J . Med. Chem. 1985,28,347-358
`3-Hydroxy-3-methylglutaryl-coenzyme A Reductase Inhibitors. 1. Structural
`Modification of 5-Substituted 3,5-Dihydroxypentanoic Acids and Their Lactone
`Derivatives
`
`347
`
`G. E. Stokker,*f W. F. Hoffman,*t A. W. Alberts,* E. J. Cragoe, Jr.,t A. A. Deana,? J. L. Gilfillan,* J. W. Huff,$
`F. C. Novello,+ J. D. Prugh,t R. L. Smith,t and A. K. Willardt,$
`Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486, and Rahway, New Jersey 07065.
`Received June 21, 1984
`
`A series of 5-substituted 3,5-dhydroxypentanoic acids and their derivatives have been prepared and tested for inhibition
`of HMG-CoA reductase in vitro. In general, unless a carboxylate anion can be formed and the hydroxy groups remain
`unsubstituted in an erythro relationship, inhibitory activity is greatly reduced. Furthermore, only one enantiomer
`of the ring-opened form of lactone 6a(*) possesses the activity displayed by the racemate. Insertion of a bridging
`unit other than ethyl or (E)-ethenyl between the 5-carbinol moiety and an appropriate lipophilic moiety (e.g.,
`2,4-dichlorophenyl) attenuates activity.
`
`acid (l), the dihydroxy acid form of mevinolin, is the most
`potent HMG-CoA reductase inhibitor (Ki = 0.6 nM) re-
`ported to date? Of even greater interest are the findings
`that compactin11J2 and mevinolin9J3 are highly effective
`hypocholesterolemic agents in several animal species and
`man. Subsequent to the first reports disclosing the
`structure14 and biological activity8 of compactin, a series
`of studies directed toward the development of structurally
`simplified HMG-CoA reductase inhibitors were initiated
`in these Laboratories. Described in this paper are the
`results of our initial s t ~ d y , ' ~ which served to delineate key
`
`Formation of the atheromatous plaque or atheroma is
`accompanied by the localized deposition of plasma lipids,
`primarily cholesteryl esters, in the intima of the arterial
`wa11.lp2 Growth of the atheroma eventually leads to con-
`striction of the coronary arterial lumen and ultimately
`results in atherosclerosis and coronary heart disease (CH-
`D), the major cause of death and disability in Western
`countries. These observations, coupled with the compelling
`epidemiological evidence implicating hypercholesterolemia
`as a primary risk factor for CHD,3s4 have stimulated re-
`search on the development of therapeutic agents for pre-
`venting and treating atherosclerosis based on the attenu-
`ation of plasma cholesterol level^.^ The results of the
`recently completed Lipid Research Clinics Coronary
`Primary Prevention Trial (LRC-CPPT)6 provide strong
`support for the basis of this approach. The LRC-CPPT
`clearly demonstrated that reduction of low-density lipo-
`protein cholesterol (LDL-C) through dietary modification
`and treatment with the bile acid sequesterant cholestyr-
`amine, either alone or in combination, diminished the
`incidence of CHD morbidity and mortality in hypercho-
`lesterolemic men at high risk for CHD. Nevertheless, the
`reduction of dietary cholesterol and saturated fat intake
`and the use of bile acid sequesterants often fail to lower
`elevated plasma LDL-C levels to the desired extent, par-
`ticularly in patients with familial hypercholesterolemia
`(FH).'
`An attractive and potentially more efficacious way to
`lower plasma cholesterol levels would be to control de novo
`cholesterogenesis by selectively inhibiting an early bio-
`synthetic step. The highly functionalized fungal metab-
`olites compactin (ML-236B, CS-500)8 and mevinolin
`(MK-803)9 are potent inhibitors of cholesterol biosynthesis
`at the level of the major rate-limiting enzyme 3-hydroxy-
`3-methylglutaryl-coenzyme A reductase [HMG-CoA re-
`ductase; mevalonate:NADP+ oxidoreductase (CoA acy-
`lating), EC 1.1.1.34],10 which catalyzes the conversion of
`HMG-CoA to mevalonic acid (eq 1). Indeed, mevinolinic
`C H 3
`
`(1) Ross, R. Annu. Rev. Med. 1979, 30,l.
`(2) Fuster, V. Scand. J. Haematol. 1981, 27 (Suppl. 38), 1.
`(3) U.S. Department of Health and Human Services, National
`Institutes of Health, National Heart, Lung, and Blood Insti-
`tute, "Arteriosclerosis", Vol. l and 2, NIH Publication No.
`81-2034 and 81-2035, U.S. G.P.O., Washington, DC, 1981.
`(4) Hamburg, D. A.; Elliott, G. R. Arteriosclerosis 1982, 2, 357.
`(5) Prugh, J. D.; Roonej, C. S.; Smith, R. L. Annu. Rep. Med.
`Chem. 1983,18,161.
`(6) (a) LRC-CPPT, J. Am. Med. Assoc. 1984,251,351. (b) LRC-
`CPPT, Zbid. 1984,251, 365.
`(7) Havel, R. J.; Kane, J. P. Annu. Rev. Med. 1982,33,417.
`(8) Endo, A.; Kuroda, M.; Tsujita, Y. J. Antibiot. 1976 29, 1346.
`(9) Alberta, A. W.; Chen, J.; Kuron, G.; Hunt, V.; Huff, J.; Hoff-
`man, C.; Rothrock, J.; Lopez, M.; Joshua, H.; Harris, E.;
`Patchett, A.; Monaghan, R.; Currie, 5.; Stapley, E.; Albers-
`Schonberg, G.; Hensens, 0.; Hirshfield, J.; Hoogsteen, K.;
`Liesch, J.; Springer, J. Proc. Natl. Acad. Sci. U.S.A. 1980, 77,
`3957.
`(10) Rodwell, V. W.; Nordstrom, J. L.; Mitschelen, J. J. Adv. Lipid
`Res. 1976, 14, 1.
`(1 1) (a) Tsujita, Y.; Kuroda, M.; Tanzawa, K.; Kitano, N.; Endo, A.
`Atherosclerosis (Shannon, Zrel.) 1978,32,307. (b) Kuroda, M.;
`Tsujita, Y.; Tanzawa, K.; Endo, A. Lipids 1979, 14, 585.
`(12) (a) Yamamoto, A.; Sudo, H.; Endo, A. Atherosclerosis (Shan-
`non, Zrel.) 1980,35,259. (b) Mabuchi, H.; Haba, T.; Tatami,
`R.; Miyamoto, S.; Sakai, Y.; Wakasugi, T.; Watanabe, A.;
`Koizumi, J.; Takeda, R. N. Engl. J. Med. 1981, 305, 478.
`(13) (a) Tobert, J. A.; Hitzenberger, G.; Kukovetz, W. R.; Holmes,
`I. B.; Jones, K. H. Atherosclerosis (Shannon, Zrel.) 1982,41,
`61. (b) Tobert, J. A.; Bell, G. D.; Birtwell, J.; James, I.; Ku-
`kovetz, W. R.; Pryor, J. S.; Buntinx, A.; Holmes, I. B.; Chao,
`Y.-S.; Bolognese, J. A. J. Clin. Inuest. 1982, 69, 913.
`(14) The X-ray crystal structure of compactin was first reported by
`Brown, A. G.; Smale, T. C.; King, T. J.; Hansenkamp, R.;
`Thompson, R. H. J. Chem. SOC., Perkin Trans. 1 1976,1165.
`Merck Sharp & Dohme, West Point, PA.
`Note that the relative configuration in Figure 1 of the cited
`t Merck Sharp & Dohme, Rahway, NJ.
`*Present address: Stuart Pharmaceuticals, Division of IC1
`reference does not agree with the crystal coordinates; we
`present here the correct relative and absolute stereochemical
`Americas, Wilmington, DE 19897.
`configuration of compactin.
`0022-2623/85/l828-0347$01.50/0 0 1985 American Chemical Society
`
`I
`SCoA
`HMG-CoA
`
`mevalonic acid
`
`Mylan Exhibit 1040, Page 1
`
`

`

`348 Journal of Medicinal Chemistry, 1985, Vol. 28, No. 3
`SARs for compactin-like mimics and afforded a series of
`moderately effective HMG-CoA reductase inhibitors typ-
`ified by the ring-opened form of lactone 6a(+).
`HO
`V C O d +
`
`How
`
`B
`
`QC'
`
`CI
`6a(+)
`Chemistry. The compounds prepared for this study
`are listed in Tables 1-111.
`Their syntheses from the
`corresponding aldehydes, exemplified by 2, are shown in
`Schemes 1-111. Condensation of aldehyde 2 with the
`dianion of ethyl acetoacetate16 followed by borohydride
`reduction, basic hydrolysis, acidification, and azeotropic
`removal of water provided a mixture of the trans (sa(*))
`and cis (6b(*)) lactones which subsequently was separated
`by chromatography (Scheme I). The use of MeOH in the
`borohydride reduction step was found to be advantageous;
`replacement of MeOH by EtOH produced some of the
`corresponding ethyl ester which was more resistant to
`hydrolysis. The resolution of sa(*) was accomplished via
`formation and chromatographic separation of the diaste-
`reomeric (R)-a-methylbenzylamines followed by basic
`hydrolysis and relactonization to yield sa(+) and 6a(-).
`Hydrolysis and acidification of 5-hydroxy-3-keto ester
`3 without prior reduction resulted in spontaneous lacton-
`ization to enol lactone 4. Numerous attempts to reduce
`4 to hydroxy lactone 6, either catalytically or via metal
`hydrides, were unsuccessful. Treatment of lactone 6a( *)
`with NHB provided the erythro amide 10. Catalytic re-
`duction of lactone 6a(*) provided compound 7 containing
`a saturated bridging unit. The lactol ethers 8 and 9 were
`prepared by diisobutylaluminum hydride (Dibal) reduction
`of sa(*) followed by treatment with MeOH in the pres-
`ence of pyridinium p-toluenesulfonate (PPTS).
`The syntheses of methyl lactones 13, 16, and 17 are
`illustrated in Scheme 11. A tin-mediated aldol condensa-
`tion of aldehyde 2 with 2-acetoxypropene following a
`
`During the course of this study, a series of mevalonolactone
`derivatives of the general structure were reported to inhibit
`
`cn3v
`
`(CHZ)"
`
`&x
`HMG-CoA reductase by Sat0 et al.: Sato, A,; Ogiso, A.; No-
`guchi, H.; Mitsui, s.; Kaneko, I., Shimada, Y. Chem. Pharm.
`Bull. 1980,28, 1509.
`Huckin, S. N.; Weiler, L. Tetrahedron Lett. 1971, 4835.
`
`Stokker et al.
`slightly modified procedure of Noltes et al.17 provided
`4-hydroxy ketone 11 in high yield. Acylation of 11 with
`2-bromoacetyl bromide in the presence of pyridine fur-
`nished bromo acetate 12 which was ring closed to 4-
`hydroxy-4-methyl lactone 13 via an intramolecular Re-
`formatsky reaction.ls Substitution of triethylamine for
`pyridine in the acylation step resulted in elimination of
`the 2-bromoacetoxy moiety and isolation of the resultant
`dienone.
`An alternate route to lactone 13 starting from 4-hydroxy
`ketone 11 was investigated. This route involved sequential
`acylation, intermolecular Reformatsky reaction with ethyl
`2-bromoacetate, basic hydrolysis, acidification, and lac-
`tonization. This route was abandoned in favor of the more
`efficient two-step route (vide supra). Ethylene lactone 13a
`was reduced in the same manner as sa(&) to provide the
`corresponding ethyl-bridged compound 16. Refluxing a
`solution of 13a and toluene in the presence of PTSA (trace)
`resulted in the smooth conversion of 13a to 17.
`5-Methoxy-3-hydroxyheptenoic acid 20 was prepared by
`treating the dimethyl acetal of 2 with diketene (1 equiv)
`in the presence of Tic& by using the general procedure
`of Izawa and Mukaiyamalg followed by borohydride re-
`duction, basic hydrolysis, and acidification of the resultant
`3-keto ester 19 as shown in Scheme 111. A similar con-
`densation of aldehyde 2 provided a mixture of 3-keto ester
`3 and dihydro lactone 4 and, thus, was a less expeditious
`route to target lactone 6 than was the dianion procedure
`(Scheme I).
`The previously undescribed requisite aldehydes were
`prepared as shown in Schemes IV-VIII. The synthesis
`of the a,@-unsaturated aldehyde 55a needed for elaborating
`lactone 55 is shown in Scheme IV with phenanthrene-4-
`carboxaldehyde as starting material. This procedure was
`also used to prepare the known a,@-unsaturated aldehyde
`precursors for lactones 56-58.
`The 3-(decahydronaphthy1)propanals 25 and 27 (pre-
`cursors to 51 and 52) were elaborated from 21 as shown
`in Scheme V. After high-pressure hydrogenation of 22, the
`acid 23 was converted to aldehyde 25 by the Burgstahler
`modification of the Rosenmund reduction.20 The ethyl
`ester of 23 (24, isolated in about an equal amount during
`the workup of 23) was isomerized with AlC1, (2 equiv) at
`room temperature and subsequently hydrolyzed to acid
`26, which was converted to aldehyde 27 in the same
`
`manner used for 23 - 25.
`
`The Dibal reduction of nitriles 29, 32, and 34 (Scheme
`VI) provided the aldehydes requisite for preparing lactones
`47, 48, and 53, respectively. Aldehyde 37 (precursor to
`44)21 was prepared by alkylation of phenol 36 with the
`diethyl acetal of 2-bromoacetaldehyde followed by hy-
`drolysis (Scheme VII).
`Finally, conversion of cinnamaldehyde 2 to propargyl-
`aldehyde 41 (precursor to 42) was effected via the four step
`
`sequence 2 - 38 - 39 - 40 - 41 by using the general
`
`method of Allen and Edens22 as shown in Scheme VIII.
`
`Noltes, J. G.; Verbeek, F.; Creemers, H. M. J. C. Organometal.
`Chem. Synth. 1970-1971, 1, 57. In the present case, (tri-
`butylstanny1)acetone was prepared in situ.
`For a general method, see: Maruoka, K.; Hashimoto, S.; Ki-
`tagawa, Y.; Yamamoto, H.; Nozaki, H. J. Am. Chem. Soc. 1977,
`99, 7705.
`Izawa, T.; Mukaiyama, T. Chem. Lett. 1975, 161.
`Burgstahler, A. W.; Weigel, L. 0.; Shaefer, C. G. Synthesis
`1976, 767.
`During the course of this investigation, the 4R,6R dechloro
`analogue was prepared from tri-0-acetyl-D-glucal as a possible
`HMG-CoA reductase inhibitor by Yang et al.: Yang, Y. L.;
`Falck, J. R. Tetrahedron Lett. 1982, 23, 4305.
`
`
`
`
`
`Mylan Exhibit 1040, Page 2
`
`

`

`HMG-CoA Reductase Inhibitors
`
`Journal of Medicinal Chemistry, 1985, Vol. 28, No. 3 349
`
`Table I. Effects of Lactone Modification and Stereochemistry
`
`&R
`
`no.
`q c d
`
`6a(k:)e
`
`6b(*.)
`
`sa(+)
`
`sa(-:)
`
`8c
`
`9 C
`
`1 oc
`
`13a
`
`13b
`
`17
`
`2w
`
`CI
`
`recryst
`solvent
`
`R
`
`OH
`I
`
`EtOAc
`
`yield,
`7 0
`52
`
`mp, OC
`168-169
`
`formula'
`
`acetone/ hexane
`
`33
`
`148-150
`
`acetone/ hexane
`
`17
`
`115-117
`
`n-BuC1
`
`25
`
`114-116
`
`n-BuC1
`
`44
`
`114-116
`
`chromat. acetone/CHzClz
`
`33
`
`wax
`
`chromat. acetone/CHzClz
`
`64
`
`88-93
`
`acetone/ hexane
`
`63
`
`117-118
`
`n-BuC1
`
`35f
`
`136-138
`
`n-BuCl/ hexane
`
`5.4
`
`135-137
`
`chromat. CHC13/MeOH
`
`90
`
`108-110
`
`chromat. CH2Clz/HOAc
`
`42h
`
`gum
`
`concn,
`%
`inhibb
`pg/mL
`0
`2
`12
`10
`15
`50
`13
`1
`41
`5
`64
`10
`84
`20
`4
`2
`6
`5
`27
`10
`17
`20
`18
`50
`26
`1
`45
`2
`5
`66
`72
`10
`86
`20
`1
`0
`2
`0
`4
`0
`8
`0
`2
`0
`4
`2
`4
`6
`2
`0
`4
`0
`6
`8
`0
`10
`1
`0
`2
`0
`4
`0
`8
`0
`11
`1
`2
`21
`36
`4
`61
`10
`3
`2
`4
`7
`5
`10
`25
`15
`2
`0
`3
`4
`3
`10
`5
`25
`1
`0
`2
`0
`5
`0
`10
`0
`"Analytical results are within f0.4% of the theoretical values unless otherwise noted. *See Experimental Section for protocol. CTested
`in the form indicated since carboxylate anion could not be formed under this testing protocol. dpK, =I 5.22 (30% EtOH). e When tested in
`the lactone form only 25% inhibition was observed at 50 rg/mL. fYield from 12. gAbout a 4 3 ratio of erythro and threo. hoverall yield
`from 18. 'Anal. Calcd: C, 52.68. Found: C, 52.02.
`The synthesis of 43 (the Z isomer of 6a(h)) was accom-
`plished by the catalytic hydrogenation (Lindlar) of 42.
`Biological Results and Discussion
`The compounds listed in Tables I-IV Were evaluated for
`their ability to inhibit solubilized, partially purified rat
`liver HMG-CoA reductase. During the initial phase of this
`study, both the lactone and the ring-opened sodium di-
`
`hydroxycarboxylate forms of each compound were tested
`for intrinsic inhibitory activity. In each instance, the so-
`dium dihydroxycarboxylate form proved more active than
`the lactone form (see Table I, footnote e). Accordingly,
`subsequent tests were done exclusively on the sodium
`dihydroxycarboxy~ate forms unless noted otherwise.
`The contributions of lactone moiety stereochemistry and
`functionality to intrinsic inhibitory activity in compound
`6 are illustrated in Table I. Separation-of the lactone
`mixture 6 into the racemic cis (6bWt)) and trans (6a(*:))
`isomers showed that activity resided principally in the
`
`(22) Allen, C. F. H.; Edens, C. O., Jr. "Organic Syntheses"; Wiley,
`New York, 1955; Collect. Vol. 111.
`
`
`
`Mylan Exhibit 1040, Page 3
`
`

`

`350 Journal of Medicinal Chemistry, 1985, Vol. 28, No. 3
`
`Table 11. Effects of Bridge Modification
`
`no.
`
`A
`
`recryst
`solvent
`
`Stokker et al.
`
`yield,
`%
`
`OH
`1
`
`mp, OC
`
`formula‘
`
`concn,
`pg/mL
`
`%
`inhibb
`
`7
`
`,CH~CH~”
`
`n-BuC1
`
`60
`
`50
`
`96-98
`
`87-88
`
`C13H14C1203
`
`C14H16C1203
`
`chromat. CHzC12/acetone
`
`/ C H ~ C H /
`
`-ccc-
`
`H>C=C<H
`
`,OCHz’
`
`42
`
`43
`
`44
`
`45
`
`1
`2
`5
`10
`1
`2
`4
`8
`0.625
`3.125
`0.625
`3.125
`1
`2
`4
`10
`5
`10
`20
`50
`
`29
`38
`57
`80
`14
`26
`37
`63
`0
`8
`0
`0
`21
`24
`18
`21
`4
`26
`27
`23
`
`chromat. CH2C12/acetone
`
`chromat.e IPA/hexane
`
`chromat. CH2C12/acetone
`
`lld
`50
`4.2d
`
`viscous oil
`95-98
`oil
`
`C13HloC1203
`
`C13H12C1203
`
`C12H12C1204
`
`n-BuC1
`
`C11H10C1203
`
`13d
`
`133-136
`
`6% &
`
`CH2C12/n-C4H&l
`
`24d
`
`104-107
`
`C15H1403
`
`46
`
`47
`
`48
`
`/ C H Z C H /
`
`chromat. CH2C12/acetone
`
`9.5d
`
`gum
`
`C17H1803’1/2H20
`
`/CHzCHzCHz /
`
`chromat. CH2C12/acetone
`
`16d
`
`oil
`
`C18H2003’1/20C3H60
`
`5
`16
`9
`10
`20
`7
`50 5
`18
`19
`29
`10 20
`45
`61
`50 2
`11 7
`5
`25
`10
`35
`25
`aAnalytical results are within f0.4% of the theoretical values unless otherwise noted.
`See Experimental Section for protocol.
`CEquatorial 4-Me in lactone by reduction of 13a. dOverall yield from aldehyde. eHPLC purification on Dupont silica 10/30 with i-
`PrOH-hexane (1:19, v/v) at 2 mL/min. Times of elution are 13.2 min for 42 and 21.8 min for 43.
`
`racemic trans lactone (sa(*)). Resolution of 6a(f) af-
`forded enantiomers sa(+) and sa(-); their evaluation
`showed that the activity displayed by the racemate re-
`sulted solely from the dextrorotatory isomer. The addition
`of a methyl group to the 4-position of 6a(f) to give trans
`lactone 13a, a compound which more closely resembles the
`HMG moiety of the substrate HMG-CoA, did not alter
`activity appreciably. However, cis lactone 13b, which
`possesses the opposite relative stereochemistry at (2-4, was
`much less active as anticipated from the results obtained
`for sa(*) and 6b(f). Interestingly, oxidation of the 4-
`hydroxyl group of 6a(f) to provide enol 4 greatly reduced
`activity. This result is a likely consequence of the fact that
`4 readily forms the sodium salt of the enolate and,
`therefore, fails to undergo ring opening to afford the re-
`quired carboxylate anion under alkaline conditions. Re-
`placement of the enolic hydroxyl group in 4 with a methyl
`group to provide 17 further reduced activity. Replacement
`of the carboxyl group in the ring-opened form of 6a(*)
`with a carboxamido group (10) ablated activity as did
`conversion of the 5-hydroxl group to the corresponding
`
`methyl ether (20). These results demonstrate the im-
`portant contributions of the carboxylate and 5-hydroxyl
`groups to activity. Finally, it should be noted that lactol
`ethers 8 and 9 displayed greatly diminished activities.
`The effects of altering the moiety bridging the aromatic
`and the lactone fragments in sa(*) on intrinsic inhibitory
`activity are shown in Table 11. Saturation of the ethenyl
`bridge in 6a(h) and its 4-methyl derivative Ita(&) gave
`ethyl-bridged compounds 7 and 16, respectively, with little
`change in activity. However, other modifications of the
`bridge such as replacement with the ethynyl (42), cis-
`ethenyl(43), and oxymethylene (44) groups resulted in loss
`of activity as did complete removal (45) of the bridging
`moiety. In a companion series of naphthalene analogues
`(Table 11), compound 47 containing the saturated two-
`carbon bridge proved superior. Increasing the length of
`the bridge to three carbons (48) reduced activity and
`elimination of the bridge to provide 46 further reduced
`activity.
`The results of various carbocyclic moieties substituted
`at the 6-position of the lactone ring are shown in Table
`
`
`
`Mylan Exhibit 1040, Page 4
`
`

`

`HMG-CoA Reductase Inhibitors
`
`Table 111. Effects of 6-Substitution
`
`Journal of Medicinal Chemistry, 1985, Vol. 28, No. 3 351
`
`yield,' 6%
`
`x \\"
`
`%
`-8-10
`
`mp, "C
`56-58
`
`formulab
`
`X
`
`recryst
`solvent
`EtzO/pet. ether
`
`no.
`49
`
`50dqe
`
`51eJ
`
`520,'
`
`53ej
`
`54
`
`55
`
`56k
`
`57'
`
`58"'
`
`concn,
`pg/mL
`5
`10
`20
`50
`5
`10
`20
`50
`5
`10
`20
`50
`1
`5
`12.5
`1
`5
`12.5
`5
`10
`20
`50
`0.35
`0.75
`1.0
`
`0.1
`0.2
`0.4
`0.8
`
`0.1
`0.2
`0.4
`
`0.1
`0.2
`0.4
`0.8
`
`%
`inhibC
`1
`0
`6
`36
`23
`6
`17
`34
`15
`22
`44
`70
`1
`13
`28
`18
`16
`28
`3
`15
`14
`31
`39
`58
`64
`
`25
`48
`61
`81
`
`20
`34
`42
`
`22
`36
`14
`20
`
`c H , C H ~
`
`A 9
`
`Et20/ hexane
`
`-8-10
`
`69-70.5
`
`Et20/ hexane
`
`-8-10
`
`68-71
`
`EtzO/ hexane
`
`-8-10
`
`102-110
`
`EtzO/ hexane
`
`-8-10
`
`122-129
`
`Et20
`
`n-BuC1
`
`n-BuC1
`
`n-BuC1
`
`23
`
`25
`
`3
`
`96-98
`
`140-142
`
`151-152
`
`C1gH1603
`
`C21H1803
`
`n-BuC1
`
`10
`
`143-144
`
`C19H1803
`
`a Overall yield from aldehyde. *Analytical results are within k0.4% of the theoretical values unless otherwise noted. See Experimental
`Section for protocol. dMixture of trans/&; 1.4/1. ePercent inhibition calculated as if contribution of cis isomer was zero. fAnal. Calcd: C,
`68.99. Found C, 68.54. BMixture of trans/cis; 28/1. hAnal. Calcd: C, 72.82. Found: C, 72.27. 'Mixture of trans/&; 3.8/1. jMixture of
`trans/&; 2/1. kPreparation of aldehyde, mp 112-116 OC; Hennion, G. F.; Fleck, B. R. J. Am. Chem. SOC. 1955, 77,3253. 'Preparation of
`aldehyde, mp 96-98 OC; Bergmann, E. D.; Weiler-Feilchenfeld, H.; Mandel, N. Vietnamica Chim. Acta 1966, 129; Chem. Abstr. 1972, 72,
`3276s. "Preparation of aldehyde, mp 42-44 "C; Kohler, E. P.; Larsen, R. G. J. Am. Chem. SOC. 1935,57, 1452.
`111. The decahydronaphthalenes 51 and 52 and the ada-
`binding to HMG-CoA reductase is sensitive (a) to the
`mantyl compound 53 all possessed similar activity, while
`stereochemistry of the lactone moiety, (b) to the ability
`the cyclohexanes 49 and 50 were less active. Aromatization
`of the lactone moiety to be opened to a dihydroxy acid,
`of the cyclohexane ring to give the phenyl derivative 54
`(c) to the length of the moiety bridging the lactone and
`had little effect on activity. However, substitution of the
`the lipophilic groups, and (d) to the size and shape of the
`bridging moiety with larger aromatic groups such as those
`lipophilic group. Further modifications of the lipophilic
`in compounds 55-57 increased activity about 10-fold, i.e.,
`group leading to more potent inhibitors will be described
`to about 1% of the inhibitory activity of compactin.
`in subsequent papers from these Laboratories.
`Scission of the 4a-4b bond of compound 56 provided a less
`Experimental Section
`compact molecule 58 with diminished activity.
`Melting points were determined on a Thomas-Hoover capillary
`The ICM and relative potency values of the most active
`melting point apparatus and are uncorrected. Proton NMR
`compounds evaluated in this study are compared in Table
`spectra were recorded in CDCl,, unless noted otherwise, on either
`IV. Although the compounds described above are only
`a Varian T-60, EM-390, or NT-360 spectrometer. Chemical shifts
`moderately active HMG-CoA reductase inhibitors, analysis
`are reported in parb per million relative to Me,Si as the internal
`of their intrinsic inhibitory activities suggests that inhibitor
`standard. Elemental analysis for carbon, hydrogen, and nitrogen
`
`
`
`Mylan Exhibit 1040, Page 5
`
`

`

`362 Journal of Medicinal Chemistry, 1985, Vol. 28, No. 3
`Scheme I
`
`Stokker et al.
`
`OH
`
`li \
`
`OH on
`
`o
`
`8
`H'.
`a ?!H,CO~H,CO,CH,.
`j (R)-( + )-C,H,CH(CH,)NH,.
`
`9
`OH-. NaBH,, EtOH. e C,H,CH,, A .
`
`IV
`
`f H,, Rh/C. Dibal.
`
`CH,OH, PPTS. NH,.
`
`Table IV, In Vitro Inhibitory Potencies against HMG-CoA
`Reductase
`
`re1
`potencJP
`100
`0.08
`0.16
`0.09
`0.08
`
`ICSO,a,*
`PM
`no.
`compactin
`0.01
`sa(&)
`22
`10.8
`6a(+)
`15.2
`7
`20
`13a
`19.8
`16
`129
`47
`51
`107
`90
`52
`1.9
`55
`1.1
`1.5
`0.89
`56
`0.9
`1.6
`57
`"Relative precision is *lo%. *ICw is the micromolar concen-
`tration of the inhibitor required to give 50% inhibition under the
`conditions of the assay system.
`
`were determined with a Perkin-Elmer Model 240 elemental an-
`alyzer and are within &0.4% of theory unless noted otherwise.
`Optical rotations were determined with a Perkin-Elmer Model
`141 polarimeter. AU starting materials were commercially available
`unless indicated otherwise.
`3-(2,4-Dichlorophenyl)-2-propenal (2) was prepared by
`modification of the procedure of Baker.23 A solution of NaOH
`
`(23) Baker, B. R.; Janson, E. E.; Vermeulen, N. M. J. J. Med.
`Chen. 1969,12,898.
`
`(0.125 g) in CHBOH (2 mL) was added dropwise to a stirred
`suspension of 2,4-dichlorobenzaldehyde (7.5 g, 0.043 mol) in ac-
`etaldehyde (30 mL) cooled in an ice bath. The resulting solution
`was stirred 30 rnin with cooling, diluted with acetic anhydride
`(25 mL), and heated at 120 "C for 1 h. This mixture was cooled,
`diluted with HzO (60 mL) and 6 N HCl(25 mL), and heated at
`100 "C for 0.5 h. The light brown, oily product solidified upon
`cooling. It was collected, dried, and triturated with EhO to provide
`2 (8.5 g, 98%), mp 106-108 "C. An analytical sample was prepared
`by recrystallization from hexane to provide 2 as a pale yellow solid
`mp 107-108 "C; NMR 6 6.70 (H, dd, J = 15,6 Hz), 7.20-7.73 (3
`H, m), 7.87 (H, d, J = 15 Hz), 9.8 (H, d, J = 6 Hz). Anal.
`(CBH&lzO) C, H.
`Methyl (E)-7-(2,4-dichlorophenyl)-5-hydroxy-3-oxo-6-
`heptenoate (3) was prepared by a modification of the procedure
`of Weiler.le Methyl acetoacetate (23.2 g, 0.2 mol) was added
`dropwise to a stirred suspension of sodium hydride (50% oil
`suspension) (10.5 g, 0.22 mol) in anhydrous THF (500 mL) at 0
`"C under a Nz atmosphere. The resulting solution was stirred
`15 min at 0 "C and then treated with a 2.2 M solution (95.4 mL,
`0.21 mol) of n-butyllithium in hexane over 10 min. The yellow
`solution was stirred 15 min at 0 "C and then was treated with
`a solution of 2 (44.2 g, 0.22 mol) in anhydrous THF (250 mL).
`The resulting orange solution was stirred 15 min at 0 "C and then
`quenched by dropwise addition of 12 N HCl(48 mL). The reaction
`mixture was diluted with H20 (300 mL) and extracted with EtzO
`(3 X 300 mL). The organic extracts were combined, washed with
`brine (2 X 200 mL), dried over MgSO,, and filtered. The filtrate
`was evaporated in vacuo, leaving a red oil. The red oil was stirred
`in petroleum ether (200 mL) in order to remove the mineral oil.
`The mixture was cooled and the petroleum ether decanted to
`provide 62.8 g (90%) of 3: NMR 6 2.83 (2 H, d, J = 6 Hz), 3.47
`
`
`
`Mylan Exhibit 1040, Page 6
`
`

`

`HMG-CoA Reductase Inhibitors
`
`Scheme I1
`
`CI
`I
`
`OH 0
`I
`II
`
`Journal of Medicinal Chemistry, 1985, Vol. 28, No. 3 353
`Scheme I11
`
`11
`
`/ \
`
`.A c
`
`p.
`
`V
`
`CI
`
`12
`
`14
`
`18
`
`O
`
`C
`
`H
`
`3
`
`
`
`19 ~o~
`
`CI
`
`a MeOH,PTSA.
`f H'.
`
`Scheme IV
`
`20
`TiCl,. MeOH. NaBH,.
`
`e OH-.
`
`CHO
`
`a LiCH=CHOC,H,.
`
`Silica gel.
`
`5%
`
`Scheme V
`
`C02H
`
`CHO
`I
`
`21
`
`22
`
`CH3 bo h0
`
`\
`
`15
`
`t cis isomer
`13b
`
`130
`
`CI
`
`HO
`
`Y
`
`17
`
`CI
`
`16
`HO,CCO,H.
`a (n-Bu),SnOCH,, CH,=C(OAc)CH,.
`Zn, CuBr, Et,AlCl. e Ac,O,
`BrCH,COBr, C,H,N.
`C,H,N.
`f BrCH,CO,Et. g OH-. HC. A , C,H,CH,.
`
`I H,, Rh/C. ' PTSA, C,H,CH,, A ,
`(2 H, s), 3.70 (3 H, s), 4.76 (H, m), 6.13 (H, dd, J = 15,6 Hz), 6.90
`(H, d, J = 15 Hz), 7.0-7.5 (3 H, m).
`(E)-6-[ 2-(2,4-DichlorophenyI)ethenyl]-5,6-dihydro-4-
`hydroxy-2H-pyran.2-one (4). The ester 3 (2.0 g, 6.3 mmol) was
`stirred in 0.1 N NaOH (200 mL) for 4 h. The resulting solution
`was acidified with 6 N HC1 to provide a yellow solid which was
`recrystallized to analytical purity: yield 0.93 g; NMR (MezSO-d6)
`6 2.60 (2 H, m), 5.00 (H, s), 5.13 (H, m), 4.67 (H, dd, J = 15, 6
`Hz), 6.97 (H, d, J = 15 Hz), 7.27-7.87 (3 H, m), 11.5 (H, br 8).
`Methyl (E)-7-(2,4-Dichlorophenyl)-3,5-dihydroxy-6-
`heptenoate (5). Sodium borohydride (1.3 g, 33.7 mmol) was
`added with stirring to a cooled solution (5 "C) of 3 (10.7 g, 33.7
`mmol) in EtOH (100 mL) at a rate sufficient to maintain the
`internal temperature at 15-20 "C. The resulting solution was
`stirred an additional 2 h with ice-bath cooling and then acidified
`with 6 N HC1. The resulting mixture was diluted with H20 (250
`mL) and extracted with EhO (3 X 200 mL). The EtzO extracts
`were combined, washed with brine, dried over MgS04, and filtered.
`The filtrate was evaporated in vacuo to provide a yellow oil (10.4
`g, 97%). A portion of the oil was purified by medium-pressure
`chromatography on a 25 X 1000 mm silica1 gel column. Elution
`
`*Ifs P (yJ \/COzH %(fj
`
`24
`
`23
`
`25
`
`\\\\\/CHO
`
`-
`H
`
`R
`
`26
`27
`SOC1,.
`H,, Ru/C, EtOH.
`CH,(CO,H),, C,H,N.
`H,, Pd/C, 2,6-(Me),C,H,N. e AlCl,.
`f OH-. H+.
`
`with CH,C12-CH30H (491, v/v; 500 mL) provided a forerun which
`was discarded. Continued elution with the same eluant (3500 mL)
`provided 5 as a yellow oil; NMR 6 1.60-1.93 (2 H, m), 2.50 (2 H,
`d, J = 6 Hz), 3.67 (3 H, s), 4.13-4.77 (2 H, m), 5.93-6.40 (H, m),
`6.93 (H, d, J = 15 Hz), 7.17-7.50 (3 H, m). Anal. (C14H16C1204)
`H; C: calcd, 52.68; found, 52.25.
`(E)-6-[2-(2,4-Dichlorophenyl)ethenyl]-3,4,5,6-tetrahydro-
`4-hydroxy-2H-pyran-2-one (6a(f) and 6b(i)). An EtOH so-
`lution (100 mL) containing 5 (8.4 g, 26.3 mmol) and l N NaOH
`
`
`
`Mylan Exhibit 1040, Page 7
`
`

`

`354 Journal of Medicinal Chemistry, 1985, Vol. 28, No. 3
`Scheme VI
`
`CN
`I
`
`CN
`I
`
`CHO
`I
`
`28
`
`29
`
`30
`
`31
`
`32
`
`33
`
`34
`NCCH,CO,H, NH,OAc, C,H,N.
`NaCN.
`Scheme VI1
`
`CI
`
`36
`H,, Pd/C. Dibal.
`
`37
`
`2N H,SO,.
`
`36
`a BrCH,CH(OEt),.
`Scheme VI11
`
`CI
`I
`
`38
`
`I
`
`CI
`
`39
`CI
`
`40
`
`OH 1
`
`42
`
`CI
`I
`
`43
`(EtO),CH, NH,C1.
`K,CO,.
`Br,.
`KOH.
`e Dilute H,SO,.
`f 5% Pd/CaCO,.
`(26.3 mL) was stirred at ambient temperature for 1 h. The
`reaction solution was acidified with 6 N HCl, diluted with HzO
`(200 mL), and extracted with Et20 (3 X 100 mL). The combined
`
`Stokker et al.
`organic extracts were washed with brine, dried over MgSO,, and
`filtered. The filtrate was evaporated in vacuo to provide a mixture
`of acid and lactone (7.8 g, 97%). A solution of this mixture in
`toluene (100 mL) was heated at reflux in a Dean-Stark apparatus.
`After 2 h, the Dean-Stark apparatus was replaced with a Soxhlet
`containing 3-1\ molecular sieves (100 8). The solution was refluxed
`for an additional 4 h and then the toluene was removed in vacuo
`leaving a yellow oil (7.2 g, 95%) which was a mixture of sa(&)
`and 6b(*). The oil was chroamtographed on a silica gel column
`(500 g). Elution with CH2C12-acetone (41, v/v; 900 mL) provided
`a forerun which was discarded. Continued elution with the same
`eluant (300 mL) gave the trans isomer 6a(*) (2.5 g). Recrys-
`tallization of the solid provided an analytical sample, as colorless
`needles: NMR (acetone-d6) 6 2.06 (2 H, m), 2.69 (2 H, m), 4.43
`(H, m), 5.42 (H, m), 6.49 (H, dd, J = 15, 6 Hz), 7.08 (H, d, J =
`15 Hz), 7.33-7.59 (2 H, m), 7.79 (H, d, J = 8 Hz). An isomeric
`purity of 99.8% was determined for 6a(&) by HPLC on a
`Whatman Partisil-5 RAC column with 15% 2-propanol/hexane
`as the eluant. The time of elution was 4.96 min at a flow rate
`of 6 mL/min.
`Further elution of the column with the same eluant (600 mL)
`gave the cis isomer 6b(*) as a solid (1.25 g). Recrystallization
`gave an analytical sample as colorless needles: NMR (acetone-d6)
`6 1.50-2.93 (4 H, m), 4.36 (H, m), 5.02 (H, m), 6.37 (H, dd, J =
`15, 6 Hz), 7.02 (H, d, J = 15 Hz), 7.16-7.50 (2 H, m), 7.67 (H, d,
`J = 8 Hz). An isomeric purity of 99.3% was determined for 6b(rt)
`by HPLC on a Whatman Partisil-5 RAC column with 15% 2-
`propanol/hexane as the eluant. The time of elution was 5.79 min
`at a flow rate of 6 mL/min.
`trans -6-[2-(2,4-Dichlorophenyl)ethyl]-3,4,5,6-tetrahydro-
`4-hydroxy-2H-pyran-2-one (7). A solution of sa(&) (1.5 g, 5.2
`mmol) in THF (100 mL) was stirred magnetically and hydro-
`genated at room temperature under atmospheric pressure in the
`presence of 5% rhodium on carbon (150 mg) until 1.25 molar equiv
`of hydrogen had been consumed. After removal of the catalyst
`by filtration, the filtrate was evaporated in vacuo, leaving a solid.
`The solid was recrystallized to provide 7 (0.9 9): NMR 6 1.67-2.17
`(4 H, m), 2.60-3.13 (4 H, m), 4.30-4.50 (H, m), 4.57-4.90 (H, m),
`7.14-7.44 (3 H, m).
`Resolution of (&)-trans-(E)-6-[2-(2,4-Dichlorophenyl)-
`ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (sa).
`A solution of sa(&) (2.87 g, 10 mmol) in (R)-(+)-a-methyl-
`benzylamine (15 mL) was stirred for 18 h at ambient temperature
`and then poured into H20 (100 mL). This aqueous mixture was
`acidified with 6 N HCl and extracted with EhO (3 X 100 mL).
`The E t 0 extracts were combined, washed with brine, dried over
`MgSO,, and fiitered. Evaporation of the filtrate in vacuo provided
`the crude diastereomeric amides as a tan viscous oil (4.1 g, 100%).
`This oil (3.1 g, 7.6 mmol) was chromatographed on a silica gel
`column (200 g). Elution with acetone-CH2C12 (1:4, v/v; 1200 mL)
`gave a forerun which was discarded. Continued elution with the
`same eluant provided the mixture of diastereomeric amides as
`a viscous oil (3.0 g, 97%). This mixture was separated by chro-
`matography on a Waters Prep LC 500. The separation was
`accomplished by using two Prep PAK-500 silica cartridges in series
`and eluting with acetone-CHzClz (1:4, v/v). Use of the shave-
`recycle technique provided diastereomer A (1.36 g) and diaste-
`reomer B (1.2 g).
`Recrystallization of diastereomer A from n-butyl chloride gave
`colorless clusters (1.0 g) which melted at 106-108 "C; NMR 6 1.47
`(3 H, d, J = 6 Hz), 1.70 (2 H, m), 2.33 (2 H, d, J = 6 Hz), 4.30
`(H, m), 4.58 (H, m), 5.13 (H, m), 6.20 (H, dd, J = 15,6 Hz), 6.33
`(H, m), 6.93 (H, d, J = 15 Hz), 7.33 (8 H, m). Anal. (CZ1H23-
`C12NO3) C, H, N.
`Recrystallization of diastereome