Lovastatin

LOVASTATIN

Gerald S. Brenner, Dean K. Ellison, and Michael J . Kaufman

Merck Sharp & Dohme Research Laboratories West Point, PA 19486

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS - VOLUME 21

277

Copyright @ 1992 by Academic Press, Inc. All rights of reproduction reserved in any form.

GERALD S. BRENNER, DEAN K . ELLISON, AND MICHAEL J. KAUFMAN

278

LOVASTAT IN Gerald S. Brenner Dean K. Ellison Michael J. Kaufman

1. History and Therapeutic Properties 2. Description 2.1 Nomenclature 2.1.l Chemical Name 2.1.2 Generic Name (USAN) 2.1.3 Laboratory Codes 2.1.4 Trade Names 2.1.5 Trivial Names 2.1.6

Chemical Abstract Services (CAS)

2.2 Structure, Formula and Molecular Weight 2.3 Appearance 3. Synthesis

4. Physical Properties Infrared Spectrum Proton Nuclear Magnetic Resonance Spectrum Carbon-13 Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Mass Spectrum Optical Rotation Thermal Behavior Solubility 4.9 Crystal Properties 4.10 Dissociation Constants 4.11 Partition Behavior

4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8

LOVASTATIN

5. Methods of Analysis

5.1 Elemental Analysis 5.2 Chromatography 5.2.1 Thin Layer Chromatography 5.2.2 High Performance Liquid Chromatography 5.3 Flow Injection Analysis 5.4 Identification Tests 6. Stability and Degradation 6.1 Solid State Stability 6.2 Solution Stability 7. Pharmacokinetics and Metabolism 7.1 Absorption and Distribution 7.2 Metabolism 7.3 Excretion 8. Determination in Biological Fluids

9. References

219

GERALD S . BRENNER, DEAN K. ELLISON, AND MICHAEL I. KAUFMAN

280

1. Historv and TheraDeutic ProDerties It was discovered by the Merck Sharp & Dohme Research Laboratories that a strain of Aspergillus terreus obtained from a soil sample produced the cholesterol lowering fungal metabolite lovastatin (initially named mevinolin). Details of the isolation, structural characterization and biochemical properties of lovastatin have been summarized by Alberts et al. (1). Lovastatin is identical to monacolin K isolated independently from Monascus ruber by Endo

(2). Lovastatin is a prodrug. After oral administration, the inactive parent lactone is hydrolyzed to the corresponding hydroxyacid form. The hydroxyacid is the principle metabolite and a potent inhibitor of 3-

Lactone

Hydroxyacid

hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase. This enzyme catalyzes the conversion of hydroxymethylglutarate to mevalonate, which is an early and rate limiting step in the biosynthesis of cholesterol. The effectiveness of lovastatin in lowering cholesterol has been confirmed clinically and it is approved for the treatment of primary hypercholesterolemia. Several review articles give a detailed account of the discovery, preclinical evaluation, mechanism of action, biological profile, and clinical evaluation of the drug (3-7).

2. DescriDtion 2.1

Nomenclature 2.1.1

Chemical Name [l S-[ 1a(R*),3a,7P,8P(2S*,4S*),8a~]]-2-Methylbutanoic acid 1,2,3,7,8,8a-hexahydro-3,7-dirnethyl-8-[2-

LOVASTATIN

(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1naphthalenyl ester; (1S,3R,7S,8SI8aR)-1 ,2,3,7,8,8ahexahydro-3,7-dimethyl-8-[2-[(2R,4R)-tetrahydro-4hydroxy-6-0~0-2H-pyran-2-ylJethyl]1-naphthatenyl (S)2-methylbutyrate; 1,2,6,7,8,8a-hexahydro-P,6dihydroxy-2,6-dimethyl-8-(2-methyl-l-oxobutoxy)-1naphthaleneheptanoic acid Glactone; PP,Ga-dimethyl8a-(2-methyl-l -oxobutoxy)-mevinicacid lactone.

2.1.2

Generic Name (USAN1 Lovastatin

2.1.3

Laboratow Codes L-l54,803-000G MK-0803

2.1.4

Trade Names Mevacor; Mevinacor; Mevlor

2.1.5

Trivial Names Mevinolin Monacolin K 3-Methyl Compactin

2.1.6

Chemical Abstracts Services GAS1 Registry Number: 75330-75-5

2.2

Structure, Formula, and Molecular Weiaht Structure:

28 I

GERALD S . BREWER, DEAN K. ELLISON, AND MICHAEL I. KAUFMAN

282

Molecular Formula: C O H ,, Molecular Weiaht: 40&5

Lovastatin is a white, crystalline powder. 3. Synthesis There have been numerous approaches to the total synthesis of lovastatin (8-10); however, lovastatin is produced commercially via a multi-stage fermentation process which originates from cultures of a strain of Aspergilks ferreus. The complete details of the isolation and identification of lovastatin from the fermentation media have been described (1). Synthetic approaches have been reviewed (1 1). 4. Phvsical Properties 4.1

Infrared Spectrum The infrared spectrum of lovastatin is shown in Figure 1 (12). The spectrum was obtained as a potassium bromide pellet using a Nicolet Model 7199 FT-IR spectrophotometer. Assignments for the characteristic absorption bands are shown below.

Wavenumber (cm-’ 1 3542 3016 296 7 2929 2866 1725 1711 1700 1460 1384 1359 1260 1222 1072 1056 969 87 1

Assignment Alcohol 0-H stretch Olefinic C-H stretch Methyl C-H asymmetric stretch Methylene C-H asymmetric stretch Methyl and methylene C-H asymmetric stretch Lactone and ester carbonyl stretch (hydrogen bonded for 1711 and 1700 cm-’ ) Methyl asymmetric bend Methyl symmetric bend Methylene symmetric bend Lactone C-0-C asymmetric bend Ester C-0-C asymmetric bend Lactone C-0-C symmetric stretch Ester C-0-C symmetric stretch Alcohol C-OH stretch Trisubstituted olefinic C-H wag

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283

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1

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4000 3600 3200 2800 2400 2000 1600 1200 800 Wavenumbers Figure 1. Infrared Absorption Spectrum of Lovastatin

2R4

4.2

GERALD S. BRENNER. DEAN K. ELLISON. AND MICHAEL J. KAUFMAN

Proton Nuclear Mametic Resonance Spectrum The proton magnetic spectrum is shown in Figure 2 (13). This spectrum was obtained on a Bruker Instruments Model AM-300 NMR spectrometer using a 4% w h solution of lovastatin in deuterated chloroform. Chemical shifts (6) are expressed as ppm downfield from tetramethylsilane (internal standard). The tabulated signal assignments refer to the numbered structure of lovastatin shown below.

6 (mml 0.88 0.89 1.08 1.11 1.20-2.05 2.20-2.50 2.55-2.77 4.37 4.64 5.38 5.53 5.78 6.00 7.27

'

Multiplicity /J

Assianment

t/J = 7.6 HZ d/J = 7.3 HZ d/J = 7.4 HZ d/J = 7.0 HZ Overlapping Multiplets Overlapping Multiplets Overlapping Multiplets m m m Broad t d Of d/J = 6.1, 9.6 Hz dN = 9.6 HZ S

1 Multiplicity: s, singlet; d, doublet; t, triplet; m, multiplet

PPM Figure 2. Proton Nuclear Magnetic Resonance Spectrum of Lovastatin

286

4.3

GERALD S. BRENNER, DEAN K . ELLISON, AND MICHAEL J. KAUFMAN

Carbon-13 Nuclear Maanetic Resonance Spectrum The carbon-13 nuclear magnetic resonance spectrum of lovastatin shown in Figure 3 was obtained using a Bruker Instruments Model AM-300 NMR spectrometer and an approximately 4% w/v solution of the compound in deuterochloroform. Signal assignments are tabulated below and refer to the numbered structure shown in Section 4.2 Chemical Shift (61, Dpm

Assianment

11.69 13.83 16.21 22.79 24.23 26.78 27.39 30.63 32.62 32.90 36.06 36.55 37.24 38.55 41.46 62.52 67.86 76.37 77.00 128.26 129.58 1315 3 133.03 170.50 176.88

In recent publications, the 1H and 13C NMR spectra of lovastatin were fully assigned by the use of selective homonuclear and heteronuclear decoupling and two dimensional techniques (14,15).

Figure 3. Carbon-13 Nuclear Magnetic Resonance Spectrum of Lovastatin

288

4.4

GERALD S. RRENNER. DEAN K . ELLISON. AND MICHAEL J . KAUFMAN

Ultraviolet Spectrum The ultraviolet (UV) absorption spectrum of lovastatin is characterized by absorption maxima at 231,238, and 247 nm with A l % values of 538, 629, and 424, respectively. The absorption maxima at 238 nm is typical for a trisubstituted heteroannular diene chromophore (16). A UV spectrum of lovastatin (c = 0.015 mg/mL in acetonitrile) is shown in Figure 4.

4.5

Mass Spectrum The mass spectrum of lovastatin is shown in Figure 5. This spectrum was obtained by the direct probe electron impact (90 eV) method using a Finnigan MAT 212 mass spectrometer (17). The spectrum exhibits a weak molecular ion signal at m/z = 404 (C H 0 , exact mass calculated = 404.2563; observed = 461.2%6lf. Other pertinent fragment ions are at m/z = 302, 284, and 159; these ions can be rationalized by the fragmentation pattern shown in Figure 6.

4.6

ODtical Rotation Lovastatin has eight chiral centers and is optically active. The specific rotation a [2 ,]5 is +330" for a 5.0 mg/mL solution in acetonitrile.

4.7

Thermal Behavior The differential scanning calorimetry (DSC) curve for lovastatin at a heating rate of 2"/min under a nitrogen atmosphere is shown in Figure 7. The thermogram is characterized by a single melting endotherm with an extrapolated onset temperature for melting of 175°C which is independent of heating rate from 2-2O0C/min. In contrast, the DSC thermogram for lovastatin obtained at a heating rate of 2"/min in air (Figure 8) exhibits an exotherm at 154°C which is attributed to oxidative reactions occurring in the non-inerted atmosphere. The thermal properties of lovastatin, in particular those derived from DSC experiments, have been used to assess the oxidative stability of the compound (18,19).

Wavelength (nm) Figure 4. Ultraviolet Absorption Spectrum of Lovastatin

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172

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100

150

200

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300

1

350

Figure 5 . Direct Probe Electron Impact Mass Spectrum of Lovastatin

400

29 I

LOVASTATIN

m/z 302

mlz 159

m/z 284

Figure 6. Proposed Fragmentation Pattern to Explain the Mass Spectrum of tovastastin

292

Figure 7.

DSC Therrnogram for Lovastatin under Nitrogen

293

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294

4.8

GERALD S . BRENNER. DEAN K. ELLISON, AND MICHAEL J. KAUFMAN

Solubility Lovastatin is insoluble in water, and is sparingly soluble in the lower alcohols (methanol, ethanol, and i-propanol). Solubility data obtained at room temperature are tabulated below (20). Solvent Acetone Acetonitrile n-Butanol i-Butanol Chloroform N,N-dimethylformamide Ethanol Methanol n-Octanol n-Propanol i-Propanol Water

4.9

Solubility ImalmL) 47 28 7 14 350 90 16 28 2 11 20 0.4

Crvstal Properties Lovastatin is a white, crystalline, non-hygroscopic solid. Single crystal X-ray diffraction experiments on a sample crystallized from ethanol indicate that the space group is P2,2 2, with a = 5.974A, b = 17.337A, and c = 22.148A. The calculated density is 1.17 g/cm3. (1) The X-ray powder diffraction pattern for lovastatin is shown in Figure 9. This spectrum was obtained on a Phillips APD 3720 X-ray diffractometer using CuKa irradiation. No crystal forms (polymorphs) other than that represented by the X-ray pattern in Figure 9 have been observed.

4.1 0 Dissociation Constants

Consistent with the structure, lovastatin exhibits no acidhase dissociation constants. Potentiometric titration of a sample in 50% aqueous methanol revealed no observable buffering action in the pH range of 2-1 1.

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GERALD S. BRENNER, DEAN K. ELLISON, AND MICHAEL J. KAUFMAN

296

4.11 Partition Behavior

In the n-octanol/water test system, lovastatin partitions quantitatively into the organic phase. At room temperature, the = 1.2 x 104. The partition coefficient is approximately K partition coefficient for the hydroxyaci82erivative (opened lactone form of lovastatin) between n-octanol and a pH 7.4 phosphate buffer is KO,, = 14.1 (21). 5. Methods of Analysis 5.1

Elemental Analysis Analysis of Merck Sharp & Dohme reference lot L-154,803000G102 for carbon and hydrogen gives values compared to calculated values as given below: Calculated Carbon Hydrogen

5.2

71.25 8.97

Found 71.26 9.18

Chromatoqraphy 5.2.1

Thin-Layer Chromatoaraphv Table I lists the thin layer chromatographic systems which have been used for the analysis of lovastatin. Table 1

Thin-Layer ChromatographicSystems for Lovastatin (221 Solvent System Toluenehnethanol

Plate Type

Rf

System

70/30

Analtech@ Silica Gel GF

0.77

1

Toluenelacetone 70BO

Analtech@ Silica Gel GF

0.48

2

Cyclohexaneh-butanoI/ethyl acetate

Analtech@ Silica Gel GF

0.43

3

Cyclohexane/chloroformlisopropanol 5:2:1

E. Merck Silica 0.60 Gel 60 F254 High Performance

4

4:l:l

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291

Visualization is either by viewing the developed plate under ultraviolet light or by spraying the developed plate with a dilute methanolic sulfuric acid solution and application of heat. System 4 with sulfuric acid spray detection is the most useful system because non-UV absorbing impurities are detectable.

5.3.2

Hiqh Performance Liquid Chromatoaraphv (HPLC)

A variety of gradient and isocratic reverse phase HPLC systems have been used to chromatograph lovastatin (see Table 2). Table 2 Hiqh Performance Liquid Chromatoqraphic Systems Application

System No.

Column

Mobile Phase

nm Detection

238

Ref

Drug substance purity

1

Whatrnan Partisil C-8

A = Acetonitrile B = 0.1% (v/v%) H3P04 aqueous A:B 70:30

Measurement of low level impurities in drug substances

2

Whatman Partisil C-8

Gradient 238 and A = Acetonitrile 200 nrn B = 0.1% (vW/O) H3P04 aqueous

(24)

Measurement in plasma and bile

3

Sepralyte C-18 lsocratic and 238 nm Gradient A = 0.05 ( NH4)3P04 and 0.01 H3P04 Buffer B = acetonltrile A:B 5050 (isocratic)

(25)

260 nrn

(26)

An

Measurement of low levels in fermentation broth'

4

DuPont Zorbax C-8

Measurement in tablets

5

Waters

M

A = acetonitrile B = methanol A:B:C

(23)

62229

C = water

A = acetonitrile 238 nm B = water (0.04M KH2P04 pH s 4) 60:40 A:B

(27)

GERALD S. BRENNER. DEAN K. ELLISON, AND MICHAEL J . KAUFMAN

298

Table 2 (Cont'd) High Performance Liquid Chromatographic Systems Application

System No.

Measurement in tablets

6

nm Detection

Ref

230 nm

(28)

Column

Mobile Phase

Hypersil 5 micron ODS

A = 0.025M NaH2P04

pH = 4 B = CH CN C = MebH 33:55:12 A:B:C Derivatization of lovastatin described.

5.3

Flow lniection Analysis A flow injection analysis system has been described by Mazzo

-et al. to simultaneously monitor lovastatin and antioxidants in tablets (29).

5.4

Identification Tests Three methods are routinely used to identify lovastatin: 1. the infrared spectrum; 2. the ultraviolet spectrum; and 3. the chromatographic retention time.

6. Stability and Degradation 6.1

Solid State Stability Crystalline lovastatin stored at room temperature yields with time trace amounts of oxidation products. The oxidative pathway for degradation has been supported with data generated by chromatography, degradate isolation, and identification, differential scanning calorimetry and heat conduction calorimetry. No products of nonoxidative degradation have been detected. HPLC and TLC studies have demonstrated that samples stored in air generate a complex mixture of largely unidentified trace polar products (30). These products are essentially absent and drug loss prevented in samples stored under nitrogen. For samples stored in air, all isolated and identified degradates result from oxidation and include the 4'-oxolactone which is the major

LOVASTATIN

299

degradate retaining the diene of the parent. The ultraviolet absorption spectra of air degradates indicate

Oxolactone that more than half of the mass has lost diene, suggesting that oxidation takes place primarily at this site (e.g., epoxidation and subsequent reactions of the resultant epoxides). Heat conduction calorimetry (19) and differential scanning calorimetry (18) also demonstrate the enhanced reactivity of the compound in an air vs. a nitrogen atmosphere. 6.2

Solution Stability The hydrolysis of the lactone ring of lovastatin occurs readily in aqueous solution especially under acidic or alkaline conditions (31). The acid catalyzed hydrolysis is reversible leading to a mixture of lactone and hydroxyacid, the equilibrium ratio of the two species being pH dependent. The rate to equilibrium is also pH dependent, being more rapid at acidic pH than near neutrality. In alkaline solution, the lactone ring is irreversibly converted to the hydroxyacid. Solutions of the hydroxyacid demonstrate good stability. Kaufman (32) has studied and determined the rate and equilibrium constants for the acid catalyzed hydrolysis of mevalonolactone, lovastatin and other structurally related HMG CoA reductase inhibitors in pH 2.0 buffer at 37°C. Under these conditions lactone concentrations decrease with time but do not approach zero indicating that the hydrolysis is reversible. The equilibrium nature of the reaction was further confirmed by repeating the experiment with hydroxyacid as starting material in the same system and demonstrating that an equilibrium composition is achieved that is identical to that achieved starting with lactone. Kinetic points in all studies were carried out to 15 hours and data obtained indicate that

GERALD S. BRENNER, DEAN K . ELLISON. A N D MICHAEL J. KAUFMAN

300

there are no side reactions (e.g., oxidation) competing with hydrolysis/lactonization during this time frame. The solution phase oxidation of a number of HMG CoA reductase inhibitors, including lovastatin, was studied in aqueous surfactant solutions at 40°C (33). Reaction rate constants were determined by monitoring oxygen consumption using an oxygen electrode. In the absence of a free radical initiator, there was no oxygen uptake indicating that the spontaneous rate of oxidation at 40°C was too slow to be detected. With an initiator present, all analogs consumed oxygen with the exception of the one in which the diene is saturated, demonstrating the diene functionality to be most labile to oxidation. Oxidation of lovastatin in aerated ethylene dichloride solution at 35", containing a free radical initiator, has been monitored kinetically using HPLC (34). The degradates formed in this complex solution system, different from those in the solid state, are primarily oligomers, with peroxide groups within the backbone chain and hydroperoxide end groups. Also, some monomeric epoxides are formed. 7. Pharmacokinetics and Metabolism Lovastatin is an inactive prodrug which undergoes in vivo lactone hydrolysis to give the hydroxyacid derivative which is an inhibitor of HMG-CoA reductase. The pharmacokinetic and metabolic profile of lovastatin has been described in detail (33,3537). In the sections below, the absorption, distribution, metabolism, and excretion of lovastatin are briefly reviewed. For this discussion it is helpful to distinguish between active inhibitors (defined as the sum concentration of the hydroxyacid derivative of lovastatin plus other active hydroxyacid metabolites) and total inhibitors (the total concentration of active inhibitors plus lactones and conjugates). Active and total inhibitors can be separately quantitated by assaying samples before and after ex vivo hydrolysis of plasma samples. 7.1

Absorption and Distribution

In studies in laboratory animals, the absorption of lovastatin following oral administration is approximately 30% complete as estimated relative to an intravenous dose of the hydroxyacid. An intravenous formulation of lovastatin for human studies is

LOVASTATIN

30 I

not feasible due to its low aqueous solubility. In all species studied, lovastatin is converted to the hydroxyacid form in viva This conversion is apparently reversible since lovastatin is found in the biological fluids of rats and dogs following administration of the hydroxyacid. In animals, lovastatin is more efficiently extracted by the liver where it is converted to the active enzyme inhibitor. Accordingly, the systemic bioavailability of active inhibitors is less than 5% of an oral dose of lovastatin. The high hepatic extraction and low systemic availability are desirable features since the liver is the primary site of cholesterol biosynthesis. Peak plasma concentrations of both active and total inhibitors occur between 2-4 hours post dose, and the area under the curve (AUC) increases proportionally with dose. The hydroxyacid is rapidly cleared; plasma clearance and half-life range from 300-1248 mUmin and 1.1-1.7 hrs, respectively. When lovastatin is administered with food, a 50% increase in AUC for inhibitory activity is attained relative to administration in the fasted state. The plasma protein binding of lovastatin and the hydroxyacid form has been determined by equilibrium dialysis. Both forms are greater than 95% protein bound. 7.2

Metabolism Lovastatin is extensively metabolized to give both active and inactive compounds. The major active metabolites present in human plasma are the hydroxyacid of lovastatin and its 3hydroxy-, 3-hydroxymethyl, and 3-exomethylenederivatives. The 3-hydroxylated metabolite is approximately 70% as active as the non-hydroxylated metabolite. In human bile, the 3hydroxylated metabolite undergoes an allylic rearrangement to give the 6-hydroxy isomer which is inactive (38):

"3C

GERALD S. BRENNER. DEAN K . ELLISON. A N D MICHAEL J . KAUFMAN

302

All of the hydroxyacid metabolites also exist in their corresponding inactive lactone forms. After base hydrolysis to convert lactones to active inhibitors, about 80% of the total enzyme inhibitory activity in human plasma is accounted for by these four lactonelhydroxyacidpairs.

7.3

Excretion The excretion of lovastatin has been assessed following an oral dose of 14C-labeled compound in man. Total recovery of drug equivalents in urine and feces averaged 10% and 83%, respectively. A substantial amount of radioactivity is also recovered in the feces following intravenous dosing of 14Clabeled hydroxyacid, indicating that biliary excretion is an important elimination for orally administered lovastatin.

8. Determination in Bioloaical Fluids

An enzyme inhibition assay capable of measuring total HMG-CoA reductase inhibitors in biological fluids has been described in the literature (1). The basis of this assay is the in vitro inhibition of the HMG-CoA reductase catalyzed conversion of 14C-HMG-CoA to 14Cmevalonic acid. The concentration of inhibitors can be measured before and after base hydrolysis of plasma samples. The measurement before hydrolysis gives the concentration of inherently active species (active inhibitors). Base hydrolysis irreversibly converts inactive but potentially active species (lactones and conjugates) to their corresponding active forms; the inhibition assay of hydrolyzed samples thus provides the concentration of total inhibitors. The enzyme inhibition assay is sensitive (detection limit of ca. 5 ng/mL), but is not specific for lovastatin. The determination of lovastatin and its hydroxyacid metabolite in plasma and bile can be accomplished by high performance liquid chromatography (25). Plasma samples are prepared for analysis by solid phase extraction and are analyzed using isocratic elution on a C18 column. Bile samples do not require any sample clean-up prior to HPLC analysis, but do require the use of a gradient elution method to separate the compounds of interest. The HPLC assay has a limit of detection of 25 ng/mL. An analytical method for the determination of lovastatin in serum based on gas chromatography/massspectrometry has recently been reported (39).

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Acknowledclements The authors wish to thank Mrs. Laurie Rittle for typing the manuscript and Ms. Agnes Hendrick for performing the literature search. 9. References 1.

A.W. Alberts, J. Chen, G. Kuron, V. Hunt, J. Huff, C. Hoffman, J. Rothrock, M. Lopez, H. Joshua, E. Harris, A. Patchett, R. Monaghan, S. Currie, E. Stapley, G. Albers-Schonberg, 0. Hensens, J. Hirshfield, K. Hoogsteen, J. Liesch and J. Springer, Roc. Natl. Acad. Sci. USA 77,3957 (1980).

2.

A. Endo, J. Antibiot. 32, 852 (1979).

3.

J.S. MacDonald, R.J. Gerson, D.J. Kornburst, M.W. Kloss, S. Prahalada, P.H. Berry, A.W. Alberts and D.L. Bokelman, Am. J. Cardiol. 62, 16J (1988).

4.

E.E.Stater and J.S. MacDonald, Drugs 36 (Suppl. 3), 72 (1988).

5.

J.M. McKenney, Clin. Pbarm. 7, 21 (1988).

6.

A.W. Alberts, Am. J. Cardiol. 62, 1OJ (1988).

7.

J.A. Tobert, Circulation 76, 534 (1987).

8.

S.J. Hecker and C.H. Heathcock, J. Org. Chem. 50,5159

9.

M. Hirama and M. Iwashita, Tetrahedron Letters 24, 1811 (1983).

10.

D.L.J. Clive, K.S.K. Murthy, A.G. Wee, J.S. Prasad, M. Majewski, P.C. Anderson, C.F. Evans, R.D. Hauger, L.D. Heerz and J.R. Barrie, J. Am. Cbern. SOC.112, 3018 (1990).

11.

T. Rosen and C.H. Heathcock, Tetrahedron 42, 4909 (1986).

12.

R. Cervino, Merck Sharp & Dohme Research Laboratories, personal communication.

(1985).

304

GERALD S.BRENNER, DEAN K. ELLISON, AND MICHAEL J. KAUFMAK

13.

R. Reamer, Merck Sharp & Dohme Research Laboratories, personal communication.

14.

J.K. Chan, R.N. Moore, T.T. Nakashima, J.C. Vederas, J. Am. Chem. SOC.105, 3334 (1 983).

15.

R.N. Moore, G. Bigam, J.K. Chan, A.M.Hogg, T.T. Nakashima, J.C. Vederas, J. Am. Chem. SOC.107,3694 (1985).

16.

A.I. Scott, Interpretation of the Ultraviolet Spectra of Natural Products, Pergamon Press, Oxford, 1964.

17.

D. Zink, Merck Sharp & Dohme Research Laboratories, personal communication.

18.

J.P. Elder, Thermochim. Acta 134, 41 (1988).

19.

L.D. Hansen, E.A. Lewis, D.J. Eatough, R.G. Bergstrom, D. Degraft-Johnson, Pharm. Res. 6, 20 (1989).

20.

A.Y.S. Yang, Merck Sharp & Dohme Research Laboratories, personal communication.

21.

M.J. Kaufman, Merck Sharp & Dohme Research Laboratories, personal communication.

22.

A.Y.S. Yang, L. Pierson and J. Baiano, Merck Sharp & Dohme Research Laboratories, personal communication.

23.

A.H. Houck, Merck Sharp & Dohme Research Laboratories, personal communication.

24.

A.H. Houck, S. Thomas and D.K. Ellison, Pittsburgh Conference (1990),manuscript in preparation.

25.

R.J. Stubbs, M. Schwartz and W.F. Bayne, J. Chromatog. 383, 438 (1986).

26.

V.P. Gullo, R.T. Goegelrnan, I. Putter and Y. Lam, J. Chromatog. 212, 234 (1 98 1).

27.

L.L. Ng, Anal. Chem. 53, 1142 (1981).

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28. C.V. Bell and J.C. Wahlich, Merck Sharp & Dohme Research Laboratories, personal communication.

29.

D.I. Mazzo, S.E. Biffar, K.A. Forbes, C. Bell and M.A. Brooks, J. Pharm. Biomed. Anal. 6, 271 (1 988).

30.

M. Baum, G.Dezeny, L. DiMichele, R. Reamer and G.B. Smith, Merck Sharp & Dohme Research Laboratories, personal communication.

31.

A.Y.S. Yang, Merck Sharp & Dohme Research Laboratories, personal communication.

32.

M.J. Kaufman, Int. J. Pharm. 66, 97 (1990).

33.

M.J. Kaufman, Pharm. Res. 7, 289 (1990).

34.

G. Dezeny and G.B. Smith, Merck Sharp & Dohme Research Laboratories, personal communication.

35. J.J. Krukemeyer and R.L. Talbert, Pharmacotherapy 7, 198 (1 987). 36.

D.E. Duggan, I.W. Chen, W.F. Bayne, R.A. Halpin, C.A. Dunca, M.S. Schwartz, R.J. Stubbs and S. Vickers, Drug Metab. Dispos. 17,166 (1989).

37.

D.E. Duggan and S. Vickers, Drug Metab. Rev. 22, 333 (1990).

38.

R.A. Halpin, K.P. Vyas, P. Kari, B.H. Arison, E.H. Ulrn and D.E. Duggan, Pharmacologist 29, 238 (1987).

39.

D. Wang-lverson, E. Ivashkiv, M. Jemal and A.I. Cohen, Rapid Comm. Mass. Spectrom. 3 , 132 (1989).