In Vitro Metabolism of Phenoxypropoxybiguanide Analogues in

Drugs for Parasitic Infections: Advances in the Discovery of New Antimalarials. M.A. Avery , D.J. Weldon , K.M. Muraleedharan. 2016, ...
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In Vitro Metabolism of Phenoxypropoxybiguanide Analogues in Human Liver Microsomes to Potent Antimalarial Dihydrotriazines Todd. W. Shearer,*,† Michael. P. Kozar,† Michael T. O’Neil,† Philip L. Smith,† Guy A. Schiehser,‡ David. P. Jacobus,‡ Damaris S. Diaz,† Young-Sun Yang,† Wilbur. K. Milhous,† and Donald. R. Skillman† Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, Maryland 20910, and Jacobus Pharmaceutical Company, 37 Cleveland Lane, P.O. Box 5290, Princeton, New Jersey 08540 Received April 29, 2004

Phenoxypropoxybiguanides, such as 1 (PS-15), are prodrugs analogous to the relationship of proguanil and its active metabolite cycloguanil. Unlike cycloguanil, however, 1a (WR99210), the active metabolite of 1, has retained in vitro potency against newly emerging antifolateresistant malaria parasites. Unfortunately, manufacturing processes and gastrointestinal intolerance have prevented the clinical development of 1. In vitro antimalarial activity and in vitro metabolism studies have been performed on newly synthesized phenoxypropoxybiguanide analogues. All of the active dihydrotriazine metabolites exhibited potent antimalarial activity with in vitro IC50 values less than 0.04 ng/mL. In vitro metabolism studies in human liver microsomes identified the production of not only the active dihydrotriazine metabolite, but also a desalkylation on the carbonyl chain, and multiple hydroxylated metabolites. The Vmax for production of the active metabolites ranged from 10.8 to 27.7 pmol/min/mg protein with the Km ranging from 44.8 to 221 µM. The results of these studies will be used to guide the selection of a lead candidate. Introduction It has been estimated that there are 300 million cases of malaria resulting in approximately 2 million deaths per year.1,2 The majority of these cases occur in developing countries with the fatalities predominantly among young children and pregnant women.3 With the absence of an effective vaccine and the global emergence of multidrug resistance, malaria continues to be a worldwide epidemic. Resistant strains of malaria have been identified for most of the antimalarial drugs in clinical use today, including antifolate drugs such as pyrimethamine and cycloguanil, the active metabolite of proguanil. In recent years, the molecular mechanisms conferring resistance to antifolate drugs have been elucidated. Resistance to these compounds is caused by a series of point mutations on the Plasmodium falciparum dihydrofolate reductase thymidylate synthase (pfDHFR-TS) enzyme that leads to a decreased binding affinity for the drugs.4-9 The drug 1a (WR99210), however, was demonstrated by Rieckmann10 in 1973 to be potent in vitro against a highly chloroquine- and pyrimethamine-resistant strain of malaria. Over the 30 years since this discovery, 1a continues to demonstrate a high level of efficacy against mutant pfDHFR-TS enzymes and is equally effective against antifolateresistant and antifolate-sensitive isolates of the malaria parasite.11-17 The crystal structures of wild-type and mutant forms of the pfDHFR-TS enzyme have been solved and provide insight into the effectiveness of 1a against the mutant enzymes.17 This recent work has established the mo* Corresponding author. Present address: Alcon Laboratories, 6201 South Freeway, Fort Worth, TX 76134. Phone: 817-615-2435, Fax: 817-568-7202. E-mail: [email protected]. † Walter Reed Army Institute of Research. ‡ Jacobus Pharmaceutical Company.

lecular mechanisms responsible for the success of 1a against a series of mutant antifolate-resistant strains of the P. falciparum parasite. In summary, the flexible oxygen bridge found in 1a, as first proposed by Warhurst,18 allows the molecule to contort and thus escape the steric hindrance caused by the various mutations. Specific mutations in the pfDHFR enzyme greatly diminish the binding of the more rigid structures of cycloguanil and pyrimethamine. Cycloguanil, the active metabolite of proguanil, is not well absorbed and locally toxic. However, the parent biguanide proguanil is well tolerated and has been the drug of choice for malaria prophylaxis in pregnant women and children for many years prior to the occurrence of resistance.19 Unfortunately, development of 1a into an antimalarial chemotherapy was halted because of significant gastrointestinal intolerance in animal studies and poor bioavailability. The inherent potency of 1a and the need to obviate the gastro-intestinal (GI) intolerance and bioavailability problems, however, led to the development of the phenoxypropoxybiguanide prodrug 1 which metabolized in vivo into 1a (analogous to the conversion of proguanil to cycloguanil).16,20 Development of this compound for human use was terminated primarily due to regulatory issues surrounding toxic manufacturing materials.21 Jensen et al.21 previously reported the synthesis of a series of DHFRinhibiting diaminotriazine antimalarial prodrugs modeled after 1. Like 1, these analogues were presumed to be converted to an active dihydrotriazine metabolite. Several of these analogues showed good activity in animal malaria models, and their presumed active metabolites exhibited a similar level of potency to 1a in vitro.21 Since 2001, Jacobus Pharmaceutical Company in collaboration with the Walter Reed Army Institute of

10.1021/jm049683+ CCC: $30.25 © 2005 American Chemical Society Published on Web 03/18/2005

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Research has selected a group of seven potential antimalarial drug candidates based on the previously described phenoxypropoxybiguanide analogues. Each of these new analogues, parent or active metabolite, have displayed a high level of antimalarial activity in either an in vivo mouse model (data not shown) or in in vitro efficacy testing, respectively. The purpose of this study was to quantify the production of active metabolite and identify the production of other metabolites from these analogues in human liver microsomes in order to guide selection of a lead candidate for further development. Experimental Section Compounds. Proguanil, cycloguanil, 1, 1a, and the novel phenoxypropoxybioguanide analogues, 2 (PS-26), 3 (JPC-2005), 4 (JPC-2028), 5 (JPC-1084), 6 (JPC-2056), 7 (JPC-2058), and 8 (JPC-2059) and their respective putative active metabolites (2a, 3a, 5a, 6a, 7a, and 8a, respectively) were obtained from Jacobus Pharmaceutical Company (Princeton, NJ). 4a could not be synthetically derived and was therefore not available for these studies. The structures of proguanil and its active metabolite cycloguanil are presented in Figure 1A. Figure 1A also shows the general structure of the prodrug analogues and their putative active dihydrotriazine metabolites. In addition, the structures of the phenyl R-groups containing the various ring substitutions for each analogue are exhibited in Figure 1B. In Vitro Activity. The in vitro potency of each of the analogues was tested using the tritiated hypoxanthine method as described by Milhous et al.22 with minor modifications. The parent analogues were dissolved in DMSO (Sigma, St. Louis, MO) as 50.0 mg/mL stock solutions. The active metabolites were dissolved in DMSO as 10.0 mg/mL stock solultions. The stock solutions were diluted 1000-fold in folate-free media (RPMI-1640, Invitrogen Corp., Carlsbad, CA) to get the 50.0 or 1.0 µg/mL starting concentrations, respectively. Two-fold dilutions of the starting concentration were made in folatefree media and 20 µL/well was added to a 96-well culture plate. A 0.5% parasite concentration was diluted 4-fold in folate-free media and 180 µL/well was added to each culture plate. Five 10-fold serial dilutions were made to evaluate a range from 5000 ng/mL (or 2500 ng/mL depending on analogue solubility) to 5 pg/mL for the parent compounds or 5 ng/mL to 5 fg/mL for the active metabolites. The plates were maintained at 37 °C for 48 h in a chamber charged with 90% N2, 5% CO2, 5% O2. At 48 h, 25 µL of [3H]-hypoxanthine (15 µCi/mL) (American Radio labeled Chemicals, Inc., St. Louis, MO) was added, and the parasite plates were incubated for an additional 24 h. At 72 h, parasites were harvested onto Unifilter-96 microplates (PerkinElmer, Wellesley, MA). The filter plates were air-dried and 50 µL/well scintillation fluid was added. Radioactive emissions were counted in a TopCount NXT (PerkinElmer, Wellesley, MA). Each analogue was assayed against each parasite strain in triplicate on three separate occasions. Parasite growth inhibition was measured by the decreased accumulation of [3H]-hypoxanthine used in the purine nucleotide salvage pathway. The percentage of growth inhibition was determined by comparing radioactive emissions in drug free controls versus parasites incubated with various concentrations of drug. The IC50 value was determined by sigmoid dose-response nonlinear regression analysis using Prism 4.0 (GraphPad Software Inc., San Diego, CA). The equation used to determine percentage of growth inhibition was percent reduction ) 100 × [(mean counts per minute (cpm) drug free control samples minus mean cpm test samples)/mean cpm drug free control samples]. Parasite growth inhibition was also determined for DMSO. DMSO concentrations as high as 0.1% had no inhibitory effect on parasite growth, and in this study, the DMSO concentration was never above 0.002% in any drug experiment. Two well characterized P. falciparum clones W2 (Indochina III/CDC) and D6 (Sierra Leone I/CDC), representing antifolate-resistant and -sensitive strains, respectively, were assayed.23,24

Figure 1. Structures of proguanil (1), the new phenoxypropoxybiguanide analogues (1, 2, 3, 5, 6, 7, and 8), and their respective active dihydrotriazine metabolites (1a, 2a, 3a, 5a, 6a, 7a, and 8a). 1A. Conversion of proguanil (1) to its active metabolite cycloguanil (1a) by cytochrome P450s 2C19 and 3A4 as previously described. Also shown is the proposed mechanism for the conversion of the phenoxypropoxybiguanide analogues to the respective active metabolites. 1B. General structure of the aromatic (Ar) ring portion of the phenoxypropoxybiguanide analogues and their putative active metabolites. Microsomal Assay. Each compound was added to a mixture containing NADPH regeneration buffer [1.25 mM βNADP+, 3.3 mM glucose-6-phosphate, and 3.3 mM MgCl2] and 1 mg/mL pooled human liver microsomes (BD Biosciences., San Jose, CA) to a final volume of 225 µL. The mixture was incubated for 5 min at 37 °C and then initiated by the addition of 25 µL of glucose-6-phosphate dehydrogenase (Sigma) [1 unit/ mL final concentration]. Each reaction was maintained at 37 °C until termination at the specified time point by the addition of an equal volume of ice cold acetonitril. Metabolic profiles were determined at 50 µM for each compound and terminated following 30, 60, 90, and 120 min incubation periods. For Vmax studies, the acetonitrile stop solution contained 10 µM of Jacobus Pharmaceutical analogue PS-33 (4-chloro analogue similar to the structure of 2) as an internal standard. The Vmax experiments were conducted at 25, 50, 100, 150, 200, 250, and 500 µM for each compound and were terminated following a 90 min incubation period. Vmax and Km values were determined by Michaelis-Menton nonlinear regression analysis (Y ) bx/K1 + x) performed using Prism

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Table 1. In Vitro Antimalarial Activity of Phenoxypropoxybiguande Prodrugs (1-8) and the Corresponding Putative Active Metabolites (1a-8a) parent drug IC50a (ng/mL)

M1 dihydrotriazine IC50a (ng/mL)

drug

D6b

W2c

pyrimethamined proguanil/cycloguanile 1/1a 2/2a 3/3a 4g 5/5a 6/6a 7/7a 8/8a

1.2 >5000h 877 ( 83 845 ( 43 >2500h >2500h >2500h 2232 ( 376 >2500h >2500h

130 >5000h 3786 ( 385 2551 ( 608 >5000h >5000h >5000h >5000h >5000h >5000h

D6b NAf 0.328 ( 0.015 0.02 ( 0.002 0.009 ( 0.001 0.01 ( 0.001 NDg 0.01 ( 0.007 0.01 ( 0.001 0.022 ( 0.010 0.022 ( 0.005

W2c NAf >2.5h 0.016 ( 0.001 0.028 ( 0.003 0.052 ( 0.007 NDg 0.063 ( 0.005 0.031 ( 0.002 0.053 ( 0.005 0.081 ( 0.01

a Calculated IC 50 values were determined by nonlinear regression analysis (sigmoid dose response in GraphPad Prism 4.0) ( 95% confidence interval. b D6 is a chloroquine-, DHFR wild-type pyrimethamine-sensitive strain. c W2 is a chloroquine-, DHFR mutant pyrimethamine-resistant strain. d Pyrimethamine is an DHFR inhibitor and antimalarial drug used as a reference compound. e Cylcoguanil) is the DHFR inhibiting, active metabolite of a the antimalarial drug proguanil. f NA ) not applicable. g ND ) not determined, 4a was not available and could not be tested. h Indicates IC50 value exceeded the highest concentration tested. The value following the (>) symbol indicates the highest concentration tested in the assay.

Figure 2. Representative LC-MS chromatograms of 2 and its putative metabolites. Panel A shows the parent compound. Panel B shows the putative active metabolite 2a (M1), -2 mu. The lower two panels depict representative LC-MS chromatograms in which the two additional metabolites were identified. These metabolites were determined to be a desalkylation (M2), -42 mu, and several potential hydroxylated metabolites (M3aryl 1-3 and M3β), +16 mu, Panels C and D, respectively. These chromatograms are representative of those observed for each of the analogues. The desalkyl metabolite (M2) was identified for each analogue with the exception of 4. Hydroxylated metabolites were identified for Proguanil, 2, 4, 5, 6 and 7 but not 1, 3, or 8. 4.0 (GraphPad Software, Inc.). A minimum of six reactions were conducted for each compound. Production of metabolites was initially evaluated at 0.25, 0.5, 1.0, and 2.0 mg/mL and at time points ranging from 10 to 120 min. Production of all observed metabolites was determined to be linear within these parameters. LC-MS and LC-MS/MS. Metabolic profiling of the various analogues was determined by LC-MS and LC-MS/MS. Chromatography conditions consisted of a slow gradient ranging from 75:5:20 (dH2O:acetonitrile:0.5% formic acid, pH 3.2) to 5:75:20 (dH2O:acetonitrile:0.5% formic acid, pH 3.2) over 60 min using a 2.1 mm × 50 mm, 3.5 µm Xterra C18 column (Waters Corp., Milford, MA) with a 0.2 mL/min flow rate using a Waters 2690 HPLC system. Metabolite identification was performed on a Thermofinnigan LCQ Classic ion trap mass spectrometer (Thermoelectron, San Jose, CA) tuned and calibrated according to the manufacturer’s procedure in the positive ion, atmospheric pressure ionization mode. Detection and mass scanning was performed in the full scan mode (150 m/z to 650 m/z) with the electrospray voltage set to 4.5 kV, capillary temperature of 250 °C, sheath gas flow of 90 abu, auxiliary flow at 5 abu, injection waveforms off and automatic gain control on. Preliminary metabolite structure

elucidation was conducted using MS/MS analysis of suspected metabolites and the Metabolite ID software from Thermo Electron. Due to the lack of authentic standards for the hydroxylated and desalkylated metabolites exact quantitation in in vitro microsomal incubations could not be determined. However, assuming similar extraction recoveries and similar ionization potential for these compounds, the relative production of the metabolites was estimated.25-27 Production of the active triazine metabolite was determined using LC-MS/MS on a Thermo Electron Surveyor HPLC coupled to a TSQ Quantum AM triple-quadrupole mass spectrometer (Thermo Electron, San Jose, CA). The chromatographic separation was performed using a 2.1 mm × 50 mm, 3.5 µm Waters Xterra C18 column (Waters Corp, Milford, MA). A rapid gradient was employed using 20% A (0.5 formic acid), 75% C (H2O) and 5% D (MeOH) which was held for one minute, then ramped to 20% A, 5% C, 75% D in 5 min and held for 4 additional minutes, followed by returning to the starting conditions and equilibrating for 3 min. Selected reaction monitoring (SRM) was employed by selecting the parent ion of the drug (M + 1) in the first quadrupole followed by collision induced disassociation in the collision cell (99.999% argon at

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Figure 3. Tandem mass spectra of analogue 2 hydroxylated metabolites (M3aryl 1-3 and M3β) and a proposed fragmentation pathway. A. Tandem mass spectrometry (LC-MS/MS) ion spectra of analogue 3 hydroxylated metabolites m/z 378. The MS/MS ion spectrum for the aryl hydroxylated metabolites (M3aryl 1-3) contain a unique fragment at m/z 361 and a common fragment at m/z 294. Fragmentation of the M3β metabolite resulted in a unique ion at m/z 331. B. A proposed fragmentation pathway for the major ion fragments of the M3aryl and M3β metabolites of analogue 2. The m/z 361 fragment is consistent with aryl hydroxylation and a loss of NH2. The 331 fragment is consistent with a predictable fragment ion containing alkyl hydroxylation of the isopropyl group. The m/z 294 fragment is common in all of the spectra suggesting that the hydroxylation has been lost from both the M3aryl 1-3 and M3β metabolites. a pressure of 1.0 mTorr and collision energy experimentally determined) to produce the most intense, characteristic product ion. The mass spectrometer was tuned and calibrated according to the manufacturer’s procedure in the positive ion, atmospheric pressure ionization mode with the electrospray voltage set at 4.7 kV, sheath gas pressure at 30 psi with no auxiliary gas and the heated capillary at 325 °C. The peak area ratios (PARs) were determined by the peak area of drug or metabolite to the IS and were calculated for each sample. The PARs of the standard curve samples were fit by 1/y weighted least squares linear regression to the equation for the straight line (y ) mx + b), where y ) PAR and x ) drug or metabolite concentrations. Drug or metabolite concentrations in the microsomal incubations were calculated using the drug/metabolite to IS PARs obtained by LC-MS/MS

and processed by Xcalibur Quan Browser Software (Thermo Electron). Each run was checked for accuracy and precision of the method using internal quality control (QC) samples spiked with known concentrations of each analogue and metabolite as appropriate. Two QC samples and standard curves were injected at the beginning and end of each sample run. The run was accepted if the coefficient of variation (%CV) of the QC samples was less than 20%.

Results In Vitro Activity. The structures of the prodrugs 1-8 and the active dihydrotriazine metabolites 1a-8a are depicted in Figure 1, panels A and B. The predicted active metabolites of each of the newest analogues were

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Figure 4. Proposed pathway of metabolite formation. The structures in brackets (I and II) are proposed and have not been observed. M1 is the active, dihydrotriazine metabolite; M2 is the N-dealkylated metabolite; M3aryl are collectively the [M + 16] metabolites whose MS/MS fragmentation patterns are consistent with aryl hydroxylation; M3β is the proposed structure of the metabolite whose MS/MS fragmentation pattern is consistent with alkyl hydroxylation of the isopropyl group and whose stability to elimination and hydrolysis under the acidic chromatographic and detection conditions suggests hydroxylation at one of the two methyls. This observed chemical stability is in contrast to I which is unobserved under the analytical conditions.

tested against two laboratory adapted strains of P. falciparum (D6 and W2) and were all equally potent to 1a. IC50 values for the in vitro parasite assays are presented in Table 1. All of the active dihydrotriazines exhibited IC50 values less than 0.04 ng/mL against the chloroquine/pyrimethamine-sensitive strain (D6) and the chloroquine/pyrimethamine-resistant DHFR mutant strain (W2). Activity of the predicted metabolite of 5 could not be assessed due to unavailability of the compound. The predicted metabolites showed similar activity to the previously reported active metabolites in this series.21 Metabolic Profiling. In general, LC-MS/MS analysis of the analogues metabolized by human liver microsomes identified three primary metabolites. Initial characterization of these metabolites by LC-MS and/or LC-MS/MS followed by metabolite ID analysis predicted these metabolites to be the active dihydrotriazine (-2 mass units (mu)) M1, a desalkylation on the carbonyl chain (-42 mu) M2, and multiple possible hydroxylations (+16 mu) M3 R, β, etc., respectively. With the exception of 4a, the predicted active metabolites (analogous to 1a) of each analogue were used to confirm the presence of the active dihydrotriazine metabolite in the metabolized incubations. In each case, the synthetic standards of the active metabolites and the -2 mu metabolites found in the microsomal incubations exhibited identical analytical characteristics including retention time and MS/MS product ion spectral analysis (data not shown). Synthetically derived standards were not available for the other putative metabolites; therefore, LC-MS and LC-MS/MS was used to elucidate the probable structures. A representative chromatogram showing the primary metabolites of 2 following separation on a slow gradient is exhibited in Figure 2. Figure 3 exhibits typical product ion spectra and corresponding predicted structures used to identify different sites of hydroxylation on 2. Figure 4 represents the proposed metabolic pathway and putative structures for the primary metabolites observed in the reaction for

six of the seven analogues. Due to the significant structural difference, the putative metabolic profile of 4 is depicted separately in Figure 5. Note that hydroxylated metabolites (-16 mu) were not detected for 1, the 4-trifluoromethoxy analogue (3), or the 2-chloro-4-trifluoroethoxy analogue (8). Because no standards were available for the desalkyl or hydroxylated metabolites for these analogues, the actual quantities of these compounds could not be determined. To compare these analogues based on their ability to preferentially produce the active metabolite, it was important to obtain a relative level of each of the metabolites produced. Assuming relatively similar ionization potential for these metabolites under the given highly acidic analytical conditions, semiquantification of each metabolite based on peak area was considered acceptable for this application.25-27 Summary results of these comparisons are displayed in Figure 6. The metabolic profile of the majority of the analogues, with the exception of 4 and 7, was similar with the most abundant metabolite being the active dihydrotriazine. The active metabolite (-2 mu) represented between 60 and 80% of the total metabolites while the desalkyl metabolite (-42 mu) appeared to be the second most abundant, representing between 15 and 30%, and the hydroxylated metabolite(s) (+16 mu) representing less than 5%. In the case of 1, 3, and 8, no hydroxylated metabolite was detected. For 4 and 7, however, the metabolic profiles were dramatically different. For these analogues, the hydroxylated metabolite was the most abundant, representing greater than 65% of the total metabolites. In addition, for these two analogues, the active dihydrotriazine represented only about 30-35% followed by the desalkyl with 0% and 8% of the total metabolites for 4 and 7, respectively. Interestingly, proguanil also showed a relatively high level of hydroxylated metabolite. Relative amounts of each of the primary metabolites as determined by area under the curve are presented in Figure

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Figure 5. Proposed pathway of metabolite formation for the N-cyclopropyl derivative 4. The formation of aryl hydroxylated metabolites (M3aryl) or an N-dealkylated metabolite (M2) was not observed. However, a stable [M + 16] metabolite 4b, also labeled M3β, was observed whose MS/MS fragmentation pattern suggested hydroxylation of the cyclopropyl ring at one of the methylene carbons.

Figure 6. Metabolic profile of phenoxypropoxybiguanide analogues in human liver microsomes. Because reference standards were only available for the putative active metabolites, comparisons were made based on the area of the chromatographic peaks for each metabolite divided by the total area of all metabolites for each analogue. If multiple hydroxylated metabolites were identified, the sum of the areas of all significant peaks was used. Values are means ( SEM, n ) 6.

5. Values are presented as a percentage of the total metabolites identified. Microsomal Kinetic Studies. To determine if the different microsomal metabolic profiles of the individual analogues would effect the overall production of active dihydrotriazine metabolite, a maximum velocity (Vmax) study was initiated using human liver microsomes. Increasing concentrations ranging from 25 µM to 500 µM of each drug were incubated with 1 mg/mL microsomal protein for 90 min. Production of active dihydrotriazine metabolites were quantified using the standard curves produced from the synthetically derived dihydrotriazine standards for each analogue. Quantification of the active dihydrotriazine metabolite for 4 could not be performed due to the unavailability of a synthetically derived standard. The summary results of the Vmax study are presented in Figure 7. Michaelis-Menton nonlinear regression analysis of the production of active metabolite revealed similar Vmax and Km values for each of the analogues (Table 2). At

lower concentrations, production of the active metabolite was similar among the analogues while some significant differences were observed at higher concentrations. For example, 1 exhibited a significant decrease in active metabolite production at concentrations above 200 µM, indicating possible inhibition or saturation of the enzymes involved (data not shown). For this reason, only values resulting from the concentrations of 200 µM and below were used to calculate Vmax and Km. Additional experiments are necessary to determine the mechanism(s) involved in this response. Interestingly, even though the metabolic profile of some of the analogues are dramatically different (i.e. significantly higher production of hydroxylated metabolite(s)), there did not appear to be any significant differences in the Vmax for production of the active dihydrotriazine. These results indicate the possible involvement of different enzymes in the production of the various metabolites. Additional studies are necessary to determine the role of various enzymes in the metabolism of these analogues. Conclusions and Summary This investigation was undertaken in order to evaluate a series of phenoxypropoxybiguanide analogues for their potential as new antimalarial chemotherapeutic agents. As a class, these compounds are known to be prodrugs that require metabolic oxidation and cyclization to form the active antimalarial dihydrotriazines M1. These compounds were chosen for evaluation in order to help identify those candidates that displayed maximum production of the desired dihydrotriazine and minimum formation of other, nonefficacious metabolic products. In the presence of human liver microsomes, each of the analogues displayed a relatively characteristic metabolic profile for this class of drug. Conversion to the active dihydrotriazines M1 was typically in the range of 60 to 80% based on starting drug except for 4 and 7 in which the active species constituted only 30-35%. The rest of these metabolites were tentatively identified by MS/MS fragmentation patterns. Figure 4 shows the proposed pathway of metabolite formation that is consistent with the known metabolism

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Figure 7. Production of active metabolites in human liver microsomes. Michaelis-Menton plot showing the production of active metabolites from third generation antifolates in human liver microsomes. Reactions were terminated following a 90 min incubation. Production of active metabolites by each analogue was determined to be linear for up to 2 h for the conditions described. Values are presented as means ( SEM, n ) 6. Vmax and Km values Michaelis-Menton nonlinear regression analysis (Y ) bx/K1 + x) are presented in Table 2. Table 2. Michaelis-Menton Enzyme Kinetic Valuesa for the Conversion of Phenoxypropoxybiguanide Prodrugs to Active Dihydrotriazine Metabolites Vmax (nmol/min/mg) Km (µM) a

proguanil

1

2

3

5

6

7

8

19.6 ( 3.1 111 ( 34.1

11.6 ( 1.4 44.8 ( 17.3

27.7 ( 6.4 176 ( 69.5

13.5 ( 1.5 77.6 ( 19.6

10.8 ( 1.2 131 ( 29.8

15.4 ( 1.8 129 ( 31.0

13.3 ( 1.3 184 ( 32.1

18.7 ( 3.2 221 ( 62.4

Values are means ( standard error of the mean. N ) 6.

of proguanil28,29 and with LC-MS/MS fragmentation data obtained during the course of these experiments. Initial hydroxylation of the parent drug at the methine carbon of the isopropyl group would yield a hemiaminal I that may dealkylate to form M2 or eliminate water to form an imine intermediate II. Both I and II are proposed structures of plausible intermediates, neither of which were observed during these experiments. Cyclization of the biguanide N1 onto the isopropyl imine generates the active dihydrotriazine M1. Oxidation at other sites on the parent drug produce [M + 16] hydroxylation products collectively labeled M3. Most of the analogues examined here were differently substituted at the aryl ring in order to decrease the occurrence of aryl hydroxylation with the intent of improving production of M1 levels. For the series of chlorinated analogues, the extent to which ring hydroxylation occurred followed the expected trend of proguanil (30%) > 2 (5%) > 1 (0%) in which the number of available Ar-H sites were reduced by halogen substitution. The remainder of the analogues incorporated a trifluoromethyl substituent connected either directly to the ring (as in 5), as trifluoromethoxy (3, 6, and 7), or as a 2,2,2-trifluoroethoxy (8). The substitution of any of these CF3-containing substituents at the para-position completely or mostly prevented aryl hydroxylation on the aromatic ring. However, positioning trifluoromethoxy at the ortho-position resulted in formation of >65% hydroxylation products, presumably all aryl hydroxylation, based on starting drug. This result suggests that the CF3 group in the para- position likely results in an unfavorable steric interaction between the aryl region of the drug and the binding pocket of oxidizing enzymes. The formation of M2 N-dealkylation products was observed with each of the analogues examined except 4

and ranged from 5% to 30% of the total metabolites observed. Among the N-isopropyl derivatives, no trend could be discerned for relating their abundance (%-formation) to chemical structure. Interestingly, nonaryl hydroxylation products were observed whose MS/MS fragmentation patterns suggested that oxidation had occurred on the isopropyl group but did not render the resulting molecule susceptible to N-dealkylation. Unlike the proposed R-hydroxylated hemiaminals which are expected to be unstable to hydrolysis/N-dealkylation or elimination/cyclization, these metabolites were stable and are therefore believed to be 1,2-amino alcohol derivatives of the parent drug. These metabolites are labeled M3β. Unlike the other analogues, 4 was prepared as an N-cyclopropyl derivative of 3 with the expectation that a sterically less encumbering cyclopropyl group might promote increased conversion to the corresponding cyclic dihydrotriazine (see Figure 5). Like 3, 4 also incorporated the p-trifluoromethoxy moiety on the aromatic ring in order to minimize or prevent aryl hydroxylation. Indeed, M3aryl-type hydroxylation products were not observed following microsomal incubations, and N-dealkylation was also not observed. Unfortunately, MS/MS experiments revealed that the primary metabolite (65% abundance) was alkyl hydroxylation at one of the two methylene carbons of the cyclopropyl group (4-M3β). The active metabolite 4-M1 represented only 30-35% of the total metabolites. When compared to proguanil, these analogues appear to have a similar metabolic profile and produce comparable levels of active metabolite. Proguanil metabolism and production of its active metabolite cycloguanil have previously been shown to involve primarily cytochrome P450 2C19 with possibly minor contributions by cytochrome P450 3A4.30-32 Especially at lower concentra-

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tions (less than 100 µM), these analogues appear to have a similar Vmax and Km to that of cycloguanil production from proguanil. The Vmax of the novel analogues ranged between 10.8 and 27.7 pmol/min/mg microsomal protein with Km ranging from 44.8 to 221 µM, while proguanil conversion to cycloguanil exhibited a Vmax and Km of 19.6 pmol/min/mg microsomal protein and 111 µM, respectively. On the basis of these results, it can be hypothesized that metabolism of these new analogues to their active triazines involve similar enzyme systems. Additional studies are currently underway to identify which cytochrome P450 enzymes are involved in the production of the active triazines and other metabolites. A further understanding of the enzymes involved will be an integral part in the downstream development of one of these analogues into an antimalarial drug. In addition, a further understanding of the different metabolic profiles of these compounds will be necessary for the interpretation of toxicological and efficacy studies in animal models. Identification of the significant level of hydroxylated metabolite produced from 4 and 7 in human liver microsomal assays may play a key role in selection of the final lead compound. Additional studies will be needed to determine if a similar profile is produced in the microsomes of other animal species and what occurs in vivo. Although the different metabolic profiles of these drugs in vitro does not appear to affect the rate of production of active metabolite by human liver microsomes, these differences may be more dramatic in vivo. Because the parent analogue is not an active antimalarial but depends on enzyme metabolism to form the active dihydrotriazine, it will be important to understand the in vivo effect of the different possible metabolic profiles. Further studies are needed, however, to determine if the metabolic characteristics of these analogues will have any effect on the production of active metabolite, half-life, or toxicity of these analogues in vivo. Although many questions remain concerning the metabolism of these compounds, this study has provided a basis by which these analogues can be assessed in the future. Given the similarities of the in vitro potency of this series of analogues, the metabolic characteristics of the individual compounds will be an important distinction when selecting a lead candidate for further development. Acknowledgment. The authors would like to thank Ms. Lucia Gerena for her technical assistance and continued extraordinary service conducting the in vitro parasite assay. Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publications. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting true views of the Department of the Army or the Department of Defense. References (1) Report of the Ad Hoc Committee on Health Research Relating to Future Intervention Options. Investing in health research and development; World Health Organization: Geneva, Switzerland, 1996. (2) World Health Report; World Health Organization: Geneva, Switzerland, 1999. (3) Wirth, D. F. Malaria: A Third World Disease in Need of First World Drug Development. Annu. Rev. Med. Chem. 1999, 34, 349-358.

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