Design and Synthesis of Potent Inhibitors of Plasmepsin I and II: X-ray

Design and Synthesis of Potent Inhibitors of Plasmepsin I and II: X-ray Crystal. Structure of Inhibitor in Complex with Plasmepsin II. Per-Ola Johanss...
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J. Med. Chem. 2005, 48, 4400-4409

Design and Synthesis of Potent Inhibitors of Plasmepsin I and II: X-ray Crystal Structure of Inhibitor in Complex with Plasmepsin II Per-Ola Johansson,† Jimmy Lindberg,‡ Michael J. Blackman,§ Ingemar Kvarnstro¨m,† Lotta Vrang,| Elizabeth Hamelink,| Anders Hallberg,⊥ Åsa Rosenquist,*†,| and Bertil Samuelsson*,|,X Department of Chemistry, Linko¨ ping University, S-581 83 Linko¨ ping, Sweden, Department of Cell and Molecular Biology, BMC, Uppsala University, Box 596, S-751, 24, Uppsala, Sweden, Division of Parasitology, National Institute for Medical Research, The Ridgeway, Mill, Hill, London NW7 1AA, UK, Medivir AB, Lunastigen 7, S-141 44 Huddinge, Sweden, Department of Medicinal Chemistry, Uppsala University, BMC, Box 574, S-751 23 Uppsala, Sweden, and Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106, 91 Stockholm, Sweden Received October 5, 2004

New and potent inhibitors of the malarial aspartic proteases plasmepsin (Plm) I and II, from the deadliest malaria parasite Plasmodium falciparum, have been synthesized utilizing Suzuki coupling reactions on previously synthesized bromobenzyloxy-substituted statine-like inhibitors. The enzyme inhibition activity has been improved up to eight times by identifying P1 substituents that effectively bind to the continuous S1-S3 crevice of Plasmepsin I and II. By replacement of the bromo atom in the P1 p-bromobenzyloxy-substituted inhibitors with different aryl substituents, several inhibitors exhibiting Ki values in the low nanomolar range for both Plm I and II have been identified. Some of these inhibitors are also effective in attenuating parasite growth in red blood cells, with the best inhibitors, compounds 2 and 4, displaying 70% and 83% inhibition, respectively, at a concentration of 5 µM. The design was partially guided by the X-ray crystal structure disclosed herein of the previously synthesized inhibitor 1 in complex with plasmepsin II. Introduction Malaria is the most serious parasitic disease in the world with mortality and morbidity rates only exceeded by tuberculosis.1 Almost half of the world’s population lives in malaria-endemic areas, and the disease afflicts 500 million people yearly, killing as many as 2 million of them, mostly children.2 Plasmodium falciparum, the most lethal of the four parasites infecting humans, is increasingly becoming resistant to existing therapies, underscoring the paramount need for new treatment paradigms and for new efficacious drugs.2a,3 An important step of the parasite’s life cycle occurs when the parasite invades the red blood cells of its host and starts consuming the hemoglobin as a source of amino acids necessary for growth and maturation. Hemoglobin is degraded by a number of proteases in an acidic food vacuole of the parasite.4a Four aspartic proteases have been found to be active in the food vacuole: plasmepsin I (Plm I), plasmepsin II (Plm II), plasmepsin IV (Plm IV), and HAP, a histidine aspartic protease.2e,5,6 Plasmepsin I and II have been studied in detail, and inhibitors of the plasmepsins have shown efficacy in cell and animal models of malaria, suggesting that these enzymes may be suitable targets for the development of new antimalarial drugs.7 Plm I and II show a high degree of sequence homology (73%), indicating that * Corresponding authors. Phone: +46 8 54683100. Fax: +46 8 54683199. E-mail: [email protected]; asa.rosenquist@ medivir.se. † Linko ¨ ping University. ‡ Department of Cell and Molecular Biology, Uppsala University. § National Institute for Medical Research. | Medivir AB. ⊥ Department of Medicinal Chemistry, Uppsala University. X Stockholm University.

molecules can be designed to target both enzymes. The plasmepsins also show similarity to human cathepsin D, making selectivity an important factor in inhibitor design.3,8 Several structural motifs have been employed in the design and synthesis of Plm I and II inhibitors including hydroxyethylamines,3,9 reversed statines,10 allophenylnorstatines,6c,11 and C2-symmetric dipeptide mimetics.8,12 Moreover, the statine core, incorporated in the naturally occurring aspartyl protease inhibitor pepstatin A, has successfully been used in the development of potent inhibitors of Plm I and II (Figure 1).4,13,14 We have previously reported on the synthesis of a series of highly promising p-bromobenzyloxy-substituted statine-like inhibitors,14 where inhibitor 1 (Figure 1) represents the most potent inhibitor in that series displaying Ki values of 0.5 and 2.2 nM for Plm I and II, respectively. We have now obtained the X-ray crystal structure of compound 1 in complex with Plm II, which has provided new and important information on the key interactions utilized by the inhibitor in the enzyme active site. Moreover, it has revealed conformational changes in the plasmepsin II enzyme which rationalize the potency observed for inhibitor 1 (Figures 2, 3, 4). The detailed structure analysis will be published elsewhere.15 Herein we report a series of potent inhibitors with extended P1 substituents. The structural information gained from the crystal structure of inhibitor 1 bound to Plm II guided the design, and room-temperature Suzuki couplings16 with three selected aryl boronic acids to modify the bromobenzyloxy moiety served as key reactions in the lead optimization process (Scheme 1, Table 1). The best inhibitors identified exhibit Ki values

10.1021/jm040884n CCC: $30.25 © 2005 American Chemical Society Published on Web 06/02/2005

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Figure 1. Two potent plasmepsin inhibitors encompassing a modified statine (A) and a hydroxyethylamine (B) motif. Compound 1 represents the most potent inhibitor in our previous series of p-bromobenzyloxy-substituted statine-like inhibitors. Figure 3. Ribbon diagram of the substrate cavity of Plm II (gray) in complex with compound 1 (gold) showing the specific hydrogen bonds to residue side chains (D34, D214, Y192, and S79). The omit electron density map indicates a unique configuration of the inhibitor in the substrate cavity where the central hydroxyl group donates and accepts a hydrogen in two hydrogen bonds to the catalytic aspartic acids (D34 and D214). In addition, the water-coordinated S2′ subsite is indicated as well as the sandwich stacking of P1 and P3 against F111 and T114 of the S1-S3 subsite. The image was compiled using the molecular graphics program O21 and the web-based POV-Ray program MOLRAY.22 The omit map was contoured at 0.4 e/Å3.

Figure 2. Surface plot of the substrate cavity of Plm II (gray) in complex with compound 1 (gold) showing the flap in a closed state. The image was compiled using the molecular graphics program O21 and the web-based POV-Ray program MOLRAY.22

in the range of 0.3 to 16 nM for both Plm I and II. Some of the inhibitors also show efficacy by attenuating parasite growth in red blood cells,17 e.g. compound 2 and 4, which at 5 µM display an inhibition of 70% and 83%, respectively (Table 1). Results and Discussion Chemistry. We have previously published on the synthesis of the p-bromobenzyloxy-substituted inhibitors 1, 5, 9, 13, and 16 (Table 1).14 Several conditions were explored in order to introduce the novel p-benzyloxy heterocycles or aryl substituents in the P1 position. Heck-like reactions using thiazole, tetrakis(triphenylphosphine)-palladium(0), and potassium acetate in N,N-dimethylacetamide at 110 °C to 160 °C18 resulted in poor yields and epimerization of the starting material. Negishi cross-coupling conditions to introduce a thiaz-

Figure 4. Ribbon diagram of the flap (gray) and the S1-S3 helix (gray) showing the sandwiched stacking of the P1 and P3 aromatic rings and the bromine substituent of compound 1 (gold) in the S1-S3 subsites. The 4-bromobenzyloxy moiety is making face-to-face van der Waals contacts to residues F111, T114, and the P3 substituent. The majority of the protein is omitted for clarity. The image was compiled using the molecular graphics program O21 and the web-based POV-Ray program MOLRAY.22

ole19 and palladium-catalyzed aryl aminations20 were then examined but again found to give very low and unsatisfactory yields. Finally, using room-temperature

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Scheme 1a

a Reagents: (i) R -B(OH) , Pd(OAc) , 2-(di-tert-butylphosphino3 2 2 )biphenyl, KF, dry THF, rt.

Suzuki-couplings with selected boronic acids, palladium acetate, 2-(di-tert-butylphosphino)-biphenyl, and potassium fluoride in dry THF16 the targeted P1 modified compounds 2-4, 6-8, 10-12, 14, 15, 17, and 18 were delivered in modest to excellent yields ranging from 21% to 96% (Scheme 1, Table 1). The reaction time was optimized by monitoring the reaction using MALDI-TOF spectrometry, and after 48 h this analysis indicated that all starting material had been consumed. The boronic acids were phenylboronic acid, 3-thiopheneboronic acid, and 3,4-methylenedioxyphenylboronic acid selected based on commercial availability, synthetic feasibility, and the diverse properties of the substituents regarding for example hydrogen bonding possibilities and polarity. The m-bromobenzyloxy-substituted compound 19 was prepared in 40% total yield over six steps from 3-deoxy1,2-O-isopropylidene-D-glucose using m-bromobenzyl bromide as alkylating agent, according to the procedure for the synthesis of the corresponding benzyloxysubstituted compound in ref 14. The meta-substituted inhibitors 20 and 21 were prepared in 30% and 85% yields, respectively, using the same Suzuki coupling conditions as for the synthesis of the corresponding para-substituted inhibitors (Scheme 1, Table 1). Biological Data and Structure-Activity Relationships All products were screened against both Plm I and II and the Ki values are shown in Table 1. Table 1 also displays the synthesis yields of the inhibitors, Ki values for the inhibition of human cathepsin D, and the percentage inhibition of parasite growth in red blood cells (RBC)17 at an inhibitor concentration of 5 µM, for selected inhibitors. X-ray Crystal Structure of the Inhibitor 1-Plm II Complex. Compound 1 represents the most potent inhibitor in the previous series of p-bromobenzyloxysubstituted statine-like inhibitors.14 To determine the key interactions and molecular features that contribute to the impressive inhibitory properties of inhibitor 1, this compound was cocrystallized with Plm II providing an X-ray crystal structure determined to 2.2 Å resolution and refined down to 20% and 24% in R and Rfree, respectively. The X-ray images (Figures 2-4) were compiled using the molecular graphics program O21 and the web-based POV-Ray program MOLRAY.22 The structural analysis reveals a compact binding between inhibitor and the substrate cavity similar to the “closed state” binding of pepstatin A reported by Silva et al.4a (Figure 2). Examination of the Plm II-inhibitor 1 complex reveals a network of nine hydrogen bonds that coordi-

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nate the inhibitor with the β-hairpin structure, known as the flap, and the residues at the bottom of the binding cleft, including the catalytic aspartic acids, D34 and D214. Two hydrogen bonds are formed between the central hydroxyl group and the carboxyl groups of D34 (2.8 Å) and D214 (2.6 Å). Two additional hydrogen bonds between residue side chains and the inhibitor are observed between S79 and Y192 side chains and the P3 and P2′ inhibitor moieties, respectively (Figure 3). The P1-P3 substituents are well accommodated for in the continuous S1-S3 subsite. The elongated pbromobenzyloxy substituent adopts an orientation that efficiently fills the S1 and S3 subsites. There are several beneficial hydrophobic close contacts associated with the P1 substituent that contribute to the overall good binding properties, including van der Waals contacts between the methyl-O-methyl linker and residues D34, Y77, G216 and between the p-bromobenzyloxy moiety and residues I32, S79, F111, T114, F120. The bromosubstituted benzyloxy side chain has van der Waals contacts to residues F111 (3.8 Å) and T114 (3.6 Å) on one side and to the P3-pyridinyl substituent (4.0 Å) on the other side in a sandwich manner (Figure 4). In addition, the edges of the p-bromobenzyloxy substituent are in close contact with the S1 subsite residues I32, I123, and F120 on one side and G80 of the flap on the other side resulting in efficient utilization of S1 subsite contacts. The optimal interactions between the P1 moiety and the S1 and S3 subsites contribute to the impressive inhibitory efficacy of this class of compounds. In contrast to the P1, the P3 substituent has fewer close contacts to the S3 subsite residues, and the configuration of the pyridinyl ring is mainly stabilized by stacking to the P1 aromatic ring and by a hydrogen bond interaction to S79 of the flap. The similarity between the P1′-P3′ side to that of pepstatin A imposes a similar hydrogen bond network and side group configuration associated with few contacts to the S1′-S3′ subsites. Examination of the structure reveals an inefficient utilization of the accessible volumes of the S1′ and S2′ subsites by the P1′ and P2′ substituents. Thus, the hydrophobic S1′ (I300 I212, T218, F294) and the more hydrophilic S2′ (M75, S37, T77, N39, A38) subsites are potential targets for inhibitor modification. We have previously shown, based on molecular modeling, that increased potency against Plm II and in particular against Plm I can be achieved from elongation of the P1 residue of statine-like inhibitors, thus utilizing the continuous S1-S3 crevice.14 In the present work we have continued this line of investigation exploring the effect on potency from introducing large P1 elongations. The p-bromobenzyloxy-substituted inhibitors 1, 5, 9, 13, and 16 (Table 1) have been employed as convenient advanced precursors for this investigation. The novel inhibitors 2-4 are all more potent against both Plm I and II than the parent compound 1, with compound 2 and 3 displaying the very impressive Ki value of 0.3 nM against Plm I. Compounds 2 and 4 also show an increased inhibition of blood stage parasite growth in vitro with 70% and 83% inhibition, respectively, at a concentration of 5 µM. The inhibitors 2-4 are, however, somewhat less selective over human cathepsin D than compound 1 (Table 1).

Potent Inhibitors of Plasmepsin I and II Table 1

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Table 1 (Continued)

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Table 1 (Continued)

a

Total yield over five steps. b Yield for the final step (Suzuki coupling). c Total yield over three steps.

Inhibitors 6-8 are also more potent than the corresponding p-bromo-substituted compound 5, with inhibitor 7 being more than eight times as potent against Plm I than compound 5. In addition, compounds 6-8 also maintain the relatively high selectivity over cathepsin D, displayed by the parent compound 5. Compounds 11 and 12 show a slight increase in potency against Plm I but are to some extent less potent against Plm II and somewhat less selective over cathepsin D than the parent compound 9. Compound 10 is the least promising in this series, being less active against both plasmepsins and less selective over cathepsin D than compound 9. Compounds 14 and 15 are substantially more potent against Plm I than the corresponding p-bromo compound 13. The effect on Plm II inhibition upon elongation is less pronounced with 14 being slightly better and 15 not as good as the parent compound 13. The selectivity over cathepsin D is, however, relatively good for inhibitor 14 and 15. Elongation of the P1 moiety in compound 16, one of the smallest inhibitors in the p-bromo series, also has the most pronounced effect on the Plm I inhibition with compounds 17 and 18 being 3-4 times as potent as 16 (Table 1). In addition compounds 17 and 18 are selective inhibitors displaying Ki values over 3600 nM against cathepsin D. Based on the current X-ray structure of inhibitor 1 complexed with Plm II, the observed improvement in Plm II inhibition by elongation of the P1 residue, that can be seen in the Suzuki coupled inhibitors 2-4, 6-8, 14, and 17 (Table 1), excludes an increased number of contacts to S1, but rather suggests that the inhibitors gain access to a hydrophilic cleft between the flap and the S3 R-helix. In the present structure the cleft is partly occupied by a water molecule coordinated between the main chain carbonyl atoms of F111 and E112 that help to stabilize the S1 and S3 loop region. Thus, the distance

d

ND ) not determined.

to the water molecule from the p-bromo substituent (3.7 Å) determines the length of a potential P1 substituent for efficient removal of the water molecule. However, even longer flexible substituents may be accommodated for by penetration of the cleft between the flap and the S3 R-helix and thereby reaching out into the solvent. To evaluate the effect of the position of substitution on the benzyloxy moiety the m-bromo compound 19 and the corresponding meta-elongated compounds 20 and 21 were also synthesized (Table 1). The m-bromo compound 19 is somewhat less potent against Plm I and II compared with the corresponding p-bromo compound 1. Interestingly, inhibitor 19 displays better selectivity over cathepsin D, with a Ki of 32 nM compared to 4.9 nM, and is slightly more effective in attenuating parasite growth in vitro than the p-bromo compound 1. The meta-elongated compounds 20 and 21 are more potent against the plasmepsins than the parent compound 19, but less potent than the corresponding p-elongated compounds 2 and 3. They are also less selective over cathepsin D than 19. An interesting observation is, however, that the meta-substituted compound 21 is more effective in the inhibition of parasite growth than the para-substituted compound 3, with 65% compared to 46% inhibition at 5 µM concentration, respectively (Table 1). Since elongation of the P1 in general seems to have a larger positive effect on the Plm I inhibition one can speculate that the continuous S1-S3 crevice in Plm I is larger or more flexible than in Plm II, thus accommodating the bulkier P1 substituents better. A general observation is also that the elongated inhibitors become slightly less selective over cathepsin D than the parent bromo compounds indicating that cathepsin D also can accommodate bulky P1 substituents. However, a number of the elongated inhibitors particularly those containing the 2,4,6-trifluorophenyl capping group in the P2 position (compounds 10, 11, 12, 14, 15, 17, and 18)

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still exhibit high cathepsin D selectivity. This supports our earlier conclusion that the S2 pocket in cathepsin D is smaller than in Plm II,14 which can be utilized in the design of selective inhibitors by incorporation of bulky arylbenzoic acids as capping groups for the N-terminus of the statine-like core.

to the dose response data (Grafit), where vi and vo are the initial velocities for the inhibited and uninhibited reaction respectively and [I] is the inhibitor concentration.24 The Ki was subsequently calculated by using Ki ) IC50/(1 + [S]/Km)25 and a Km value determined according to Michaelis-Menten. For each compound the % inhibition was initially measured at two different concentrations (5 µM and 0.5 µM). Then the Ki value was determined, and if the obtained value was in correlation with the % inhibition data, a single determination of the Ki was performed, which was the case with the inhibitors in this report. Plasmodium Falciparum Growth Inhibition Assay. Culture and synchronization of the asexual blood stages of Plasmodium falciparum (clone 3D7) was performed as previously described.26 Assays evaluating the effects of compounds on parasite growth were performed using a modification of the [3H]hypoxanthine incorporation assay described by Chulay et al.17 Briefly, a highly synchronous culture containing early trophozoite stage parasites at ∼1% haematocrit and 1% parasitaemia was supplemented with [3H]hypoxanthine (Amersham Biotech) to 10 µCi mL-1, then 50 µL aliquots were dispensed into wells of flat-bottomed 96-well microtiter plates. Wells were supplemented with an equal volume of medium containing various concentrations of test compound (1-5 µM final) or DMSO only (maximum final concentration 1% v/v). Plates were transferred to gassed boxes and cultured at 37 °C for 30 h to allow parasite development through to mature schizont stage. Cultures were then harvested onto glass fiber filters (Filtermat A, Wallac, Turku, Finland) using a cell harvester. Filters were wetted with scintillation cocktail and bound radioactivity quantified in a β-counter. Control cultures containing established growth-inhibitory compounds or without parasites were included in each experiment. The amount of radioactivity in each sample was expressed relative to that in the control wells containing DMSO only. Four independent experiments were performed for each concentration of each test compound. General Methods. NMR spectra were recorded on a Varian 300 MHz instrument using CDCl3 and CD3OD as solvents. TMS was used as reference. TLC was carried out on Merck precoated 60 F254 plates using UV light and charring with ethanol/sulfuric acid/p-anisaldehyde/acetic acid 90:3:2:1, and a solution of 0.5% ninhydrin in ethanol for visualization. Flash column chromatography was performed using silica gel 60 (0.040-0.063 mm, Merck). Organic phases were dried over anhydrous magnesium sulfate. Concentrations were performed under diminished pressure (1-2 kPa) at a bath temperature of 40 °C. MALDI-TOF-spectra were recorded on a VoyagerDE STR Biospectrometry Workstation using R-cyano-4-hydroxycinnamic acid as a matrix and reference. Preparative HPLC was performed on a Gynkotek system (pump: P580; detector: UVD 170S (detection wavelengths: 214, 230, 254, and 280 nm simultaniously); Software: Chromeleon) using a Kromasil 100-10-C18 (250 × 20 mm) column. p-Bromobenzyloxy-Substituted Compounds (1, 5, 9, 13, and 16). The p-bromobenzyloxy-substituted compounds (1, 5, 9, 13, and 16, Table 1) were prepared according to literature procedure by Johansson et al.14 General Synthetic Procedures. Procedure A. RoomTemperature Suzuki Couplings (Typical Procedure). The aryl bromide (0.037 mmol), the boronic acid (0.375 mmol), 2-(di-tert-butylphosphino)biphenyl (0.050 mmol), Pd(OAc)2 (0.031 mmol), potassium fluoride (0.637 mmol), and dry THF (1.5 mL) were added to an oven-dried Schlenk flask under argon atmosphere, and the suspension was stirred at room temperature for 48 h. The solvent was evaporated, and the crude product was purified by flash column chromatography or HPLC. The boronic acids used in the synthesis of the target compounds were phenylboronic acid, 3-thiopheneboronic acid, and 3,4-methylenedioxyphenylboronic acid. Pyridine-2-carboxylic Acid ((S)-1-{(1S,2S)-1-(Biphenyl4-ylmethoxymethyl)-3-[(S)-1-((S)-1-carbamoyl-3-methylbutylcarbamoyl)-ethylcarbamoyl]-2-hydroxy-propylcarbamoyl}-2-methyl-propyl)-amide (2). Compound 2 was

Conclusion In this work we are presenting the design and synthesis of novel and highly potent inhibitors of plasmepsin I and II. In addition we also report the X-ray crystal structure of Plm II in complex with one of our most promising previous inhibitors, compound 1, displaying Ki values of 0.5 and 2.2 nM for Plm I and II, at a resolution of 2.2 Å with R and Rfree refined down to 20% and 24%, respectively. The X-ray crystal structure reveals the important interactions leading to the impressive potency displayed by this series of p-bromobenzyloxy-substituted statinelike inhibitors. We have in this work continued our inhibitor design investigation to look at P1 substituents that effectively can utilize the potential offered by the continuous S1S3 crevice of plasmepsin I and II. By replacement of the bromo atom in the P1 bromobenzyloxy-substituted inhibitors 1, 5, 9, 13, 16, and 19 with three selected aryl groups, using room-temperature Suzuki coupling conditions, a number of more potent inhibitors were delivered exhibiting Ki values in the low nanomolar range for both Plm I and II. Several inhibitors, i.e., 2, 3, 4, 6, 7, 8, 11, 12, 20, and 21, exhibit Ki values below 4 nM for Plm I. The most promising inhibitors identified are compounds 2 and 4, where the bromo atom has been replaced with phenyl and 3,4-methylendioxyphenyl substituents. Compound 2 displays Ki values of 0.3 nM and 0.9 nM for Plm I and II, respectively, and inhibits parasite growth in red blood cells with 70% at a concentration of 5 µM. Compound 4 exhibits Ki values of 0.4 nM and 1.1 nM for Plm I and II and attenuates parasite growth with 83% inhibition at 5 µM concentration. Experimental Section Enzyme Inhibition Measurements. Pro-plasmepsin II was a generous gift from Helena Danielson (Department of Biochemistry, Uppsala University, Uppsala, Sweden). The expression and purification of plasmepsin I will be published elsewhere.23 Human liver cathepsin D was purchased from Sigma-Aldrich, Sweden. The activities of plasmepsin I (Plm I), plasmepsin II (Plm II), and cathepsin D were measured essentially as described previously,3 using a total reaction volume of 100 µL. The concentration of pro-Plm II was 3 nM, the amount of Plm I was adjusted to give similar catalytic activity, and 50 ng/mL pro-cathepsin D was used. The prosequence of Plm II was cleaved off by preincubation in assay reaction buffer (100 mM sodium acetate buffer (pH 4.5), 10% glycerol, and 0.01% Tween 20) at room temperature for 40 min, and cathepsin D was activated by incubation in the same reaction buffer at 37 °C for 20 min. The reaction was initiated by the addition of 3 µM substrate (DABCYL-Glu-Arg-Nle-PheLeu-Ser-Phe-Pro-EDANS, AnaSpec Inc, San Jose, CA), and hydrolysis was recorded as the increase in fluorescence intensity over a 10 min time period, during which the fluorescence increased linearly with time. Stock solutions of inhibitors in DMSO were serially diluted in DMSO and added directly before addition of substrate, giving a final DMSO concentration of 1%. IC50 values were obtained by assuming competitive inhibition and fitting a Langmuir isotherm (vi/vo ) 1/(1 + [I]/IC50))

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synthesized from compound 1 according to Procedure A. Purification by column chromatography using chloroform/ ethanol 13:1 + 1.5% methanol sat. with NH3 gave 2 in 21% yield. 2: 1H NMR (CD3OD, 300 MHz) δ 0.87 (d, J ) 6.3 Hz, 3H), 0.90 (d, J ) 6.1 Hz, 3H), 1.04 (d, J ) 5.5 Hz, 3H), 1.07 (d, J ) 5.2 Hz, 3H), 1.42 (d, J ) 7.4 Hz, 3H), 1.59-1.83 (m, 3H), 2.23-2.36 (m, 1H), 2.44 (app. d, J ) 7.2 Hz, 2H), 3.66 (app. d, J ) 6.6 Hz, 2H), 4.17-4.31 (m, 3H), 4.35-4.58 (m, 4H), 7.307.58 (m, 10H), 7.90-7.98 (m, 1H), 8.11 (d, J ) 7.7 Hz, 1H), 8.62-8.67 (m, 1H). MS calcd for C38H50N6O7Na (M + Na)+: 725.4. Found: 725.3. MS calcd for C38H50N6O7K (M + K)+: 741.5. Found: 741.3 Anal. (C38H50N6O7) C, H, N. Pyridine-2-carboxylic Acid {(S)-1-[(1S,2S)-3-[(S)-1-((S)1-Carbamoyl-3-methyl-butylcarbamoyl)-ethylcarbamoyl]2-hydroxy-1-(4-thiophen-3-yl-benzyloxymethyl)-propylcarbamoyl]-2-methyl-propyl}-amide (3). Compound 3 was synthesized from compound 1 according to Procedure A. Purification by column chromatography using chloroform/ ethanol 13:1 + 1.5% methanol sat. with NH3 gave 3 in 77% yield. 3: 1H NMR (CD3OD, 300 MHz) δ 0.87 (d, J ) 6.1 Hz, 3H), 0.90 (d, J ) 6.1 Hz, 3H), 1.05 (d, J ) 5.2 Hz, 3H), 1.06 (d, J ) 5.2 Hz, 3H), 1.41 (d, J ) 7.4 Hz, 3H), 1.60-1.84 (m, 3H), 2.21-2.34 (m, 1H), 2.44 (app. d, J ) 7.1 Hz, 2H), 3.65 (app. d, J ) 6.6 Hz, 2H), 4.15-4.32 (m, 3H), 4.35-4.57 (m, 4H), 7.29 (d, J ) 8.0 Hz, 2H), 7.38 (d, J ) 5.0 Hz, 1H), 7.43-7.59 (m, 5H), 7.91-7.99 (m, 1H), 8.11 (d, J ) 8.0 Hz, 1H), 8.64 (app. d, J ) 4.1 Hz, 1H); 13C NMR (CD3OD, 75.5 MHz): δ 17.6, 18.7, 20.2, 21.5, 23.6, 26.1, 32.3, 41.0, 41.1, 52.1, 53.1, 53.2, 61.0, 68.8, 71.1, 73.6, 121.1, 123.3, 127.1, 127.2, 128.1, 129.3, 136.5, 138.3, 138.9, 143.3, 149.9, 150.4, 166.7, 173.9, 174.2, 175.7, 177.8. MS calcd for C36H48N6O7SNa (M + Na)+: 731.3. Found: 731.3. MS calcd for C36H48N6O7SK (M + K)+: 747.4 Found: 747.2. Anal. (C36H48N6O7S) C, H, N. Pyridine-2-carboxylic Acid ((S)-1-{(1S,2S)-1-(4-Benzo[1,3]dioxol-5-yl-benzyloxymethyl)-3-[(S)-1-((S)-1-carbamoyl-3-methyl-butylcarbamoyl)-ethylcarbamoyl]-2-hydroxy-propylcarbamoyl}-2-methyl-propyl)-amide (4). Compound 4 was synthesized from compound 1 according to Procedure A. Purification by HPLC using methanol/water 4:1 + 0.2% TFA gave 4 in 41% yield. 4: 1H NMR (CD3OD, 300 MHz) δ 0.87 (d, J ) 6.1 Hz, 3H), 0.90 (d, J ) 6.0 Hz, 3H), 1.05 (d, overlapped, 3H), 1.07 (d, overlapped, 3H), 1.41 (d, J ) 7.2 Hz, 3H), 1.59-1.86 (m, 3H), 2.22-2.35 (m, 1H), 2.44 (app. d, J ) 7.4 Hz, 2H), 3.65 (app. d, J ) 6.3 Hz, 2H), 4.13-4.32 (m, 3H), 4.34-4.56 (m, 4H), 5.98 (s, 2H), 6.86 (d, J ) 8.5 Hz, 1H), 6.97-7.04 (m, 2H), 7.30 (d, J ) 8.1 Hz, 2H), 7.38 (d, J ) 8.1 Hz, 2H), 7.47-7.61 (m, 1H), 7.92-8.01 (m, 1H), 8.12 (d, J ) 7.4 Hz, 1H), 8.59-8.66 (m, 1H). MS calcd for C39H50N6O9Na (M + Na)+: 769.4. Found: 769.2. MS calcd for C39H50N6O9K (M + K)+: 785.5. Found: 785.2. Anal. (C39H50N6O9) H: calcd, 6.75; found, 6.19; C: calcd, 62.72; found, 58.22; N: calcd, 11.25; found, 8.85. Purity LC-MS: 96%. Pyridine-2-carboxylic Acid {(S)-1-[(1S,2S)-1-(Biphenyl4-ylmethoxymethyl)-3-butylcarbamoyl-2-hydroxy-propylcarbamoyl]-2-methyl-propyl}-amide (6). Compound 6 was synthesized from compound 5 according to Procedure A. Purification by column chromatography was performed using chloroform + 1.5% methanol sat. with NH3 giving 6 in 40% yield. 6: 1H NMR (CD3OD, 300 MHz): δ 0.93 (t, J ) 7.3 Hz, 3H), 1.02 (d, J ) 6.9 Hz, 3H), 1.06 (d, J ) 6.9 Hz, 3H), 1.291.43 (m, 2H), 1.44-1.55 (m, 2H), 2.19-2.30 (m, 1H), 2.33 (app. d, J ) 6.6 Hz, 2H), 3.18 (t, J ) 7.0 Hz, 2H), 3.54-3.70 (m, 2H), 4.11-4.20 (m, 1H), 4.21-4.30 (m, 1H), 4.48-4.60 (m, 3H), 7.29-7.56 (m, 10 H), 7.88-7.97 (m, 1H), 8.09 (d, J ) 7.7 Hz, 1H), 8.63 (d, J ) 4.1 Hz, 1H); 13C NMR (CD3OD, 75.5 MHz): δ 14.1, 18.5, 20.1, 21.1, 32.5, 32.7, 40.2, 41.8, 54.3, 60.3, 68.5, 70.7, 73.6, 123.2, 127.8, 127.9, 128.0, 128.3, 129.8, 138.6, 138.9, 141.7, 142.1, 149.9, 150.5, 166.4, 173.5, 173.7. MS calcd for C33H42N4O5Na (M + Na)+: 597.3. Found: 597.3. MS calcd for C33H42N4O5K (M + K)+: 613.4. Found: 613.2. Anal. (C33H42N4O5) C, H, N. Pyridine-2-carboxylic Acid {(S)-1-[(1S,2S)-3-Butylcarbamoyl-2-hydroxy-1-(4-thiophen-3-yl-benzyloxymethyl)propylcarbamoyl]-2-methyl-propyl}-amide (7). Compound

7 was synthesized from compound 5 according to Procedure A. Purification by column chromatography was performed using chloroform + 1.5% methanol sat. with NH3 giving 7 in 82% yield. 7: 1H NMR (CD3OD, 300 MHz): δ 0.93 (t, J ) 7.1 Hz, 3H), 1.02 (d, J ) 6.6 Hz, 3H), 1.06 (d, J ) 6.6 Hz, 3H), 1.30-1.42 (m, 2H), 1.43-1.55 (m, 2H), 2.19-2.31 (m, 1H), 2.33 (app. d, J ) 6.6 Hz, 2H), 3.18 (t, J ) 7.0 Hz, 2H), 3.53-3.68 (m, 2H), 4.12-4.20 (m, 1H), 4.21-4.29 (m, 1H), 4.44-4.56 (m, 3H), 7.29 (d, J ) 8.2 Hz, 2H), 7.36 (dd, J ) 1.4, 4.9 Hz, 1H), 7.42-7.56 (m, 5H), 7.88-7.97 (m, 1H), 8.09 (d, J ) 7.7 Hz, 1H), 8.60-8.65 (m, 1H); 13C NMR (CD3OD, 75.5 MHz): δ 14.1, 18.5, 20.1, 21.1, 32.5, 32.6, 40.2, 41.7, 54.3, 60.3, 68.5, 70.7, 73.7, 121.2, 123.2, 127.1, 127.2, 127.2, 128.0, 129.3, 136.5, 138.3, 138.9, 143. 2, 149.8, 150.5, 166.4, 173.5, 173.8. MS calcd for C31H40N4O5SNa (M + Na)+: 603.3. Found: 603.2. MS calcd for C31H40N4O5SK (M + K)+: 619.4. Found: 619.2. Anal. (C31H40N4O5S) C, H, N. Pyridine-2-carboxylic Acid {(S)-1-[(1S,2S)-1-(4-Benzo[1,3]dioxol-5-yl-benzyloxymethyl)-3-butylcarbamoyl-2hydroxy-propylcarbamoyl]-2-methyl-propyl}-amide (8). Compound 8 was synthesized from compound 5 according to Procedure A. Purification by column chromatography was performed using chloroform + 1.5% methanol sat. with NH3 giving 8 in 47% yield. 8: 1H NMR (CD3OD, 300 MHz): δ 0.93 (t, J ) 7.1 Hz, 3H), 1.02 (d, J ) 6.9 Hz, 3H), 1.06 (d, J ) 6.9 Hz, 3H), 1.29-1.42 (m, 2H), 1.43-1.56 (m, 2H), 2.19-2.30 (m, 1H), 2.33 (app. d, J ) 6.6 Hz, 2H), 3.18 (t, J ) 7.0 Hz, 2H), 3.53-3.68 (m, 2H), 4.11-4.19 (m, 1H), 4.20-4.28 (m, 1H), 4.46-4.56 (m, 3H), 5.98 (s, 2H), 6.85 (d, J ) 8.5 Hz, 1H), 6,94-7.01 (m, 2H), 7.30 (d, J ) 8.2 Hz, 2H), 7.37 (d, J ) 8.2 Hz, 2H), 7.50-7.57 (m, 1H), 7.88-7.98 (m, 1H), 8.09 (d, J ) 7.7 Hz, 1H), 8.59-8.66 (m, 1H); 13C NMR (CD3OD, 75.5 MHz): δ 14.1, 18.5, 20.1, 21.1, 32.5, 32.7, 40.2, 41.8, 54.3, 60.3, 68.6, 70.7, 73.6, 102.5, 108.3, 109.4, 121.5, 123.3, 127.6, 128.0, 129.2, 136.5, 138.2, 138.9, 141.5, 148.6, 149.9, 150.5, 166.4, 173.5, 173.8. MS calcd for C34H42N4O7Na (M + Na)+: 641.3. Found: 641.3. MS calcd for C34H42N4O7K (M + K)+: 657.4. Found: 657.3. Anal. (C34H42N4O7) C, H, N. N-{(1S,2S)-1-(Biphenyl-4-ylmethoxymethyl)-3-[(S)-1((S)-1-carbamoyl-3-methyl-butylcarbamoyl)-ethylcarbamoyl]-2-hydroxy-propyl}-2,4,6-trifluoro-benzamide (10). Compound 10 was synthesized from compound 9 according to Procedure A. Purification by HPLC using methanol/water 4:1 + 0.2% TFA gave 10 in 48% yield. 10: 1H NMR (CD3OD, 300 MHz) δ 0.84 (d, J ) 6.1 Hz, 3H), 0.85 (d, J ) 6.3 Hz, 3H), 1.37 (d, J ) 7.1 Hz, 3H), 1.56-1.78 (m, 3H), 2.54 (dd, J ) 7.0, 15.1 Hz, 1H) 2.63 (dd, J ) 7.4, 15.1 Hz, 1H), 3.67-3.80 (m, 2H), 4.22 (app. q, J ) 7.2 Hz, 1H), 4.32-4.47 (m, 3H), 4.57 (d, J ) 11.9 Hz, 1H), 4.63 (d, J ) 11.9 Hz, 1H), 6.95 (d, J ) 7.7 Hz, 1H), 6.98 (d, J ) 7.7 Hz, 1H), 7.28-7.36 (m, 1H), 7.39-7.49 (m, 4H), 7.55-7.66 (m, 4H); 13C NMR (CD3OD, 75.5 MHz): δ 17.5, 21.4, 23.4, 26.0, 40.9, 40.9, 51.7, 53.2, 53.9, 68.7, 70.9, 73.8, 101.4, 101.7, 102.1, 127.9, 128.3, 129.4, 129.8, 138.7, 141.8, 142.2, 160.9, 162.6, 165.0, 166.6, 170.9, 174.5, 175.7, 177.9. MS calcd for C34H39F3N4O6Na (M + Na)+: 679.3. Found: 679.2. MS calcd for C34H39F3N4O6K (M + K)+: 695.4. Found: 695.2. Anal. (C34H39F3N4O6) C, H, N. N-[(1S,2S)-3-[(S)-1-((S)-1-Carbamoyl-3-methyl-butylcarbamoyl)-ethylcarbamoyl]-2-hydroxy-1-(4-thiophen-3yl-benzyloxymethyl)-propyl]-2,4,6-trifluoro-benzamide (11). Compound 11 was synthesized from compound 9 according to Procedure A. Purification by HPLC using methanol/ water 4:1 + 0.2% TFA gave 11 in 96% yield. 11: 1H NMR (CD3OD, 300 MHz) δ 0.84 (d, J ) 6.0 Hz, 3H), 0.85 (d, J ) 6.1 Hz, 3H), 1.37 (d, J ) 7.4 Hz, 3H), 1.55-1.78 (m, 3H), 2.53 (dd, J ) 6.9, 15.1 Hz, 1H) 2.62 (dd, J ) 7.7, 15.1 Hz, 1H), 3.673.77 (m, 2H), 4.24 (app. q, J ) 7.3 Hz, 1H), 4.31-4.47 (m, 3H), 4.54 (d, J ) 11.8 Hz, 1H), 4.60 (d, J ) 11.8 Hz, 1H), 6.95 (d, J ) 7.6 Hz, 1H), 6.98 (d, J ) 7.6 Hz, 1H), 7.39 (d, J ) 8.0 Hz, 2H), 7.42-7.51 (m, 2H), 7.58-7.67 (m, 3H); 13C NMR (CD3OD, 75.5 MHz): δ 17.5, 21.4, 23.4, 26.0, 40.9, 51.8, 53.2, 53.9, 68.6, 70.9, 73.8, 101.4, 101.7, 102.1, 121.2, 127.1, 127.3, 129.4, 136.6, 138.4, 143.3, 161.8, 162.6, 163.3, 168.1, 170.0, 174.5, 175.7, 177.9. MS calcd for C32H37F3N4O6SNa (M + Na)+: 685.2.

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Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13

Johansson et al.

Found: 685.2. MS calcd for C32H37F3N4O6SK (M + K)+: 701.3. Found: 701.1. Anal. (C32H37F3N4O6S) C, H, N. N-{(1S,2S)-1-(4-Benzo[1,3]dioxol-5-yl-benzyloxymethyl)3-[(S)-1-((S)-1-carbamoyl-3-methyl-butylcarbamoyl)-ethylcarbamoyl]-2-hydroxy-propyl}-2,4,6-trifluoro-benzamide (12). Compound 12 was synthesized from compound 9 according to Procedure A. Purification by HPLC using methanol/ water 4:1 + 0.2% TFA gave 12 in 60% yield. 12: 1H NMR (CD3OD, 300 MHz) δ 0.84 (d, J ) 6.0 Hz, 3H), 0.85 (d, J ) 6.1 Hz, 3H), 1.37 (d, J ) 7.1 Hz, 3H), 1.54-1.77 (m, 3H), 2.53 (dd, J ) 6.7, 15.3 Hz, 1H) 2.62 (dd, J ) 7.4, 15.3 Hz, 1H), 3.673.77 (m, 2H), 4.22 (app. q, J ) 7.3 Hz, 1H), 4.31-4.46 (m, 3H), 4.55 (d, J ) 11.7 Hz, 1H), 4.61 (d, J ) 11.7 Hz, 1H), 5.97 (s, 2H), 6.88 (d, J ) 8.5 Hz, 1H), 6.95 (d, J ) 7.7 Hz, 1H), 6.98 (d, J ) 7.7 Hz, 1H), 7.06-7.11 (m, 2H), 7.40 (d, J ) 8.2 Hz, 2H), 7.51 (d, J ) 8.2 Hz, 2H); 13C NMR (CDCl3, 75.5 MHz): δ 17.5, 21.1, 22.8, 24.9, 39.4, 39.6, 51.0, 51.4, 70.1, 71.7, 73.5, 100.8, 101.2, 101.5, 107.5, 108.6, 110.4, 120.6, 127.1, 128.4, 134.9, 135.5, 140.9, 147.3, 148.2, 160.5, 162.0, 165.4, 172.1, 173.2, 176.9. MS calcd for C35H39F3N4O8Na (M + Na)+: 723.3. Found: 723.1. MS calcd for C35H39F3N4O8K (M + K)+: 739.4. Found: 739.1. Anal. (C35H39F3N4O8‚0.16 TFA) C, H, N. N-{(1S,2S)-1-(Biphenyl-4-ylmethoxymethyl)-2-hydroxy3-[2-(3-methoxy-phenyl)-ethylcarbamoyl]-propyl}-2,4,6trifluoro-benzamide (14). Compound 14 was synthesized from compound 13 according to Procedure A. Purification by column chromatography using toluene/ethyl acetate 1:1 gave 14 in 79% yield. 14: 1H NMR (CDCl3, 300 MHz) δ 2.29 (dd, J ) 3.8, 15.1, 1H), 2.45 (dd, J ) 9.3, 15.1 Hz, 1H), 2.78 (t, J ) 7.1 Hz, 2H), 3.41-3.57 (m, 2H), 3.75 (d, overlapped, 2H), 3.77 (s, 3H), 4.22-4.32 (m, 1H), 4.36 (bs, 1H), 4.37-4.45 (m, 1H), 4.59 (s, 2H), 6.10 (b, 1H), 6.56 (b, 1H), 6.66-6.79 (m, 5H), 7.34-7.48 (m, 6H), 7.54-7.62 (m, 4H). MS calcd for C34H33F3N2O5Na (M + Na)+: 629.2. Found: 629.3. MS calcd for C34H33F3N2O5K (M + K)+: 645.3. Found: 645.3. Anal. (C34H33F3N2O5) C, H, N. N-{(1S,2S)-2-Hydroxy-3-[2-(3-methoxy-phenyl)-ethylcarbamoyl]-1-(4-thiophen-3-yl-benzyloxymethyl)-propyl}2,4,6-trifluoro-benzamide (15). Compound 15 was synthesized from compound 13 according to Procedure A. Purification by column chromatography using toluene/ethyl acetate 1:1 gave 15 in 73% yield. 15: 1H NMR (CDCl3, 300 MHz) δ 2.28 (dd, J ) 3.8, 15.1, 1H), 2.44 (dd, J ) 9.3, 15.1 Hz, 1H), 2.78 (t, J ) 7.0 Hz, 2H), 3.43-3.57 (m, 2H), 3.73 (d, J ) 5.2 Hz, 2H), 3.77 (s, 3H), 4.21-4.29 (m, 1H), 4.31 (bs, 1H), 4.36-4.45 (m, 1H), 4.55 (s, 2H), 6.03 (b, 1H), 6.53 (b, 1H), 6.67-6.79 (m, 5H), 7.14-7.22 (m, 1H), 7.31-7.46 (m, 5H), 7.56 (d, J ) 8.2 Hz, 2H); 13C NMR (CDCl3, 75.5 MHz): δ 35.7, 39.9, 40.7, 52.7, 55.4, 68.4, 70.7, 73.5, 100.9, 101.3, 101.6, 112.2, 114.7, 120.6, 121.2, 126.5, 126.8, 128.5, 129.3, 135.8, 136.6, 140.4, 142.1, 160.1, 160.1, 172.0. MS calcd for C32H31F3N2O5SNa (M + Na)+: 635.2. Found: 635.1. MS calcd for C32H31F3N2O5SK (M + K)+: 651.3. Found: 651.1. Anal. (C32H31F3N2O5S) C, H, N. N-[(1S,2S)-1-(Biphenyl-4-ylmethoxymethyl)-3-cyclohexylcarbamoyl-2-hydroxy-propyl]-2,4,6-trifluoro-benzamide (17). Compound 17 was synthesized from compound 16 according to Procedure A. Purification by column chromatography using toluene/ethyl acetate 2:1 gave 17 in 59% yield. 17: 1H NMR (CDCl3, 300 MHz) δ 1.06-1.24 (m, 3H), 1.251.44 (m, 3H), 1.55-1.70 (m, 2H), 1.82-1.95 (m, 2H), 2.30 (dd, J ) 3.7, 14.8 Hz, 1H), 2.46 (dd, J ) 9.3, 14.8 Hz, 1H), 3.673.81 (m, 1H), 3.76 (d, overlapped, 2H), 4.26-4.35 (m, 1H), 4.38-4.46 (m, 1H), 4.53 (bs, 1H), 4.60 (s, 2H), 5.95 (b, 1H), 6.58 (b, 1H), 6.70 (d, J ) 8.2 Hz, 1H), 6.72 (d, J ) 8.2 Hz, 1H), 7.33-7.46 (m, 5H), 7.54-7.62 (m, 4H); 13C NMR (CDCl3, 75.5 MHz): δ 24.8, 25.4, 27.5, 32.8, 32.9, 39.8, 48.3, 52.5, 68.1, 70.4, 73.2, 100.7, 101.0, 101.4, 127.2, 127.3, 128.8, 136.6, 140.8, 140.9, 159.9, 170.9. MS calcd for C31H33F3N2O4Na (M + Na)+: 577.2. Found: 577.2. MS calcd for C31H33F3N2O4K (M + K)+: 593.3. Found: 593.2. Anal. (C31H33F3N2O4) C, H, N. N-[(1S,2S)-3-Cyclohexylcarbamoyl-2-hydroxy-1-(4thiophen-3-yl-benzyloxymethyl)-propyl]-2,4,6-trifluorobenzamide (18). Compound 18 was synthesized from compound 16 according to Procedure A. Purification by HPLC

using methanol/water 4:1 + 0.2% TFA gave 18 in 23% yield. 18: 1H NMR (CDCl3, 300 MHz) δ 1.06-1.44 (m, 6H), 1.541.72 (m, 2H), 1.84-1.94 (m, 2H), 2.28 (dd, J ) 3.7, 15.0 Hz, 1H), 2.47 (dd, J ) 9.5, 15.0 Hz, 1H), 3.68-3.79 (m, 1H), 3.76 (d, overlapped, 2H), 4.18-4.25 (m, 1H), 4.26-4.34 (m, 1H), 4.39-4.47 (m, 1H), 4.59 (s, 2H), 5.75 (b, 1H), 6.70 (d, J ) 7.7 Hz, 1H), 6.73 (d, J ) 7.7 Hz, 1H), 7.35 (d, J ) 8.2 Hz, 2H), 7.37-7.40 (m, 2H), 7.43-7.46 (m, 1H), 7.57 (d, J ) 8.2 Hz, 2H). MS calcd for C29H31F3N2O4S Na (M + Na)+: 583.2. Found: 583.2. Anal. (C29H31F3N2O4S‚0.45H2O) C, H, N. Pyridine-2-carboxylic Acid ((S)-1-{(1S,2S)-1-(3-bromobenzyloxymethyl)-3-[(S)-1-((S)-1-carbamoyl-3-methyl-butylcarbamoyl)-ethylcarbamoyl]-2-hydroxy-propylcarbamoyl}-2-methyl-propyl)-amide (19). The m-bromo scaffold employed in the synthesis of compound 19 was prepared in 72% yield over three steps from 3-deoxy-1,2-O-isopropylideneD-glucose using m-bromobenzyl bromide as alkylating agent according to the method for the preparation of compound 10 in ref 14. Compound 19 was then obtained in 56% total yield over three steps according to the method for the synthesis of compound 15 in ref 14. Purification was performed by HPLC using methanol/water 4:1 + 0.2% TFA. 19: 1H NMR (CD3OD, 300 MHz) δ 0.87 (d, J ) 6.0 Hz, 3H), 0.90 (d, J ) 6.0 Hz, 3H), 1.03 (d, J ) 2.5 Hz, 3H), 1.05 (d, J ) 2.2 Hz, 3H), 1.42 (d, J ) 7.1 Hz, 3H), 1.62-1.84 (m, 3H), 2.23-2.36 (m, 1H), 2.43 (app. d, J ) 7.4 Hz, 2H), 3.64-3.70 (m, 2H), 4.15-4.30 (m, 3H), 4.36-4.52 (m, 4H), 7.14 (app. t, J ) 7.7 Hz, 1H), 7.24 (d, J ) 7.7 Hz, 1H), 7.34 (d, J ) 7.7 Hz, 1H), 7.45 (s, 1H), 7.54-7.61 (m, 1H), 7.92-8.01 (m, 1H), 8.12 (d, J ) 8.0 Hz, 1H), 8.65 (d, J ) 4.7 Hz, 1H); 13C NMR (CD3OD, 75.5 MHz): δ 17.6, 18.8. 20.1, 21.5, 23.6, 26.0, 32.1, 40.9, 41.0, 52.1, 53.0, 61.2, 68.7, 71.3, 73.0, 123.2, 123.3, 127.3, 128.1, 131.0, 131.4, 131.5, 138.9, 142.2, 149.8, 150.4, 166.8, 173.9, 174.1, 175.7, 177.7. MS calcd for C32H45BrN6O7Na (M + Na)+: 727.2. Found: 727.1. MS calcd for C32H45BrN6O7K (M + K)+: 743.4. Found: 743.1. Anal. (C32H45BrN6O7‚0.42H2O) C, H, N. Pyridine-2-carboxylic Acid ((S)-1-{(1S,2S)-1-(biphenyl3-ylmethoxymethyl)-3-[(S)-1-((S)-1-carbamoyl-3-methylbutylcarbamoyl)-ethylcarbamoyl]-2-hydroxy-propylcarbamoyl}-2-methyl-propyl)-amide (20). Compound 20 was synthesized from compound 19 according to Procedure A. Purification by HPLC using methanol/water 4:1 + 0.2% TFA gave 20 in 30% yield. 20: 1H NMR (CD3OD, 300 MHz) δ 0.85 (d, overlapped, 3H), 0.87 (d, overlapped, 3H), 1.00 (d, J ) 6.9 Hz, 3H), 1.02 (d, J ) 6.9 Hz, 3H), 1.41 (d, J ) 7.1 Hz, 3H), 1.57-1.83 (m, 3H), 2.19-2.34 (m, 1H), 2.43 (app. d, J ) 7.1 Hz, 2H), 3.68 (app. d, J ) 6.0 Hz, 2H), 4.13-4.30 (m, 3H), 4.36-4.43 (m, 2H), 4.51 (d, J ) 11.7 Hz, 1H), 4.59 (d, J ) 11.7 Hz, 1H), 7.25-7.47 (m, 7H), 7.52-7.63 (m, 3H), 7.92-8.00 (m, 1H), 8.10 (d, J ) 8.0 Hz, 1H), 8.63 (app. d, J ) 3.3 Hz, 1H). MS calcd for C38H50N6O7K (M + K)+: 741.5. Found: 741.2. Anal. (C38H50N6O7‚0.10TFA) C, H, N. Pyridine-2-carboxylic Acid {(S)-1-[(1S,2S)-3-[(S)-1-((S)1-Carbamoyl-3-methyl-butylcarbamoyl)-ethylcarbamoyl]2-hydroxy-1-(3-thiophen-3-yl-benzyloxymethyl)-propylcarbamoyl]-2-methyl-propyl}-amide (21). Compound 21 was synthesized from compound 19 according to Procedure A. Purification by column chromatography using chloroform/ ethanol 13:1 + 1.5% methanol sat. with NH3 gave 21 in 85% yield. 21: 1H NMR (CD3OD, 300 MHz) δ 0.86 (d, overlapped, 3H), 0.87 (d, overlapped, 3H), 1.00 (d, J ) 6.9 Hz, 3H), 1.02 (d, J ) 6.9 Hz, 3H), 1.41 (d, J ) 7.4 Hz, 3H), 1.59-1.83 (m, 3H), 2.19-2.33 (m, 1H), 2.43 (app. d, J ) 7.4 Hz, 2H), 3.67 (app. d, J ) 6.3 Hz, 2H), 4.13-4.31 (m, 3H), 4.34-4.46 (m, 2H), 4.48 (d, J ) 11.5 Hz, 1H), 4.56 (d, J ) 11.5 Hz, 1H), 7.177.31 (m, 2H), 7.41-7.61 (m, 6H), 7.91-7.99 (m, 1H), 8.10 (d, J ) 7.7 Hz, 1H), 8.59-8.66 (m, 1H); 13C NMR (CD3OD, 75.5 MHz): δ 17.6, 18.7, 20.1, 21.5, 23.6, 26.1, 32.2, 40.9, 41.1, 52.1, 53.1, 53.1, 61.2, 68.8, 71.2, 74.0, 121.4, 123.3, 126.5, 126.7, 127.2, 127.5, 128.1, 129.8, 137.2, 138.9, 140.1, 143.4, 149.9, 150.4, 166.8, 173.9, 174.2, 175.8, 177.8. MS calcd for C36H48N6O7SNa (M + Na)+: 731.3. Found: 731.1. MS calcd for C36H48N6O7SK (M + K)+: 747.4. Found: 747.1. Anal. (C36H48N6O7S) C, H, N.

Potent Inhibitors of Plasmepsin I and II

Acknowledgment. We gratefully thank Medivir AB, the Swedish Foundation for Strategic Research (SSF), and Knut and Alice Wallenberg’s Foundation for financial support. Work in M.B.’s laboratory was supported by the Medical Research Council, U.K. Supporting Information Available: Combustion analyses; LC-MS purity measurements. This material is available free of charge via the Internet at http://pubs.acs.org.

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