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SHORT COMMUNICATION

ANTI-AIDS AGENTS 65: INVESTIGATION OF THE IN VITRO OXIDATIVE METABOLISM OF 3',4'-DI-O-(–)-CAMPHANOYL-(+)-CIS-KHELLACTONE DERIVATIVES AS POTENT ANTI-HIV AGENTS

Division of Medicinal Chemistry, School of Pharmacy (M.S., S.L.M.-N., K.-H.L), Department of Environmental Science and Engineering, School of Public Health (Y.L., J.A.S), and Division of Drug Delivery and Disposition, School of Pharmacy (P.C.S.), University of North Carolina, Chapel Hill, North Carolina; and Panacos Pharmaceuticals Inc., Gaithersburg, Maryland (D.E.M.)

(Received February 14, 2005; accepted August 8, 2005)

	Abstract

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3',4'-Di-O-(–)-camphanoyl-(+)-cis-khellactone (DCK) is a synthetic khellactone ester that exhibits potent in vitro anti-human immunodeficiency virus (HIV) activity with a mechanism distinct from clinically used anti-HIV agents. Several series of mono- and di-substituted DCK derivatives (DCKs) have previously been synthesized, and their structure-activity relationships are well established. To optimize DCK as a drug lead and to guide further structural modifications, metabolic stabilities and metabolite structures were analyzed. In vitro metabolic stabilities of DCKs in human liver microsomes were assessed using high performance liquid chromatography (HPLC) with UV detection to establish structure-metabolism relationships (SMRs). HPLC coupled with ion trap mass spectrometry was used to identify the metabolite structures. The results indicated that DCKs undergo rapid oxidation on the lipophilic camphanoyl moieties and the substituents on the khellactone do not alter the rate or the metabolic pathways for this compound type. Our SMR and metabolite analysis study suggested that the two camphanoyl ester moieties are the determinants of the low metabolic stability and that structural alteration in the two esters may be necessary to improve metabolic profiles of DCKs.

View larger version (25K): [in this window] [in a new window]   FIG. 1. Chemical structures of DCK derivatives and their in vitro metabolic stabilities in human liver microsomes.

	Materials and Methods

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View larger version (31K): [in this window] [in a new window]   FIG. 2. Tandem mass spectra of khellactone esters. A, 4-methyl DCK; B, 3'-O-(S)-(–)-camphanoyl-4'-O-adamantanecarbonyl-4-methyl-cis-khellactone; and C, 3'-O-adamantanecarbonyl-4'-O-(S)-(–)-camphanoyl 4-methyl-cis-khellactone.

Sample Preparation. Stock solutions of DCKs (1 mg/ml) were prepared by dissolving the pure compound in acetonitrile and stored at 4°C in the dark until use. For measurement of metabolic stability, DCKs (1-14) were brought to a final concentration of 1 µM with 100 mM sodium phosphate buffer at pH 7.4, which contained 0.1 mg/ml human liver microsome and 5 mM MgCl₂. Incubation volumes were 1.5 ml. Reactions were started by adding 150 µl of NADPH (final concentration of 1.5 mM) after a 10-min incubation at 37°C, and stopped by taking the aliquots over time, then adding to 3 volumes of ice-cold acetonitrile. Incubations of all samples were run in duplicate, and for control incubations, NADPH was omitted. For each sample, 200-µl aliquots were taken out at 0-, 5-, 10-, 15-, 30-, and 60-min time points. After addition of acetonitrile and before centrifugation, 15 µl of a 0.01 mg/ml acetonitrile solution of 4-methyl-DCK (compound 3, for incubation of 1) or DCK (compound 1, for incubations of 2-14) was added to supernatants as an internal standard. The mixture was centrifuged at 15,000 rpm for 5 min. Supernatants were then evaporated to dryness under nitrogen using an N-Evap analytic evaporator (Organomation Associates Inc., Berlin, MA), and the residue was reconstituted with 150 µl of mobile phase (acetonitrile and water with 0.1% acetic acid, 1:1 v/v); then, an aliquot (75 µl) was injected onto HPLC with UV detector. For liquid chromatographic-mass spectrometric analysis of unknown metabolites, DCKs (1 and 10) were brought to a final concentration of 1 µM or 10 µM with 0.02 mg/ml or 0.2 mg/ml microsomal protein, respectively. Time-dependent formation of metabolites was followed in 1 µM samples, and reactions were stopped at 0, 5, 10, 15, 30, and 60 min. To analyze fragmentation patterns of metabolite structures, the reaction was scaled up to 10 µM concentration in 0.02 mg/ml microsomes and stopped at 60 min without intermediate sampling points. Dried supernatants were evaporated, diluted with 50 µl of acetonitrile, cleaned with an Oasis SPE cartridge (Waters), dried, and reconstituted with 50-µl mobile phase (acetonitrile and water with 0.1% acetic acid, 3:7 v/v), and an aliquot (15 µl) was injected onto the LC-ITMS.

HPLC-UV Conditions. Analysis was carried out on an HP Series 1100 (Hewlett Packard, Palo Alto, CA), consisting of an autosampler, degasser, binary pump, and variable wavelength UV detector. An Axxiom ODS column, 150 x 4.6 mm i.d., 5 µm (Thomson Instrument Co., Clear Brook, VA) connected to an RP-18 guard column (15 x 3.2 mm i.d., 7 µm) (Brownlee Precision Co., San Jose, CA) was isocratically eluted with acetonitrile and 25 mM acetic acid in water (55:45 v/v) at a flow rate of 1.5 ml/min. Detection wavelength was set at 320 nm, which was previously determined to be _max for the DCKs. Chromatograms were recorded on a Hewlett-Packard Chemstation A.05.01. Standards for calibration graphs were prepared ranging from 0.020 to 100 µg/ml.

Calculations. In the determination of the in vitro half-life (t_1/2), the analyte/internal standard peak height ratios were converted to percentage drug remaining, using the initial time (0 min) peak height ratio values as 100%. The slope of the linear regression from log percentage remaining versus incubation time relationships (–k) was used in the conversion to in vitro t_1/2; in vitro t_1/2 = 0.693/k. Conversion to in vitro CL_int (in units of ml/min/mg protein) was done using the following formula (Obach et al., 1997): CL_int = (0.693/in vitro t_1/2) x (ml incubation/mg microsomes).

HPLC-MS Conditions. Analysis was carried out on a Surveyor LC system (Thermo Electron Corporation, Waltham, MA) interfaced to an LCQ Deca ion trap mass spectrometer (Thermo Electron Corporation) with an electrospray ionization source. A microbore Zorbax column SB-C18, 2.1 x 150 mm, 5 µm (Agilent Technologies, Palo Alto, CA), was eluted with 0.1% acetic acid in water (A) and acetonitrile with 0.1% acetic acid (B) at a flow rate of 200 µl/min. Initially, B was held at 30% and increased linearly to 50% B at 5 min, then to 95% B at 23 min. The mobile phase was then returned to the initial condition and re-equilibrated for 7 min. The MS conditions were optimized with DCK to sheath gas at 50 arbitrary units, auxiliary gas at 15 arbitrary units, capillary heated to 330°C, spray voltage at 5 kV, and tube lens offset at 25 V. Electrospray ionization was operated in the positive ion mode. Full-scan spectra were acquired over the range of 180 to 800 m/z. Collision-induced dissociation (CID) was applied to determine fragmentation of DCK 1 (C₃₄H₃₈O₁₁) and 3-chloro-4-methyl DCK 10 (Cl-Me-DCK, C₃₅H₃₉ClO₁₁) using 30 and 35% relative collision energy with isolation width (the width of the ion isolation band for the selected mass) of 1 and 7 (to observe Cl pattern in the fragment ion), respectively. For objective 2, fragmentation of khellactone esters 3 (C₃₅H₄₀O₁₁), 15 (C₃₆H₄₂O₉), and 16 (C₃₆H₄₂O₉) was analyzed using direct infusion in a solution of acetonitrile/water (1:1 v/v) via a syringe pump at a flow rate of 10 µl/min.

	Results

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Metabolic Stabilities of DCKs in Human Liver Microsomes. In vitro metabolic stabilities of various DCK compounds were investigated using human liver microsomal preparations under oxidative conditions. The incubation mixtures of DCKs were analyzed by analytical HPLC with UV detection to measure the consumption of the parent compound. The structures of DCKs containing khellactone modifications and results of in vitro metabolic rates are shown (Fig. 1). Introduction of substituents in the coumarin ring had no significant effect on the metabolic rates, regardless of the substituent type or the positions of the modification, except for 3-bromomethyl DCK (12). Compound 12 could not be detected even at time 0 min, due to the rapid conversion of the compound to 3-hydroxymethyl DCK (13). The hydrolysis of 12 to 13 was independent of the presence of NADPH or microsomes. The remaining DCKs were rapidly consumed in human liver microsomes with half-life values of the parent compound between 1.5 and 5.7 min.

Characterization of Khellactone Ester Fragmentation. The proposed fragmentation pattern and the observed mass fragment values of the khellactone esters 3, 15, and 16 are shown (Fig. 2). The product-ion spectrum of 4-methyl DCK (3, m/z 659, Na adduct) showed initial loss of a fragment corresponding to a camphanic acid from either the 3' or 4' position, which generates a chromene system (m/z 461) as a result of CID. Loss of CO₂ (m/z 615) was also observed, as is characteristic of many other coumarin compounds (Concannon et al., 2000). The remaining camphanoyl moiety could be further fragmented by subsequent CID, giving a product ion at m/z 303. Mass spectra of khellactone esters 15 and 16 allowed identification of the positions of fragment sources, which could not be achieved with compound 3. These two compounds contained two different bulky acyl groups, camphanoyl on the 3'- and adamantanecarbonyl on the 4'-position (15), or vice versa (16). In both cases, an abundant parent ion m/z 641, [M + Na]⁺, was observed which dissociated to produce ions (m/z 461 for 15 and m/z 443 for 16) attributable to the loss of the 4'-position acyloxy group. Subsequently, loss of the alkyl group from the remaining 3'-position ester generated ions at m/z 303, identical for the three khellactones. The patterns observed from 4-methyl DCK (3) and the two khellactone esters (15 and 16) were similar; therefore, the same pattern is expected for other DCKs and also for their possible metabolites, i.e., neutral loss of the 4'-position ester under MS/MS, followed by the sequential loss of alkyl from the 3'-position ester.

Characterization of DCKs Metabolites. The metabolism of DCKs 1 and 10 in human hepatic microsomes was investigated using the positive mode with low (1 µM) and high (10 µM) substrate concentration incubations. At low concentration, a time-dependent increase in multiple ion peaks corresponding to hydroxylated metabolites, [M + 16 + Na]⁺, was observed. To identify the site of hydroxylation, the reaction was scaled up 10-fold in concentration. Similar to the low concentration incubates, the major metabolites were monohydroxylated. Representative extracted ion chromatograms of mono-hydroxylated metabolites of compounds 1 and 10 (Fig. 3, B and D, respectively) showed multiple peaks that were not baseline resolved, and MS/MS and MS³ were conducted for these mono-hydroxylated metabolites. Fragmentation patterns of the monohydroxy metabolites (Fig. 4) suggested two types of metabolite structures: hydroxylation on the 4'-position camphanoyl (M2-M4 and M9-M10) or on the 3'-position camphanoyl (M1, M5, M6-M8, and M11). Hydroxylation on the khellactone moiety was not detected under the experimental conditions, corresponding to the SMR result showing that substituents on the coumarin did not impact the metabolic stability. Compound 10 has one Cl on the 3-position, and a Cl pattern was observed for both the parent and metabolites (when isolation width was set at 7), which greatly aided confirmation of metabolite structures. No ion peaks corresponding to ester-hydrolyzed khellactones, [M–180 + Na]⁺ or [M–360 + Na]⁺, were detected under the experimental conditions or in plasma (not shown). In addition, no ion peaks expected from general coumarin metabolism were observed: [M–26 + Na]⁺, [M–24 + Na]⁺, and [M–10 + Na]⁺, corresponding to o-hydroxyphenyl acetaldehyde, o-hydroxyphenylethanol, and o-hydroxyphenylpropionic acid, respectively. At higher concentration, multiple peaks corresponding to dihydroxylated [M + 32 + Na]⁺, ketone [M + 14 + Na]⁺, and hydroxy ketone/carboxylic acid [M + 30 + Na]⁺ metabolites were also observed. These metabolites were likely formed sequentially from mono-hydroxylated compounds.

View larger version (31K): [in this window] [in a new window]   FIG. 3. Extracted ion chromatograms of DCK (A), hydroxylated DCK metabolites (B), Cl-Me-DCK (C), and hydroxylated Cl-Me-DCK metabolites (D).

View larger version (20K): [in this window] [in a new window]   FIG. 4. Tandem mass spectra fragments of the hydroxylated DCK metabolites M1 to M5 (isolation width of 1 and CID = 30) and the hydroxylated Cl-Me-DCK metabolites M6 to M11 (isolation width of 7 and CID = 35).

	Discussion

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This is the first investigation, to our knowledge, on the metabolism of DCKs. In the present study using LC-UV, the rapid depletion of the parent compound did not allow for precision; however, the half-life values observed for DCKs (1.5–5.7 min) are shorter than reported values of highly cleared drugs despite the lower protein concentration (0.1 mg/ml) used. Obach (1999) reported in vitro half-life values between 8.0 and 11.0 min for the high clearance drugs propafenone, verapamil, and diclofenac at microsomal protein concentrations between 0.3 and 0.5 mg. Therefore, the rapid in vitro metabolic rates observed for the tested DCKs are predictive of high hepatic clearance in vivo and a high first pass effect. LC-UV chromatograms also indicated the production of multiple metabolites, regardless of the position or the type of the substituent on the khellactone (data not shown), and suggested similar metabolic pathways for various DCK derivatives. Subsequently, metabolites of selected DCK compounds were structurally characterized using LC-ITMS. No hydrolysis of either camphanoyl ester moiety was observed under the hepatic microsomal incubation conditions used. The absence of hydrolytic metabolism may be due to the steric hindrance of the bulky camphanoyl structure. Instead, hydroxylated metabolites were observed as primary oxidative metabolites in microsomal incubations and were attributed mostly to hydroxylation of the two lipophilic camphanoyl moieties. This type of oxidation parallels that of camphor, a terpene that is structurally similar to camphanic acid, reported with rabbit P450 (White et al., 1984) and more commonly with prokaryotic P450_CAM (Grogan et al., 2002), although we know of no report of human P450 oxidation of camphor.

In summary, we have demonstrated that DCK derivatives are rapidly and extensively metabolized by human liver microsomes under oxidative conditions. Although the substituents on the khellactone are reported to greatly affect the in vitro pharmacological activity, the results from both SMR and metabolite analysis indicate that these substituents are unlikely to influence the metabolic stability or the metabolic pathways of the compounds. The results suggest that incorporating alternative structures at the 3'- and/or 4'-ester position is the most viable approach to change the metabolic stability of these compounds.

	Footnotes

 
This investigation was supported primarily by Grant AI-33066 from the National Institute of Allergy and Infectious Diseases, awarded to K.-H.L. The mass spectrometric work was also supported in part by the Center for Environmental Health and Susceptibility Pilot Project Program, University of North Carolina at Chapel Hill University Research Council Grant P30-CA16086 from the National Institutes of Health.

Current address (Y.L.): Merck Research Laboratories, Department of Safety Assessment, West Point, Pennsylvania.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.004218.

ABBREVIATIONS: DCK, 3',4'-di-O-(–)-camphanoyl-(+)-cis-khellactone; DCKs, DCK derivatives; HPLC, high performance liquid chromatography; SMR, structure-metabolism relationship; ITMS, ion trap mass spectrometry; HIV, human immunodeficiency virus; CL_int, intrinsic clearance; t_1/2, half-life; MS/MS, tandem mass spectrometry; MSⁿ, multiple stage mass spectrometry where n refers to the generation number of fragment ions being analyzed; Cl-Me-DCK, 3-chloro-4-methyl DCK; P450, cytochrome P450.

Address correspondence to: Dr. Kuo-Hsiung Lee, Division of Medicinal Chemistry, University of North Carolina, Chapel Hill, NC 27599-7360. E-mail: khlee{at}email.unc.edu

	References

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This Article Abstract Full Text (PDF) All Versions of this Article: dmd.105.004218v1 33/11/1588    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Suzuki, M. Articles by Lee, K.-H. Search for Related Content PubMed PubMed Citation Articles by Suzuki, M. Articles by Lee, K.-H.