.4 mmole) in dry (15 ml). The mixture was stirred at room .temperature under argon for 30 minutes, then Scooled back to 0°C and quenched by sequential dropwise addition of 260 pl H2 0, 260 pl of s 15 NaOH and 780 pl H20. The suspension was diluted with EtOAc, filtered through anhydrous Na 2 S 4 over packed Celite bed) and the colorless filtrate evaporated in vacuo to give 2.99 g of a white solid. Trituration of the crude solid with 20 cold hexane and drying in vacuo afforded 2.467 g a4 of desired title alcohol as a white solid with m.p. 102-1030 C. TLC: Hex-EtOAc, Rf 0.24, U.V. and PMA.

E. (S)-3-[((l,l-Dimethylethyl)diphenyl- silyl]oxy]-4-(hydroxymethoxyhosphinyl)- butanoic acid, methyl ester (S)-4-Bromo-3-hydroxybutanoic acid, methyl ester

[R-(R*,R*)]-2,3,4-trihydroxy- butanoic acid, calcium salt, hydrate_

fi iiX8b

Ref. Carbohydrate Research 72, pp. 301-304 (1979). Calcium carbonate (50 g) was added to a solution of D-isoascorbic acid (44.0 g, 250 mmol) in H2 0 (625 ml), the suspension cooled to 0 C (ice bath) and treated portionwise with 30% H2 02 (100 ml). The mixture was stirred at 30 0 -40 0 C (oil bath) for 30 minutes. Darco (10 g) was added and the black suspension heated on a steam bath until evolution of 02 ceased. The suspension was filtered through Celite, evaporated in vacuo (bath temperature 40 0 The residue was taken up in H20 (50 ml), warmed on a steam bath and CH3 OH was added until the solution was turbid. The gummy *a precipitated solid was collected by filtration and 15 ai dried to give 30.836 g of desired *t calcium salt as a powdery white solid. TLC iPrOH-NH40H-H 0, Rf 0.19, PMA.

1-2,4-Dibromo-3- 20 hydroxybutanoic acid, methyl o ester SRef. Bock, K. et al., Acta Scandinavica 37, pp. 341-344 (1983) Part calcium salt (30 g) was 25 dissolved in 30-32% HBr in acetic acid (210 ml) and stirred at room temperature for 24 hours. .Methanol o* (990 ml) was then added to the brown solution and it was stirred overnight. The mixture was evaporated to an orange oil, taken up in CH3 0H (75 ml), refluxed for 2.0 hours and evaporated. The residue was partitioned between EtOAc (100 ml) and H20, the organicphase washed with H20 (2x) and brine then dried over anhydrous Na 2 SO4 and

S-

HX8b -51-

evaporated to give 22.83 g of crude dibromide as a light orange oil. TLC (1:1) EtOAc-Hex, Rf 0.69, UV PMA.

(S)-4-Bromo-3-hydroxybutanoic acid, methyl ester Ref. the same as for preparation of An argon purged solution of the dibromide (20.80 g, 75.4 mmol) and anhydrous NaOAc (21.0 g) in EtOAc (370 ml) and glacial HOAc (37 ml) was treated with 5% Pd/C (1.30 g) and the black suspension stirred under of H 2 (1 atm) while monitoring H2 uptake. After n 2

o hours H2 uptake was complete, the mixture was 15 filtered through Celite, the filtrate washed with saturated NaHCO 3 and brine then dried over anhydrous MgSO 4 and evaporated to give crude dibromoester as a brown oil. The crude oil was combined with another batch (starting from 36.77 g 20 of the dibromide) and vacuum distilled to give p.0* 25.77 g of desired title bromoester as a clear oil with b.p. 790 -80°C (1.0 mm Hg). 4 -4 TLC EtOAc-Hex, Rf 0.44, PMA.

Anal Calcd for C5 H03Br; C, 30.48; H, 4.60; 4 0- Br, 40.56 4009 Found: C, ,29.76; H, 4.50; Br, 39.86

(S)-4-Bromo-3-[[(1,1-dimethylethyl)- diphenylsilyl]oxy]butanoic acid, methyl ester A solution of part E(l) bromohydrin (4.0 g, 20.4 mmol), imidazole (6.94 g, 5.0 and

.t

HX8b -52-

4-dimethylamino pyridine (4-DMAP) (12 mg, 0.005 eq.) in dry DMF (40 ml) was treated with t-butyl- diphenylsilyl chloride (5.84 ml, 1.1 eq.) and the homogeneous mixture stirred overnight under argon at room temperature. The mixture was partitioned between 5% KHSO 4 and EtOAc, the organic phase washed with H2 0 and brine, dried over anhydrous Na2 SO4 and evaporated to give 9.32 g (100%) of crude silyl ether as a clear, viscous oil. TLC Hex-EtOAc, Rf silyl ether 0.75, UV. and PMA.

(S)-4-Iodo-3-[[(1,l-dimethylethyl)- diphenylsilyl] oxy]butanoic acid, methyl ester A solution of the crude Part E(2) bromide (9.32 g, 201 mmole) in methyl ethyl ketone' (60 ml, dried over 4A sieves) was treated with sodium iodide (15.06 g, 100.5 mmole, 50 eq.) and the yellow suspension refluxed for 5.0 hours under 4$ 5 argon. The mixture was cooled, diluted with EtOAc, filtered, the filtrate washed with dilute NaHSO 3 (until colorless) and brine then dried over anhydrous Na2 SO4 and evaporated in vacuo to give 10.17 g of a yellow oil. The crude oil was no,. purified by flash chromatography on silica gel (600 g) eluting with Hexane-CH 2 Cl2 Product fractions were combined and evaporated to give 7.691 g overall yield for both steps) of desired title iodide as a clear, colorless, viscous oil, TLC Hex-EtOAc, product. Rf 0,75, UV, and PMA. (Note: product iodide co-spots with starting bromide).

a t

HX8b -53-

(S)-4-(Diethoxyphosphinyl)-3-[[(1,l- dimethylethyl )diphenylsilyl Ioxy] butanoic acid, methyl ester A solution of the iodide (7.691 g) in' triethyl phosphite (20 was heated at 1551C -(oil bath) for 3.5 hours under arg~on. The mixture was cooled and excess phosphite distilled off in vac~io (0.5 mmn Hg, 7511C) to leave a yellow oil The crude oil was purified by flash chrom~atography on silica gel (4(23 g) eluting with Hexane-acetone. fracti6ns were evaporated to give 3.222 g of desired title phosphonate as a clear, colorless, viscous oil. TLC Hex-acetone, Rf 0.51, UV. and PMA. 15 Additionally 2.519 g corrected yield) of starting Paft iodide wati recovered.

(S)-3-[[(l,l-Dimethylethyl)diphenyl- silyl~oxyl-4-phosphonobutanoic acid, methyl ester A solution of the Part phosphonate (9,85 g, 20.0 mmole) in dry CH C12 (60 ml) was *treated secqt.ntially with bistrimethylsilyltri- fluoroacetamide (BSUTFA) (5.31 ml, 32.0 mm'ole, 1.6 eq,) and trimethylsilyl bromide (TMSBr) (6.60 n'l 50.0 imole, 2.5 eq.) and th6 clear mixture stirred :overnight under argon at room temperature. KHSQ 4 (80 ml) was added and the mixciure was extracted with EtOAc. The aqueous phase was saturated with NaCl and re-extracted with ttOAc. The combined organic layers were waehed with brine,. dried over anhydrous Na 2 so 4 and evapora.td in vacuo to give crude title phosphonic~ acid as a

HX8b -54-

VISCOUS Oil. TLC iPr0F -iH4 OH-H 2 0, Rf 0.30, U. V. and PMA.

(S)-3-[[(l,l-Dimethylethyl)diphenyl- silyl IIoxyl-4- (hydroxymethoxyphosphinyl.) butanoic acid, methyl ester___ Part crude phosphonic acid (N20.0 mmole) in dry pyridine (25 ml) was treated with dried CH3 0H (over 3A sieves, 1.62 ml, 40.0 mmole, eq.) and dicyclohexyl carbodiimide (DCC) (4.54 gin, 22.0 mmole, 1.10 eq.) and the resulting white suspension stirred under argon at room temperature overnight., Pyridine- was removed in vacuo, then azeotroped with benzene (2 x 15 ml). The residual oil was dissolved in EtOAc, filtered and washed with 1.0 N HCl and brine, dried over anhydrous Na2 SO4 and evaporated in vacuo to give 0 8.272 g of crude title ester as an oil containing a small amount of precipitated dicyclohexy. urea~ at0 le0 20 (DCU). TLC iPrOH-NH -OH H 0, Rf =0.60, U.V. aldPMA.

[1,1 -biphenyl] -2-yl iimethoxyphosphinyl] 3-t-butyldiphenylsilyloxybutanok~ acid, methyl este. 0. Part E crude phosphonic acid mono methyl ester (6.595 gin, n-14.7 mmole) was dissolved in dry CH2 C12 (30 ml), treated with distilled trimethyl- silyldiethylamine (5.60 ml, 29.4 inmole, 2.0 eq.) and stirred under argon at room temperature for 1 hour, The mixture was evaporated in vacuo, chased with benzene (1 x 30 ml) and dried in vacuo.

I t HX8b

5'1 light yellow viscous oil was dissolved in dry CH2 Cl 2 (30 ml) and DMF (dried over 4A sieves, 2 drops), the clear solution cooled to -100 C (salt/ice bath) and treated dropwise via syringe with distilled oxalyl chloride (1.41 ml, 16.2 mmole, 1.1 Vigorous gas evolution was evident and the solution became deeper yellow in color. The mixture was stirred under argon at for 15 minutes then allowed to stir at room temperature for 1 hour. The mixture was evaporated in vacuo, chased with benzene (1 x 30 ml) and dried S, in vacuo'to give crude phosphonochloridate as a c. yellow oil. *To a solution of the crude phosphono- 15 chloridate (N 14.7 mmole) in dry CH2 Cl2 (10 ml) was added dropwise a solution of the Part D t biphenyl alcohol (2.06 g, 8.43 mmole) in dry pyridine (15 ml) and the resulting mixture stirred at room temperature under argon for 16 hours. 20 The mixture was evaporated to dryness and the

9* residue partitioned between 5% KHSO 4 and EtOAc. The organic phase washed with saturated NaCO3 and brine then dried over anhydrous Na 2 SO4 and evaporated in vacuo to give 8.290 g of a brown oil. The crude product was purified by flash chromatography on silica gl (370 g) eluting with (70:30) Hexane-acetone. Product fractions were Scombined and evaporated to give 3.681 g (66%) of the desired title phosphonate as a pale yellow oil. TLC Hexane-acetone, Rf 0.59, U.V. and PMA.

L 4 .f

S l t7..

HX8b -56-

G. (S)-4-[[[4'-Fluoro-3,3',5-trimethyl-

[1,1'-biphenyl]-2-yl]methoxyphosphinyl]- 3-hydroxybutanoic acid, methyl ester A mixture of the Part F silyl ether (1.103 g, 1.66 mmole) in dry THF (20.0 ml) was treated with glacial acetic acid (380 pi, 6.64 mmole, eq.) and a 1.0 M tetrabutylammonium fluoride solution (4.98 ml, 4.98 mmole, 3.0 eq.) and the clear yellow solution stirred overnight at room temperature under argon. The mixture was partitioned between cold H2 0 and EtOAc, the organic phase washed with saturated NaHCO3 and brine, dried over anhydrous Na2 SO4 and evaporated to a viscous yellow oil (1.174 The 15 crude oil was purified by flash chromatography on silica gel (47 g) eluting with (85:15) CH 2 Cl2-Acetone. Product fractions were evaporated *to give 679 mg of desired title alcohol as a clear viscous oil. TLC Hexane-acetone, Rf 0.41, U.V. and PMA.

H. Ot* [1,1'-biphenyl -2-yl methoxy]methoxy- phosphinyl]-3-hydroxy-butanoic acid, monolithium salt A solution of the Part G methyl ester (184 mg, 0.420 mmole) in dioxane (5.0 ml) was treated with 1.0 N LiOH (0.50 ml, 1.2 eg.) and the mixture stirred at room temperature under argon for 3 hours. The mixture was diluted with H 2 0, filtered through a 0.4 pm polycarbonate membrane and evaporated in vacuo. The residue dissolved in H90 ml), frozen and lyophilized. The crude acid

AI

HX8b -57-

was dissolved in a minimum amount of H 2 0 and chromatographed on a 100 ml bed of CHP-20 resin eluting with a H 2 /CH 3 CN linear gradient system. Product frac-tions were evaporated, dissolved in H 2 0 (50 ml), filtered through 0.4 jrn polycarbonate mc;mbrane and lyophilized -Eo give 174 riag (89.1% based on weight of hydrate) of desired title mono-lithiwim salt as a white solid. TLC (7:2:1) iPrOH-NH4 OH-H 2 0, Rf 0.58 U.V. and PMA. .0 Anal Calcd for C21 H25 06 PFLi 1.95 moles H 2 0 (MW 4-65.46): C, 54.19; H, 6.26; F, 4.08; P. 6.65 Found: C, 54.19; H, 6.21; F, 4.29; P, 6.43

H 1 NMkZ (400 HNz): 6 1.74-2.08 ppm (2H, M, -PO(OCH3 )2CH 2 0.4. 2.30 (3H, s, aromatic methyl) 9 40 2.32 (3H, d, aromatic methyl a~ to fluorine, J HF 2 2 Hz) 64 4 20 2.35-2.62 (2H, m, -CH 2Co2 Li) 04 4 2.46 (3H, s, aromatic methyl) 3.57 3 .63 (3H, 2 doublets, -OP(OCH3 2 diastereomers, J 10.3 Hz) 4.28 (1H, m, -CH 2 CH(OH)CH 2 CO2 L) 0 4.97 (2H, i, PhCH2 OP(OCH3 )R) 6.87-7.25 (5H, m, aromatic H's)

HX8b -58-

Example 2 (S)-4-[[[4'-Fluoro-3,3',5-trimethyl[l,1'- biphenyl]-2yll]methoxy]hydroxyphosphiny -3- hydroxybutanoic acid, dilithium salt A solution of the Example 1 diester (374 mg, 0.853 mmole) in dioxane (8.0 ml) was treated with 1.0 N LiOH (2.6 ml, 3.0 eq.) and heated at 0 C (oil bath) for 5.0 hours under argon. A white precipitate was evident. The mixture was diluted with H2 0 and filtered. The aqueous solution was extracted once with Et20, filtered through a 0.4 pm polycarbonate membrane and concentrated in vacuo. The crude product was chromatographed on CHP-20 resin (100 ml bed) 15 eluting with a H2 O/CH 3 CN linear gradient system. Product fractions were evaporated in vacuo, taken up in H2 0 (50 ml), filtered through a 0.4 pm polycarbonate membrane and lyophilized to give 260 mg (67.1% based on hydrate weight) of desired title 20 di-lithium salt as a white solid. TLC SPrOH-NH 4 OH-HO0, Rf=0.47, TJ.V. and PMA.

Anal Calcd for C2 0 H22 06PFLi2 1.77 moles C, 52.88, H, 5.67; F, 4.18; P, 6.82 Found: C, 52.88; H, 5,26; F, 4.24; P, 6.43 i

SH 1 NMR (400 MHz, CD OD): 13

0 It 6 1.69 ppm (2H, m, -OPCH2 CH(O0)-) 2.26-2.42 (2H, m, CH2 CO2 Li) 2.30 (3H, s, aromatic methyl) 2.31 (3H, d, aromatic methyl a to F, JHF=1. 9 Hz)

r"

H~ -59-

2.38 O3H, s, aromatic methyl) 4.22 (1H, m, -CH(OH)CH 2 0 4.75 (2H, m, PhCH2 OP-) 51 6.8.6-7.23 aromatic protons)

Example 3 (3S)-4-[[[4'-Fluoro-3,3' ,5-trimethyl [1.1 hanyl] 2-.yl Imethoxy] methyiphosphinyl] -3-hydroxybutano,4c acid, monolithium salt A. (S)-4-[(Chloro)methylphosphinyll-3- 1-dimethylethyl )diphenylsilyl] oxy] butanoic acid, methyl -ester 15 The titl.e phosphinochioridate compound is a prepared as described in Example 6 Part B first three paragraphs.

B. (3S)-4,-rj[4'-Fluoro-3,3'

[1,1'-biphenyl]-2-yl]methoxy]mkethyl- ~*phosphinyl] -3-t-butyldiphenylsilyloxy- butanoic acid, methyl ester A cooled (0 0 C, ice bath) solution of Part A phosphinochl ori date (N2.2 uuole) and Exam~ple 1 Part C(2) biphenyl alcohol (429 mg, 2.2 mmole, *eq) in dry CH2 C12 (10 ml) was treated with Et 3 N (425 p1, 3.04 mmole, 1.4 eq) and 4-DMAP (27 mg, 0.22 mmole) and the orange solution stirred at room temperature overnight under argon. The mixture was partitioned between 5% KHSO4 and EtOAc, the organic layer washed with brineo dried over anhydrous Na 2 so4 and evaporated to give 1 1 g of an orange oil. The crude oil was purified by flash

HX8b

chromatography on LPS-1 silica gel (44 g) eluting with EtOAc:Hexane. Product fractions were combined and evaporated to give 298 mg of desired coupled title product as a pale yellow oil. Also 460 mg (67% corrected yield) of starting Example 1 Part C(2) biphenyl alcohol was recovered. TLC EtOAc:Hex, Rf=0.18 UV and PMA.

C. (3S)-4-[[[4'-Fluoro-3,3',5-trimethyl-

[1,1'-biphenyl]-2-yl]methoxy]methyl- phosphinyl]-3-hydroxybutanoic acid, S* methyl ester A solution of the Part B silyl ether (298 mg, 0.46 mmole) in dry THF (6.0 ml) was treated 15 with glacial HOAc (110 pl, 1.84 mmole, 4.0 eq) and a 1.0 M in THF solution of tetrabutylammonium fluoride (1.43 ml, 3.1 eq) and the resulting solution stirred overnight under argon at room o temperature. The mixture was partitioned between S 20 cold H 0 and EtOAc, the organic phase washed with saturated NaHCO 3 and brine, dried over anhydrous NaSO04 and evaporated to a yellow oil 0o (273 mg). The crude oil was purified by flash chromatography on LPS-1 silica gel (11 g) 25 eluting with Hex-acetone. Product fractions were combined and evaporated to give 150 mg St of desired title alcohol as a viscous oil. TLC Hex:acetone, Rf=0.23, UV and PMA.

r (3S)-4-[[[4'-Fluoro-3,3',5-trimethyl-

[l,1'-biphenyl]-2-yl]methoxy]methyl- phosphinyl]-3-hydroxybutanoic acid, monolithium salt

.7

HX8b -61-

A solution of the Part C methyl ester (150 mg, 0.367 mmole) in dioxane (3.0 ml) was treated with 1.0 N LiOH (0.44 ml, 1.2 eq.) and the resulting white suspension was stirred at room temperature under argon for 2 hours. The mixture was diluted with H2 0, filtered through a 0.4 pm polycarbonate membrane and evapor.ated in vacuo to a colorless glass. The crude product was taken up in a minimum amount of H2 0 and chromatographed on HP-20 (100 ml bed) eluting with a H 2 O/CH 3 CN linear gradient. Product fractions were evaporated, taken up in ml), filtered through a 0.4 pm polycarbonate '"ir membrane and lyophilized to give 130 mg (79% based 15 on hydrate weight) of desired title'lithium salt as a white solid. TLC CH 2 Cl 2 -CH3 OH-HOAc, Rf=0.52, UV and PMA.

Anal Calcd for C2 1 H2 5 05FLiP 1.73 moles H 20 20 (MW 445.49): C, 56.61; H, 6.44; F. 4.26; P, 6.95 a* Found: C, 56.67; H, 6,36; F, 4.31; P, 7.43

l. H1 NMR (400 MHz): 0 II 6 1.49 ppm (3H, d, -OP(CH 3 JH-p= 1 4 7 Hz) 4 O

1.83-2.0 (2H, m, P-(CH3 )CH2 2.27-2.40 (2H, m, CH2 CO2 Li) 2.30 (6H, s, 2 aromatic methyl's) 2.44 (3H, s, aromatic methyl) 4.26 (1H, m, -CH2 CH(OH)CH2CO Li)

HX8b -62-

0 4.87 (2H, m, ArgH 2 0P (CH3 6.90-7.20 (5H, m, aromatic HIS)

Example 4

[[2,4-Dichloro-6-[(4-fluorophenyl)methoxy]- phen~yl ]methoxy] methoxypho sphinyl -3 -hydroxy- butanoic acid, monolithium salt A. 2 ,4-Dichloro-6- (4-fluorophenylmethoxy) benzaldehyde (Ref. J. Med Chem., 1986, 29, 167) **Asolution of 13.77 g (72.5 mmol) of 4,6-dichloro-2-hydroxybenzaldehyde in 100 ml of DMF was stirred and 12.02 g (87 mmol) of K2 C03 was added. This mixture was heated to N~70 0C for fir- minutes, then 11.7 ml of 4-fluorobenzyl bromide was added. The resulting solution was stirred at 0 C for 3.5 hours, then this was poured onto ice

H20 5 filtered and washed with H12 0, and t_1tit 20 recrystallized from Et2 /petroleum ether. Yield: 17.88 g of off-white crystals, m.p. 107-108'C.

B. 2,4-Dichloro-6- [(4-fluorophenyl) methoxylbenzenemethanol Cold (00 C, ice bath), dry Et2 0 (10.0 ml) was treated With LiAlH 4 (158 mg, 4.16 mmole, 0.6 eq.) and the grey suspens'ion treated dropwise with a solution of Part A aldehyde (2.06 g, 6.93 mmole) in 10 ml dry THF. The mixture was warmed to room temperature and stirred for an hour under argon, The mixture was cooled back to 00 C (ice bath) and quenched by sequential dropwise addition

HX8b -63-

of H2 0 (160 pl), 15% NaOH (160 pi) and H2 0 (475 pl). Precipitated salts were removed by filtration through anhydrous Na2 SO4 over packed Celite (s" bed). The clear filtrate was evaporated to give 2.052 g of crude alcohol as-white crystals. One trituration with cold hexane afforded 1.892 g of pure title alcohol as a white crystalline solid with m.p. 72-730 C. TLC Hex-acetone, Rf=0.31, UV and PMA. *I

si.t Anal Calcd for C1 4 H11 02C2F (MW 301.142): SC, 55.84; H, 3.68; Cl, 23.55; F, 6.31 15 Found: C, 55.97; H,.3.71; Cl, 23.42; F, 6.30

(S)-4-[[[2,4-Dichloro-6-[(4-fluoro- C. phenyl)methoxy]phenyl]methoxy]methoxy- phosphinyl]-3-t-butyldiphenylsilyloxy- 20 butanoic acid, methyl ester A solution of the Example 1 Part E(6) methyl ester (N3.84 mmole) in dry CH2CI 2 (10 ml) was treated with distilled trimethylsilyl diethylamine (1.46 ml, 7.68 mmole, 2.0 eq,) and the resulting solution stirred at room temperature under argon for 1.0 hour. The mixture was evaporated in vacuo, chased with benzene (1 x ml) and dried in vacuo to give crude silylated phosphonic acid mono methyl ester as a colorless oil. A solution of the crude ester (,3.84 mmole) in dry CH2Cl 2 (10 ml) and dry DMF (1 drop) was cooled to -100 C (salt, ice bath) and treated

L!

HX8b -64-

dropwise with distilled oxalyl chloride (368 pIl, 4.22 mmole, 1.1 Gas evolution was evident from the clear yellow mixture. The mixture was stirred at room temperature under argon for one hour, evaporated in vacuo, chased with benzene (2 x ml) to give crude phosphonochloridate as a viscous yellow oil. The crude phosphonochloridate ('3.84 mmole) in dry CH2 C1 2 ml) at 0 C (ice bath) was treated with Part B alcohol (1.15 g, 3.84 mmole, 1.0 eq.) followed by Et 3 N (805 pl. 5.76 mmole, 1.5 eq.) and 4-DMAP (47 mg, 0.384 inmole, 0.1 eq.) and the brpwn 0* mixture stirred overnight at roqm temperature under argon. The mixture was partitioned between 4 15 5% KHSO 4 and EtOAc, the organic phase washed with brine, dried over anhydrous Na2 SO4 and evaporated to give 3.197 g of a dark brown oil. The crude product was purified by flash chromatography on silica gel (160 g) eluting with Hex-EtOAc. o 20 Product fractions were combined and evaporated to give 594 mg of desired title phosphonate as a yellow oil. Additionally, 688 mg (52.4% corrected 1 o yield of starting Part B alcohol was recovered. TLC Hex-acetone, Rf=0.29, UV and PMA.

•r r D. (r)-4-[[[2,4-Dichloro-6-[(4-fluoro- phenyl)methoxy]phenyl]methoxy]methoxy- Sphosphinyl]-3-hydroxy butanoic acid, methyl ester A solution of the Part C silyl ester (578 mg, 0.788 mmole) in dry THE (8 ml) was treated with glacial HOAc (180 pl, 3.2 mmole, 4.0 eq.) followed by 1.0 M solution of n-Bu NF in THF (2.36

fl. 1 4 t i

HX8b

ml, 2.36 mmole, 3.0 eq.) and the resulting pale yellow solution stirred overnight under argon at room temperature. The mixture was poured into cold H2 0 and extracted with EtOAc The organic phase was washed with saturated NaHCO 3 and brine, dried over anhydrous Na 2 SO4 and evaporated tc give 625 mg of a yellow oil. The crude product was purified by flash chromatography on silica gel (31 g) eluting with Hexane-acetone. Product fractions were combined and evaporated to give 339 mg of desired title alcohol as 9 0 So a clear, colorless, viscous oil. O 999, TLC Hex-acetone, Rf 0.25, UV and PMA. «9

0 999 9 6 15 E. (S)-4-[[[2,4-Dichloro-6-((4-fluoro- a phenyl)methoxy]phenyl]methoxymethoxy- phosphinyl]-3-hydroxybutanoic acid, 0- monolithium salt O 6 9 A solution of the Part D phosphonate (132 9 o9a 9,49 20 mg, 0.267 mmole) in dioxane (2.5 ml) was treated a 9 99 with 1.0 N LiOH (0.32 ml, 1.2 eq.) and the mixture 9 stirred under argon at room temperature for 99 9. -99 14 hours. A white precipitate was noted. The 9 9 OII 99 9* mixture was diluted with H20, filtered and the 99 9 filtrate evaporated to dryness in vacuo. The 4 t residue was chromatographed on HP-20 resin (100 ml bed) eluting with a H2 0/CH 3 CN linear gradient system. Product fractions were combined and evaporated, taken up in H20, filtereA through a 0.4 pm polycarbonate membrane and lyophilized to give 108 mg (79% based on hydrate weight) of desired title lithium salt as a white solid.

I- W h 4

-79- HX8 b

Ai-

HX8b -66-

TLC (20:1:1) CH2 C1 2 CH3 OH-HIOAc, Rf 0.41, UV and PMA.'

Anal C cd for C1 9 H1 8 07 C12 Fli 2 p 1.42 moles H2 0 (MW 511.72): C, 44-'.59; H, 4.10; Cl, 13.86; F, 3.71; P, 6.05 Found: C, 44.22; H, 4.09; Cl, 13.91; 3.72; P, 6.11

a1 NMR (400 MHz): 0 11 6 1.98-2.11 ii (2H, m, QO(0CH3 )CH2 CH(0OH)- 2.26-2.45 ppm (21H, m, -CH(OH )CH2 CO2 Li) 3.63 3.62 (3H, 2 doublets,, 2 diastereojners, 0 0* 04 OP(OCH3 )CH2 JP-11 Hz) 4.23 (1H, m, (-CH2 CH(OH)CH 2CO2 Li) 0 00 04 5.116 (2H, s, F-PhCH 2 O0) 000.

5.24 (2H, d, ArCH2 OP, J, =6.2 Hz) 7.13-7.53 (6H, m, aromatic H's)

Exajmpe 2,4-Dichoro-6-[(4-fluorophenyl)- methoxy]phenyl]methoxy] hydroxyphosphinyl] -3- hydroxybutanoic acid, dilithium salt A mixture of the Example 4 Part D diester (210 mg, 0.424 nimole) in dioxane (4.0 ml) was treated with 1.0 N LiOH (1.30 ml, 3.0 eq.) and the colorless solution heated at 50 0 C (oil bath) under argon for 3.5 hours. white precipitate was evident after 15 minutes. The mixture was diluted with H2 0, filtered and the filtrate evaporated

/1

HX8b -67-

in veduo. The residue was dissolved in a. mainimum amount of H2 0 and chromatographed on HP-20 resin (100 ml bed) e'luting with a H2 /CH.,CN linear gradient. Product fractions were combined and evaporated. The residue. was taken -up in H2 0 ml), filtered through a 0.4 pm polycarbonate membrane and lyophilized to give 175 mg (81% based on hydrate weight) of desired title dilithiumn salt as a white solid. TLC CH 2 C11 2 -CH3 H-H0Ac, Rf

UV and FIMA.

Anal Cacld for C 1 8 H 1 6 0 7 C12 FLiP 1.70 moles H 2 0 (MW 509.62): C, 42.42; H, 3.84, F, 3.73; Cl, 13.91; P, 6.08 Found: C, 42.46; H, 3.90; F, 3.93; Cl, 13.42; P, 5.66

H NMR (400 MHz); 0 6 1.73-1.92 ppm (2H, m, -cAP(0Li)-CH2 CH(OH)- 2.27 (1Hi, dd, -CHIO)H 2 OLi, JHH8.8 Hz) 2.39 (1H, dd, -CH(QH )CH2 CO2 Li, JH=4. 4 25 Hz) a.4.26 (1H, m, CH2 CHOH)CH2 COLi) 5.08 (2H, s, F-Ph-CH 2 OAr) 7.03-7.53 (6H, aromnatic H's).

JA, U,

HX8b -68-

Example 6 2 4 -Dichloro-6-(4-flvorophenylmethoxy]-

phenyl lmethoxy]metiiylphosphinyl] -3-hydroxybutanoic acid, methyl ester A. (S)3-([(l,l-Dimethylethyl)diphenylsilyl]- oxy] (ethoxymethyiphosphinyl )butanoic acid, methyl ester____ A mixture of the Example 1 Part E(3) iodide (4.68 g, 9.18 mmole) in methyl diethoxy- phosphine (Strem Chemicals, 5.0 g, 36.7 mmole) was heated at 100 0 C (oil. bath) for 2.5 hours, then at 150 0 C for three additional hours under argon. A **white precipitate slowly formed in the yellow ~.solution. Excess phosphine was distilled off 15 in vacuo, (0.5 mm Hg) and the crude product pu iiie!d by flash chromatography on silica gel eluting, with (65:35) Hexane-acetone. Product fractions were combined and evaporated to give 1,590 g (38%) of desired title phosphinic ester (mixture of diastereomers) as a clear viscous oil, TLC Hex-acetone, RE (2 diastereomers) 0.19 and 0.22, UV and PMA. too 4

B. (3S)-4-([2,4-Dichloro.6-[(4-fjluoro- #06 25 phenyl )methoxy]phenyl ]methoxy)methyl- phosphinyl] -3-t-butyldiphenylsilyl- butanoic acid, methyl ester A solution of Part A phosphinic ester (605 mg, 1.3 mmole) in dry WT2 C1 2 (6.0 ml) was treated with bis (trimethylsilyl )trifluoroacetanide (BSTFA) (280 p1, 1.05 mmoJle, 0.8 eq.) and trimethylsilyl bromide (TMSBr) (210 p1, 1.57 mmole, 1.2 eq.) and the resulting solution stirred at room temiperature'

1 r

HX8b -69-

under argon overnight. 5% KHSO 4 (15 ml) was added and the mixture extracted with EtOAc. The organic phase was washed with brine, dried over anhydrous Na2 SO4 and evaporated in vacuo to -ive crude phosphinic acid as a colorless oil. A solution of the crude phosphinic acid mmole) in ,dry CH2C12 (6.0 ml) was treated with distilled trimethylsilyl diethylamine (270 pl, 1.44 mmole, 1.1 eq.) and the clear mixture was stirred at room temperature under argon for *0 hour. The mixture was evaporated in vacuo, 0 0 p

*000 *0 chased with benzene (1 x 15 ml), and dried *000 in vacuo. 0 000 0 A cooled (C0C, ice bath) solution of the crude silylated phosphinic acid (N1.3 mmole) in dry CH2 Cl2 (6.0 ml) and DMF (1 drop) was treated drcpwise via syringe with distilled oxalyl chloride rl

(130 pl, 1.44 mmole, 1.1 Gas evolution was 00 tO 0 evident. The mixture was stirred at room 00 0 20 temperature under argon for one hour then *4

evaporated in vacuo, chased with benzene (2 x 15 ml) and dried in vacuo to give crude phosphino- 00 4 Opoi 0

chloridate as a yellow oil. A cooled ice bath) solution of phosphinochoridate (1l,3 mmole) and Example 1 Part E(6) alcohol (392 mg, 1.3 mmole) in dry CH2 Cl2 ml) was treated with Et3 N (275 pl, 1.97 amole, eq.) and 4-DMAP (16 mg, 0.13 mmole, 0.1 eq) and the resulting yellow mixture stirred under argon at room temperature overnight. The mixture was partitioned between 5% KHSO 4 and EtOAc, the organic phase washed with brine, dried over anhydrous Na2 SO4 and evaporated to give 908 mg of ff i- 11: i r i

HX8b

crude product as a dark yellow oil. The crude product was purified by flash chromatography on silica gel (45 g) eluting with Hex-EtOAc. Product fractions were combined and evaporated to give 266 mg of desired title product as a clear, colorless oil. Also 197 mg corrected yield) of the starting alcohol was recovered.

C. (3S)-4-[[2,4-Dichloro-6-[(4-fluoro- phenyl)methoxy]phenyl]methoxy]methyl- *i I phosphinyl]-3-hydroxybutanoic acid, methyl ester A solution of the Part B silyl ester (275 It

mg, 0.38 mmole) in dry THF (6.0 ml) was treated with glacial HOAc (90 pl, 1.53.mmole, 4.0 eq.) and St a 1.0 M solution in THF of tetrabutylammonium fluoride (1.2 ml, 3.1 The resulting solution was stirred overnight under argon at room (I 9 II #4 temperature. The mixture was partitioned between cold H2 0 and EtOAc, the organic phase washed with 44 saturated NaHCO 3 and brine, dried over anhydrous Na 2 SO4 and evaporated to give 258 mg of a yellow oil. The crude product was purified by flash chromatography on LPS-I silica gel (8 g) t 25 eluting with Hexane-acetone. Product 4 a~ fractions were combined and evaporated to give 142 mg of desired title alcohol as a clear, colorless, oil. TLC Hexane-acetone, Ri 0.20, UV and PMA.

i' i i

HX8b i -71-

D. (3S)-4-[[2,4-Dichloro-6-[(4-fluoro- phenyl)methoxy]phenyl]methoxy]methyl- phosphinyl]-3-hydroxybutanoic acid, monolithium salt A solution of the Part C methyl ester (142 mg, 0.296 mmole) in dioxane (3.0 ml) was treated with 1.0 N LiOH (0.36 ml, 1.2 eq.) and the resulting white suspension stirred under argon at room temperature for 2.0 hours. The mixture was dilute4 with H2 0, filtered through a 0.4 pm polycarbonate membrane and the filtrate evaporated in vacuo. The crude product was dissolved in a minimum ,oo* °amount of H20 and chromatographed on a 100 ml bed 15 of HP-20 resin eluting with a H 2 0/CH 3 CN linear gradient. Product fractions were combined and evaporated. The residue was taken up in 2 0, filtered through a polycarbonate membrane and lyophilized to give 93 mg (63% based on hydrate 20 weight) of desired title lithium salt as a white solid. Io." TLC CH2C1 2 2 CHO3H-HOAc, Rf 3 0.51, UV and PMA.

tt* 25 Anal Calcd for C1 9 H 90OCl 2 FLiP 1.38 moles 2 0 (MW 495.94): C, 46.01; H, 4.42; F, 3.83; Cl, 14.30; P, 6.24 Found: C, 46.10; H, 4.49; F, 3.82; Cl, 14.32; P, 6.43

H 1 NMR (400 MHz):

I I,

HX8b -72-

0 11 6 1. 53 ppm (3H, d, OP(CH3 )CH 2 JH-P= 14.6 Hz)

1.87-2.10 (2H, m, -OP(OCH3 )CH2 2.27 (1H, dd, -CH(OH )CH2 CO2 Li, 2.38 jHH= 4 Hz, JHP=1 HZ)

4.29 (1H, m, -CH2 CH(O-H)CH2 CO2 Li) *9 5.16+5.18 (4H, m, ArCH2 OP 'and F-PhCH 2 O0)-) .9 '4

0*99 99 7.11-7.52 (6H1, m, aromatic) 99

99 9* 9*9 9 9 Example 7 9 *9 .J 99* 9 9 15 ,5-trimethy1[l1,1'-b-4henyl- *9'4 0 2-y. ]methyl'] amino] methoxyphosphinyl]j -3 -hydroxy- 6

butaoicacid, monolithium salt A. 41-Fluoro-3,3' 99 9 99 9 biphenyll-2-carboxylic acid 9 99 9 20 A solution of the E;natple 1 Part C(2) 99 aldehyde (1.0 g, 4.13 nunole) in acetone 9 09 99 ~94 9 9 (10.0 ml) at 00 C (ice bath) was treated dropwise with 8.0 N Jones reagent (4.1 ml, excess) and the resulting brown-green suspension stirred overnight *49.

*49* 25 under argon at room temperature. Excess oxidant 9* *9 9 was destroyed by adding isopropanol (10.0 ml) and 9 *4 precipitated chromium salts removed by filtration through a 4" pad of Celite. The filtrate was evaporated, taken up in EtOAc, washed with 1.0 N HCI saturated NH4 C1 (2X) and brine, then dried over anhydrous Na2 SO4 and evaporated to give 1.011 g of a green solid with m.p. 153-1541C.

x-I i Y"~rrn*_

t

HX8b -73-

The crude acid was purified via the dicyclohexylamine salt. To a solution of the crude acid in EtOAc (5.0 ml) was added dicyclohexyl amine (DCHA) (823 pl, 1.0 The solution diluted with hexane and precipitated crystalline salt was collected to give 997 mg (55% from aldehyde, m.p. 181-1830 C) of desired product as an off-white crystalline DCHA salt. The title free acid was regenerated from the DCHA salt by partitioning the salt between SKHSO 4 and EtOAc. The organic phase was washed CL C with brine, dried over anhydrous Na2 SO4 and C C cC C evaporated in vacuo to give 554 mg (52% from dC, aldehyde) of desired title acid. TLC CH2 C12-CH3OH, Rf=0.37, UV and PMA.

B. 4'-Fluoro-3,3',5-trimethyl[1,1'- biphenyl]-2-carboxamide A suspension of the Part A acid (554 mg, 2.14 mmole) in dry CH2 C12 (6.0 ml) and dry DMF (1 drop) at 0 C (ice bath) was treated dropwise via t C syringe with distilled oxalyl chloride (205 pi, ICC C 2.35 mmole, 1.1 eq.) and the clear yellow solution stirred under argnn at room temperature for one hour. The mixture was evaporated in vacuo, chased with benzene (2X) and dried in vacuo to give crude acid chloride as a yellow oil. A cooled ice bath) mixture of THF ml) and concentrated NH4 OH (2.0 ml, excess) was treated dropwise with a THF solution of the crude acid chloride and'the bright orange solution stirred at room temperature under argon for 1.0 hour. The mixture was partitioned

HX8b -74-

between H2 0 and EtOAc, the organic phase washed with saturated NaHCO 3 H2 0 and brine, then dried over anhydrous Na2S04 and evaporated to give 528 mg of crude amide as a light orange solid. One recrystallization from EtOAc-hexane afforded 435 mg of purified title' amide as pale yellow needles with m.p. 197-198 0 C. TLC (1:1) Rf=0.83, UV and PMA.

C. 4'-Fluoro-3,3',5-trimethyl[1,1'- biphenyl]-2-methanamine A-cooled (0 C, ice bath) solution of dry t: THF (5.0 ml) was treated with solid LiAlH 4 (125 4 I 44. 4 mg, 3.3 mmole) and the gray suspension treated dropwise over five minutes with a solution of the Part B amide (424 mg, 1.65 mmole) in THF (5.0 ml). 9r The resulting suspension was stirred at room temperature under argon for 2.5 hours, then refluxed for 45 minutes. The mixture was cooled to 20 0°C (ice bath) and quenched by sequential dropwise I c addition of 125 pl H2 0, 125 pl of 15% NaOH and 375 p1 H2 0. Precipitated aluminum salts were removed by filtration through anhydrous Na2 SO4 over packed 4i i 4444s Celite. The clear filtrate was evaporated 4. I 25 in vacuo to give the crude amine as a clear oil. I 4 4; TLC Et20-acetone, Rf=0.60, UV and PMA. The amine was purified as the HC1 salt. A solution of the crude amine (N1.65 mmole) in absolute EtOH (8.0 ml) was treated with concentrated HC1 (152 pl, 1.82 mmole) and the mixture stirred for 15 minutes at room temperature under argon. The mixture was evaporated in vacuo to a white crystalline solid.

HX8b

The solid was triturated with cold Et20, collected by filtration and dried in vacuo to give 426 mg of title amine-HC1 as fine white crystals.

D. -Fluoro-3,3 1

[1,1-biphenyl]-2-yl]methyl]amino]- methoxyphosphinyll-3-t-butyldiphenyl- silyloxy butanoic acid, methyl ester A solution of Example 1 Part E(6) methyl ester (N2.0 mmole) in dry CH2 Cl 2 (5.0 ml) was treated with distilled trimethylsilyl diethylamine (758 pi, 4.0 mmole, 2.0 eq.) and the clear mixture stirred at room temperature under argon for one to* hour. The mixture was evaporated in vacuo, "15 chased with benzene (1.x 15 ml) and dried in vacuo. Lt,.t A cooled (0 C) solution of the c'ude silyl phosphonate in dry CH2 C12 (7.0 ml) and DMF (1 drop) was treated dropwise with distilled oxalyl chloride (192 pl, 2.2 mmole, 1.1 Gas 20 evolution was evident from the clear yellow mixture. The solution was stirred at room temperature for one hour, evaporated in vacuo, chased with benzene (2 x 15 ml), and dried in vacuo to give the crude phosphonochloridate as 25 a yellow, viscous oil. A cooled solution of the phosphonochloridate and Part C biphenyl amine HCl (416 mg, 1.49 mmole) in dry CH2 Cl2 (10 ml) was treated with Et3 N (641 pl, 4.6 mmole, 2.3 eq.) and 4-DMAP (24 mg,. 0.2 mmole, 0.1 eq.) and the clear yellow mixture stirred i:ght at room temperature under argon. The mixtu e was partitioned between 5% KHSO 4 and EtOAc, the

HX8b -76-

organic phase washed with brine, dried over anhydrous Na2 SO4 and evaporated in vacuo to give 1.19 g of a yellow oil. The crude product was purified by'flash chromatography on silica gel (60 g) eluting with hexane-acetone. Product fractions were evaporated to give 588 mg of desired title phosphonamide as a pale yellow, viscous oil. TLC Hexane-acetone, Rf=0.20, UV and PMA.

E. 4' -Fluoro-3,3 -biphenyl )-2-yl]methyl ]amino]- t* methoxyphosphinyl] -3-hydroxybutanoic (i acid, methyl ester f t 15 A solution of Part D silyl ether (588 mg, 0.888 mmole) in dry THF (10,0 nl) was treated with glacial HOAc (203 pl, 3.55 mmole, 4.0 eq.) and a e;

If 1.0 M solution in THF of tetrabutylammonium fluoride (2.66 ml, 2.66 mmole, 3.0 eq.) and the resulting solution stirred overnight under argon I t at room temperature. The mixture was poured into cold H2 0 and extracted with EtOAc. The organic phase was washed with saturated NaHC03 and brine, then dried over anhydrous Na2 S04 and evaporated ti 25 in vacuo to give 605 mg of an orange oil. The crude product was purified by flash chromatography on silica gel (36 g) eluting with Hexane- acetone. Product fractions were combined and evaporated to give 196 mg of desired title alcohol as a light orange oil. TLC Hexane-acetone, Rf=0.16, UV and PMA.

HX8b -77-

F. 1'-biphenyl] -2--yl]methyl] amino] methoxyphosphinyl] -3-hydroxybutanoic acid, mb'nolithiun salt A solution of the Part E diester (105 mg, 0.240 mmole) in dioxane (2.0 ml) was treated with N LiOH (288 p1, 1.2 eq.) and the white suspension stirred under argon at room temperature for 4.0 hours. The mixture was diluted with H2 0, filtered, the filtrate evaporated'in vacuo. The residue was chromatographed on HP-20 (100 ml bed) .0#41 resin eluting with a H2 O/CH3 CN linear gradient. Product fractions were combined and evaporated. 9..The residue -was taken up in H820 (50 ml), filtered through a 0.4 pm polycarbonate membrane and lyophilized to give 70 mg (62.7% based on weight of hydrate) of desired titl~e lithium salt as a white 4 solid. TLC (20;1:1l) CH2 C12 -CH3OH-HlOAc, Rf=0.19, UV and PMA. 949

Anal. CaJlcd for C2 1 H2 NO PFUi 2.41 moles H2 (MW 47f2.75): C, 53.35; H, 6.57; N, 2.96; F,

25 round-, C, 53.35; Re, 6,52; N, 2.98; F, 4.05- P, 6.59

H-X8b -78-

1 1 (400 MHz): 0 6 1.79-1.97 ppm (2H, m, -P(0CH3 )2H2 2.26-2.44 ppm (2H, m, -CH2 CO 2 Li 29 P3H, s, aromatic methyl) 2.31, (3H, d, aromatic met-,hyl a to fluorine, JHF 1l 4 Hz) 2.47 (3H, aromatic methyl) 3.46& I. 3.50 (3Hf 2 doublets, 2 9 9 4 9 *4 diastereomers, JHP=10.5 Hz) 4 9.99 0 ~y 9 4 9 9 3.96 (2H, m, -PhCH2 NHP(OCH 3 49 9 *994 4 4.17 (lH, M, (-CH2 CH(OH)CH 2CO 2Li) I 6.84-7.21 (5H, m, aromatic protons) 4

Examples 8 to 4 9 9 IF F Following the procedures as outlined 4 I 99 4 heretofore and as described in the previous I 4-9 working Examples, the following additional s94 4 4 compounds may be prepared.

0 H 149 9

99.4 4 R-P-CH2 2 RX 99 99 9 X OH 9~4

C2 n

CC.

HX8 b -79-

No. R 0 x Rx

8. OH H 1 0 012 0

9. CIHO5 Cif 3 0 2 NH CH 3

0 253H

*4 4 0 4 0 Li

04 0

to 44 2 NH H

12. OH 2 0 H

13,, C4HO9 1 0 Li CH -0O

c

I

HX~b

n X R

14. C5H 1 1 NH CH 3

CH I C2H

S. a *5 CH sq., 15. OK C2 Hn 5 -t 2 0 OK ft.,. at ft a 0 a. a ft..

ft H CH3

CH3 a ft a f&~

ft #t a. 16. ONa ft 2 0 H -10 a a~ 2C a, t ft

ft. ft. ft I ''ft. ft. CH3 0 1 NH H ft ft I C

HO C H L

HX8b

-81-

No. R n -X R

C H -C-C- 18. CH 3 1~a H CH 3 CH 3

49 4 44 4 19. HO 2 0 Li 9499 4 9999

4 *9 99 S *99 9

9 99 4 9 949.

999.99 20. CHO3 NH H 9 0

'9 .4 4 ~0 4 94

4 94 4. 4 99

4 94 49 4 499 9

4499 I 9999

94 4 44

HX8b -82-

Example 21 (S)-4-Diisopropyloxyphosphinyl)-3- [(1,l-dimethyl- ethyl)diphenylsilyl] oxy]-butanoic acid, methyl ester The Example 1 Part E(3) iodide (45.1 mmol., 21.70 g) was stirred under high vacuum for minutes. Freshly distilled triisopropyl phosphite (0.451 mol., 93.92 g, 113.37 ml.) was added in one portion and the reaction mixuture was stirred under 10 argon and heated in a 155 oil bath for 16.5 42: hours. The mixture was then c.s to room temperature. Excess triisopropyl phosphite and •volatile reaction products were removed by short o path distillation (10 mm Hg) followed by Kugelrohr 15 distillation (0.50.mm Hg, 100 0 C, 8 hours). The product was further purified via flash chroma- tography (95 mm diam. column, 6"/Merck silica gel, 6/3/1 Hexane/acetone/toluene eluent, 2"/min flow rate, 50 ml fractions) to afford 17.68 g (33.96 mmol, 75% yield) of the title Got isopropylphosphonate as a clear viscous oil. TLC: Silica gel Rf=0. 3 2 (6:3:1 Hexane/acetone toluene) HNMR: (270 MH Z CDCl.) 25 6 7.70-7.65 (m,4H) 7.45-7.35 (m,6H) 4.57-4.44 (m,3H) 3.59 (s,3H) 2.94 and 2.88 (2xd, 1H J=3.7 Hz) 2.65 and 2.60 (2xd, 1H J=7.4 Hz) 2.24-1.87 (Series of m, 2H) 1.19 and 1.12 (2xd, 12H J=6.3 Hz) 1.01 9H)

L !l

HX8b -83-

Example 22 S)-4-(Hydroxymethoxyphosphinyl)-3- [[(1,1-dimethyl- ethyl)diphenylsilyl]oxy]butanoic acid, methyl ester, dicyclohexylamine salt The Example 21 isopropyl phosphonate (30.5 mmol, 10.66 g) was stirred under argon, at room temperature, In 80 ml of dry CH2 Cl2 This solution was treated dropwise (5 min) with bistrimethylsilyltrifluoroacetamide (BSTFA) (32.8 mmol, 8.44 g, 8.71 ml), followed by dropwise addition (10 min)' of trimethylsilylbromide (TMSBr) (51.3 mmol, 7,84 g, 6.75 ml). After stirring at room temperature for 20 hours, the reaction mixture was quenched with 200 ml of 5% aqueous S 15 KHSO 4 and stirred vigorously for 15 minutes. The aqueous layer was extracted 3 times with ethylacetate. The organic extracts were combined, S washed once with brine, dried over Na22 SO44 and concentrated in vacuo. The residue was azeotroped 2 times with 50 ml of toluene. The precipitate which formed was suspended in toluene and filtered. The filtrate was concentrated and the azeotrope/filter process repeated. The resulting filtrate was evaporated in vacuo and then pumped 25 under high vacuum for 5 hours. The resulting viscous clear oil was stirred under argon, at room temperature, in 50 ml of dry pyridine. This solution was treated in one portion with dicyclohexylcarbodiimide (DCC) (22.6 mmol, 4.65 g), followed by addition of methanol (41.0 mmol, 1.31 g, 1.67 ml). After stirring at room temperature for 20 hours, the reaction mixture was filtered through a celite pad in a sintered glass funnel.

I M ,i

EX8b -84-

The celite was washed with ethyl acetate and the combined filtrates were evaporated in vacuo. The residue was redissolved in ethyl acetate and washed 2 times with 5% aqueous KHSO 4 and once with brine. The organic extract was dried over Na2 SO 4 filtered, the filtrate concentrated and azeotroped 2 times with toluene, suspended in toluene and filtered. The resulting filtrate was again

vacuum for 6 hours to afford the phosphonate t monoester as a clear viscous oil (10.2 g, >100% yield). TLC: silica gel Rf=0.50 (7:2:1 he nPrOH/NH 4 OH/H 2 The phosphonate monoester

[1.21 g was pumped under high vacuum for 4 hours, affording 1.16 g (2.57 mmol)] was dissolved in ml of dry ethyl ether and treated dropwise with dicyclohexylamine (2.65 mmol, 0.481 g, 0,528 ml). The resulting homogeneous solution sat at room temperature for 7 hours resulting in significant crystal formation. The mixture was stored at 0 C for 16 hours and then warmed to room temperature and filtered. The crystals were washed with cold, dry ethyl ether and then pumped *w 25 under high vacuum over P2 0 5 for 18 hours. The crystals were subsequently pumped under high vacuum at 45 0C for 4 hours, affording 1.25g (1.98 mmol, 77% yield) of the title dicyclohexylamine salt as a white powdery solid, m.p. 155-156 0 C. 4 30 TLC: Silica gel Rf=0. 5 7 (20% MeOH/CH 2C1 2 NMR: crsa omto.Temitr a trda (270 MH2, CDC1

HX8b

6 7.71-7.65 (i,4H)

7.40-7.32 (i,6H) 4.02 (mn, 1H) 3.52 3H,) 3.28 and 3.22 (mn, 1H) 3.11 3H J=11 Hz) 2.77-2.64 (in,2H) 2.62-2.56 (mn, 1H) 1.92-1.08 (Series of mn, 22H) 1.00 9H) M'ass Spec: (FAB) 632 (MEH) IR:(KBr) 3466-3457 (broad) 3046, 3016, 2997; 2937, 2858, 2836, 2798, 2721, ~2704, 2633, 2533, 2447, 1736, 1449, 1435, 1426, 1379, 1243, 1231, 1191, 1107, 1074, 1061, 1051., 820 CM-i Anal Calcd for C 2 2 H 3 1 0 6 P si C1 2 H 2 3 N *t C~,64.63; H,8.61; N,2.22 SFound; C, 64.51; H, 8.49; N, 2.18

I

HX8b -86-

The claims defining the invention are as follows:

1. A compound useful in inhibiting the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase having the moiety

0 I -P-CH 2 -CBH-CH-CO- X. OH (CH)n

t

wherein X is or n is 1 or 2 and Z is a S.I hydrophobic anchor. O,t 2. The compound as defined in Claim 1 wherein the hydrophobic anchor is a lipophilic group which when linked to the HMG-like upper side chain of the molecule by the appropriate linker binds to a hydrophobic pocket of the enzyme not utilized in binding the substrate HMG CoA, resulting in enhanced potency relative to I I. compounds where Z=H.. 3. The compound as defined in Claim 1 having the structure

4 II R-P-CH 2 CO 2R X OH

z

including salts thereof, wherein R is OH, lower X k alkoxy ao or lower alkyl; and R i H or lower alkylt or

V<ii

HX8b -87-

X is or -NH-; n is 1 or 2 Z is a hydrophobic anchor which is

2a ~t I I *1

*414 4 Is4t $4 ~0 0

*0 0 0 q*00

0 @4440 0 6a

4 I wherein the dotted lines represent optional double 40

-0 40 4 .4 wheei R ,R 2 R2 a adRbmay be the same 04 or different and are H, halogen, lower alkyl, 4 4 *44 0 haloalkyl, phenyl, substituted phenyl or ORY in which Ry is H, aikanoyl, benzoyl, pheniyl, 0044 halophenyl, phenyl-lower alkyl, lower alkyl, I *4t1 04 C cinnamyl, haloalkyl, allyl, cycloalkyl-lower 1 4 I~ alkyl, adamantyl-lower alkyl or substituted phenyl-lower alkyl; wherein Z is

(R6a

Claims (1)

1. r HX8b -88-

   R5 and R5 1 are thie same or different and are H, lower alkyl or OH,. one or R5 and R5 1 being present when the carbon to which it is attached is linked to a double bond; 0 6 R is alkyl-C-or arylCH2 R6ais lower alkyl, hydroxy, oxo or halogen, and q is 0, 1, 2 or wherein Rx is hydrogen or lower alkyl in .4 a 4. 4 free acid form or in the form of a physiologically- 4 4 hydrolysable and -acceptable ezter in salt form. 4 a a 44 4. The compound as defined in Claim 3 9.9 a *4 wherein X is R is alkoxy, and Z is 4. a *4~4 a 44 a

   0* 4

   12a 4 *t~ 4 4 4 44

   a. a 4 a 4 5. The compound as defined in Claim 3 wherein X is R is alkoxy and (CH2 )n is CH2 0*4* and Z isI a *44 I 04 0 0 0 a 4*

   'R2 a

   6. The compound as" defined in Claim 4 having the name irimethyl (1,1 '-biphenyl J-2-yl]methoxy]methoxy-

   HX8b -89-

   phosphinyl] -3-hydroxy-butanoic acid, met hyl ester or its monolithium salt, (S)-4-[C[4t-fluoro-3,31,5-trimethyl[l,l- biphenyl] -2-yl]methoxy) hydroxyphosphinyl] -3- hydroxybutanoic acid, dilithium salt,

   biphenyl] -2-yl ]methoxylmethylphosphinyl hydroxybutanoic acid, monolithium salt, ([2,4-dichloro-6- ((4-fluoroph.enyl 9. methoxyjphenyl ]methoxy~methoxyphosphinyl) hydzoxybutanoic acid,-monolithium salt, (3S 4-dichloro-6-(4-fluorophenyl) methoxy] phenyl ]methoxy] hydroxyphosphinyl] -3- hydroxybuitanoic acid, dilithium salt,

   [[2,4-dichloro-6- [,(4-fluorophenyl methoxy]phenyl ]mechoxy] methyiphosphinyl] -3- hydroxybutanoic acid, or its methyl ester, or

   .4 biphenyl, -yl ]methyl) amino) methcxyphosphinyl hydroxybutanoic acid, monolithium salt. 7. A hypocholesterolemic or hypofipemi~c composition comprising a c~ompound as defined in ,*Claim 3 and a pharmaceutically acceptable carrier there for. 8. A compound havilng the structure 0 Ra 1-Oal1 Rr-P-CH 2-CH-CH2-C2aly Oalkyl OSi-CCH3 3 C6 H wherein Ra is lower alkyl or lower alkoxy, including all stereoisomers thereof.

   'ILL

   RX8b

   9. A compound havinig the stracture 0 Ra--cC-CH-C O d".1 OH Osi-C(CB3 3a

   whri al C6 E5 C6 E1 wherin R is lower alkyl, or OH, including all stercoisomers thereof. use of a compound as defined in Claim 3 for inhibiting cholesterol biosynthesis.

   e.DATED: 6 May 1988

   PHILLIPS ORMONDE FITZPATRI "II Attorneys for: E.R. SQUIBB SONS, INC.