HMG-CoA Reductase Inhibitors: An Exciting
`Development in the Treatment of
`Hyperlipoproteinemia
`
`F. G. Kathawala
`Preclinical Research Department, Sandoz Research Institute, Route 10, East Hanover, New Jersey 07936
`
`I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`II. Design Aspect for HMG-CoA Reductase Inhibitors at Sandoz Research Institute
`Leading to Fluvastatin (XU 62-320) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`III. General Chemistry Approach. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`A. Synthesis of Synthon 1 and 2, Fig. 6 (Scheme 1 and Scheme 2) . . . . . . . . . . . . . . .
`B. Choice of R and Synthesis of Intermediates 3, Fig. 6, and 4, Fig. 7. . . . . . . . . . . .
`C. Synthesis of Indole Intermediates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`D. Synthesis of Indene Intermediates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`E. Synthesis of Naphthalene Intermediates....................................
`F. Synthesis of Imidazole Intermediates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`G. Synthesis of Pyrazole Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`H. Synthesis of HMG-CoA Reductase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`IV. Biological Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`A. Results in in vitro HMG-CoA Reductase Microsomal Assay and in in vivo
`Cholesterol Biosynthesis Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`B. SAR of Fluvastatin (XU 62-320) Analog. . . . . . . . . .. . . . .. . . . .. . . . . .. . . . . . . . . . .
`C. SAR of Indene Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`D. SAR of Naphthalene Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`E. SAR of Pyrazole Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`F. SAR of Imidazole Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`V. Effects of Fluvastatin (XU 62-320) on Plasma Lipoprotein Levels . . . . . . . . . . . . . . . . .
`VI. Toxicological, Drug Metabolism and Pharmacokinetic Studies of Fluvastatin
`(XU 62-320).................................................................
`VII. Human Studies with Fluvastatin (XU 62-320)...................................
`VIII. Overview of Published Literature on HMG-CoA Reductase Inhibitors . . . . . . . . . . . .
`IX. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`References.......................................................................
`
`121
`
`122
`124
`125
`126
`128
`128
`128
`129
`129
`129
`133
`
`133
`133
`137
`137
`138
`138
`139
`
`140
`140
`140
`143
`145
`
`I. INTRODUCTION
`
`Coronary heart disease (CHO) continues to be one of the major health
`problems in all the developed countries of the world. A considerable body
`of clinical and epidemiological data has emerged over the years linking ele(cid:173)
`vated blood levels of total cholesterol, 1ow Qensity 1ipoprotein ~holesterol
`(LDL-C), and Yery 1ow Density 1ipoprotein ~holesterol (VLDL-C) as im(cid:173)
`portant risk factors for the development of coronary heart disease. 1
`For the treatment of elevated LDL-C and VLDL-C, a judicious diet, low in
`cholesterol and fat with saturated fatty acids replaced by polyunsaturated
`fatty acids, is the recommended choice. However, for patients nonresponsive
`
`Medicinal Research Reviews, Vol. 11, No. 2, 121-146 (1991)
`© 1991 John Wiley & Sons, Inc.
`
`CCC 0198-63251911020121-26$04.00
`
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`122
`
`KATHA WALA
`
`to dietary intervention, the development of effective and safe therapeutic
`agents for the treatment of hyperlipoproteinemia remains an important need.
`This need has gained considerable support as a result of two important events:
`(1) the results of the Lipid Research Clinic's Coronary Primary Prevention
`Trial (LRC-CPPT), a multicenter, randomized, double-blind study involving
`3806 asymptomatic middle-aged men in the United States with type II hy(cid:173)
`perlipoproteinemia, that demonstrated that a statistically significant reduction
`of 19% in the rate of fatal plus nonfatal coronary heart disease was associated
`with a 9% decrease in blood cholesterol levels, 2 and (2) the recommendation
`to treat individuals with blood cholesterol above the 75th percentile, which
`emerged from the consensus panel of the December, 1984 NIH Consensus
`Development Conference on the lowering of blood cholesterol to prevent
`coronary heart disease. 3
`In recent years, to achieve this goal of finding effective and safe therapeutic
`agents to lower LDL-cholesterol, great interest has focused on potent inhib(cid:173)
`itors of the enzyme [3-Hydroxy-[3-Methyl-Glutaryl-CoA reductase (HMG-CoA
`reductase, EC 1.1.1.34), which controls a key step in the endogenous synthesis
`of cholesterol. Several studies, both in animals and humans, have been re(cid:173)
`ported with HMG-CoA reductase inhibitors: compactin (Mevastatin), CS-514
`(Pravastatin, Mevalotin®, Pravachol®), mevinolin (Lovastatin, Mevacor®) and
`Synvinolin (Simvastatin, Zocor®), 4 which are structurally very closely related
`to one another. In order to assess fully the potential of HMG-CoA reductase
`inhibitors as an effective therapeutic intervention for the treatment of hyper(cid:173)
`lipoproteinemia, it is thus desirable to study in humans a variety of these
`inhibitors derived from different structural prototypes which can be distin(cid:173)
`guished in their overall biological profile from one another. This conceptual
`framework formed the basis for initiating efforts at the Sandoz Research In(cid:173)
`stitute to develop and study a variety of HMG-CoA reductase inhibitors with
`chemical structures different in several respects from compactin, pravastatin
`(a hydroxy analog of compactin), lovastatin (a methyl analog of compactin),
`and simvastatin (a dimethyl analog of compactin), and has led to fluvastatin
`(XU 62-320), the first totally synthetic HMG-CoA reductase inhibitor currently
`in Phase III human clinical trials (Fig. 1).
`
`II. DESIGN ASPECT FOR HMG-CoA REDUCTASE INHIBiTORS AT
`SANDOZ RESEARCH INSTITUTE LEADING TO FLUVASTATIN
`(XU 62-320)
`
`Investigations by Akira Endo with compactin4 have to be largely credited
`for the resurgence of the research on cholesterol biosynthesis and the renewed
`interest in HMG-CoA reductase inhibitors, a field now almost three decades
`
`F. G. Kathawala obtained his M.Sc. from the University of Bombay, India, and his Ph.D.
`in 1961 from Technische Hochschule Braunschweig, West Germany (Prof. H. H. Inlwffen), in
`Synthetic Organic Chemistry. After a few years of postdoctoral work at Harvard (Prof. R. B.
`Woodward), Wisconsin (Prof. H. Muxfeldt), and Gottingen (Prof. F. Cramer), he joined Sandoz
`in East Hanover, New Jersey, as a Senior Scientist, in 1969. Currently, he is the Director of
`Medicinal Chemistry in the area of Lipoprotein Metabolism/Atherosclerosis. His research in(cid:173)
`terests in Medicinal Chemistn; are focused towards the discoven; of agents affecting lipoprotein
`metabolism/atherosclerosis.
`
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`HMG-CoA REDUCT ASE INHIBITORS
`
`123
`
`II
`
`Mevastatin ( Compactin )
`
`Lovastatin ( Mcvinolin )
`HO ycooNa
`.. '>r Oil
`
`~g II, Oil
`
`Simvastatin ( Synvinolin )
`F
`
`Oil OH O
`
`Pravastatin ( Eptastatin )
`
`Fluvastatin (XU 62-320)
`
`Figure 1
`
`old. While all intensive studies hitherto conducted have been with closely
`related metabolites, such as compactin, mevinolin, and CS-514 (pravastatin),
`derived from fungal broths, efforts at the Sandoz Research Institute towards
`the development of new HMG-CoA reductase inhibitors have been based on
`synthesis, guided by the following assumptions:
`(a) There are two regions at the active site of the enzyme: one with high
`specific recognition of a 5-carbon unit (C-1 to C-5 as shown below) of the ~­
`OH-~-Methyl-glutaryl portion, and the other of CoA moiety present in HMG(cid:173)
`CoA
`(Fig. 2).
`
`2
`4
`CoAS~Oll
`~~
`~;
`
`/\OH
`
`HMG-CoA
`
`Figure 2
`(b) Compactin (R = H, Fig. 3), a known inhibitor of the enzyme, may be
`regarded as a transition state analog, when in the open dihydroxy acid form.
`
`6
`4
`2
`7~011
`,,H H OH H OH o
`CH3
`
`Figure 3
`
`Compactln ( R= II )
`
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`124
`
`KATHA WALA
`
`The 5-carbon unit of the side chain present in compactin (Fig. 3) probably
`occupies the same region as the 5-carbon unit in HMG-CoA (Fig. 2); the bicyclic
`A-B-ring system, with its substituents in compactin (Fig. 3), possibly sits in the
`same region or very close to the same region the CoA portion of the substrate
`HMG-CoA occupies at the active site of the enzyme. However, it is difficult to
`see any similarity in structure between the bicyclic-ring system of compactin
`and CoA, when one examines the structure of CoA shown in Fig. 4.
`
`Figure 4
`
`In light of (a) and (b) above, one hoped that it might possible to prepare
`interesting synthetic inhibitors of HMG-CoA reductase with a very general
`structure as shown in Fig. 5, with the 5-carbon unit (C-1 to C-5) preferably
`possessing the absolute configurations of C-3-0H and C-5-0H as present in
`compactin.
`Choice of R and R1 in Fig. 5 has depended on:
`
`R
`
`7
`
`6
`
`5 4
`
`2
`
`3
`
`I OH
`
`~ R1
`
`0
`
`Figure 5
`
`Rl=H,CH3
`
`(a) Consideration of the elements of structure of CoA.
`(b) Considerations of the overall shape and assumptions of the importance
`of substituents on Ring A-B of compactin (Fig. 3), first with molecular models
`and later with computer modelling.
`(c) Exploiting the knowledge gained in structure activity relationships with
`our own Sandoz Research Institute compounds or being reported in literature
`by outside investigators.
`Efforts with the above considerations in mind have led to the development
`of a variety of novel HMG-CoA reductase inhibitors. Synthesis and Structure
`Activity Relationships (SAR) of some of these novel inhibitors are discussed
`below with emphasis on the Phase III candidate, fluvastatin (XU 62-320):
`[R*, S*-(E)]-( ± )-Sodium-3,5-dihydroxy-7-[3-( 4-fluorophenyl)-1-(1-methyl(cid:173)
`ethyl-lH-indol-2-yl]-hept-6-enoate (Fig. 1), a mevalonic acid analog more po(cid:173)
`tent than compactin and lovastatin.
`
`III. GENERAL CHEMISTRY APPROACH
`
`Guided by the conviction that the C-3, C-5 dihydroxy acid fragment was
`the key pharmacophore necessary for the inhibition of HMG-CoA reductase,
`
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`HMG-CoA REDUCTASE INHIBITORS
`
`125
`
`our synthetic approach towards the synthesis of compounds of generic struc(cid:173)
`ture (Fig. 5) involved:
`(a) A convergent synthesis coupling chiral Synthon 1 or racemic or chiral
`(3R, 5S) C-3, C-5-dihydroxy ester Synthon 2 with a variety of aryl or alkyl
`fragments 3 (Fig. 6), or
`(b) A linear synthesis of the C-3, C-5 dihydroxy acid derivatives wherein
`the aldehyde 4 is reacted with acetoacetate 5 (Fig. 7) to provide a hydroxyketo
`ester intermediate, which, with subsequent steps, gives the desired final
`products of Fig. 5.
`
`R~X
`X = pt(Ph) 3z(cid:173)
`y = P=O(OR 3J2
`
`Rt = Si(t-Bu)Phz
`
`2
`
`3
`Rz "R3 = AlkJI, z· =Cl, Br
`
`R 7 6 5 0
`~
`H
`
`4
`
`0
`0
`4 u 2 u
`~JOR2
`
`5
`
`Figure 6
`
`Figure 7
`
`A. Synthesis of Synthon 1 and 2, Fig. 6 (Scheme 1 and Scheme 2)
`
`Synthon 1 has been synthesized starting from 0-glucose via the key lithium
`aluminum hydride reductive opening of the epoxide as depicted in Scheme
`1. 5 The desired axial alcohol could be separated from the equatorial isomer
`by preparation of the silyl derivatives. The protected axial alcohol on PCC
`oxidation gave the desired lactol aldehyde.
`
`R = Sl(t-llu)Phz
`
`SCHEME 1
`
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`126
`
`KATHA WALA
`
`Ph3CO~OCH3
`
`cmo
`lt -Butylacetate
`
`LDA
`
`1 . MeOH I AcCJ
`HO~OH
`2. DBMS I NaBH4
`
`cm 0
`
`0
`
`1. Et3 B /Nalll14
`
`0
`H~Ot-Bu
`2. TllDPSCI
`IMIDAZOLE
`OR OR 0
`
`R = S I ( I • ll u ) Ph 2
`
`3 . TFA I HzO I CHzClz
`
`4 • PCC I CllzCl2
`
`SCHEME 2
`
`OH
`
`{1
`
`HO
`
`OH
`
`112/Ra:"I
`
`EtOll
`
`Oft
`
`OR
`
`H000H
`
`l\llDAZOLE
`
`TBDPSCl/D\IF ROD OH
`J:r
`
`OR
`
`OR
`
`~COOCH3
`
`CHO
`
`RO
`
`t.TFA/\-leOH
`
`2.PCCICll2Cl2
`
`RO
`
`iPCC
`c11 2c1 2
`
`\ICPBA
`
`:"allC03
`Cll2Clz
`
`OR
`
`.)io
`
`(R = TllDPS)
`
`SCHEME 3
`
`Synthesis of chiral Synthon 2 has been accomplished starting from S-malic
`acid in excellent yields via an eight-step reaction as illustrated in Scheme 2. 6
`On the other hand, an efficient route was developed for the preparation
`of racemic Synthon 2 starting from 1,3,5-trihydydroxy benzene through a
`five-step reaction sequence shown in Scheme 3. 7
`
`B. Choice of Rand Synthesis of Intermediates 3, Fig. 6, and 4, Fig. 7
`
`Our initial efforts at the synthesis, and the biological results of C-3, C-5-
`dihydroxy acid derivatives (Fig. 5) wherein choice of R was based on elements
`of substructures of coenzyme A (Fig. 4) or the decalin ring structure of com(cid:173)
`pactin (Fig. 3) were not promising. 8 This led us to question the importance
`and the necessity of the complex stereochemistry and the substituents present
`in the decalin ring of compactin and turn our attention towards the prepa(cid:173)
`ration of C-3, C-5-dihydroxy acid derivatives (Fig. 5) wherein R was a naph(cid:173)
`thalene ring. During these ongoing efforts, we were being encouraged and
`helped by two important publications9 describing mevalonolactone deriva(cid:173)
`tives of the general structure 6 and 7 as inhibitors of HMG-CoA reductase
`(Fig. 8).
`Further exploration of R in Fig. 5 led to the first interesting indolyl derivative
`(Fig. 9) comparable to compactin in its inhibitory activity against HMG-CoA
`reductase. 10<a>
`An extensive and rapid analog program allowed the choice of XU 62-320
`
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`HMG-CoA REDUCT ASE INHIBITORS
`
`127
`
`HO
`.... ,.
`H
`
`HO~
`
`6
`
`7
`
`F
`
`01-1011 0
`
`o(cid:173)
`N a+
`
`X= CH=CH , (Cll2) m
`
`01-1 OH 0
`Z= A~o~a+
`
`Either one or two of the
`atoms a,b,c, = Heteroatom
`
`Figure 8
`
`Figure 9
`
`Figure 10
`
`(Fig. 1) as a candidate for extensive biological testing. Currently, fluvastatin
`(XU 62-320) is in clinical Phase III trials.
`With the discovery of XU 62-320, the stage was set for a large number of
`variations of R in Fig. 5. Extensive work at the Sandoz Research Institute has
`led to many novel HMG-CoA reductase inhibitors, some of which are dis(cid:173)
`cussed in this paper as shown in Fig. 10, 10 and Figs. 12-14.21
`23
`-
`Synthesis of the many interesting fragments 3 (Fig. 6) and 4 (Fig. 7) needed
`for synthesis of final HMG-CoA-R inhibitors are described in Schemes 4-12
`below. 10 Since the appearance of Merck & Co., Inc. and Sandoz patents and
`publications, s, 9,IO(a) extensive efforts have followed in many laboratories
`worldwide with semi-synthetic and totally synthetic HMG-CoA reductase
`inhibitors. A brief overview of these reported activities is presented in Section
`VIII. It is no wonder that in such a feverish pursuit of finding patentable
`HMG-CoA reductase inhibitors, review of patent and published literature
`presents overlapping activities in the laboratories of competing pharmaceut(cid:173)
`ical research companies.
`
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`128
`
`KATHA WALA
`
`R1 0 NH
`
`I
`Rz
`
`+
`
`EtOH
`
`CHO
`
`1. Mc2N r
`
`2. NaOH
`
`SCHEME 4
`
`R,-rfrP
`R 1
`.-.... Rnf:'.~
`~~~~~ ~CHO
`:-iallll,i l R2
`
`N
`I
`Rz
`
`D:\1F
`
`I
`
`~R3-v
`J .SOCl2
`....._ ____ Ri-f:{_J(~r·•
`N
`c11 2011
`I
`R2
`
`SCHEMES
`
`C. Synthesis of Indole Intermediates
`
`Scheme 4 describes the preparation of cx,[3-unsaturated aldehydes readily
`obtained from a variety of 3-phenyl substituted indoles using dimethylamino(cid:173)
`acrolein and phosphorous oxychloride, while the triphenyl phosphonium
`salts of indolyl derivatives are prepared via the 2-formyl and 2-hydroxymethyl
`indoles using standard procedures (Scheme 5). 10<al
`
`D. Synthesis of Indene Intermediates
`
`A variety of indenyl-cx, [3-unsaturated aldehydes and phosphonates have
`been synthesized via a six-step reaction sequence as depicted in Schemes 6
`and 7. The synthesis of these derivatives involves the preparation of the
`desired indenes from the respective indanones followed by either formylation
`at C-2 and subsequent alkylations at C-1 or vice versa, and then processing
`the formyl group through standard reaction sequences to the desired inter(cid:173)
`mediates.10<b) .
`
`E. Synthesis of Naphthalene Intermediates
`
`For the preparation of naphthalene derivatives, a novel photochemical route 11
`was exploited to give the key hydroxy aldehyde, which on dehydration pro(cid:173)
`vides the ene aldehyde. Dehydrogenation of the ene aldehyde and chain
`
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`HMG-CoA REDUCT ASE INHIBITORS
`
`129
`
`l. R2 MgX, llOAc
`
`2. Ph'.'\ (Me )CHO
`
`POCl3
`
`l. DIBAL
`
`R3
`
`R2
`
`R1
`
`R1
`
`co 2 Me
`
`SCHEME 6
`
`l'\all
`
`-----.-
`
`l NaBH4
`
`2 SOCl2
`
`3 P(OMe)3
`
`SCHEME 7
`
`y oco
`
`~CHO
`
`88 % by G.C.
`
`l Ph '.'\(:\-le)CllO
`
`POCl3
`
`II
`
`Horisll l C6 H5 CH3
`
`Reflux
`
`0
`
`II
`
`l . DDQ, Toluene
`
`2. n·llu3SnCJl=CHOEt
`n -lluLI, TllF, -60°
`
`3. p-TsOH ,THF,1120
`
`16 hrs, r. t.
`SCHEMES
`
`H
`
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`130
`
`KATHA WALA
`
`extension of the formyl group then leads to the desired cx,(3-unsaturated
`aldehydes10(c) (Scheme 8).
`
`F. Synthesis of Imidazole Intermediates
`Highly substituted imidazole derivatives with the desired functional group
`at the desired C- or hetero- atom are not well described in the literature.
`Synthesis of the required imidazole intermediates was best accomplished
`starting from the respective glycine derivatives as shown in Scheme 9. The
`key step in the synthetic pathway involves oxidation of the methyl group
`with potassium persulfate to give the 5-formyl imidazole derivatives, which
`through standard reaction sequences give the needed a,(3-unsaturated alde(cid:173)
`hydes or the phosphonates. ID(d)
`
`J.R2COCI
`2. Ac20 I AcOH, Pyridine, .1
`
`3. R3NH2, pTsOH, MgS04, Toluene, .1
`
`4. PCl5 I CHCl3
`
`RJ, R2, R3 =ALKYL OR SUBSTITUTED ARYL
`
`SCHEME9
`
`G. Synthesis of Pyrazole Derivatives
`
`A number of pyrazole intermediates have been prepared via procedures
`dependent on whether one needs the 1,5 (Scheme 10), the 1,3 (Scheme 11),
`or the 3,4 (Scheme 12) disubstituted pyrazole intermediates. 2,3-disubstituted
`pyrazole derivatives are obtained through the reaction of the appropriate
`diketoesters with aryl-hydrazines, requiring separation from the concomitant
`formation of the corresponding 1,3 isomer (Scheme 10). 10
`(e)
`1,3-disubstituted pyrazoles can be best synthesized from the imide chloride
`on reaction with the acetoacetate derivatives (Scheme 11), while the ring
`closure of arylhydrazones give the desired 3,4 diaryl pyrazole intermediates
`(Scheme 12).
`
`H. Synthesis of HMG-CoA-R Inhibitors
`
`All of the intermediates of the many different prototypes described above
`in Schemes 4-12 could be converted to the final HMG-CoA reductase inhib-
`
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`HMG-CoA REDUCTASE INHIBITORS
`
`131
`
`F
`
`F
`
`PhNHNH2
`
`I. LIAIH4 I
`2.PCC t
`
`F
`
`F
`
`CHO
`
`J. Ph3P=CHCOOEt
`
`2.LIAIH4
`J.Mno2
`SCHEME JO
`
`F 2
`
`0
`
`HN
`I
`A-NH
`
`v
`
`PCl5 ...
`
`F
`
`2 _1. __ '_"'_'"-~_._n_12_c_oo--1E~1
`
`N,, Cl
`
`NaOEt
`
`F
`
`COO Et
`
`er~H 2.H+
`
`SCHEME 11
`
`F
`
`~6
`~ 0
`
`J. l-Pr-NHNH2 I EtOH I AcOH (80°)
`
`I.NBS
`
`SCHEME 12
`
`N-N
`
`)-
`
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`132
`
`KATHA WALA
`
`0
`0
`~OR
`
`' CHO Nall/ n-BuLI R1
`
`COOR
`
`l 1 • Et 311 , TllF
`
`2 . NaBll4, -78°
`Et
`
`COOR
`
`COON a
`
`SCHEME 13
`
`itors either using the linear route involving the "dianion chemistry," or the
`coupling of the respective phosphonates or phosphonium salts with the chiral
`Synthons 1 and 2 (Fig. 6) or with the racemic Synthon 3 (Fig. 6).
`1. Linear Route. Synthesis using the linear route is illustrated in Scheme 13
`for the preparation of the indolyl HMG-CoA reductase inhibitors. The key
`step involves the reduction of the hydroxyketoester using trialkylbor(cid:173)
`ane/THF/MeOH with sodium borohydride at - 78° (Ref. 12) to give the mixture
`of desired erythro and threo isomers in the ratio of 95-98:5-2%, respectively.
`In some cases, the boronic esters can be crystallized, which on methanolysis
`and subsequent hydrolysis with sodium hydroxide provide the desired so-.
`dium salts. Nonstereoselective reduction of hydroxyketoester with borane t(cid:173)
`butylamine complex has been used to prepare a mixture of cis and trans
`lactones separable on flash chromatography. 10<aJ
`
`R1~
`
`N
`R2
`
`CH2P+Ph3
`er
`
`1. AcOH /THF I H20
`
`SCHEME 14
`
`Rt= Si( t-Bu )Ph2
`
`NCI Exhibit 2027
`Page 12 of 26
`
`

`

`HMG-CoA REDUCT ASE INHIBITORS
`
`133
`
`LDA ff HF
`
`OHC~Ot-Bu
`
`OR OR 0
`
`~ YOt-Bu
`
`OR OR 0
`
`F~ 1. n- Bu4NF
`
`2. NaOH
`
`ON a
`
`OH
`
`R =SI (t-Bu ) Ph2
`
`SCHEME 15
`
`2. Convergent Route. For illustrative purposes, a convergent route for the
`preparation of chiral indolyl HMG-CoA reductase inhibitors using the silyl
`protected Synthon 1 is depicted in Scheme 14. The crucial step in this reaction
`pathway is the oxidation of lactol with RuC12(PPh3)J/NMM0. 10<rJ
`Scheme 15 shows the use of silyl-protected aldehyde Synthon 2 (derived
`from malic acid) for the synthesis of indenyl HMG-CoA reductase inhibitors. 13
`
`IV. BIOLOGICAL RESULTS AND DISCUSSION
`
`A. Results in in vitro HMG-CoA Reductase Microsomal Assay and in in
`vivo Cholesterol Biosynthesis Assay
`
`All initial studies to assess the inhibitory potency of various compounds
`against HMG-CoA reductase were conducted with rat liver microsomal sus(cid:173)
`pensions, freshly prepared from male Sprague-Dawley rats, using an assay
`for HMG-CoA reductase activity as described in Ref. 14. The potency of each
`compound is expressed as IC50 (in µmoles, the concentration which inhibits
`to the extent of 50% conversion of the substrate HMG-CoA to mevalonate)
`and for structure activity relationship compared either to compactin = 1 or
`to XU 62-320 = 1. Tables I-XII summarize the most salient features of structure
`activity relationships for a few of the varied structural prototypes as HMG(cid:173)
`CoA reductase inhibitors being currently studied at the Sandoz Research
`Institute. In Tables X-XIII, the Relative Potency column is derived from the
`ICso values of each compound vs. compactin in the in vitro rat microsomal
`HMG-CoA reductase assay.
`
`B. SAR of Fluvastatin (XU 62-320) Analogs
`
`Table I compares the in vitro inhibitory activity against HMG-CoA reductase
`of XU 62-320 with compactin and lovastatin and as their corresponding sodium
`salts. XU 62-320 is 146- and 52-fold more active than compactin and Lovastatin,
`respectively. As compared to the respective sodium salts of compactin and
`Lovastatin, XU 62-320 is 22- and 10-fold more potent in inhibiting HMG-CoA
`reductase. It is important to note that current clinical studies are being con(cid:173)
`ducted with XU 62-320, which is a dihydroxy acid sodium salt. In contrast,
`
`NCI Exhibit 2027
`Page 13 of 26
`
`

`

`134
`
`KATHA WALA
`
`Table I
`Comparison of Microsomal HMG-CoA Reductase Inhibitory Activity
`
`Compound
`
`ICso (µM)
`
`Relative Potency•
`
`XU 62-320
`Compactin
`Lovas ta tin
`Na Salt Compactin
`Na Salt Lovastatin
`
`0.0069
`1.011
`0.3S2
`0.154
`0.068
`
`146.1
`1.0
`2.8
`6.S
`14.8
`
`*As compared to Compactin = 1
`
`compactin used in clinical studies and Lovastatin (Mevacor®), now marketed,
`both exist as the lactone forms (Fig. 1).
`Features of the side chain are very important for maximal inhibitory activity
`as shown in Table II. Erythro configuration, as well as the double-bond con(cid:173)
`figuration, are very important [anti-isomer 17-fold less active and dramatic
`loss of activity for one (Z) diene isomer]. The dihydro derivative, as well as
`the ester and the lactone forms, are considerably less active. Maximal inhib(cid:173)
`itory activity resides in the 3R, SS antipode.
`The importance of the features of the side chain described in Table II for
`the indole series holds true as well for all the prototypes to be described later
`and hence, during the discussion of SAR of these prototypes, these aspects
`will not be reemphasized. HMG-CoA, the substrate for the HMG-CoA re(cid:173)
`ductase, has at C-3 a methyl group. It was important to determine if an analog
`of XU 62-320 carrying a methyl group at C-3 would be more potent. Surpris(cid:173)
`ingly, introduction of methyl group at C-3 in either of syn- or anti-configu(cid:173)
`ration was considerably less active (Table III).
`Studies of the effects of the substituents in the 3-phenyl ring of the indole
`moiety are given in Table IV. Either electron-withdrawing or electron-donat(cid:173)
`ing substituents in the 3-phenyl ring tend to decrease the potency, which is
`unaffected by the presence of alkyl groups.
`Electron-donating or electron-withdrawing substituents (not shown in Ta(cid:173)
`ble IV) or bulky alkyl groups at C-5 of the indole moiety led to decrease of
`potency. However, alkyl or alkoxy groups at C-4 and C-6 tend to maintain
`or enhance the potency slightly (Table V).
`
`Table II
`SAR of Variations in the Side Chain
`
`Compound
`
`ICso (µM)
`
`Relative Potency•
`
`F
`
`XU 62-320
`3R, SS
`I ~
`3S, SR
`Na Salt, ANTI
`OH OH 0 •
`Methyl Ester, SYN
`Trans Lactone
`o- CIS(f:) Double Bond
`Dihydro (Reduced
`Na+
`Double Bond)
`
`0.0069
`0.0024
`0.08
`0.12
`O.OS2
`0.029
`0.62
`0.114
`
`1.0
`2.8
`0.086
`O.OS7
`0.13
`0.23
`0.011
`0.06
`
`*As compared to XU 62-320 = 1.
`
`NCI Exhibit 2027
`Page 14 of 26
`
`

`

`HMG-CoA REDUCTASE INHIBITORS
`
`135
`
`Table III
`Comparative Activity of XU with the 3-Methyl Analogs
`
`Compound
`
`ICso (µM)
`
`Relative Potency*
`
`XU 62-320
`R = CH3, SYN
`R = CH3, ANTI
`
`0.0069
`0.14
`0.51
`
`1.0
`0.049
`0.013
`
`*As compared to XU 62-320 = 1
`
`Table IV
`SAR for the Substituents of the 3-Phenyl Ring
`
`R
`
`ICso (µM)
`
`Relative Potency*
`
`o-
`
`4-F
`2-Me
`2-Me, 4-F
`3-Me, 4-F
`3,5-diMe, 4-F
`3,5-diMe
`H
`4-CF3
`4-SCH3
`4-COONa
`
`0.0069
`0.14
`0.004
`0.009
`0.02
`0.005
`0.017
`0.09
`1.152
`>10.0
`
`1
`0.049
`1.7
`0.76
`0.345
`1.38
`0.40
`0.076
`0.006
`
`*As compared to XU 62-320 = 1
`
`Table V
`SAR for the Substituents of the Benzenoid Indole Ring
`
`F
`
`o·
`
`R
`
`ICso (µM)
`
`Relative Potency*
`
`H (62-320)
`4,6-diMe
`4,6-dii-Pr
`5-C6H11
`6-0CH2Ph
`
`0.0069
`0.011
`0.005
`24.0
`0.0026
`
`1.0
`0.62
`1.38
`0.0022
`2.65
`
`*As compared to XU 62-320 = 1
`
`Table VI
`SAR for the Substituents of Indolyl-Nitrogen
`
`F
`
`R
`
`ICso (µM)
`
`Relative Potency*
`
`i-Pr (62-320)
`CH3
`C2Hs
`C6H11
`CH2CH2Ph
`CH2CH(CH3)z
`
`0.0069
`0.62
`0.096
`50
`49.4
`0.245
`
`1.0
`0.011
`0.071
`0.0001
`0.0001
`O.o28
`
`*As compared to XU 62-320 = 1
`
`NCI Exhibit 2027
`Page 15 of 26
`
`

`

`136
`
`KATHA WALA
`
`Table VII
`SAR for Reversing Substituents at 1 and 3 Positions
`
`R1
`
`Ri
`
`ICso
`(µM)
`
`Relative
`Potency•
`
`~ i-Pr (62-320)
`
`:.-..
`
`N\
`
`R1
`
`o·
`Na+
`
`4-FC6H4
`i-Pr
`
`4-FC5H4, ~ 0.0069
`0.0016
`i-Pr,~
`4-FC~4, anti
`0.12
`
`1.0
`4.3
`0.057
`
`*As compared to XU 62-320 = 1
`
`Most sensitive to the activity is the substituent on the nitrogen of the indole
`moiety (Table VI). Optimal activity is provided by the isopropyl group, while
`marked loss in potency results with either bulky alkyl or phenethyl groups.
`Reversing the substituents on N-1 and C-3 of the indole moiety to give
`(Table VII) 3-isopropyl-N-p-fluorophenyl analog of XU 62-320 gives a 4-fold
`increase in potency.
`Most of the substances with a reasonable level of activity against HMG(cid:173)
`CoA reductase in in vitro microsomal assay were studied in vivo for their
`effects on inhibition of sterol biosynthesis. Results are expressed as EDso
`(mg/kg), effective concentration which inhibits to the extent of 50% incor(cid:173)
`poration of C14 acetate into sterols in rats when administered as appropriate
`doses of drug substances as compared to controls receiving vehicle alone.
`Table VIII shows that in vivo XU 62-320 is about 40- and 4.5-fold more potent
`than compactin and Lovastatin, respectively, in inhibiting endogenous cho(cid:173)
`lesterol synthesis in rats. For most substances, although not for all, the relative
`
`Table VIII
`Relative Potency for Inhibition of Cholesterol Biosynthesis
`
`Compound
`
`EDso (mg/kg)
`
`Relative Potency•
`
`XU 62-320
`Compactin
`Lovas ta tin
`(Monacolin)
`
`0.093
`3.5
`0.414
`
`37.6
`1.0
`8.4
`
`*As compared to Compactin = 1
`
`Table IX
`SAR for Cholesterol Biosynthesis Inhibition
`
`F
`
`o-
`Na+
`
`XU 62-320
`
`*As compared to XU 62-320 = 1
`
`Compound
`
`XU 62-320
`3R,5S
`3S, SR
`Na Salt, Anti
`Methyl ester, ~
`Trans Lactone
`Dihydro (Reduced
`Double Bond)
`
`ED so
`(mg/kg)
`
`0.093
`0.056
`>0.5
`1.37
`0.40
`0.33
`1.23
`
`Relative
`Potency•
`
`1.0
`1.66
`
`0.067
`0.23
`0.28
`0.075
`
`NCI Exhibit 2027
`Page 16 of 26
`
`

`

`HMG-CoA REDUCTASE INHIBITORS
`
`137
`
`Table X
`SAR of Indene Derivatives
`
`R1
`
`Relative Potency*
`
`Na+
`o-
`
`OH OH 0
`
`(CH2)4
`(Racemic)
`(CH2)4
`(3R, 55)
`(Clhh
`(CH2)s
`CH2CH3
`CH3
`H,iPr
`
`R1
`
`Phenyl
`3,5-Dimethylphenyl
`iPr
`Cyclohexyl
`
`RJ
`
`4-Me
`6-Me
`7-Me
`6-0Me
`4,6-(0Meh
`
`202
`
`337
`
`38
`1.5t
`<.2
`2
`8
`
`88t
`146
`<0.5
`16.5
`
`114
`181
`24
`130
`60
`
`*As compared to Compactin = 1
`tAs its Ethyl Ester
`
`potency determined in in vitro microsomal assay against HMG-CoA reductase
`parallels the in vivo activity in rats for the inhibition of 14C-acetate into sterols.
`As an example, comparison of Tables II and IX reveals the relative potency
`of several analogs of XU 62-320 when compared in in vitro and in in vivo.
`Thus, as compared to XU 62-320, the anti-isomer is - 17- (Table II) and -
`15-fold (Table IX) less active than XU 62-320 in in vitro and in in vivo assays,
`respectively. Similarly, close parallelism prevails for the ester (less active -
`7.5-fold, in vitro vs. 4.3-fold, in vivo), trans-lactone (less active 4.2-fold, in vitro
`vs. 3.5, in vivo) and the dihydro derivative (less active 16.5-fold, in vitro vs.
`13-fold in vivo).
`
`C. SAR of Indene Derivatives
`
`The structure activity relationships for the indene derivatives can be best
`summarized as follows: Maximal activity is obtained with a spiro cyclopentyl
`group at C-1, again emphasizing the importance of the bulky group in the
`vicinity of the dihydroxy acid side chain. At C-3 the best substituent is 4-F(cid:173)
`phenyl, while the optimal substituent for the benzenoid portion of the indene
`moiety is hydrogen (see Table X).
`
`D. SAR of Naphthalene Derivatives
`
`The most interesting part of the structure activity relationships for this
`group of compounds is the difference observed in the potency of 1-(4-F-
`
`NCI Exhibit 2027
`Page 17 of 26
`
`

`

`138
`
`KATHAWALA
`
`Table XI
`SAR of Napthalene Derivatives
`
`OH OH 0
`
`o-
`
`4-F-Ph
`4-F-Ph
`4-F-Ph
`4-F-Ph
`3,5-diMe-Ph
`Ph
`i-Pr
`i-Pr
`
`H
`CH3
`Et
`i-Pr
`CH3
`CH3
`4-F-Ph
`Ph
`
`Relative
`Potency*
`
`0.10
`8
`19
`22
`56
`2
`337
`144
`
`*As compared to Compactin = 1
`
`phenyl)-3-isopropyl derivative vs. 1-isopropyl-3-(4-F-phenyl) compound (22
`times more potent vs. 337 as compared to compactin) (see Table XI).
`
`E. SAR of Pyrazole Derivatives
`
`Table XII illustrates the structure activity relationships for a few of the many
`pyrazole derivatives prepared. Here, too, the optimal substituents are the 4-
`F-phenyl and isopropyl group adjacent to the dihydroxy acid side chain. The
`dihydro and the 5-keto derivatives are substantially less potent. 1,3-diaryl(cid:173)
`substituted pyrazole derivatives show decreased inhibitory activity (not shown
`in the table) in contrast to the 1,5 and 3,4-diaryl-substituted compounds,
`which tend to have comparable potency.
`
`F. SAR of Imidazole Derivatives
`
`To emphasize the most salient features of the structure activity relationships
`for the imidazole derivatives, only a few of the derivatives prepared are
`tabulated in Table XIII. Optimal activity is obtained with 1,2-diaryl derivatives
`
`Table XII
`SAR of Pyrazole Derivatives
`
`R
`
`Relative Potency*
`
`4-F
`4-F (6,7 Dihydro)
`4-F (5 Keto)
`H
`3,5 Dimethyl
`
`4-F
`
`60
`5.9
`3.5
`5.6
`4.1
`
`30
`
`o-
`
`*As compared to Compactin = 1
`
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`Page 18 of 26
`
`

`

`HMG-CoA REDUCT ASE INHIBITORS
`
`Table XIII
`SAR of lmidazole Derivatives
`
`4-F
`(Racemic)
`4-F
`(3R, SS)
`p-CI
`p-Br
`3,5-Di-Me
`3,5-Di-Cl
`
`i-Pr
`t-Butyl
`cyclohexyl
`2-Thienyl
`1,4-Biphenylyl
`p-Dimethylamino-phenyl
`p-Nitro-phenyl
`
`F
`
`i-Pr
`4-F-Phenyl
`
`13