4,450,171
`[11]
`Ulllted States Patent [19]
`Hoffman et al.
`[45]
`* May 22, 1984
`
`
`1/1979 End0 at at .
`4.137.322
`549/292
`4,231,938 11/1930 Monaghan et a1.
`562/501
`4,293,496 10/1981 Willard et al.
`549/292
`4,294,346 10/1981 Albers-Schonberg et al.
`S49/292
`4,342,761
`8/1982 Albers-Schonberg
`424/279
`4,321,311? 9/1982 Patchett et al.
`3
`,
`11 1982 T ah
`....... iafiiigiii
`$375,353
`3119113 1:11
`.....................
`X
`C13 51..
`4,381,242
`FOREIGN PATENT DOCUMENTS
`554109024
`s/1930 Japan .
`56-110618
`1/1981 Jap_a.n
`2073l99A 10/1981 Un1tedKJ.l1gd0l:I1
`OTHER PUBLICATIONS
`
`-124/311
`562/501
`
`
`
`.
`
`[54] AN'I'Il-IYPERCI-IOLESTEROLEMIC
`C01\¢[P()UN]_)S
`
`[75]
`
`Inventors: William F. I-Io1’fman;Ro1:ert L
`Smith, both o1'Lansdale. Pa.; Alvin K.
`Willard, Wilmington, Del.
`-
`[73] As-signee: Merck 8: Co» Inc-. Rahway, NJ-
`[‘] Notice:
`The portion ofthe term of-this Patent
`:»l1_1bsl<=:c,1u:r:It to Apr- 24, 2001 has been
`lsc 3.1111
`.
`
`_
`[21] Appl. No.: 388,312
`[22] Filed:
`Jun. 14’ I982
`
`[63]
`
`Related U-S- Application Data
`Continuation-in-part of Ser. No. 217,640, Dec. 18,
`1980, w11.ic11
`is a continuation-in-part of Set. No.
`175,460, Aug. 5, 1980, abandoned, which is acontinua-
`D11
`.
`-
`§0n;1'Pa1't Of 561* N0- 113.051. F€b- 4. 1930. 8b1II-
`
`1111- Cl-3 -------------------- Cmc 59/743
`[51]
`[52] U.S. C1. .................................. .. 424/279; 549/292;
`550/119; 560/256;, 424/305; 424/311
`Bf S£91'I‘.‘£12I ....................... 424/279, 305, 317;
`260/343.5; 550/107, 135,- 255,- 119; 552/501;
`- 549/291
`
`[58]
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`3,983,140 10/1977 Endoetal.
`4,04-9,495
`6/1978 Endoctal.
`
`.
`.
`
`F. M. Singer et al., Proc. Soc. Exper. Biol. Med., 102,
`379: (1959)-
`Hulcher, A1'c.h.Biochcm.Biophys. 146, 422, (1971).
`Brown et al., I. Chem. Soc., Perkin I, 1165, (1976).
`Endo et al., J. Antibiotics. XXXI1, 852, (1979).
`Primary Examiner—Ethel G. Love
`Attorney, Agent, or Fz'rm—Wi]1iam H. Nicholson; Mario
`A. Monaco
`
`ABSTRACT
`[57]
`6(R)—[2-(8'-acyloxy-2'-mathyI-6'.methyl (of ]-‘yd:-gggn}.
`polyhydronaphthyl-1’)-ethyl]-4(R)-hydroxy-3,4,5,6-tet-
`rahydro-2H-pyran-2-ones are prepared by acylation of
`the corresponding 8'-hydroxy COI12l1:l0lJJ'ldS_. T1'1e prod-
`ucts are strong inhibitors of the biosynthesis of choles-
`terol.
`
`9 Claims, No Drawings
`
`Inf]?
`
`PENN EX. 2191
`
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`1
`
`4,450, 171
`
`ANTIHYPERCHDLESTEROI.EMlC COMPOUNDS
`
`SUMMARY OF THE INVENTION
`
`This is a continuation-in-part of copending applica-
`tion Ser. No. 217,640, filed Dec. 18, 1980, which is a
`continuation-in-part of copending application Ser. No.
`175,460, filed Aug. 5, 1980, now abandoned, which in
`turn is a continuation-in-part of copending application
`Ser. No. 118,051, filed Feb. 4, 1980, (now abandoned).
`This invention relates to a group of 6(R)-[2«(8’-
`acyloxy-2'-methyl-6'-methy1(or hydrogen)-polyhydro-
`naphthyl-1')-ethyl]-4(R)-hydroxy-3,4,S,6-tetral'1ydro-
`21-I-pyran-2-ones and to the hydroxy acid form of said
`pyranones, the pharmacentically acceptable salts of said
`hydroxy acids and to the lower alkyl and phenyl, di-
`niethylamino or acetylamino substituted lower alkyl
`esters of said hydroxy acid.
`More specifically,
`this invention relates to a com-
`pound of the structure I in Table I, in which the dotted
`lines X, Y and Z represent possible double bonds, said
`double bonds being, when any are present, either X and
`2 together in combination or X, Y or Z alone; R repre-
`sents
`l—ethyl-l-rnethylpropyl,
`1,14:liethy1propyl
`1,1-
`dimethylethyl,
`1,1-diethylbutyl,
`1,1-dimethylpropyl,
`C340 cycloalkyl, C240 alkenyl, C|.1o CF;-, substituted
`alkyl, phenyl, halophenyl, phenyl-C1.3 alkyl or substi-
`tuted phenyl-C1.3 alkyl, in which the substituent is halo,
`C14 alkyl or C1-3 all-coxy; and the free hydroxy acids of
`formula II formed by opening the lactone ring of for-
`mula ] in Table I.
`
`BACKGROUND OF THE INVENTION
`It is known that certain mevalonate derivatives in-
`hibit the biosynthesis of cholesterol, c.f. F. M. Singer et
`al, Proc. Soc. Exper. Biol. Med, 102 370 (1959) and F. H.
`Hulcher, Arch. Bfochem. B1'op!2ys'., 146, 422 (I971). Nev-
`ertheless, the activity of these known compounds has
`not always been found to be satisfactory, i.e. to have
`practical application.
`Recently, Endo et al, reported (U.S. Pat. Nos.
`4,049,495, 4,137,322 and 3,983,140) the production of
`fermentation products which were quite active in the
`inhibition of cholesterol biosynthesis. The most active
`member of this group of natural products, now called '
`compactin, IIIa(R’=H) was reported by Brown et al.
`[1 Chem. Soc. Perkin I 1165 (1976)] to have a complex
`rnevalonolactone structure.
`
`More recently. Monaghan et al in U.S. Pat. No.
`4,231,938, which is incorporated herein by reference,
`reported an inhibitor, designated MK-803 and having
`the structure Illa fR'=CH3) in Table I, which was
`isolated from an entirely different fermentation. Albers-
`Schonberg et al (US. Pat. No. 4,294,846) described a
`dihydro MK-803, designated Illa (R’=CH3) in Table I,
`of about equal potency to MK-803 isolated from the
`same fermentation as was MK-803. Patchett et al (U.S.
`Ser. No. 210,826, filed Dec. 1, 1980) describe dihydro
`and tetrahydro derivatives of MIK-803 of different
`structures (I115; and, (R’=CH3) in Table I), prepared by
`the catalytic hydrogenation of MK.-803. Willard fU.S.
`Pat. No. 4,293,496), describes the preparation of the
`8-hydroxy derivatives (IVH (R’=CH3) in Table 1)
`which are the starting materials for the preparation of
`some of the novel compounds of this invention.
`
`30
`
`50
`
`55
`
`2
`A tetrahydro analog III, (R’=H), of comparztin was
`reported in published Japanese Application (Kokai)
`55009-024.
`
`Very recently a dihydro-analog of compactin of
`structure II1a(R=H) was isolated from compactin fer-
`mentation broths as reported by Gullo et al, (US. appli-
`cation Ser. No. 207,508, filed Nov. 17, 1980).
`1115,
`The preparation of
`the starting material,
`Rl=CI-I3) as mentioned previously,
`is described by
`Alhers-Schonberg et al in U.S. Pat. No. 4,294,846, and is
`the product of the following fermentation with a strain
`of Aspergillns rerreus ATCC No. 20542, designated
`MF-4845 in the culture collection of MERCK & CO.,
`Inc., Rahway, N.J.
`
`PREPARATION OF COMPOUND 111,: (R’ =CI-I3)
`A. Fermentation
`
`A tube of lyophilized culture MIF4845 was opened
`aseptically and the contents suspended in an unbaflled
`250 ml Erlenmeyer flask (seed flask) containing approx-
`imately 10 ml of the Medium which has the following
`composition:
`
`Medium
`Cornsteepliqucr
`Tomato paste
`Oatmeal
`Glucose
`Trace Element Solution
`Distilled water
`12!"! 6.8 with NaOH
`Trace Element Solution
`1000 mg
`FeSO4.'l'H;O
`1000 mg
`l\u'[nS04.4H10
`25 mg
`CnCl1.2I-I20
`100 mg
`CaCl3_.2H;;O
`56 mg
`H3303
`19 mg
`(N1*L0sMo102.|.4H2O
`2.0.’) mg
`ZnS0.1..7H30
`
`Distilled Deioniaed Water 1000 ml
`
`5 g
`40 g
`10 g
`10 g.
`10 g
`1000 1111
`
`_
`
`The inoculated flask was incubated for 24 hours at 28°
`C. on a 220 rpm shaker (2 inch throw). An unbaffled 2
`liter Erlenmeyer flask containing 500 ml of the medium
`was then inoculated with 10 ml of the first stage fermen-
`tation growth from the seed mixture. This too was
`shaken 24 hours at 28° C.
`A 200 gallon stainless steel fermentation vat was then
`charged with 485 liters of a medium comprising:
`
`Cerelose
`4.5% Wtfvol
`Peptonized Milk
`2.5% Wtfvol
`Autclyzed yeeu
`0.25% wt/vol
`
`Polyglycol P2000 0.25% vol/vol
`
`whose pH was adjusted to 7.0. This was sterilized 15
`minutes at 121° C. One liter of the second stage above
`was then charged and the mixture was incubated at 85
`rpm for 12 hours then at 130 rpm for 84 hours at 28° C.
`with an air flow of 5 cfm for 12 hours then 10 cfm for 84
`hours.
`
`B. Isolation
`
`l. Extraction
`Two batches of one hundred gallons of whole broth
`were combined, acidified with stirring to pH 4.1 by
`careful addition of 800 ml of concentrated hydrochloric
`acid, and extracted by addition of '.-'5 gal of ethyl acetate
`and further stirring for two hours.
`
`201']?
`
`PENN EX. 2191
`
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`3
`About 25 lbs of a silicaceous filter aid was then added
`and the total slurry was pumped through a 24-inch filter
`press. An additional 75 gal of ethyl acetate was used to
`wash the press cake and continue the extraction, by
`reversing the direction of pumping through the press
`four times. Then all of the wash solvent was discharged
`from the press and combined with the first filtrate. The
`two-phase filtrate was allowed to settle, and the water
`layer removed. The ethyl acetate layer was washed
`with 10 gal of deionized water. the phases were allowed
`to separate and the ethyl acetate extracts were concen-
`tratedunder vacuum to a residue of about 10 gal.
`2. Lactonization
`Ethyl acetate extracts from an additional three hun-
`dred gal of broth were added to the above extract and
`the volume was reduced to about thirty gal by vacuum
`distillation. About fifty gal of toluene was added, and
`the batch was concentrated under vacuum to 32 gal; this
`step was repeated;
`then sufiicient new toluene was
`added to bring the volume to 75 gal. Without vacuum,
`the batch was brought to reflux and maintained there
`for two hours, with a temperature over 106' C.
`This solution was then concentrated under vacuum to
`a small volume, which was further concentrated to an
`oily residue in a large rotary evaporator under vacuum.
`3. Chromatography on Silica Gel
`The extract obtained above was flushed free of other
`solvents by addition of 2 gal of methylene chloride and
`reconcentration to an oil.
`The oily -residue was dissolved inabout 5 gal of ethyl
`acetate-methylene chloride (30/70; v/v) mixture, and a
`slurry was made by addition of 2.8 kg of silica gel.
`The slurry was loaded as a level layer on the top of a
`12 in. X50 in. silica gel column packed in the same sol-
`vent mixture.
`Elution was with ethyl acetate-methylene chloride
`(40/60; v/v) at 800 ml/min. A forerun of 10 gal, then
`further fractions of 4 gal each were collected.
`Fractions 6-10 -inclusive were concentrated under
`vacuum to an oily residue which was dissolved in hot
`ethyl acetate, treated with decolorizing carbon, filtered
`hot, and cooled. Crystals of Compound Illa (R’=CI-I3)
`were filtered off and the mother liquors were concen~
`trated to an oil for further chromatography.
`4. Recbromatography on Silica Gel
`Mother liquor residues from similar broth extract
`work-ups equivalent to an additional 600 gal of fermen-
`tation production, were combined with the above in
`methylene chloride solution. One-half of this solution
`
`10
`
`20
`
`25
`
`30
`
`35
`
`45
`
`4,450, 171
`
`4
`was taken for further silica gel chromatography. A
`small aliquot showed a total solids content of 325 g. The
`solution was treated with 40 g of decolorizing carbon,
`filtered. and the cake rinsed with methylene chloride.
`The combined filtrate and washings were concentrated
`under vacuum to an oily residue. This was redissolved
`in 800 ml of ethyl acetate/methylene chloride (30/70;
`v/v) and slurried with 225 g of silica gel. The slurry was
`loaded on top of a 14x 36 cm column bed of silica: gel
`packed in the same solvent mixture. Development was
`with ethyl acetate/methylene chloride (40/60; v/v). A
`forecut of three liters was set aside; then fractions of 800
`ml each were collected.
`5. Chromatography on Reverse-phase Packing
`Forty ml from fraction 12 of the above chromatogra-
`phy were concentrated to an oil weighing 500 mg and
`the oil redissolved in 5 ml acetonitrile. This acetonitrile
`solution was charged to .a 5/8" OD by 6 ft long stainless
`steel chromatography column packed with preparative
`reverse-phase liquid chromatography coluum packing
`material “Bondapak C18/PorasilB” (Waters Associates,
`Inc., Milford, Mass. 0175?). The column was eluted
`with a mixture consisting of (v/v) 55% aeetonitrile and
`45% 0.05 M ammonium phosphate pH3. The elution
`volume between 1360 ml and 1700 ml was combined on
`the basis of refractive index detection. The organic
`solvent was removed in vacuo and the residual aqueous
`solution extracted with ethyl acetate. In vacuo removal
`of the ethyl acetate left 120 mg of compound which
`crystallized from a concentrated acetonitrile solution
`yielding crystals of Compound Illa (R‘ =CH3), m.p.
`1295131” C.
`
`PREPARATION OF COMPOUNDS I115, c_ 3
`
`III,._- and IIL, (R'=CH3) as
`Starting materials Illa,
`mentioned above are described in U.S. application, Ser.
`No. 210,826, filed Dec.
`l.-1980 by Patchett et al., in
`accordance with the following Flow Sheet and prepara-
`tive methods extracted therefrom.
`The desmethyl analogs, Ills, III; and III,(R'=H) are
`obtained substantially as described by Patchett et al. but
`starting with IIL; (R'=H) in each case.
`For the preparation of [IL it is advantageous to re-
`duoe IIL; inasmuch as the desired trans fusion of the
`perhydronaphthalene ring, present in the starting mate-
`rials, is retained in the final product, and the need to
`separate isomers is avoided.
`
`CH3
`I
`cn2—cH—cH—c’
`
`
`
`301']?
`
`PENN EX. 2191
`
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`5
`
`4,450, l 7 l
`
`-continued
`
`FLOW SHEET
`
`6
`
`H
`
`#0
`
`0 .
`
`E
`
`CH3
`
`O
`cH3—cHz—cH-cf _
`I
`9
`CH3
`g
`
`(3)
`
`
`
`cH3—cH¢—c1-I-C"
`
`111%
`
`:11,
`
`Reactions and Reagents
`
`l. Hydrogenation at about 20°-15° C. and about atmo-
`spheric pressure to about 4 atmospheres over tr'ls-(tri-
`phenylphosphine)chlororhodiu'm in an aromatic sol- 35
`vent such as benzene, toluene or xylene, preferably
`toluene. Preferred conditions are about 40’ C. ‘and
`about 2-7 atmospheres in toluene.
`2. Hydrogenation at about 20°-25° C. and about atmo-
`spheric pressure over 5% palladium on calcium car- 40
`bonate in a lower alkanol such as a C1.3 alkanol, espe-
`cially ethanol.
`3. Hydrogenation at about 20°-25" C. and atmospheric
`pressure over platinum oxide in ethyl acetate
`4. I-lydrogenation at about 20°—25“ C. and atmospheric 45
`pressure over 10% Palladium on charcoal in ethyl
`acetate.
`
`Preparation of
`6ct-[2-(8'-B-2-(S)-methylbutyrylosy-2'3, ’o'.-dimethyl-
`1',2',3',4’,6’,7',8',8'a-octahydronaphthyl- l)ethyl]-4B-
`hydroxy-3,4,5,6-tetrahydro-2H-pyran-2-one, lllg,
`(R' = CH3)
`
`so
`
`A mixture of 50 mg ((11236 mmol) of Compound Illa
`(R’=CI-I3) and an equal molar amount (I14-.35 mg. 55
`0.1236
`rnrnol)
`of
`tris(triphenylphosphine)chloror-
`hodium in 10 ml of dry toluene was hydrogenated at
`room temperature for 6 days, with a total uptake of 14.6
`ml of hydrogen. The mixture was evaporated in vacuo
`to dryness. The red residue was subjected to prepsra- 60
`tive thin-layer chromatography on silver nitrate im-
`pregnated silica plates and was developed twice in the
`10% ethyl acetate-ether system. The yield of Com-
`pound Ills (R'=CH3) was 22.3 mg.
`Mass spectrum (M/e): 406 (m+), 304 (tn-102), 286 65
`(rn-102-18)
`nn1r(CDCl3, 300 MHz): 3 4.37 (an, 11-1), 4.so(m, 11-1),
`5.34 (cl of :, J=2.S Hz, 1H), 5.41 (m,- 1H)
`
`Preparation of
`6a-[2-(8’,8-2-(S)-methylbutyryloxy-2',8,6'a-din1ethyl- _
`l',2',3',5’,6’,’l”,3',8'a-octahydronaphthyl-l)ethy1]-4B-
`hydroxy-3,4,5,6-tetrahydro-2H-pyran-2-one, III‘,-
`(R' =CH3)
`
`A solution of 80.91 mg (0.2 mmol) of Compound III‘.
`(R'=CH3) in 10 ml of absolute ethanol, in the presence
`of an equal weight of 5% Pd on C3003 was hydroge-
`nated at 1 atmosphere until an uptake of one mole
`equivalent of hydrogen was observed. The catalyst was
`then removed by filtration and the filtrate was evapo-
`rated to dryness (81 mg). After a purification by prepar-
`ative thin-layer chromatography to remove a small
`amount of by-product tetrahydro compound, 72 mg of
`the 1,4 reduction product III, (R’=CH3) was isolated.
`Mass spectrum (M/e); 406 (rn+), 304 (Io-I02), 286
`(304-1120)
`'
`umr (CDCI3, 300 MHZ): 8 4.38 (m, 1H), 4.64 (tn, 11-1).
`5.28 (d oft, J=3.5 Hz, 1H), 5.48 (m, 1H)
`
`Preparation of 6a-[2-(813-2(S)-methylbutyryloxy-2"a.,
`boL.B-dimethyl-1',2’,3’,4’,4’acr.,5',6',7',8',8'a-decahy-
`dronaphthyl-I)ethyl]-4,8-hydroxy-3,4,5,6-tetrahydro-
`2H-pyran-2-one, III.» (R’ =CI-I3)
`A solution of 80.91 mg (0.2 mmol) of Compound III“
`(R’=CH3) in 10 ml of ethyl acetate was hydrogenated
`in the presence of an equal weight of platinum oxide at
`one atmosphere. A11 exact 2 mole equivalent of hydro-
`gen was consumed within 1 hour. The catalyst was
`removed by filtration and the filtrate was concentrated
`to dryness to give an oil. The cis and trans isomers were
`separated by preparative thin-layer chromatography on.
`silica gel plates (10% ethyl acetate-ether system, bands
`detected by water spray). The tram isomer
`III,
`(R'=CI-I3) appears as the more polar spot. compared to
`the cis isomer. and 60 mg was isolated.
`Mass spectrum (Life): 408 (n:1+), 323 (m-85), 306
`(m-I02)
`
`4ofl7
`
`PENN EX. 2191
`
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`7
`nmr (CDCI3, 300 MHz): 3 4.36 (broad singlet. 11-1),
`4.59 (m, 1H), 5.19 (d of :, J=2.5 Hz, in)
`
`Fermentntive Production of Compound IIL; (R’=H)
`A. Fermentation
`
`4,450, 171
`
`8
`
`adjusted to pH 4.0 with 1 N hydrochloric acid. The
`aqueous solution was extracted with 7 liters of ethyl
`acetate and the extract was back-extracted with 3X2
`
`'-liters of aqueous sodium hydroxide solution. The com-
`bined sodium hydroxide extract was adjusted to pH 3.8
`with l N hydrochloric acid- and extracted with 2 liters
`and 1 liter of ethyl acetate The combined ethyl acetate
`solution was dried over anhydrous Na;»_SO4, filtered and
`concentrated to dryness. The oily residue was dissolved
`in toluene and refluxed for 1 hour. The toluene solution
`
`I0
`
`- was concentrated to dryness and the residue was dis-
`15
`solved in 18 ml of a mixture of n-hexane/toluene/me
`
`A natural isolate of Penfcillium cftrinuna. NRRL 8082
`was used to prepare a yeast-malt extract (YMIE) slant
`which was incubated for 2 weeks at 28° C.
`A portion (1/5) of the slant (MF-48103) was used to
`inoculate each of 5 unbafiled seed flasks (250 ml) con-
`taining44mlofKFseedn1ediumwithCaC'l2.They'
`were incubated for 3 days at 28° C., and 220 rpm. A
`portion ofthe seed growth (about 1.5 ml} was used to
`inoculate each of 1(1) production medium flasks (250 ml
`unbaffled) containing 40 ml of LM Production Medium
`Without Malt Extract. The production flasks were incu-
`bated for 4 days at 25° C.
`Another group of production medium flasks (140),
`each containing 40 ml of LM Production Medium
`Without Modification were inoculated and incubated
`under the same conditions as previously described. The
`broths from both fermentations were combined.
`The various media employed in the foregoing fer-
`mentations are:
`
`.
`
`20
`
`25
`
`thanol (4/1/l by volume). This solution was loaded
`onto a 30 mm (ID))<40 cm. Sephadex LH-20 column
`equilibrated in the same solvent system. After eluting
`with 3(1) ml of solvent, a 10 ml fraction was obtained
`which was concentrated to an oil. High performance
`liquid chromatography (HPDC) on an ES Industries
`Chromega ® column (9 mmx 50 cm) using a mixture of
`aoetonitrile/water (60/4-D by volume) as the eluting
`solvent yielded 45 mg of dihydrocompactin (Com-
`pound Illa, R'=I-I), m.w. 392.2560 by mass spectrum
`(calculated for C231-13605, 392.2553).
`_
`In KBI, the major IR peaks obtained from a Fourier
`Transform-IR (FIIR, Nicolet, Model 7199) are at 1124,
`1704, 1253, 1073 and 1070 cm“1. Of significa.nce.is-a
`peak at 3005 cm—1 and the absence of.a peak at 3030
`35 cm_]_
`was obtained
`A nuclear gnetic resonance
`in cocig, ~1 mg/0.5 111]) on a Variau sc_—3oo super-
`conducting nmr spectrometer. The followmg are the
`40 peak positions given in ppm (8) relative to internal tet-
`.
`-
`=
`rametbylsllane (TMS).
`
`_
`
`YME Slant
`
`Dem-one
`flfimmm
`Aguw
`''‘°'
`3,?‘
`
`m.__.....m2_KF5°“! M°'1i'“" “"i'-5 C391
`gzghmcp 1-:
`E
`Tumimpum
`0;;-:,,k,,,
`‘PI:-9e F-‘=3:-=__; M"
`1:}!
`T“'°° Emmi M“
`FCSO4-7H20
`m.so,,_4H2o
`CuCl;.2H2O
`CaClz
`H
`(r~iiieD)iMo-;o2t4H2o
`%d"‘§,.’f’m
`1.1»: Production Medium Without Malt
`EWW1
`
`_
`
`4 g..v'L
`‘3 fig
`10
`,$ ‘"
`
`N; g
`:3 g
`10 E _
`1% g
`as
`
`1 8
`1 5
`25 mg
`III! mg
`so
`I9 if
`fig 3‘
`
`to g
`3 mg
`Dig? mi
`1.0
`
`Anlamzne pH
`CoCl1.6I-I20
`mwmflezfm
`H
`11).!!! Production Medium Without
`Modification
`Dextrose
`Erd-WHIP“
`20 g
`Mall Extract
`8 mg
`OoC|1.6HzO
`Polyglycol p 2000
`0.12596 ml
`Dnfil'led
`our
`
`PH
`Wu”
`10
`
`20 5
`
`
`
`5
`5.52 d.d.d (2.17. 4.5, IDJJ)
`5-43 d (10)
`5.20 in
`
`mm“
`113- (:1?)
`Hr (ct)
`Ha-
`
`‘-39 "1
`2.75 an (11.5, 5.5)
`253 d.a.d (17.5, 4.0, 1.5)
`2.39 m
`
`2.29m
`1.1-id
`
`"4
`
`2.cn;
`
`it)
`
`CI-l3HCC
`
`l-I.w+H5-
`.
`éfl
`CIl3CHC
`
`.
`
`0.90 t
`CH3C1-I:
`
`0.3-Id
`_CH3H2*
`
`30
`
`45
`
`5"
`
`55
`
`60
`
`_
`B. IS0lfltlOl1
`The combined whole broth (10.3 liters) was filtered
`and the mycelia cake was washed with 2.5 liters of
`deionized water. The combined filtrate and wash was
`
`d: doublet;
`65 M mumpm;
`‘-""'|"“
`
`The evidence indicates the structure to be:
`
`5 Hf”
`
`PENN Ex. 2191
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`4,450, 17 1
`
`
`
`l:l
`
`PREPARATION OF COMPOUNDS IV“:
`
`The starting materials, the 8’a.-hydroxy compounds
`lV,,.._- (R'=CH3) described by Willard (U.S. Pat. No.
`4,293,496) are prepared from the various 8’-esters de-
`scribed by Monaghan et al (Illa, R’-—-CH3), Albers-
`Schonberg et al (Illa, R'=CH3) and Patchett et al
`(III,5,,,g, R’=CH3) by heating them with lithium hy-
`droxide solution for extended periods. The pyranone
`ring readily opens but the removal of the side chain acyl
`group is not easily effected. The heating must be pro-
`longed and/or pressure must be used. An inert atmo-
`sphere is also helpful.
`In the case of the Compounds IIIG1? (R’=H) the sa-
`ponilication of the B’-esters is much more facile pro-
`ceeding to completion in about 20 hours.
`The 3’-hydroxy products are isolated by acidification
`and extraction with organic solvents which provides
`the trihydroxy acid form, in which the pyranone ring is
`still opened. These trihydroxy acids are relactonized by
`heating a solution of the acid in an appropriate organic
`solvent such as benzene or toluene in an apparatus per-
`mitting continuous separation of the water formed.
`The Compound IV}; (R'=H) is known as ML-236A
`as reported by Endo et al in U.S. Pat. No. 3,983,140.
`In their lactone form, these alcohols are the com-
`pounds of Formula IV“; in Table I and are prepared as
`described in the following preparations.
`
`Preparation of
`6(R)-[2-(8'(S)-hydroxy—2’(S),6'(R}-dimethyl-
`1’,2’,6',7',8',8’a(R)-he.xahydronaphthy1-I'(S))ethyl]-
`4-(R)-hydroxy-3,4-,5,6-telrahydro-2H-pyran-2-one, lVa
`(R'=CH3)
`
`A mixture of 8.0 g. (19.78 mmole) of MK-803 (Illa,
`R'=CI-I3) and 8.31 g (197.8 mmole) of LiOH.H2O in
`600 ml of water was stirred at reflux under a nitrogen
`atmosphere for 56 hours. The reaction mixture was
`cooled to 0" and treated, with stirring, with 20 ml of
`concentrated hydrochloric acid. The mixture was then
`extracted with three 250-ml portions of ether and the
`combined extracts were washed successively with three
`200-ml portions of water and then 200 ml of saturated
`brine. After drying over MgS04., this organic solution
`was filtered and the solvent evaporated in vacuo to give
`an oily residue. This residue was dissolved in 200 ml of
`toluene and heated at reflux under a nitrogen atmo-
`sphere for 2 hours with continuous separation of water
`to effect relactonization. Evaporation of the toluene and
`trituration of the residue with hexane gave 5.15 g (81%)
`of the title compound IV“ (R'=CI-I3) as a white solid
`which did not require further purification.
`An analytical sample was prepared by recrystalliza-
`tion of a portion of this material from butyl chloride to
`give white
`clusters: m.p.
`l28'—l3l°
`(vacuum);
`
`_
`
`I5
`
`30
`
`40
`
`50
`
`60
`
`10
`NMR(CDCl3) 80.87 (d.3.J='.-' Hz. CH3), 1.16 (d,3,J=7
`I-lz, CH3), 2.64 (m,2,pyran C3H’s), 4.27 (‘|::rm,l, naphtha-
`lene Cal-I), 4.37 (m,l,pyran C4]-I), 4.71 (n1,l.pyran Call),
`5.56 (m,l, naphthalene C5H), 5.79 (dd,l, J =6,l0 Hz,
`naphthalene C31-I), 6.03 (d,l,.l = 10 Hz, naphthalene
`C41-X); IR (CHCI3) 3400 (OH), 1725 ((3:0), 1240, 1120,
`I08 cm‘-1.
`Anal. Calcd for C19H2sO4..0.lC4H9Cl C, 70.67; H,
`8.34. Found: C, 70.77; H, 3.75.
`
`Alternative preparation of
`6(R)-[2-[8'-(S)-hydroxy-2’(S),6’(R)-dimethyI-
`l'2',6','.-",8',8'a(R)-hexahyd.ronaphthyl—l '(S)] ethyl]-
`4-(R)-hydroxy-3.4,5,6-tetrahydro-2H-pyran-2-one, [Va
`(R’=CH3)
`
`A suspension of I88 mg (0.463 mmol) of MK-803
`(Illa, R’=CH3) in 5 ml (5 mmol) of aqueous l N Li0l-I
`solution is shaken for 12 hours at 135“ in a 30 ml stainless
`steel pressure vessel. The cooled reaction mixture is
`acidified with 1 M H3PO4 and extracted with ethyl
`acetate. The ethyl acetate solution is dried (MgSO4] and
`filtered and the solvent is evaporated. The residue is
`dissolved in 20 ml of toluene which is heated to reflux
`for 4 hours in a Dean-Stark apparatus to effect relacton-
`izationi Evaporation of the toluene gives the title com-
`pound.
`
`Preparation of alcohols IV,, (R’=H) and IV5, IV,, IV/av,
`and [V9 (R’=H or CH3)
`
`Following essentially either procedure described
`above but substituting an equivalent amount of esters
`Illa (R’=H) or III;,, IIIQ, IIL-_:, or IIL (R’=H or CH3),
`for Illa (R’=CH3) used therein the corresponding alco-
`hols IV}; (R’ = H), IV.-,, IV¢, IVdaI1d We (R' = H or CH3)
`are respectively obtained.
`
`DESCRIPTION OF THE INVENTION
`
`We have found that the 8’-hydroxy compounds of
`Structure IV can be acylated to give a new class of
`8'-acyloxy compounds of the structure defined by For-
`mulas ] and II and the definitions thereunder. These
`new compounds are not_ formed in the fermentations
`described by Endo, Monaghan, Albers-Schonberg or
`(311110. They are inhibitors of cholesterol synthesis in
`vivo.
`
`The absolute configuration of these compounds is
`known from X-ray diffraction. Table I provides a con-
`venient tabulation of these structures and their stereo-
`
`chemical relationship. The reference numerals to the
`various compounds. including those of the various se-
`ries of polyhydronaphthyl structures, remain the same
`throughout these specifications and are so used. Each of
`the esters IN (R'=CH3), of this invention contains
`seven or eight chiral centers. The relative and absolute
`configuration of these asymmetric centers is as depicted
`in Table I. More specifically, for ester 1,, (R' =CH3). the
`Cahn, Ingold, Prelog dengnations for the absolute con-
`figurations are 4-(R), 6(R), l'(S), 2'(S), 6'(R), 8'(S) and
`8a'(R) [R. S. Calm, C. Ingold and V. Prelog, Angew.
`Chem. Int. Ed, 5, 385 0966)].
`
`601']?
`
`PENN EX. 2191
`
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`4,450, 1? 1
`
`12
`TABLE I-continued
`THE COMPOUNDS or nus INVENTION AND muss.
`STEREO-RELATIONSHIP
`
`
`
`11
`
`H0
`
`‘
`
`0
`
`
`
`1:01’ = CH3)
`
`As is indicated in the formulas I”, all of these com-
`pounds have the same spatial orientation of groups at
`each chiral carbon atom and therefore belong to the
`same stereochemical series. The R-S designafion for
`each center may not be identical to that found for the
`ester Ia (R'=CIi3) because of the details of the sequence
`rules used for determining that designation. In the two
`esters Id and I; which have an additional chiral carbon
`atom not present in ester Is. the hydrogen atom at 4a’ is
`in the down (or o.) orientation as depicted in Table I,
`giving a trans ring junction.
`TABLE I
`THE‘. COMPOUNDS OF THIS INVENTION AND THIECR
`STEREO-RELATIONSHIP
`
` O
`
`
`
`HO
`
`
`
`Ho
`
`éo
`
`
`
`1!.’ = H or CH3
`3'I'EREDCH.'EM1s'I'RY OF THE HYDRONAPHTHYL
`SERIES
`Double Bonds Present
`Structure
`X and Z
`
`
`
`Series
`a
`
`b
`
`c
`
`d
`
`e
`
`' X
`
`Y
`
`2
`
`None
`
`Rr
`
`I
`
`CE)"
`r.*‘‘‘‘.
`‘E!
`Rn
`
`it
`
`The compounds of this invention are useful as antihy-
`percholesterolemic agents for the treatment of athero-
`sclerosis, hyperlipemia and like diseases in humans.
`They may be administered orally or parenterally in the
`form of a capsule, a tablet, an injectable preparation or
`the like. It is usually desirable to use the oral route.
`Doses may -be varied. depending on the age, severity,
`body weight and other conditions of human patients but
`daily dosage for adults is within a range of from about
`2' mg to 2000 mg (preferably 10 to 100 mg) given in
`three of four divided doses. Higher doses may be favor-
`ably applied as required.
`,
`The compounds of this invention also have useful
`anti-fungal activities. For example, they may be used to
`control strains of Penicillium sp., Aspergiilus niger, C1ad~
`osporium sp., Cachiliaboius miyabeames and HeIminIhos-
`parium Ljpnodncrir. For those utilities they are admixed
`with suitable formulating agents, powders, emulsifying
`agents or solvents such as aqueous ethanol and sprayed
`or dusted on the plants to be protected.
`The preparation of the compounds of this invention is
`described in Flow Sheet A.
`
`5
`
`10
`
`25
`
`30
`
`35
`
`45
`
`55
`
`65
`
`7ofl7
`
`PENN EX. 2191
`
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`4,450, 1 7 1
`
`14'
`
`13
`
`FLOW SHEET A
`CH3
`1
`‘EH3
`
`1u,,_,.1%,>1v,,_,ii%cH3-T——si—-o4aC|4(’
`
`1va—"'—->1v,,,,_,
`
`
`
`Definitions
`X, Y, Z, R and R’ as defined in specification and
`series a-e as defined in Table I.
`Reactions
`(1) Lithium hydroxide, heat, acidify, and lactonize
`(2) t-Butyldimethylchlorsilane and imidazole in DMF
`at ambient temperatures in an inert atmosphere.
`(3)
`Treatment with
`RCOCI
`and
`4-dime-
`thylaminopyricline in pyridine solution preferably
`under inert atmosphere.
`(4) Treatment with RCOOH and N,N'-dicyclohexyl-
`carbodiimide and 4-pyrrolidinopyridine in dichlo-
`romethane, preferably under an inert atmosphere.
`(5) Three equivalents of tetrabutylammonium fluo-
`ride and four equivalents of acetic acid per equiva-
`lent of ester in THF, preferably in an inert atmo-
`sphere.
`(6) Aqueous alkali followed by careful acidification
`with dilute acid.
`'
`(7) See‘ Reactions and Reagents and Flow Sheet for
`synthesis of IIIi._c,,,.
`In the novel process of this invention the 4—hydroxyl
`on the pyranone ring, of alcohols IV,” is first protected
`with a t-butyldimethylsilyl group by reaction with t-
`butyldimethylchlorosilane in an inert atmosphere at
`ambient temperatures in the presence of an acid accep-
`tor such as imidazole to provide the protected alcohols
`V“. The 8-hydroxyl on the polyhydronaphthyl ring is
`then acylated in one of two ways. The first comprises
`treatment with the acid chloride of the desired acyl
`group in
`pyridine
`in
`the presence of 4-dime-
`tliylaminopyridine as a catalyst. The second comprises
`treatment of the 8’-polyhydronaphthol with the free
`acid of the desired acyl group and a carbodiimide such
`
`4-pyr-
`with
`N,N‘-dicyclohexylcarbodiimide
`as
`rolidinopyridine as a catalyst
`in dichloromethane.
`These procedures give the protected esters V1,”. The
`removal of the silyl protecti.ng group from the'.4-
`hydroxyl of the pyranonc ring is then carried out, using
`three equivalents of tetrabutylammonium fluoride and
`four equivalents of acetic acid per equivalent of esters
`VI”, to give the desired compounds I“. The ratio of
`reagents in this last reaction is critical to the yield of the
`process and the purity of the products.
`The acyl groups thus put on the 8’-hydroxyl are those
`in which R in I,” is:
`'
`I
`1,1-
`(1) 1,1-diethylpropyl,
`l-ethyl-1-methylpropyl,
`dimethyletllyl, 1,1-dicthylbutyl, 1, 1 -dimethylpro-
`pills
`I
`
`(2) C3-lcfiycloalkyl,
`(3) C1-toalkcnyl.
`(4) C140 CF3-substituted alkyl,
`(5) phenyl,
`.
`.
`{6)halophe1-iyl, wherein halo is chloro, fluoro, bromo
`or iodo,
`'
`.
`(7) phenyl—C1.3 allay],
`(S) substituted phenyl-C1_.3 alkyl in which the substitu-
`ent is halo, such as fluoro, chloro, bromo, or iodo,
`C1-3 alkyl or C1.:; alkoxy.
`It is preferred that R’ be CH3.
`Preferred definitions of R, are: 1,1-diethylpropyl,
`l-ethyl-1-methylpropyl, 1,1-dimethylethyl, 1, 1-die-
`thylbutyl, 1,l—dimethylpropyl, C3-1o cycloalkyl,
`C340 alkenyl in which the unsaturation is not in
`conjunction with the carbonyl.
`Preferred species are those wherein R is 1,l-dime-
`thylpropyl. And it is also preferred that none of X, Y or
`Z is a double‘ bond.
`'
`
`I
`
`Compounds I“. can be hydrolyzed with bases such as
`Na0I-I to yield the salts such as the sodium salt of Com-
`pounds II,,.,,.. The use of bases with other pharmaceuti-
`cally acceptable cations affords salts of those cations.
`Careful acidification of the salts affords the hydroxy
`acids II“. which revert to Compounds 1,” at acidic pH.
`Treating Compound I“ under acidic or basic catalysis
`with methanol, ethanol, propanol, or butanol or with
`phenyl-,
`dimethylamino-,
`or
`acetylamino-alkanols
`yields the corresponding esters of Compounds II“
`which also fonna part of this invention.
`The pharmaceutically acceptable salts of this'inven-
`tion include those formed from cations such as sodium,
`potassium, aluminum, calcium,
`lithium, magnesium,
`zinc and tetramethylammonium as well as those salts"
`formed from amines such as ammonia, ethylenediamine,
`N-methylglucamine, lysine, arginine, ornithine, choline,
`N,N’-dibenzylethylenediamine, chloroprocaine, dieth-
`anolamine. procaine, N-benzylphenethylamine,
`l-p-
`chlorobenzyl-2-pyrrolidine l’-yl-methylbenzimidazole,
`diethylamine,
`piperazine,
`and
`tris(hydroxymethyl-
`)a.rninomethane.
`
`15
`
`20
`
`25
`
`35
`
`45
`
`55
`
`65
`
`8ofl7
`
`PENN EX. 2191
`
`CFAD V. UPENN
`lPR20l5-01836
`
`

`
`15
`
`EXAMPLE 1
`
`4,450, 17 l
`
`I0
`
`15
`
`5(R)-i2-(3’(S)- ".2"'di111¢thY1PT0PfiI10}‘10X¥-2'(5). TR)-
`dimetl1yl-l’,2’,6’;7',8',8'a(R)-hexahydronaphthyhl‘(S))-
`ethyl]-4{R)-hydroxy-Batfi,6-tetrahydro-21-1-pyram
`ne
`Step A: Preparation of
`5(R)-[2-(3'(S)-1lYd1'0XY-2'(S)-5'(R)-dimethY1-
`1',2',6’,7',8',8'a(R)-hexahyd1'Onaphthyl-