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Journal of Lipid Research, Vol. 46, 547-563, March 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Dual-action hypoglycemic and hypocholesterolemic agents that inhibit glycogen phosphorylase and lanosterol demethylase

H. James Harwood, Jr.^1,*, Stephen F. Petras^*, Dennis J. Hoover^*, Dayna C. Mankowski^*, Victor F. Soliman^*, Eliot D. Sugarman^*, Bernard Hulin^*, Younggil Kwon^*, E. Michael Gibbs^*, James T. Mayne and Judith L. Treadway^*

^* Departments of Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton Laboratories, Pfizer Inc., Eastern Point Road, Groton, CT 06340
Exploratory Toxicology, Pfizer Global Research and Development, Groton Laboratories, Pfizer Inc., Eastern Point Road, Groton, CT 06340

Published, JLR Papers in Press, December 16, 2004. DOI 10.1194/jlr.M400436-JLR200

² The plasma cholesterol lowering observed in experimental animals was not a consequence of interference by CP-320626 with the cholesterol oxidase–based cholesterol detection system, because the signal produced for a 400 mg/dl cholesterol standard was not altered by concentrations of CP-320626 between 0.1 µg/ml and 12 µg/ml (27 µM), concentrations that bracket plasma concentrations observed at efficacious doses.

¹ To whom correspondence should be addressed. e-mail: h_james_harwood{at}groton.pfizer.com

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Diabetic dyslipidemia requires simultaneous treatment with hypoglycemic agents and lipid-modulating drugs. We recently described glycogen phosphorylase inhibitors that reduce glycogenolysis in cells and lower plasma glucose in ob/ob mice (J. Med. Chem., 41: 2934, 1998). In evaluating the series prototype, CP-320626, in dogs, up to 90% reduction in plasma cholesterol was noted after 2 week treatment. Cholesterol reductions were also noted in ob/ob mice and in rats. In HepG2 cells, CP-320626 acutely and dose-dependently inhibited cholesterolgenesis without affecting fatty acid synthesis. Inhibition occurred together with a dose-dependent increase in the cholesterol precursor, lanosterol, suggesting that cholesterolgenesis inhibition was due to lanosterol 14-demethylase (CYP51) inhibition. In ob/ob mice, acute treatment with CP-320626 resulted in a decrease in hepatic cholesterolgenesis with concomitant lanosterol accumulation, further implicating CYP51 inhibition as the mechanism of cholesterol lowering in these animals. CP-320626 and analogs directly inhibited rhCYP51, and this inhibition was highly correlated with HepG2 cell cholesterolgenesis inhibition (R² = 0.77).

These observations indicate that CP-320626 inhibits cholesterolgenesis via direct inhibition of CYP51, and that this is the mechanism whereby CP-320626 lowers plasma cholesterol in experimental animals. Dual-action glycogenolysis and cholesterolgenesis inhibitors therefore have the potential to favorably affect both the hyperglycemia and the dyslipidemia of type 2 diabetes.

Supplementary key words cholesterol synthesis • glycogenolysis • glycogen phosphorylase • lanosterol 14-demethylase • CYP51 • enzyme inhibition • type 2 diabetes • HepG2 cells • ob/ob mice

Abbreviations: AUC, area under the curve; CYP, cytochrome-P450; DMEM, Dulbecco's modified Eagle's medium

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Type 2 diabetes is a severe and prevalent disease in the Western world and affects roughly 16 million persons in the US, and another 14 million people have impaired glucose tolerance (1). Projections indicate that the incidence of type 2 diabetes will increase to over 25 million by 2010 in the US, and to over 300 million worldwide by 2025 (1–3). The annual direct medical costs associated with type 2 diabetes, which in the United States was in excess of 44 billion dollars in 1997 (4), result primarily from secondary hyperglycemia-related complications, such as retinopathy, nephropathy, peripheral neuropathy, and cardiovascular, peripheral vascular, and cerebrovascular disease.

A high correlation exists between tighter glycemic control and reduction of these long-term complications in type 2 diabetes (5). Type 2 diabetics are currently treated with interventions to improve glycemia through a progressive regimen of diet, exercise, oral antidiabetic drugs (as monotherapy or in combination), and insulin (6). However, there is an ongoing need for additional oral antidiabetic agents that will achieve better glycemic control as monotherapy and/or work more safely or effectively in combination.

In addition to their hyperglycemia, patients with type 2 diabetes often present with a concomitant atherogenic dyslipidemia (elevated triglycerides, low HDL cholesterol, and small, dense LDL) that increases their risk of cardiovascular disease (7, 8). Because there is a high incidence of mortality for type 2 diabetics with their first myocardial infarction (7), aggressive therapy for treating diabetic dyslipidemia is recommended (7). It is suggested that initial lipid-modulating therapy be directed toward reducing LDL cholesterol levels to below 100 mg/dl through administration of a cholesterol synthesis inhibitor, such as a statin (HMG-CoA reductase inhibitor), and that this treatment be combined with a fibric acid derivative, such as fenofibrate, for patients with HDL cholesterol levels below 40 mg/dl and for patients with triglycerides that remain elevated (>150 mg/dl) after both improvement of glycemic control and initiation of statin therapy (7).

In pursuing new treatments for type 2 diabetes, we have targeted inhibition of glycogen phosphorylase (E.C. 2.4.1.1), the enzyme that catalyzes the hydrolytic release of glucose-1-phosphate from glycogen, as an approach to reducing hepatic glycogenolysis and thereby controlling plasma glucose levels (2). Through these efforts, we have identified a series of indole-2-carboxamide glycogen phosphorylase inhibitors (9, 10) that inhibit the human liver isoform of glycogen phosphorylase by binding at a unique allosteric regulatory site on the enzyme (11), reduce forskolin-induced glycogenolysis in SK-HEP-1 cells (10, 12), and exhibit glucose-lowering activity when given orally to diabetic ob/ob mice (10, 12).

Because of the potential pharmacological utility of this series of glycogen phosphorylase inhibitors, we have evaluated a representative analog, CP-320626 (Fig. 1) ; [IC₅₀ vs. human liver glycogen phosphorylase, 205 nM (10)], for its subchronic effects in diabetic ob/ob mice (10, 12), in rats, and in dogs. During the course of these studies, we discovered that CP-320626 reduced plasma cholesterol levels in a variety of normoglycemic, nondiabetic animals in a manner and magnitude inconsistent with its expected action as a glycogen phosphorylase inhibitor. Herein, we report identification of the mechanism responsible for the cholesterol-lowering action of CP-320626 as inhibition of the cholesterolgenic enzyme lanosterol 14-demethylase (CYP51), and characterize structure–activity relationships for this activity within the series.

View larger version (9K): [in this window] [in a new window]   Fig. 1. The structure of CP-320626.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Studies using experimental animals
All procedures using experimental animals were approved by the Institutional Animal Care and Use Procedures Review Board. Sprague Dawley rats, C57BL/6J-ob/ob mice, and Beagle dogs were given food and water ad libitum and treated orally at a volume of 1.0 ml/200 g body weight (rats), 0.25 ml/25 g body weight (mice), or 1.0 ml/kg body weight (dogs) with either an aqueous solution of 0.1% pluronic P-105 in 10% DMSO (vehicle) or an aqueous solution of 0.1% pluronic P-105 in 10% DMSO plus CP-320626.

Measurement of plasma, cholesterol, triglyceride, glucose, and related metabolite levels
Serum glucose triglyceride and total cholesterol concentrations were determined by the Abbott VP^TM and VP Super System^® Autoanalyzer using the A-Gent^TM Glucose-UV, A-Gent^TM Triglyceride and A-Gent^TM Cholesterol Test reagent systems. Serum insulin and glucagon concentrations were determined by radioimmunoassay (RIA) using kits from Binax (Portland, ME) and Amersham Corp. (Arlington Heights, IL), respectively. ß-Hydroxybutyrate concentration was determined spectrophotometrically using kits from Sigma. Free fatty acid concentration was determined using a kit from Wako (Richmond, VA). The serum glucose, insulin, glucagon, triglyceride, total cholesterol, ß-hydroxybutyrate, and free fatty acid–lowering activity of test compounds were determined by statistical analysis (unpaired t-test) with the vehicle-treated control group.

Measurement of plasma CP-320626 levels
To 100 µl aliquots of plasma were added 50 µl of an internal standard (2 µg/ml CP-89816 in methanol), 5 ml methyl tert-butyl ether, and 1 ml of 0.5 M sodium carbonate (pH 9). After vigorous mixing and centrifugation, the ether layers were removed and evaporated to dryness, and the resulting solid was reconstituted with 75 µl mobile phase [45% acetonitrile, 55% 50 mM sodium phosphate monobasic, and 30 mM triethylamine (pH 3)]. Aliquots (30 µl) of reconstituted samples were injected onto a 4 µm Waters Nova-Pak C-18 (Waters Corp, Bedford, MA) reverse phase column (3.9 x 150 mm), with a mobile phase flow rate of 1 ml/min. CP-320626 and internal standard were detected by fluorescence (excitation at 290 nm and emission at 348 nm). The linear dynamic range was between 0.1 µg/ml (lower limit of quantification) and 1 µg/ml (upper limit of quantification). C_max was the concentration in the blood sample in which the highest plasma concentration was measured. The area under the plasma concentration time curve (AUC) from 0 to tlast (AUC_0–tlast) was calculated using a linear trapezoidal approximation, where tlast is the time point of the last quantifiable plasma concentration.

Measurement of liver and plasma precursor sterols by gas chromatography-mass spectrometry
Nonsaponifiable lipids were isolated from liver and plasma and quantitated by gas chromatography-mass spectrometry (GC/MS). Samples of plasma (0.75 ml) and liver (0.75 g) were saponified at 70°C for 120 min in 2.5 ml of 2.5 M NaOH, then 5 ml of absolute EtOH was added to each sample and the solutions were mixed. Ten milliliters of petroleum ether was then added to each sample, and the mixtures were shaken vigorously for 2 min then centrifuged at 2,000 g in a bench-top Sorvall for 10 min. After a second petroleum ether extraction, the resultant petroleum ether layers were removed, dried under nitrogen, and dissolved in 500 µl dry pyridine solution. A 500 µl aliquot of TMSI + Pyridine, 1:4 (Sylon TP) was then added to each sample, and derivatization was allowed to continue at room temperature (RT) for 1 h. Derivatized samples were analyzed using an HP-6890 Series gas chromatograph equipped with a 6890 Series GC Injector and interfaced with an HP-5973N mass selective detector. Separation was achieved using a Supelco SAC-5 (15 m x 0.25 mm x 0.25 µm) GC column. The oven temperature was held at 250°C for 0 min, then heated to 300°C at a rate of 2°C/min. The ions monitored using full scan electron ionization (70 eV) corresponded to the molecular ions of the trimethylsilyl derivatives. Retention times of detectable precursor sterols relative to that of cholesterol were cholestanol (dihydrocholesterol), 1.03; 8-dehydrocholesterol, 1.06; desmosterol, 1.07; 7-dehydrocholesterol, 1.07; lathosterol, 1.10; 4-methylsterol, 1.20; 4,4-dimethylsterol, 1.20; lanosterol, 1.42; and dihydrolanosterol, 1.42, similar to those previously reported for C-3 hydroxyl-derivatized sterols (17, 18).

Based on GC/MS analysis, the sterol composition of control rat liver was 98% cholesterol, with 0.04% cholestanol and 8-dehydrocholesterol, 0.17% desmosterol and 7-dehydrocholesterol, 0.13% lathosterol, 1.11% monomethyl and dimethyl sterols, and 0.49% trimethylsterols (lanosterol and dihydrolanosterol). The sterol composition of control rat plasma was >99% cholesterol, with 0.57% monomethyl and dimethyl sterols and 0.38% trimethylsterols (lanosterol and dihydrolanosterol). Levels of cholestanol, 8-dehydrocholesterol, desmosterol, 7-dehydrocholesterol, and lathosterol in control rat plasma were all below the limits of detection.

Measurement of CYP51 activity
The activity of recombinant human CYP51, expressed in TOPP3 cells and partially purified by the method of Stromstedt, Rozman, and Waterman (19), was determined by measuring the conversion of lanosterol to 4,4-dimethylcholesta-8,14,24-trien-3ß-ol as previously described (19), with the following modifications: Briefly, 25 µl of a 1 mM suspension of lanosterol in a mixture of tyloxapol-acetone (1:1; v/v), dioleylphosphatidylcholine micelles (5 mg/ml), methanol, and 100 mM potassium phosphate buffer (pH 7.4) was added to 5 ml glass tubes to provide a final lanosterol concentration of 50 µM (approximate K_m). Inhibitors, dissolved in methanol for final concentrations ranging between 0.01 and 200 µM, were then added, and the lanosterol/inhibitor mixtures were allowed to dry under nitrogen for 10 min. To the residues were added 20 pmol partially purified recombinant human CYP51, 125 pmol human CYP reductase, and 50 µl rat lipid. After incubation at RT for 10–15 min for enzyme reconstitution, 540 µl of 100 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol, 0.1 mM DTT, 0.1 mM EDTA, and 0.5 mM KCN was added to each tube. Reaction mixtures were preincubated for 2 min at 37°C, then reactions were initiated by the addition of 50 µl of an NADPH regenerating system (final incubation concentration, 10 mM MgCl₂, 0.54 mM NADPH, 6.2 mM DL-isocitric acid, 0.5 U/ml isocitrate dehydrogenase). After 60 min incubation at 37°C, reactions were terminated by addition of a 25 µl volume of ethyl acetate that also contained the internal standard, ergosterol (1 mg/ml), followed by extraction with 5 ml ethyl acetate. After vigorous mixing and centrifugation to facilitate phase separation, 3–4 ml of the ethyl acetate phase was transferred to fresh tubes and evaporated to dryness under nitrogen at 50°C. Samples were reconstituted in 150 µl mobile phase (see below), and 25–50 µl was applied to an HPLC system. Ergosterol (internal standard) and 4,4-dimethylcholesta-8,14,24-trien-3ß-ol (reaction product) were separated on a Waters Novapak C18 column (4.0 µM, 150 mm x 3.9 mm), with a mobile phase consisting of methanol-acetonitrile-HPLC-grade water (45:45:10 v/v/v), at a flow rate of 1.5 ml/min. Ergosterol and 4,4-dimethylcholesta-8,14,24-trien-3ß-ol were monitored by UV detection at 248 nm using a SpectroMonitor variable wavelength detector (LDC Analytical; Riviera Beach, FL), and the Multichrom^TM data acquisiton system (Version. 2.11; Fisons Instruments, Beverly, MA) was used for data collection and analysis. Approximate retention times for 4,4-dimethylcholesta-8,14,24-trien-3ß-ol and ergosterol were 25 and 30 min, respectively.

Measurement of human liver glycogen phosphorylase a activity
The activity of recombinant human liver glycogen phosphorylase, expressed in baculovirus, purified to >95% homogeneity, and fully activated by phosphorylase kinase as previously described (20–22), was determined by measuring glycogen synthesis from glucose-1-phosphate by assessing the release of inorganic phosphate (reverse reaction) at 22°C in 100 µl of 50 mM HEPES buffer (pH 7.2) containing 100 mM KCl, 2.5 mM EGTA, 2.5 mM MgCl₂, 0.5 mM dithiothreitol, 0.63 mM glucose-1-phosphate, 1.25 mg/ml glycogen, 9.4 mM glucose, 0.7% DMSO, and up to 2 µg of partially purified, activated human liver glycogen phosphorylase, based on the method of Engers, Shechosky, and Madsen (22), as previously described (9). The inorganic phosphate released during a 60 min incubation was measured at 620 nm, 20 min after the addition of 150 µl of 1 M HCl containing 10 mg/ml ammonium molybdate and 0.38 mg/ml malachite green (23).

Measurement of sterol and fatty acid synthesis in cultured cells
Sterol and fatty acid synthesis were evaluated in HepG2 cells by measuring incorporation of [2-¹⁴C]acetate into cellular lipids as previously described (13, 14), with modifications (15, 16) to allow simultaneous assessment of both sterol and fatty acid synthesis. HepG2 cells grown in T-75 flasks as previously described (13, 14) were seeded into 24-well plates at a density of 1.2 x 10⁵ cells/well and maintained in 1.0 ml of supplemented DMEM (DMEM containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 40 µg/ml gentamicin) for 7 days in a 37°C, 5% CO₂ incubator with medium changes on days 3 and 5. On day 8, the medium was removed and replaced with fresh medium containing 1% DMSO ± effector compounds. Immediately after compound addition, 25 µl of media containing 4 µCi of [2-¹⁴C]acetate (56 mCi/mmol) was added to each incubation well. Plates were then sealed with parafilm to prevent evaporation, and cells were incubated at 37°C for 6 h with gentle shaking. After incubation, the samples were saponified by addition to each well of 1 ml of 5 N KOH in MeOH, followed first by incubation for 2 h at 70°C and then by overnight incubation at RT. Mixtures were transferred to glass conical tubes and extracted three times with 4.5 ml hexane. The pooled organic fractions (containing cholesterol, post-squalene cholesterol precursors, and other nonsaponifiable lipids) were dried under nitrogen, resuspended in 25 µl chloroform, and applied to 1 x 20 cm channels of Silica Gel 60C TLC plates. Channels containing nonradioactive cholesterol, lanosterol, and squalene were included on selected TLC plates as separation markers. TLC plates were developed in hexane-diethyl ether-acetic acid (70:30:2 v/v/v), air dried, and assessed for radioactivity using a Berthold Linear Radioactivity Analyzer (Oak Ridge, TN) that reports radioactive peak location and integrated peak area. The desmethylsterol peak (R_f = 0.27) was predominately (>99%) cholesterol and also contained traces of cholestanol, cholestenol, desmosterol, and lathosterol (24). The methylsterol peak (R_f = 0.34) contained lanosterol, dihydrolanosterol, 4-methylsterols, and 4,4-dimethylsterols (24). Squalene dioxide, squalene oxide, and squalene migrated with R_f values of 0.42, 0.65, and 0.88, respectively.

The remaining aqueous phase (containing fatty acid sodium salts) was acidified to pH<2 by addition of 0.5 ml of 12 M HCl. The resulting mixtures were then transferred to glass conical tubes and extracted three times with 4.5 ml hexane. The pooled organic fractions (containing protonated fatty acids) were dried under nitrogen, resuspended in 50 µl of chloroform-methanol (1:1; v/v) and applied to 1 x 20 cm channels of Silica Gel 60C TLC plates. Channels containing nonradioactive fatty acids were included on selected TLC plates as separation markers. TLC plates were developed in hexane-diethyl ether-acetic acid (70:30:2 v/v/v), air dried, and visualized for radioactive fatty acids by analysis using a Berthold Linear Radioactivity Analyzer that reports radioactive peak location and integrated peak area. Sterol and fatty acid synthesis are expressed as dpm [2-¹⁴C]acetate incorporated into either cholesterol, post-squalene cholesterolgenic intermediates, or saponifiable lipids during the 6 h incubation at 37°C.

Measurement of sterol biosynthesis in ob/ob mice
Hepatic incorporation of R,S-[2-¹⁴C]mevalonolactone into cholesterol and precursor sterols in C57BL/6J-ob/ob mice was assessed as previously described for hepatic incorporation of R,S-[2-¹⁴C] mevalonolactone into cholesterol and precursor sterols in golden Syrian hamsters (13), with the following modifications: Five- to eight-week-old male C57BL/6J-ob/ob mice were housed in groups of five animals each and were given food and water ad libitum for 1 week. After the 1 week acclimation period, animals weighing 50 g were administered by oral gavage 0.2 ml of either vehicle or vehicle containing CP-320626. One hour after compound administration, animals received an intraperitoneal injection of 0.1 ml of R,S-[2-¹⁴C]mevalonolactone (100 µCi/ml; 58 mCi/mmol). One hour after radiolabel administration, animals were euthanized by pentobarbital injection, and two 0.5 g liver pieces were removed. Tissue samples were saponified at 70°C for 120 min in 2.5 ml of 2.5 M NaOH, then 5 ml of absolute EtOH was added to each sample and the solutions were mixed. Ten milliliters of petroleum ether was then added to each sample, and the mixtures were first shaken vigorously for 2 min then centrifuged at 2000 g in a bench-top Sorvall for 10 min. The resultant petroleum ether layers were removed, and 5.0 ml aliquots were dried under nitrogen, resuspended in 25 µl chloroform, applied to 1 x 20 cm channels of Silica Gel 60C TLC plates, developed in hexane-diethyl ether-acetic acid (70:30:2 v/v/v), and visualized for radioactive nonsaponified lipids using a Berthold Linear Radioactivity Analyzer as described above. Sterol synthesis is expressed as dpm R,S-[2-¹⁴C]mevalonolactone incorporated into cholesterol or post-squalene cholesterolgenic intermediates per gram liver during the 1 h interval between radiolabeled mevalonate injection and pentobarbital administration.

Synthesis of inhibitors
The syntheses of CP-320626 [1], [3], and [5] are reported (10). For all other inhibitors, unless otherwise specified, NMR spectra were recorded at 20–21°C on a Varian XL-300 (Cary, NC) or a Bruker (Billerica, MA)AM-300 spectrometer at 300 mHz for proton and 75.4 mHz for carbon nuclei. NMR spectra at 400 mHz were obtained on a Varian UNITY400 spectrometer at 20–21°C. Chemical shifts are expressed in ppm downfield from trimethylsilane (external reference). Routine mass spectral data were obtained using either a Hewlett-Packard (Cambridge, MA)5989 MS Engine operated with a particle beam interface and ammonia chemical ionization (designated PBMS) or a Fisons Instruments Trio-1000 spectrometer operated with a thermospray ionization interface (ammonia, designated TSPMS). The cited ion was the base peak (100% relative intensity) unless otherwise specified. Where chlorine- or bromine-containing ions are described, the ³⁷Cl- or ⁸¹Br-containing ions were also observed in the expected ratio. High-resolution mass spectra (HRMS) were obtained on a Micromass LCT-TOF spectrometer (Waters, Millford, MA) using API electrospray ionization, acetonitrile-water-0.01% formic acid inlet mobile phase gradient, and internal calibrants (L-tryptophan and leucine enkephalin). Unless otherwise specified, reversed-phase HPLC (RP-HPLC) was performed with 214 nM detection on a 250 x 4.6 mm Rainin Microsorb (Woburn, MA) C-18 column eluted isocratically by a two-pump/mixer system supplying the indicated mixture of acetonitrile and aqueous pH 2.1 (with H₃PO₄) 0.1 M KH₂PO₄, respectively, at 1.5 ml/min, with sample injected in a 1:1 mixture of acetonitrile and pH 7.0 phosphate buffer (0.025 M in each Na₂HPO₄ and KH₂PO₄). Percent purity was generally that determined from a 10–15 min run time. Reagents and starting materials, where the preparation is not given, were used as obtained from commercial sources; dimethylformamide and CH₂Cl₂ used as reaction solvents were the anhydrous grade supplied by Aldrich Chemical Co. (Milwaukee, WI). Microanalyses were performed by Schwarzkopf Microanalytical Laboratory, Woodside, NY, or by Quantitative Technologies, Inc., Whitehouse, NJ. A reaction temperature of 0–20°C indicates that the reaction vessel was initially cooled in an insulated ice bath that was allowed to warm overnight.

Procedure A [amide formation using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride]
A 0.1–0.3 M solution of the amine (1.0 equivalent, or amine hydrochloride and 1.0–1.1 equiv of triethylamine) in CH₂Cl₂ or demethylformamide (as specified) was treated sequentially at 0°C (unless other temperature specified) with 1.0–1.1 equivalent of the specified carboxylic acid, 1.5 equivalent hydroxybenzotriazole hydrate, and, after several minutes, 1.0–1.1 equivalent (corresponding in mole ratio to the carboxylic acid) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (DEC), and the mixture was stirred for 14–20 h at RT (if initially cooled, allowed to warm to RT). If the product contained ionizable amine functionality, the acid wash was omitted. Exceptions in the use of Procedure A are noted individually. Reactions conducted at 0–25°C were conducted with initial cooling of the vessel in an insulated ice bath that was allowed to warm to RT over several hours. The mixture was diluted with ethyl acetate, washed twice with 2N NaOH and twice with 2N HCl, dried over MgSO₄, and concentrated, giving a crude product that was purified by chromatography on silica gel, trituration, or recrystallization, as specified.

1-[N-(5-chloroindole-2-carbonyl)-3-(4-fluorophenyl)-D-alanyl]4-hydroxypiperidine [2] 4-Hydroxypiperidine (374 mg, 3.7 mmol) and N-(t-Boc)-3-(4-fluorophenyl)-D-alanine (1.00 g, 3.5 mmol) were coupled according to Procedure A, giving 1-[N-(t-Boc)-3-(4-fluorophenyl)-D-alanyl]4-hydroxypiperidine as a foam that was used without further purification [940 mg, 73%; HPLC (60/40) 3.64 min (95%); MS 367 (MH+, 100%)]. This material was dissolved in 4 M HCl-dioxanes (2 ml) at 0°C, stirred at RT for 2 h, concentrated, triturated with ether, and dried, giving 920 mg (124%) of 1-(3-(4-fluorophenyl)-D-alanyl)-4-hydroxypiperidine hydrochloride as a hygroscopic solid that was homogeneous by HPLC (60/40) 2.23 min (98%). A portion of this hydrochloride (188 mg, 0.6 mmol) and 5-chloro-1H-indole-2-carboxylic acid (116 mg, 0.6 mmol) were coupled according to Procedure A using triethylamine (61 mg, 0.60 mmol) and dichloromethane (2 ml), and the crude product was purified by chromatography on silica gel eluted with 50, 75, and 100% ethyl acetate in hexanes, giving the title substance [171 mg, 65%; HPLC (60/40) 4.23 min (97%)]. This material was identical to [1] by HPLC and NMR. For [2]: ¹H NMR (CDCl₃) 9.20 (br, 1H), 7.57 (d, 1H, J = 2 Hz), 7.33 (d, 1H, J = 8 Hz), 7.3–7.2 (m, 2H), 7.14 (m, 2H), 6.97 (m, 2H), 6.85 (m, 1H), 5.34 (m, 1H), 4.05–3.80 (m, 2H), 3.7–3.3 (m, 1.5H), 3.25 (m, 1H), 3.10 (m, 2H), 2.93 (m, 0.5H), 1.9–1.7 (m, 2.5H), 1.45 (m, 2H), 1.15 (m, 0.5H); TSPMS 444/446 (MH+, 100%).

1-[N-(5-chloroindole-2-carbonyl)-D-phenylalanyl]4-hydroxypiperidine [4] N-(t-Boc)-D-phenylalanine (3.81 g, 14.0 mmol) and 4-hydroxypiperidine (2.18 g, 21.5 mmol) were coupled in dichloromethane (40 ml) using 3.30 g (21.5 mmol) hydroxybenzotriazole hydrate (HBT) and 3.03 g (15.8 mmol) at 0–25°C according to Procedure A (but washed with acid first, then base), giving 1-[N-(t-Boc)-D-phenylalanyl]4-hydroxypiperidine, which was used without further purification [4.7 g, 94%; HPLC (60/40) 3.52 min (98%)]. This amide (4.35 g, 12.5 mmol) was dissolved in 4 M HCl-dioxane at 0°C, and the resulting suspension was stirred at 25°C for 1 h. The mixture was concentrated and the residue was triturated with ether and dried, giving 1-(D-phenylalanyl)-4-hydroxypiperidine hydrochloride (3.44 g, 97%). According to Procedure A, this hydrochloride (870 mg, 3.1 mmol) and 5-chloro-1H-indole-2-carboxylic acid (660 mg, 3.4 mmol) were coupled in dichloromethane (10 ml) (0–25°C reaction temperature, 60 h reaction time), and the crude product was purified by chromatography on silica gel eluted with 50, 75, and 100% ethyl acetate in hexanes followed by trituration with 1:1 ether-hexanes, giving the title product [1.1 g, 84%, HPLC (60/40) 4.06 min (99%)]. For [4]: ¹H NMR (CDCl₃) 9.38 (s, 0.5H), 9.34 (s, 0.5H), 7.60 (d, 1H, J = 2 Hz), 7.4–7.1 m, 7H), 6.86 (m, 1H), 5.4–5.3 (m, 1H), 4.0–3.9 (m, 1H), 3.9–3.75 (m, 1H), 3.6–3.4 (m, 1.5H), 3.35–3.2 (m, 1H), 3.2–3.1 (m, 3H), 2.9–2.75 (m, 0.5H), 1.9–1.3 (m, 3.5H), 1.0–0.8 (m, 0.5H); PBMS 426/428 (MH+, 100%). Anal. calcd for C₂₃H₂₄ClN₃O₃ + 0.25 H₂O: C, 64.18; H, 5.74; N, 9.76. Found: C, 64.28; H, 5.94; N, 9.41.

1-[N-(5-fluoroindole-2-carbonyl)-3-(4-fluorophenyl)-L-alanyl]4-hydroxypiperidine [6] 1-[3-(4-Fluorophenyl)-L-alanyl]4-hydroxypiperidine hydrochloride (10) (159 mg, 0.52 mmol) and 5-fluoro-1H-indole-2-carboxylic acid (90 mg, 0.5 mmol) were coupled according to Procedure A using triethylamine (52 mg, 0.52 mmol), HBT (115 mg, 0.75 mmol), and DEC (100 mg (0.52 mmol) in dichloromethane (2 ml), and the crude product was triturated once with 1:1 ether-hexanes and once with hexanes. The resulting solid was boiled in ethyl acetate, the resulting suspension filtered, and the collected solid dried [103 mg, 48%; HPLC (60/40) 3.69 min (95%)]. PBMS 428 (MH+, 100%). Anal. calcd for C₂₃H₂₃F₂N₃O₃ + 0.25 H₂O: C, 63.95; H, 5.48; N, 9.73. Found: C, 63.93; H, 5.66; N, 9.87.

1-[N-(Indole-2-carbonyl)-L-phenylalanyl]4-hydroxypiperidine [7] Indole-2-carboxylic acid (598 mg, 3.71 mmol) and 1-(L-phenylalanyl)-4-hydroxypiperidine hydrochloride (10) (961 mg, 3.37 mmol) were coupled according to Procedure A (0–25°C reaction temperature, 48 h reaction time) in dichloromethane (10 ml) using triethylamine (340 mg, 3.37 mmol), HBT (780 mg, 5.06 mmol), and DEC (711 mg, 3.71 mmol). The product was purified by chromatography on silica gel eluted with 50, 75, and 100% ethyl acetate in hexanes, followed by trituration with 1:1 ether-hexanes [1.14 g, 86%, HPLC (60/40) 3.52 min (98%)]. For [7]: PBMS 392 (MH+, 100%). Anal. calcd for C₂₃H₂₅N₃O₃ + 0.25 H₂O: C, 69.77; H, 6.49; N, 10.61. Found: C, 69.99; H, 6.72; N, 10.47.

1-[N-(5-chloroindole-2-carbonyl)-3-(2-fluorophenyl)-L-alanyl]4-hydroxypiperidine [8] N-(t-Boc)-L-3-(2-fluorophenyl)alanine (1.0 g, 3.5 mmol) and 4-hydroxypiperidine (0.39 g, 3.9 mmol) were coupled in dichloromethane (10 ml) using 0.72 g (21.5 mmol) HBT and 0.81 g (4.2 mmol) 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) at 0–25°C according to Procedure A, giving (S)-[1-(2-fluoro-benzyl)-2-(4-hydroxy-piperidin-1-yl)-2-oxo-ethyl] carbamic acid tert-butyl ester, which was used without further purification (0.92 g, 72%). This amide (0.92 g, 3.0 mmol) was dissolved in ethyl acetate and cooled to 0°C. The solution was saturated with HCl, and the resulting suspension was stirred at 0–25°C for 1 h. The mixture was concentrated to approximately one-half its volume and diluted with ether, and the solid was collected and dried, giving (S)-2-amino-3-(2-fluoro-phenyl)-1-(4-hydroxy-piperidin-1-yl)-propan-1-one (0.76 g, 100%). According to Procedure A, this hydrochloride (760 mg, 2.5 mmol) and 5-chloro-1H-indole-2-carboxylic acid (760 mg, 2.5 mmol) were coupled in dichloromethane (6 ml) (0–25°C reaction temperature, 20 h reaction time), and the crude product was purified by chromatography on silica gel eluted with chloroform-methanol, 8:1, giving the title product (0.815 g, 73%). Mp 127–129°C. For [8]: ¹H NMR (CDCl₃, 400 MHz) 9.40 (s, 0.5H), 9.37 (s, 0.5H), 7.57 (s, 1H), 7.43 (d, J = 7.1 Hz, 1H), 7.29–7.13 (m, 5H), 7.04–6.98 (m, 2H), 6.83 (s, 1H), 5.42 (m, 1H), 4.05–3.85 (m, 2H), 3.8–3.7 (m, 0.5H), 3.7–3.6 (m, 0.5H), 3.56–3.11 (m, 4H), 1.86–1.60 (m, 4.5H), 1.55–1.45 (m, 1.5H), 1.25–1.20 (m, 1H); PBMS 444 (MH+, 100%). HRMS m/e 444.1504 (calcd for C₂₃H₂₃N₃O₃FCl + H, 444.1490).

1-[N-(5-chloroindole-2-carbonyl)-3-(2-chlorophenyl)-L-alanyl]4-hydroxypiperidine [9] N-(t-Boc)-L-3-(2-chlorophenyl)alanine (1.05 g, 3.5 mmol) and 4-hydroxypiperidine (0.39 g, 3.9 mmol) were coupled in dichloromethane (10 ml) using 0.72 g (21.5 mmol) HBT and 0.81 g (4.2 mmol) EDC at 0–25°C according to Procedure A, giving (S)-[1-(2-chloro-benzyl)-2-(4-hydroxy-piperidin-1-yl)-2-oxo-ethyl] carbamic acid tert-butyl ester, which was used without further purification (1.26 g, 94%). This amide (1.26 g, 3.3 mmol) was dissolved in ethyl acetate and cooled to 0°C. The solution was saturated with HCl, and the resulting suspension was stirred at 0–25°C for 1 h. The mixture was concentrated to approximately one-half its volume and diluted with ether, and the solid was collected and dried, giving (S)-2-amino-3-(2-chloro-phenyl)-1-(4-hydroxy-piperidin-1-yl)-propan-1-one (1.0 g, 100%). According to Procedure A, this hydrochloride (1.0 g, 3.3 mmol) and 5-chloro-1H-indole-2-carboxylic acid (650 mg, 2.5 mmol) were coupled in dichloromethane (8 ml) (0–25°C reaction temperature, 20 h reaction time), and the crude product was purified by chromatography on silica gel eluted with chloroform-methanol, 8:1, giving the title product (0.825 g, 54%). Mp 127–129°C. For [9]: ¹H NMR (CDCl₃, 400 MHz) 9.42 (s, 0.5H), 9.38 (s, 0.5H), 7.57 (s, 1H), 7.45 (m, 1H), 7.45 (m, 1H), 7.37–7.10 (m, 4H), 6.85 (s, 1H), 5.53 (m, 1H), 4.05–3.60 (m, 2.5H), 3.40–3.22 (m, 3H), 3.02 (m, 0.5 H), 1.90–1.70 (m, 3.5H), 1.38–1.55 (m, 2H), 1.25–1.05 (m, 0.5H); PBMS 460/462 (MH+, 100%). HRMS m/e 460.1195 (calcd for C₂₃H₂₃N₃O₃Cl₂ + H, 460.1201).

1-[N-(5-chloroindole-2-carbonyl)-3-(4-chlorophenyl)-L-alanyl]4-hydroxypiperidine [10] N-(t-Boc)-3-(4-chlorophenyl)-L-alanine (749 mg, 2.5 mmol) and 4-hydroxypiperidine (263 mg, 2.6 mmol) were coupled according to Procedure A using 574 mg (3.75 mmol) HBT, 503 mg (2.60 mmol) DEC, and 11 ml dichloromethane, and the product was purified by chromatography on silica gel eluted with 1:1 and 3:1 ethyl acetate-hexanes, giving 1-[N-(t-Boc)-3-(4-chlorophenyl)-L-alanyl]4-hydroxypiperidine as an off-white foam (662 mg, 69%). A portion of this material (475 mg, 1.2 mmol) was dissolved in 4 M HCl-dioxanes (5 ml) at 0°C. The mixture was stirred for 1.5 h at 25°C and concentrated, and the residue was triturated with ether, giving 1-[3-(4-chlorophenyl)-L-alanyl]4-hydroxypiperidine hydrochloride [422 mg, 105%; TSPMS 283 (MH+, 100%)]. A portion of the preceding hydrochloride (310 mg, 0.98 mmol) and 5-chloro-1H-indole-2-carboxylic acid (0.92 mmol) were coupled according to Procedure A using triethylamine (93 mg, 0.92 mmol), HBT (223 mg, 1.5 mmol), and DEC (186 mg, 0.97 mmol) in dichloromethane (5 ml), and the crude product was purified by chromatography on silica gel eluted with 50, 75, and 100% ethyl acetate in hexanes, giving the title product (362 mg) in 86% yield. For [10]: HPLC (60/40) 5.06 min (97%); mp 227–229°C; TSPMS 460/462 (MH+, 100%). Anal. calcd for C₂₃H₂₃Cl₂N₃O₃: C, 60.01; H, 5.04; N, 9.13. Found: C, 59.83; H, 5.18; N, 9.16.

1-[N-(5-chloroindole-2-carbonyl)-3-(4-methoxyphenyl)-L-alanyl]4-hydroxypiperidine [11] N-t-Boc-O-methyl-L-tyrosine (2.5 g, 8.47 mmol) and 4-hydroxypiperidine (0.94 g, 9.3 mmol) were coupled in dichloromethane (50 ml) using 1.15 g (8.5 mmol) HBT and 2.1 g (11 mmol) DEC at 0-25°C according to Procedure A, giving (S)-[1-(4-methoxy-benzyl)-2-(4-hydroxy-piperidin-1-yl)-2-oxo-ethyl]carbamic acid tert-butyl ester, which was used without further purification (3.1 g, 98%). This amide (3.1 g, 8.2 mmol) was dissolved in ethyl acetate and cooled to 0°C, the solution was saturated with HCl, and the resulting suspension was stirred at 0–25°C for 1 h. The mixture was concentrated to dryness, and the residue was triturated with ether. The solid was collected and dried, giving (S)-2-amino-3-(4-methoxy-phenyl)-1-(4-hydroxy-piperidin-1-yl)-propan-1-one hydrochloride (1.25 g, 48%). This hydrochloride (630 mg, 2.0 mmol), 5-chloroindole-carboxylic acid (391 mg, 2.0 mmol), triethylamine (0.28 ml, 2.0 mmol), and 4-dimethylaminpyridine (122 mg, 1.0 mmol) were placed in dichloromethane (20 ml), and DEC (497 mg, 2.6 mmol) was added. The mixture was stirred at RT overnight then diluted with chloroform, washed with water, 2 N HCl, and water and brine, dried over magnesium sulfate, and concentrated. The resulting solid was purified by flash chromatography (5% methanol in dichloromethane), and the product was isolated as a white solid (334 mg, 37%). Mp 105–120°C. For [11]: ¹H NMR (DMSO-d₆, 300 MHz) 11.8 (s, 1 H), 8.90 (t, J = 8 Hz, 1H), 7.70 (s, 1H), 7.3–7.1 m, 4H), 6.86 (m, 2H), 5.1 (m, 1H), 4.7 (d, J = 2 Hz, 1H), 4.1–3.55 (m, 3H), 3.65 (s, 3H), 2.85–3.3 (m, 3H), 1.75–1.55 (m, 2H), 1.25–1.1 (m, 2H); MS 460/462 (MH+, 100%). Anal. calcd for C₂₄H₂₆N₃O₄Cl + 0.5 H₂O: C, 62.00; H, 5.85; N, 9.04. Found: C, 62.32; H, 5.93; N, 8.83.

N-[N'-(5-chloroindole-2-carbonyl)-3-(3-pyridyl)-L-alanyl]N,O-dimethylhydroxylamine [12] N-(t-Boc)-3-(3-pyridyl)-L-alanine (422 mg, 1.6 mmol) and N,O-dimethylhydroxylamine hydrochloride (163 mg, 1.7 mmol) were coupled using triethylamine (175 mg, 1.7 mmol), HBT (367 mg, 2.4 mmol), and DEC (326 mg, 1.7 mmol) according to Procedure A (0–25°C reaction temperature, 4 ml dichloromethane and 2 ml dimethylformamide, acid wash omitted, Na₂SO₄ used for drying). The crude product was triturated with ether, giving 428 mg (86% yield) of 1-[(N-t-Boc)-3-(3-pyridyl)-L-alanyl]4-hydroxypiperidine. This substance (450 mg, 1.45 mmol) was dissolved in 4 M HCl-dioxanes at 0°C, and the resulting solution was stirred for 2 h at 25°C and concentrated, and the residue was triturated with ether, giving (3-(3-pyridyl)-L-alanyl)-4-hydroxypiperidine dihydrochloride (390 mg, 95%). This substance (370 mg, 1.3 mmol) and 5-chloro-1H-indole-2-carboxylic acid (244 mg, 1.25 mmol) were coupled according to Procedure A [0–25°C reaction temperature, 1:1 dichloromethane-dimethylformamide (DMF) reaction solvent] using triethylamine (167 mg, 1.25 mmol), HBT (287 mg, 1.9 mmol), and DEC (249 mg, 1.3 mmol), and the product was purified by chromatography on silica gel eluted with ethyl acetate, giving the title substance [313 mg, 65%, HPLC (60/40) 2.84 min (99%)]. For [12]: ¹H NMR (CDCl₃) 9.1 (br, 1H), 8.48 (dd, 1H), 8.43 (m, 1H), 7.60 (d, 1H), 7.50 (m, 1H, J = 8 Hz), 7.37 (d, 1H, J = 8 Hz), 7.23 (d, 1H), 7.18 (dd, 1H, J = 8 Hz), 7.10 (d, 1H, J = 8 Hz), 6.82 (d, 1H), 5.42 (m, 1H), 3.78 (s, 3H), 3.25 (s, 3H), 3.32 (dd, A of AB, 1H, J = 7, 14 Hz), 3.10 (dd, B of AB, 1H, J = 7, 14 Hz); TSPMS 387/389 (MH+, 100%). Anal. calcd for C₁₉H₁₉ClN₄O₃ + 0.4 H₂O: C, 57.91; H, 5.07; N, 14.22. Found: C, 58.19; H, 5.23; N, 13.82.

1-[N-(5-chloroindole-2-carbonyl)-L-tyrosyl]4-hydroxypiperidine [13] 4-Hydroxypiperidine (395 mg, 3.9 mmol) and Boc-L-tyrosine (1.05 g, 3.7 mmol) were coupled according to Procedure A [850 mg, (5.6 mmol) HBT, 750 mg (3.9 mmol) DEC, 0–25°C reaction temperature, 60 h reaction time] with the following workup: The reaction mixture was diluted with ethyl acetate and washed once with base, the base layer was acidified with 2N HCl and extracted three times with chloroform, and the chloroform extracts were concentrated. The resulting foam was purified by chromatography on silica gel eluted with 1–8% ethanol in dichloromethane containing 0.5% NH₄OH, giving 1-[N-(t-Boc)-L-tyrosyl]4-hydroxypiperidine [550 mg, 41%; HPLC (40/60) 5.02 min (87%)], which was used without further purification. A portion of this material (450 mg, 1.2 mmol) was dissolved in 4 M HCl-dioxanes (2 ml) at 0°C. The mixture was stirred at 25°C for 1 h and concentrated, and the residue was triturated with ether, giving 1-(L-tyrosyl)-4-hydroxypiperidine hydrochloride (400 mg, 107%) and showing MS 265 (MH+, 100%). Coupling of this hydrochloride (204 mg, 0.68 mmol) and 5-chloro-1H-indole-2-carboxylic acid (126 mg, 0.65 mmol) according to Procedure A was performed using triethylamine (69 mg, 0.65 mmol), HBT (149 mg, 0.98 mmol), and DEC (130 mg, 0.68 mmol) in dichloromethane (4 ml) at 0–25°C reaction temperature but substituting the following workup: The reaction mixture was diluted with ethyl acetate, the resulting solution was washed with 1N NaOH (2 ml), the aqueous layer was extracted three times with ethyl acetate, and the combined organic extracts were washed with 1N HCl, dried, and concentrated. The residue was purified by chromatography on silica gel eluted with 1, 2, 4, 8, and 16% ethanol in dichloromethane, giving the title substance in 52% yield (150 mg). For [13]: HPLC (60/40) 3.53 min (99%); PBMS 442/444 (MH+, 100%). Anal. calcd for C₂₃H₂₄ClN₃O₄ + 0.5 H₂O: C, 61.26; H, 5.59; N, 9.32. Found: C, 61.52; H, 5.89; N, 8.98.

1-[N-(5-chloroindole-2-carbonyl)-L-lysyl]4-hydroxypiperidine hydrochloride [14] 4-Hydroxypiperidine (227 mg, 2.24 mmol) and N-(Fmoc)-N-(t-Boc)-L-lysine (1.00 g, 2.13 mmol) were coupled according to Procedure A using HBT (490 mg, 3.2 mmol) and DEC (429 mg, 2.24 mmol) in dichloromethane, and the crude product was purified by chromatography on 29 g silica packed with 1:1 ethyl acetate-hexanes and eluted with 3:1 ethyl acetate-hexanes followed by ethyl acetate, giving a colorless foam. A portion of this material (111 mg, 0.20 mmol) was dissolved in dimethylformamide containing diethylamine (20 µl, 0.20 mmol) for 75 min at RT and concentrated. The residue was chromatographed on silica (15 g in 5, 10, 20, and 30% ethanol-dichloromethane containing 0.5% (v/v) concentrated NH₄OH, giving 1-(L-lysyl)-4-hydroxypiperidine as an oily solid [25 mg, 38%, m/e 330 (MH+)]. This material (0.076 mmol) and 5-chloroindole-2-carboxylic acid (15 mg, 0.076 mmol) were coupled according to Procedure A in dichloromethane (2 ml), giving an oil (50 mg), which was chromatographed on 5 g silica eluted with 1, 2, and 4% ethanol in dichloromethane to give the amide as a foam [36 mg, HPLC (60/40) 3.6 min, (99.6%), and having consistent NMR and mass spectra]. This material (31 mg) was dissolved in 4 M HCl-dioxanes, stirred at RT for 1.5 h, concentrated, and dried, giving 18 mg of a red-orange solid, which was triturated with ether [HPLC (50/50) 2.19 min, (94%)]. ¹H NMR (D₂O) 7.74 (s, 1H), 7.49 (d, 1H, J = 7 Hz), 7.32 (d, 1H, J = 7 Hz), 5.1 (m, 1H), 4.1–3.9 (m, 2H), 3.5–2.9 (m, 5H), 2.1–1.8 (m, 4H), 1.8–1.6 (m, 2H), 1.6–1.4 (m, 4H); TSPMS 407 (MH+, 100%). HRMS m/e 407.1855 (calcd for C₂₀H₂₇N₄O₃Cl + H, 407.1850).

1-[N-(5-chloroindole-2-carbonyl)-L-aspartyl]4-hydroxypiperidine [15] N-(9-fluorenylmethoxycarbonyl)-L-aspartic acid ß-t-butyl ester (795 mg, 2.0 mmol) and 4-hydroxypiperidine (212 mg, 2.2 mmol) were coupled using Procedure A [10 ml dichloromethane, 460 mg (3.0 mmol) HBT, 400 mg (2.1 mmol) DEC], and the crude product (1.34 g) was purified by chromatography on silica gel eluted with 1, 2, and 4% ethanol-dichloromethane, giving 1-[N-(Fmoc)-L-aspartyl(O-t-Bu)]4-hydroxypiperidine [516 mg, 52%, HPLC (60/40) 5.33 min, 93%], and showing TSPMS 495 (MH+). This material was dissolved in dimethylformamide (5 ml) containing diethylamine (0.10 ml) for 1 h at RT, evaporated, and the residue suspended in ether-dichloromethane and filtered. The filtrate was concentrated (328 mg) and chromatographed on 8 g silica eluted with 1, 2, 4, 10, 20, and 50% ethanol-dichloromethane containing 0.5% (v/v) concentrated NH₄OH and the requisite fractions concentrated, giving 1-[L-aspartyl(O-t-Bu)]4-hydroxypiperidine (217 mg, 80%). This material (213 mg, 0.78 mmol) was coupled to 5-chloroindole-2-carboxylic acid (153 mg, 0.78 mmol) in dichloromethane (5 ml) according to Procedure A, and the crude product (373 mg) was purified by chromatography on 9 g silica eluted with 25, 40, 50, 75, and 100% ethyl acetate-hexanes, giving 1-[N-(5-chloroindole-2-carbonyl)-L-aspartyl(ß-O-t-Bu)]4-hydroxypiperidine as a colorless foam [330 mg, 94% yield, HPLC (60/40) 4.18 min (97%), TSPMS 450 (MH+)]. This ester (293 mg, 0.65 mmol) was dissolved in trifluoroacetic acid at RT for 2 h and concentrated to a yellowish foam, which was partitioned between 2 M NaOH and ethyl acetate. The aqueous layer was separated and extracted twice with ethyl acetate, then stirred in an ice bath with ethyl acetate and acidified to pH 2 with 6N HCl. The organic layer was separated, the aqueous layer extracted with ethyl acetate, and the organic layers were combined and dried, giving the title product as a colorless solid in quantitative yield. For [15]: HPLC (60/40) 2.47 min (96%); ¹H NMR (DMSO-d₆) 11.85 (br, 1H), 9.1 (d, 0.5H), 9.05 (d, 0.5H), 7.70 (s, 1H), 7.42 (d, 1H, J = 8.7 Hz), 7.2–7.15 (m, 2H), 5.53–5.52 (m, 1H), 4.1 (m, 0.5H), 3.8–3.6 (m, 3–4H), 3.3–3.1 (m, 1H), 2.95–2.8 (m, 2H), 1.88–1.6 (m, 2H), 1.4–1.2 (m, 2H). Anal. calcd for C₁₈H₂₀N₃O₅Cl + 1.25 H₂O: C, 51.93; H, 5.45; N, 10.09. Found: C, 52.09; H, 5.05; N, 9.69.

1-[N-(5-chloroindole-2-carbonyl)-L-glycyl]4-hydroxypiperidine [16] A mixture of glycine methyl ester hydrochloride (6.25 g, 50.0 mmol), 5-chloro-1H-indole-2-carboxylic acid (9.75 g, 50.0 mmol), and 1-hydroxybenzotriazole hydrate (HBT, 11.5 g, 75.0 mmol) in dichloromethane (100 ml) at RT was treated sequentially with triethylamine (5.0 g, 49.6 mmol) and DEC (9.55 g, 50.0 mmol) and stirred overnight. The mixture was concentrated, the residue was stirred vigorously with ethyl acetate (250 ml), hexanes (50 ml), and aqueous 1N NaOH (50 ml) and filtered, and the filtered solid was washed successively, twice with 1N NaOH and twice with 2N HCl-water-ethyl acetate (V/v/v), and dried, giving N-(5-chloroindole-2-carbonyl)-glycine methyl ester as a colorless powder (11.5 g, 86% yield) and showing the following properites: mp 252–254°C with decomposition; ¹H NMR (DMSO-d₆) 11.87 (br, 1H), 9.05 (t, 1H, J = 6.0 Hz), 7.72 (d, 1H, J = 2.0 Hz), 7.45 (d, 1H, J = 8.7 Hz), 7.19 (dd, 1H, J = 2.0, 8.7 Hz), 4.05 (d, 2H, J = 6.0 Hz), 3.91 (s, 3H). Anal. calcd for C₁₂H₁₁ClN₂O₃: C, 54.05; H, 4.16; N, 10.50. Found: C, 54.11; H, 4.23; N, 10.56. A suspension of N-(5-chloroindole-2-carbonyl)-glycine methyl ester (8.0 g, 30 mmol) in tetrahydrofuran (THF) (100 ml) was treated with 1N NaOH (35 ml), and the resulting mixture was stirred for 18 h at 25°C. The solution was acidified with 6N HCl (7 ml) and concentrated. The residue was suspended in water and filtered, and the filtered solid washed with water, giving N-(5-chloroindole-2-carbonyl)-glycine as a colorless powder [7.42 g, 98%, HPLC (60/40) 2.89 min (100%)] and showing the following properies: ¹H NMR (300 mHz, DMSO-d₆) 12.68 (br, 1H), 11.85 (br, 1H), 8.95 (t, 1H, J = 5.9 Hz), 7.72 (d, 1H, J = 2.0 Hz), 7.44 (d, 1H, J = 8.7 Hz), 7.19 (dd, 1H, J = 2.0, 8.7 Hz), 7.14 (d, 1H, J = <2 Hz), 3.96 (d, 2H, J = 5.9 Hz). Anal. calcd for C₁₁H₉N₂O₃Cl: C, 52.29; H, 3.59; N, 11.09. Found: C, 52.26; H, 3.73; N, 11.20. 4-Hydroxypiperidine (84 mg, 0.83 mmol) and N-(5-chloro-1H-indole-2-carbonyl)-glycine (200 mg, 0.8 mmol) were coupled according to Procedure A using dichloromethane (2 ml) and dimethylformamide (0.3 ml) but substituting the following workup: The reaction mixture was stirred with ethyl acetate and aqueous 2N HCl, the resulting suspension was filtered and the collected solid was washed successively with aqueous 2N HCl, aqueous 2N NaOH-water-ether (V/v/v) and dried, giving the title substance (180 mg, 68%). For [16]: ¹H NMR (DMSO-d₆) 11.84 (br, 1H), 8.68 (br, 1H), 7.71 (d, 1H), 7.43 (d, 1H), 7.17 (dd, 1H), 7.14 (s, 1H), 4.80 (br, 1H), 4.15 (m, 2H), 3.91 (m, 1H), 3.72 (m, 2H), 3.20 (m, 1H), 3.05 (m, 1H), 1.75 (m, 2H), 1.48 (m, 1H), 1.38 (m, 1H); TSPMS 336/338 (MH+, 100%). HRMS m/e 336.1115 (calcd for C₁₆H₁₈N₃O₃Cl + H, 336.1115).

1-[N-(5-chloroindole-2-carbonyl)-L-histidyl]4-hydroxypiperidine [17] A mixture of N-(t-Boc)-N_im-(p-tosyl)-L-histidine (1.32 g, 3.90 mmol), 4-hydroxypiperidine (303 mg, 3.0 mmol), and triethylamine (300 mg, 3.0 mmol) in dichloromethane was treated with diethylphosphoryl cyanide (700 mg, 3.90 mmol) and stirred at RT overnight. The mixture was diluted with ethyl acetate, and the resulting suspension was washed with aqueous sodium bicarbonate and brine, dried, concentrated, and chromatographed on silica eluted with 1, 2, 4, and 8% ethanol-dichloromethane, giving 1-[N-(t-Boc)-N_im-(p-tosyl)-L-histidyl]4-hydroxypiperidine as a colorless foam [517 mg, 35%, HPLC (50/50) 4.75 min, (97%)], ¹H NMR (CDCl₃) 7.91 (m, 2H), 7.81 (d, 1H, J = 8 Hz), 7.6 (d, 1H, J = 8 Hz), 7.35 (d, 2H, J = 8 Hz), 7.07 (s, 1H), 5.4 (br, 1H), 4.9 (m, 1H), 4.1–3.6 (m, 3–4H), 3.3–3.0 (m, 2H), 2.85 (dd, 1H), 2.78 (dd, 1H), 2.45 (s, 3H), 1.8–1.4 (m, 5H), 1.38 (s, 9H). This material was dissolved in 4 M HCl-dioxanes (3 ml), stirred at RT for 1 h, concentrated, and triturated with ether, giving 314 mg of 1-(L-histidyl)-4-hydroxypiperidine dihydrochloride, ¹H NMR (D₂O, partial) 8.72 (d, 1H, J = 9 Hz), and 7.44 (d, 1H, J = 9 Hz). The dihydrochloride (200 mg, 0.73 mmol) and 5-chloroindole-2-carboxylic acid (135 mg, 0.69 mmol) were coupled using triethylamine (74 mg, 0.73 mmol), HBT (168 mg), and DEC (140 mg, 0.73 mmol) in dichloromethane (4 ml) according to Procedure A but omitting the acid wash, and the crude product was chromatographed on silica (7 g) eluted with 5, 10, and 20% ethanol-dichloromethane containing 0.5% (v/v) concentrated NH₄OH, to give the title substance as a colorless powder [232 mg, 81%, HPLC (40/60) 2.57 min, (98%)]. For [17]: ¹H NMR (DMSO-d₆) 11.8 (br, 1H), 8.87 (d, 0.5H), 8.83 (d, 0.5H), 7.72 (d, 1H, J = 2 Hz), 7.53 (m, 1H), 7.40 (d, 1H, J = 8.6 Hz), 7.23 (m, 1H), 7.18 (dd, 1H, J = 2, 8.7 Hz), 6.79 (m, 1H), 5.25–5.15 (q, 1H), 4.8 (m, 1H), 4.1–3.6 (m, 3–4H), 3.2–3.1 (m, 1H), 3.0–2.85 (m, 2H), 1.7–1.5 (m, 2H), 1.3–1.15 (m, 2H). HRMS m/e 416.1479 (calcd for C₂₀H₂₂N₅O₃Cl + H, 416.1489).

1-[N-(5-chloroindole-2-carbonyl)-L-alanyl]4-hydroxypiperidine [18] 4-Hydroxypiperidine (101 mg, 1.0 mmol) and Boc-L-alanine (189 mg, 1.0 mmol) were coupled according to Procedure A using EDC (200 mg, 1.05 mmol) and HBT (229 mg, 1.5 mmol) in dichloromethane (5 ml), giving 1-[N-(t-Boc)-L-alanyl]4-hydroxypiperidine (118 mg, 44%). This material (118 mg, 0.43 mmol) was dissolved in 4 M HCl-dioxane (2 ml) and stirred at 25°C for 2 h, concentrated, and the residue triturated with ether, giving 1-(L-alanyl)-4-hydroxypiperidine hydrochloride (90 mg, 100%). Coupling of this hydrochloride (58 mg, 0.28 mmol) and 5-chloro-1H-indole-2-carboxylic acid (55 mg, 0.28 mmol) according to Procedure A was performed using triethylamine (37 µl, 0.28 mmol), EDC (56 mg, 0.29 mmol), and HBT (51 mg, 0.33 mmol) in dichloromethane (5 ml) at 25°C overnight. The product was purified by radial chromatography (ethyl acetate) and obtained as a foam (81 mg, 83%). For [18]: PBMS 350/352 (MH+, 100%). ¹H NMR (DMSO-d6, 400 mHz) 11.75 (br, 1H), 8.74 (d, 0.5H, J = 8 Hz), 8.72 (d, 0.5H, J = 8.5 Hz), 7.65 (s, 1H), 7.37 (m, 1H), 7.15 (m, 1H), 7.13 (m, 1H), 4.95 (m, 1H), 4.71 (m, 1H), 4.0 (m, 0.5H), 3.8–3.6 (m, 2.5H), 3.3–3.25 (m, 0.5H), 3.2–3.05 (m, 1H), 2.9–2.8 (m, 0.5H), 1.8–1.6 (m, 2H), 1.3–1.2 (m, 2H), 1.24 and 1.23 (d, J = 5–6 Hz, 3H total). HRMS m/e 350.1271 (calcd for C₁₇H₂₀ClN₃O₃ + H, 150.1271).

1-[N-(5-chloroindole-2-carbonyl)-L-valyl]4-hydroxypiperidine [19] 4-Hydroxypiperidine (101 mg, 1.0 mmol) and t-Boc-L-valine (217 mg, 1.0 mmol) were coupled according to Procedure A using EDC (200 mg, 1.05 mmol) and HBT (229 mg, 1.5 mmol) in dichloromethane (5 ml), giving 1-[N-(t-Boc)-L-valyl]4-hydroxypiperidine (213 mg, 73%). This material (205 mg, 0.68 mmol) was dissolved in 4 M HCl-dioxane (4 ml) and stirred at 25°C for 1.5 h, concentrated, and the residue triturated with ether, giving 1-(L-valyl)-4-hydroxypiperidine hydrochloride (160 mg, 100%). Coupling of this hydrochloride (83 mg, 0.35 mmol) and 5-chloro-1H-indole-2-carboxylic acid (68 mg, 0.35 mmol) according to Procedure A was performed using triethylamine (45 µl, 0.35 mmol), EDC (70 mg, 0.38 mmol), and HBT (64 mg, 0.42 mmol) in DMF (4 ml) at 25°C. The product was purified by radial chromatography (1:1 hexanes-ethyl acetate, then ethyl acetate) and obtained as a foam (115 mg, 87%). For [19]: PBMS 378/380 (MH+, 100%). Anal. calcd for C₁₉H₂₄N₃O₃Cl + 3 H₂O: C, 52.84; H, 7.00; N, 9.73. Found: C, 52.80; H, 6.70; N, 9.73.

N,N-dimethyl-[N'-(5-chloroindole-2-carbonyl)-3-(2-fluorophenyl)-L-alaninamide [20] N-(t-Boc)-L-3-(2-fluorophenyl)alanine (0.280 g, 1.0 mmol) and dimethylamine hydrochloride (82 mg, 1.0 mmol) were coupled in dichloromethane (5 ml) using 0.16 g (1.2 mmol) HBT, 0.165 ml (1.2 mmol) triethylamine, and 0.23 g (1.2 mmol) EDC at 0–25°C according to Procedure A, giving (S)-[1-dimethylcarbamoyl-2-(2-fluoro-phenyl)-ethyl]carbamic acid tert-butyl ester, which was used without further purification. This amide (0.92 g, 3.0 mmol) was dissolved in a solution of 1 N hydrogen chloride in ethyl acetate (5 ml), and the solution was stirred overnight, then concentrated to dryness. The residue was dissolved in dichloromethane, and 1/5 of this solution was used for coupling with 5-chloro-1H-indole-2-carboxylic acid according to Procedure A, giving the title product as a solid. For [20]: ¹H NMR (CDCl₃) 9.14 (s, 1H), 7.59 (d, J = 1.7 Hz, 1H), 7.32–7.13 (m, 4H), 7.01–6.97 (m, 2H), 6.82 (s, 1H), 5.41–5.35 (m, 1H), 3.19–3.04 (m, 2H), 2.99 (s, 3H), 2.96 (s, 3H). MS 388 (MH+). Anal. calcd for C₂₀H₁₉N₃O₂ClF + 0.1 H₂O: C, 61.65; H, 4.97; N, 10.78. Found: C, 61.34; H, 4.61; N, 10.99.

1-[N-benzoyl-L-phenylalanyl]4-hydroxypiperidine [21] Benzoic acid (86 mg, 0.70 mmol) and 1-(L-phenylalanyl)-4-hydroxypiperidine hydrochloride (10) (173 mg, 0.70 mmol) were coupled according to Procedure A in dichloromethane (5 ml) using triethylamine (73 mg, 0.73 mmol), HBT (161 mg, 1.5 mmol), and DEC (141 mg, 0.74 mmol), and the product was purified by chromatography on silica eluted with ethyl acetate, giving the title substance (190 mg, 77%), HPLC (50/50 acetonitrile-0.1% aqueous formic acid, 5 µM Kromasil C-8 150 x 4.6 mm, 1.0 ml/min) retention time 2.58 min (99% purity, 280 nM detector wavelength). ¹H NMR (DMSO-d₆, 400 mHz) 8.72 (d, 0.5H, J = 8.5 Hz), 8.70 (d, 0.5H, J = 8.3 Hz), 7.78 (d, 2H, J = 7.5 Hz), 7.49–7.42 (m, 1H), 7.40–7.38 (m, 2H), 7.27–7.12 (m, 5H), 5.07 (q, 1H), 4.67 (m, 1H), 3.95–3.85 (m, 0.5H), 3.8–3.5 (m, 2.5H), 3.3–3.2 (m, 0.5H), 3.1–2.9 (m, 3.5H), 1.65–1.55 (m, 1.5H), 1.55–1.45 (m, 0.5H), 1.15–1.10 (m, 1.5H), 1.10–1.00 (m, 0.5H). HRMS m/e 353.1892 (calcd for C₂₁H₂₄N₂O₃ + H, 353.1865).

1-[N-(2-pyrrolyl)-L-phenylalanyl]4-hydroxypiperidine [22] Pyrrole-2-carboxylic acid (78 mg, 0.70 mmol) and 1-(L-phenylalanyl)-4-hydroxypiperidine hydrochloride (10) (173 mg, 0.70 mmol) were coupled according to Procedure A in dichloromethane (5 ml) using triethylamine (73 mg, 0.73 mmol), HBT (161 mg, 1.05 mmol), and DEC (141 mg, 0.735 mmol), and the product was purified by chromatography on silica eluted with 4:1 ethyl acetate-hexanes followed by ethyl acetate, giving the title substance (210 mg, 62%), HPLC (50/50 acetonitrile-0.1% aqueous formic acid, 5 µM Kromasil C-8 150 x 4.6 mm, 1.0 ml/min) retention time 2.2 min (99% purity, 280 nM detector wavelength). ¹H NMR (DMSO-d₆, 400 mHz) 11.35 (br, 1H), 8.27 (d, 0.5H, J = 8.3 Hz), 8.24 (d, 0.5H, J = 8.3 Hz), 7.25–7.10 (m, 5H), 6.84 (d, 1H, J = 1 Hz), 6.79 (d, 1H, J = 1 Hz), 6.02 (d, 1H, J = 2.5 Hz), 5.04 (q, 1H), 4.65 (m, 1H), 3.95–3.85 (m, 0.5H), 3.8–3.7 (m, 1H), 3.7–3.5 (m, 1.5H), 3.3–3.1 (m, 0.5H), 3.1–2.9 (m, 1.5H), 2.9–2.8 (m, 2H), 1.65–1.55 (m, 1.5H), 1.5–1.4 (m, 0.5H), 1.25–1.1 (m, 1.5H), 1.05–0.95 (m, 0.5H). Anal. calcd for C₁₉H₂₃N₃O₃: C, 66.84; H, 6.79; N, 12.31. Found: C, 66.47; H, 6.71; N, 12.17.

1-[N-(benzofuran-2-carbonyl)-L-phenylalanyl]4-hydroxypiperidine [23] Benzofuran-2-carboxylic acid (81 mg, 0.50 mmol) and 1-(L-phenylalanyl)-4-hydroxypiperidine hydrochloride (10) (124 mg, 0.50 mmol) were coupled according to Procedure A in dichloromethane (3 ml) using triethylamine (51 mg, 0.50 mmol), HBT (115 mg, 0.75 mmol), and DEC (101 mg, 0.525 mmol), and the product was purified by chromatography on silica eluted with 4:1 ethyl acetate-hexanes followed by ethyl acetate, giving the title substance (170 mg, 86%), HPLC (50/50 acetonitrile-0.1% aqueous formic acid, 5 µM Kromasil C-8 150 x 4.6 mm, 1.0 ml/min) retention time 3.6 min (99% purity, 280 nM detector wavelength). ¹H NMR (DMSO-d₆, 400 mHz) 8.86 (d, 0.5H, J = 8.3 Hz), 8.81 (d, 0.5H, J = 8.3 Hz), 7.73 (d, 1H, J = 7.5 Hz), 7.62 (d, 1H, J = 8.3 Hz), 7.58 (m, 1H), 7.43 (t, 1H, J = 7 Hz), 7.29 (t, 1H, J = 8 Hz), 7. 25–7.35 (m, 4H), 7.30–7.10 (m, 1H), 5.12 (q, 1H), 4.68 (d, 1H, J = 4 Hz), 3.95–3.89 (m, 0.5H), 3.8–3.7 (m, 1H), 3.7–3.5 (m, 1.5H), 3.3–3.15 (m, 0.5H), 3.15–2.9 (m, 3.5H), 1.65–1.55 (m, 1.5H), 1.55–1.45 (m, 0.5H), 1.3–1.0 (m, 2H). Anal. calcd for C₂₃H₂₄N₂O₄+0.33 H₂O: C, 69.34; H, 6.24; N, 7.03. Found: C, 69.70; H, 6.26; N, 6.50.

1-[N-(5-chlorobenzofuran-2-carbonyl)-L-phenylalanyl]4-hydroxypiperidine [24] 5-Chlorobenzofuran-2-carboxylic acid (137 mg, 0.70 mmol) and 1-(L-phenylalanyl)-4-hydroxypiperidine hydrochloride (10) (173 mg, 0.70 mmol) were coupled according to Procedure A in dichloromethane (5 ml) using triethylamine (73 mg, 0.73 mmol), HBT (161 mg, 1.05 mmol), and DEC (141 mg, 0.74 mmol), and the product was purified by recrystallization from ether, giving the title substance (198 mg, 66%), HPLC (50/50 acetonitrile-0.1% aqueous formic acid, 5 µM Kromasil C-8 150 x 4.6 mm, 1.0 ml/min) retention time 4.7 min (99% purity, 280 nM detector wavelength). ¹H NMR (DMSO-d₆, 400 mHz) 8.96 (d, 0.5H, J = 8.3 Hz), 8.92 (d, 0.5H, J = 8.3 Hz), 7.83 (m, 1H), 7.66 (d, 1H, J = 8.7 Hz), 7.56 (d, 1H, J = 3 Hz), 7.44 (dd, 1H, J = 2.5, 9 Hz), 7.25–7.15 (m, 5H), 7.15–7.1 (m, 1H), 5.09 (q, 1H), 4.73 (m, 1H), 3.95–3.85 (m, 0.5H), 3.8–3.7 (m, 1H), 3.7–3.5 (m, 1.5H), 3.1–2.9 (m, 4H), 1.7–1.55 (m, 1.5H), 1.55–1.45 (m, 0.5H), 1.3–1.1 (m, 1.5H), 1.1–1.0 (m, 0.5H). Anal. calcd for C₂₃H₂₃N₂O₄Cl + 0.3 H₂O: C, 63.90; H, 5.50; N, 6.48. Found: C, 63.67; H, 5.40; N, 6.36.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Antihyperlipidemic effects of CP-320626 in ob/ob mice
Evidence that CP-320626 exhibits antihyperlipidemic properties was first obtained in the diabetic ob/ob mouse, where significant reductions in plasma cholesterol² [31%; 128 ± 12 vs. 186 ± 6 (SEM; n = 10) mg/dl; P < 0.05] and triglycerides [26%; 202 ± 17 mg/dl vs. 273 ± 32 mg/dl (SEM; n = 10); P < 0.06] were observed following 10 mg/kg b.i.d. administration for 15 days (12). These reductions were observed in the absence of changes in plasma insulin, glucagon, free fatty acids, or ß-hydroxybutyrate (12), indicating that the observed reductions in plasma triglycerides and cholesterol were not a tertiary consequence of secondary alterations in glycemic control mechanisms induced by the hypoglycemia resulting from glycogen phosphorylase inhibition.

Hypolipidemic effects of CP-320626 in rats and dogs
The antihyperlipidemic properties of CP-320626 observed in ob/ob mice were confirmed in a series of two week studies in normoglycemic rats and dogs in which treatment at doses between 30 and 400 mg/kg (rats) and 3 and 30 mg/kg (dogs) was associated with substantial reductions in serum cholesterol and triglycerides (Tables 1 and 2) in the absence of notable glucose lowering (not shown). For example, male rats treated with CP-320626 displayed a dose-dependent up to 60% decrease in serum cholesterol² and triglycerides for doses between 30 and 400 mg/kg (Table 1). The magnitude of these reductions was related to exposure to CP-320626, with significant reductions occurring at peak plasma drug levels (C_max) in excess of 3 µM (Table 1). Female rats treated with CP-320626 displayed similar effects (not shown).

Direct inhibition of cholesterol biosynthesis by CP-320626
To determine whether the plasma cholesterol and triglyceride lowering observed in experimental animals was a consequence of direct inhibition of de novo sterol and/or triglyceride synthesis, we evaluated the ability of CP-320626 to inhibit cholesterol and fatty acid synthesis in cultured HepG2 (human liver) cells. As shown in Fig. 2 , when CP-320626 and [2-¹⁴C]acetate were added simultaneously to near-confluent cultures of HepG2 cells, and simultaneous incorporation of radiolabel into sterols and fatty acids was permitted to proceeded for 6 h as outlined in Experimental Procedures, CP-320626 inhibited cholesterol synthesis in a dose-dependent fashion, exhibiting an IC₅₀ of 4 µM. Fatty acid synthesis was not substantially affected by CP-320626 at concentrations that markedly inhibited cholesterol synthesis (Fig. 2), indicating that the reduction in cholesterol formation was not a consequence of altered acetate pools resulting from generalized effects on intermediary metabolism.

View larger version (23K): [in this window] [in a new window]   Fig. 2. CP-320626 inhibits cholesterol but not fatty acid synthesis in HepG2 cells. HepG2 cells were incubated for 6 h at 37°C in supplemented Dulbecco's modified Eagle's medium (DMEM) containing 5 mM glucose, either 1% DMSO (control) or 1% DMSO containing CP-320626 sufficient to produce the indicated final inhibitor concentrations, and 4 µCi of [2-14C]acetate. After incubation, samples were saponified, the nonsaponifiable lipids extracted with hexane and separated by silica gel TLC, and the cholesterol and cholesterol precursors quantitated as described in Experimental Procedures. The remaining aqueous phase was then acidified to pH <2, the protonated fatty acids extracted with hexane and separated by silica gel TLC, and the fatty acids quantitated as described in Experimental Procedures. Data for cholesterol and fatty acid synthesis are the mean of triplicate determinations ± SD and are expressed as cpm [2-14C]acetate incorporated into cholesterol (open circles) or fatty acids (closed circles) during the 6 h incubation at 37°C.

As shown in Fig. 3 , reduction of cholesterol synthesis from [2-¹⁴C]acetate by CP-320626 occurred concomitantly with an enhanced formation of isotopically labeled methylsterols. Consistent with this observation, the chromatographic pattern of accumulated cholesterol precursors appeared identical to that observed following treatment of HepG2 cells with the known CYP51 inhibitor, ketoconazole (not shown), suggesting that cholesterol synthesis inhibition by CP-320626 was potentially a consequence of inhibition of the cholesterolgenic enzyme, CYP51.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Methylsterol accumulation after cholesterol synthesis inhibition by CP-320626. HepG2 cells were incubated for 6 h at 37°C in supplemented DMEM containing 5 mM glucose, either 1% DMSO (control) or 1% DMSO containing CP-320626 sufficient to produce the indicated final inhibitor concentrations, and 4 µCi of [2-14C]acetate. After incubation, samples were saponified, the nonsaponifiable lipids extracted with hexane and separated by silica gel TLC, and the cholesterol and cholesterol precursors quantitated as described in Experimental Procedures. Data for cholesterol and methylsterol production represent the mean of triplicate determinations ± SD and are expressed as cpm [2-14C]acetate incorporated into cholesterol (closed circles) and methylsterols (open circles) during the 6 h incubation at 37°C. No increases in squalene, squalene oxide, or squalene dioxide relative to control levels were noted following CP-320626 treatment.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Comigration of the accumulating sterol with lanosterol. HepG2 cells were incubated for 6 h at 37°C in supplemented DMEM containing 5 mM glucose, either 1% DMSO (control; top panel), 1% DMSO containing CP-320626 (final inhibitor concentration of 10 µM; center panel), or 1% DMSO containing ketoconazole (final inhibitor concentration of 1.0 µM; bottom panel), and 4 µCi of [2-14C]acetate. After incubation, samples were saponified, and the nonsaponifiable lipids were extracted with hexane, mixed with an authentic [3H]lanosterol chromatographic standard (15.8 nmol, 86 mCi/mol), and separated by silica gel TLC as described in Experimental Procedures. Channels were then dried, sectioned into 2.0 mm slices, immersed in 5 ml of Aquasol-2 liquid scintillation fluid (New England Nuclear, Boston, MA), and assessed for radioactivity using a dual-channel program of a Beckmann LS6500 liquid scintillation counter. Shown are the [14C]cpm (open circles) and [3H]cpm (lanosterol standard; solid circles) as a function of distance from the bottom of the chromatogram, with cholesterol migrating to 58 mm (Rf = 0.26) and lanosterol migrating to 69 mm (Rf = 0.33).

View larger version (23K): [in this window] [in a new window]   Fig. 5. Effect of CP-320626 and ketoconazole on sterol synthesis in ob/ob mice. Male C57BL/6J-ob/ob mice (5–7 weeks old; 50–60 g) were administered a 0.2 ml oral bolus of either vehicle, or vehicle containing either CP-320,626 or ketoconazole at a concentration sufficient to give a dose of 30 mg/kg (upper panel) or the indicated doses of CP-320626 (lower panel). One hour after compound administration, animals received an intraperitonal injection of 0.1 ml R,S-[2-14C]mevalonolactone (100 µCi/ml; 58 mCi/mmol). One hour after radiolabel administration, animals were euthanized by pentobarbital injection, and two 0.5 g liver pieces were removed and saponified as outlined in Experimental Procedures. The nonsaponifiable lipids were then extracted with petroleum ether and separated by silica gel TLC, and the cholesterol and cholesterol precursors were quantitated as described in Experimental Procedures. Data for hepatic cholesterol and methylsterol production represent the mean ± SD for n = 6 mice per group and are expressed as dpm [2-14C]mevalonolactone incorporated into cholesterol (black bars, upper panel; closed circles, lower panel) and methylsterols (gray bars, upper panel; open circles, lower panel) during the 1 h interval between [2-14C]mevalonolactone injection and pentobarbital administration.