Disubstituted Bis-THF Moieties as New P2 Ligands in Nonpeptidal HIV

Article pubs.acs.org/jmc

Disubstituted Bis-THF Moieties as New P2 Ligands in Nonpeptidal HIV‑1 Protease Inhibitors (II) Konrad Hohlfeld,† Jörg Kurt Wegner,‡ Bart Kesteleyn,‡ Bruno Linclau,*,† and Johan Unge*,§ †

University of Southampton, School of Chemistry, Highfield, Southampton SO17 1BJ, United Kingdom Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340 Beerse, Belgium § Lund University, MAX-lab, Ole Römers väg 1, SE-223 63 Lund, Sweden ‡

S Supporting Information *

ABSTRACT: A series of darunavir analogues featuring a substituted bis-THF ring as P2 ligand have been synthesized and evaluated. Very high affinity protease inhibitors (PIs) with an interesting activity on wild-type HIV and a panel of multiPI resistant HIV-1 mutants containing clinically observed, primary mutations were identified using a cell-based assay. Crystal structure analysis was conducted on a number of PI analogues in complex with HIV-1 protease.

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INTRODUCTION

HIV-1 protease (PR) is essential for the virus life cycle, as the generation of mature infectious virus particles relies on the sequential cleavage of the gag-pol precursor polyproteins by this enzyme.1 HIV-1 PR recognizes at least nine different cleavage sites, which share a superimposable secondary structure, the “substrate envelope”.2 However, some side chain residues protrude out of the envelope, allowing the enzyme to distinguish the different substrates and contributing to the highly ordered cleavage process of the precursor polyproteins.3 Inhibition of the PR by protease inhibitors (PIs) results in the formation of immature, noninfectious virus particles and suppresses the HIV-1 replication in the patient.4 Since their introduction in 1996, HIV-1 PIs played a crucial role in combined anti-retroviral therapy (cART) regimes and led to significant progress in AIDS chemotherapy.5 However, the clinical usefulness of most first generation PIs was diminished by their poor bioavailability and a high pill-burden. In addition, the lack of proofreading capacity of HIV-1 reverse transcriptase and drug-induced pressure resulted in the occurrence of multiPI resistant mutant strains.6 Based on the observation that the conformation of the protein backbone in the active site of mutant proteases remains largely unaltered, structure-based design led to the development of a stereochemically defined fused bis-tetrahydrofuran (bis-THF) moiety as high-affinity nonpeptidal P2 ligand.7 The bis-THF system was subsequently combined with a (R)(hydroxyethylamine)sulfonamide isostere, resulting in the selection of darunavir (1, DRV, Figure 1) as a conceptually new HIV-1 protease inhibitor.8 DRV has proven to be highly effective against the wild-type virus as well as a broad range of multi-PI-resistant HIV-1 mutant strains.9 The high resolution X-ray crystal structure of the enzyme−inhibitor complex revealed a tight network of hydrogen-bonding interactions between DRV and the protease backbone. In particular, both © 2015 American Chemical Society

Figure 1. Structures of darunavir (1, DRV) and protease inhibitors 2− 4.

ring-oxygens of the bis-THF ligand form strong hydrogen bonds (HBs) with the backbone NH of Asp29 and Asp30.10 Subsequent research on darunavir analogues mainly focused on alterations around the P1′ phenyl group and the P2′ sulfonamide substituent, as the bis-THF moiety was deemed Received: March 4, 2015 Published: April 21, 2015 4029

DOI: 10.1021/acs.jmedchem.5b00358 J. Med. Chem. 2015, 58, 4029−4038

Journal of Medicinal Chemistry

Article

crucial for the superior resistance profile.11 However, our comparison studies of HIV-1 protease bound DRV and several recognition sequences suggested that additional substituents on the bis-THF B-ring could result in additional interactions with the enzyme backbone.12 In particular, substituents with HB acceptor and/or donor ability on the C4-position of the bisTHF B-ring could establish direct or water-mediated HB interactions with the Gly48 backbone. In 2011, Ghosh reported the synthesis of a C4-exomethoxylated darunavir analogue (2, Figure 1) and its activity on wild-type HIV-1LAI (EC50 = 2.4 nM).13 In the X-ray crystal structure of the corresponding inhibitor−enzyme complex a water-mediated hydrogen bond between the methoxy oxygen and the backbone NH of Gly48 was observed. In addition, the methyl group forms CH···O interactions with the carbonyl oxygen of Gly48. Based on the X-ray crystal structure of the C4-hydroxylated PI 3a (Figure 1),14 which also revealed contacts to the amide of Gly48 (NH and CO), we developed a series of darunavir analogues featuring a disubstituted bisTHF moiety, which included alkoxyalkyl, aryl, and benzyl substituents on the C4-position.12 Interestingly, a number of these analogues exhibited equivalent or greater activity on HIV1 mutant strains when compared to the wild-type virus. The methoxyethoxy substituted PI 4b (Figure 1) proved to be of particular interest, as additional molecular modeling studies indicated a displacement of the water molecule, observed with PIs 2 and 3a, by the second oxygen resulting in a direct hydrogen-bonding interaction with the invariant Gly48 amide NH.12 On the basis of these results, we sought to enhance the antiviral activity by further exploring the structure−activity relationships (SAR) at the bis-THF C4-substituent and P2′ aryl-sulfonamide. Herein, we report the synthesis and biological evaluation of further C4-exo substituted bis-THF containing PIs, as well as the X-ray crystal structures of selected analogues. It was primarily envisaged to synthesize analogues that could specifically interact with the backbone amide of Gly48 and possess HB acceptor/donor ability, namely, N-substituted derivatives and acetamides. On the other hand, it was attempted to mimic these interactions with halogenated isosteres.

Scheme 1. Retrosynthesis of PIs 3−5

Scheme 2. Synthesis of Carbamates 6c/da

Reagents and conditions: (a) SO3·py, Et3N, DMSO, CH2Cl2, −10 °C. (b) NaBH4, EtOH. (c) MsCl, Et3N, CH2Cl2. (d) TMSN3, TBAF, THF, reflux. (e) H2, Pd/C. (f) ClCO2Bn, DMAP, py, CH2Cl2. (g) DDQ, CH2Cl2/H2O. (h) DSC, Et3N, CH2Cl2; then BnNH2. a

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ing exo-azide using TMSN3/TBAF/THF.16 Reduction of the azide group afforded amine 14, which was then functionalized with benzyl chloroformate and deprotected via DDQ oxidation to yield the O-benzylated carbamate 6c. Similarly, the “inverted” N-benzylated carbamate 6d was obtained in two steps from alcohol 8. Here, the carbamate group was introduced in a one-pot, two-step reaction involving initial activation of the hydroxy group with disuccinimidyl carbonate (DSC),17 followed by the nucleophilic attack of benzylamine. The synthesis of the acetamides 6e−g is illustrated in Scheme 3. As the direct alkylation of 8 with a preformed 2chloroacetamide only gave low yields (not shown), a three-step strategy was applied to obtain analogues 6e−g. The reaction of deprotonated 8 with preformed sodium bromoacetate in refluxing THF afforded the corresponding acetic acid analogue 15 in excellent yield, which was then transformed to acetamides 6e−g under peptide-coupling conditions using hydroxybenzotriazole (HOBt) and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDCI) followed by endo-alcohol deprotection. Fluoroalkene 6h was obtained via a bromofluorinationdehydrobromination sequence18 from the intermediate allyl ether 16, while trifluoropropyl ether 6i was derived from

RESULTS Chemistry. The novel PIs 3−5 were obtained by coupling the functionalized bis-THF ligands 6 with the hydroxyethylsulfonamide isosteres 7 (Scheme 1). In most cases, the DRV para-aminophenyl-sulfonamide 7a8b and the (2-methylamino)benzoxazole-sulfonamide 7b11a were selected as P2′ ligands based on their pronounced hydrogen-bonding in the S2′ subsite. Other aryl P2′ substituents, like para-methoxyphenylsulfonamide 7c,8a were only incorporated in a few analogues. The functionalized bis-THF ligands were derived from the previously described endoalcohol protected intermediate 8, which in turn can be synthesized in multigram scale from Larabitol 10 via the selective protection of bis-THF diol 9.12,15 The synthesis of carbamates 6c/d is shown in Scheme 2. Parikh-Döring oxidation of alcohol 8 to ketone 11 was followed by stereoselective reduction with sodium borohydride and treatment with methanesulfonyl chloride to afford the activated endo-alcohol 13 in 63% yield over three steps. Interestingly, this sequence carried out on the corresponding TBDMS-protected analogue of 8 led to silyl migration and hence partial racemisation. Mesylate 13 was transformed to the correspond4030



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Scheme 3. Synthesis of Acetamides 6e−ga

Scheme 5. Synthesis of Chloride 6j and Fluoride 6ka

a Reagents and conditions: (a) SOCl2, py. (b) DDQ, CH2Cl2/H2O. (c) DAST, DMAP, CH2Cl2.

Reagents and conditions: (a) NaH, THF, 0 °C, 20 min; then BrCH2CO2Na, 80 °C, 16 h. (b) NHR1R2, EDCI, HOBt, Et3N, CH2Cl2. (c) DDQ, CH2Cl2/H2O. (d) H2, Pd/C, MeOH. a

Scheme 6. Synthesis of Inhibitors 3−5a

Scheme 4. Formation of Fluoroalk(en)yl Ethers 6h/ia

a Reagents and conditions: (a) Et3N, CH2Cl2. (b) 7a, b, or c, Et3N, CH2Cl2. (c) TBAF, THF (only for analogues a).

known amines 7a,8b 7b,11a or 7c8a with the activated carbonates 23 in the presence of triethylamine afforded PIs 3, 4, and 5. For the synthesis of analogue 5a additional treatment with TBAF was required to remove the silyl protection on the exo-hydroxy group. Structure−Activity Relationships. All synthesized PIs were tested for their antiviral activity (EC50) against the wildtype virus (HIV-1IIIB) and a panel of recombinant clinical isolates (M1−3), which were selected because of their high level of cross-resistance against several approved PIs (as exemplified by the activity of lopinavir and atazanavir). Every isolate was identified with 6−8 amino acid substitutions in the protease gene that are associated with resistance to protease inhibitors, including the primary mutations M46I, G48V, I50V, L76V, V82A, I84V, and L90M (see footnote Table 1 of for details).6b Isolates M1 and M2 are of particular interest, as they were identified with primary mutations (I50V, I84V, and L76V) that are associated with resistance to darunavir. The results are shown in Table 1, including the PIs atazanavir, lopinavir, and darunavir, as well as the previously reported darunavir analogue 2411a as reference compounds.12 The majority of the novel PIs inhibits HIV-1IIIB and the mutant strains at low nanomolar or even subnanomolar concentrations. Reduced activity of the PIs was noted against clinical isolate M2, which contains two primary mutations known to be associated with decreased susceptibility to DRV (I50V and L76V), while the potency was retained or even improved against the remaining mutants. Especially isolate M3, which includes the favorable V82A mutation,22 was found to be hypersusceptible to the new PIs. Hypersusceptibility is defined as a lower-fold change on a mutant virus when being compared to the wild-type virus (FC < 1) and has only been reported for a few cases.12,22,23 The hypersusceptibility characteristic was

Reagents and conditions: (a) NaH, THF/DMF, 0 °C, 20 min; then allyl bromide or BrCH2CH(OEt)2, TBAI. (b) NBS, Et3N·3 HF, CH2Cl2, 0 °C to rt. (c) tBuOK, THF. (d) DDQ, CH2Cl2/H2O. (e) conc. HCl, THF, 2 h. (f) TMSCF3, cat. TBAF, THF; then TBAF. (g) nBuLi, PhOCSCl, THF, −78 °C to rt. (h) Bu3SnH, AIBN, toluene, reflux. a

diethylacetal 19 (Scheme 4). Reaction of 16 with Nbromosuccinimide in the presence of triethylamine tris(hydrofluoride) afforded intermediate 17, which was treated with potassium tert-butoxide to give fluoroalkene 18 and deprotected with DDQ to yield alcohol 6h. On the other hand, formation of diethylacetal 19 was followed by acid-catalyzed hydrolysis and treatment with trifluoromethyltrimethylsilane (TMSCF3) in the presence of tetrabutylammonium fluoride (TBAF) to give secondary alcohol 20.19 Barton-McCombie deoxygenation to ether 21 was achieved in two steps: (1) formation of a intermediate thionocarbonate with PhOCSCl and (2) radical deoxygenation using tributyltin hydride and azobis(isobutyronitrile) (AIBN) as radical initiator in refluxing toluene. DDQ oxidation subsequently furnished the alcohol 6i. Chloride 6j and fluoride 6k were generated from endoalcohol 12 using thionyl chloride in pyridine20 and DAST,21 respectively. Finally, the synthesized ligands 6c−k, as well as the previously described exosilylated intermediate 22,12 were converted to the corresponding mixed carbonates 23 using DSC and triethylamine (Scheme 6).17b Treatment of the 4031



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Table 1. Antiviral Activity (EC50) of Compounds 3, 4, and 5 against Wild-Type (HIV-1IIIB) and Mutant Virusesa

a

The following PI resistance-associated mutations were identified in the isolates: M1: L10I, M46I, I64V, I84V, L90M, I93L; M2: L10I, I13V, M46I, I50V, L63P, L76V; M3: L10I, K20R, M36I, G48V, I62V, A71V, V82A, I93L; primary resistance mutations are in bold and mutations associated with resistance to DRV according to the IAS are underlined (ref 6b). bThe EC50 values were determined using MT4 cells and all assays were conducted in quadruplicate. The numbers in parentheses represent the fold change in EC50 value for each isolate relative to the wild-type HIV-1IIIB.

very pronounced with PIs 4 containing the benzoxazole sulfonamide.

Polar substituents (e.g., carbamates or acetamides) on the bis-THF moiety usually resulted in a better resistance profile 4032



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against the mutant strains when compared to the more hydrophobic analogues. This is in accordance with previously reported C-4 substituted PIs, where the benzyl and fluorobenzyl analogues also failed to show enhanced activity against the clinical isolates.12 For the acetamide series (entries 8 and 15−17) an activity profile similar to the corresponding hydroxy analogues was observed. Again, the para-methoxy phenyl substitution in PIs 5e−g (entries 23−25) significantly improves the absolute potency of these ligands. The increased polarity, in particular, of the methylacetamide containing PI 4f (entry 16), appears to impair the permeability of these compounds. In contrast, the fluorinated analogues 4h and 4i (entries 18, 19) exhibit a significantly improved activity on HIV-1IIIB when compared to 4f and 4b, respectively. Especially the absolute potency of these PIs is notable. The chloride and fluoride substituted PIs 3j, 4j, and 4k (entries 9, 20, 21) exhibit also an excellent activity on the wild-type virus.

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DISCUSSION In order to understand the obtained results on a molecular level and to gain further information about possible ligand−enzyme interactions, X-ray crystal structure analysis on a series of the above PIs was performed. As previously reported for similar compounds, the inhibitor is bound to the protease dimer in two orientations, which are related by a 180° rotation. The relative occupancy of the two orientations was approximately 50/50 for all structures. All obtained X-ray crystal structures show the characteristic hydrogen bonding interactions between the bisTHF-ring oxygens and the backbone NH of Asp28 and Asp29, respectively. The bis-THF B-ring substituents are situated in a solvent exposed area of the binding cleft. Hence, they are not as tightly bound to the enzyme as other more buried parts of the inhibitor and usually show more than one possible conformation. Interestingly, alternative conformations have been observed for the Gly48 in the majority of the enzyme−inhibitor complexes. Alterations of Gly48 are very unusual, due to its proximity to the conserved flap region of the HIV-1 protease, which is a crucial part of the active site.24 However, the alternative conformations of Gly48 may simply arise from the steric hindrance induced by the tight interactions of the substituted bis-THF moiety as previously described by Ghosh and co-workers.25 The observations presented below refer to the major conformation as identified from electron density maps of the corresponding enzyme−inhibitor complexes. The position of the methoxyethoxy extension in PIs 3b and 4b has previously been modeled based on ultrahigh-resolution X-ray crystal data.12 We originally proposed a displacement of a structural water molecule and a direct hydrogen bond from the backbone of Gly48 (NH) to the outer oxygen. However, analysis of the obtained X-ray crystal structure of PI 4b (PDB ID: 5AHA) reveals that the water molecule is retained, and in addition to the interaction with the Gly48 (NH) backbone there are two hydrogen bonds to the methoxy ethoxy substituent (Figure 2). In fact, this flexible substituent appeared to have various conformations (most likely conformation shown here), while the bis-THF moiety and the exo-oxygen at the 4 position are in fixed positions. The distances between the two oxygens of the substituent and the water molecule are 2.8 and 3.3 Å, respectively, while the water-backbone-contact is 3.1 Å. We then evaluated a number of PI analogues with different bis-THF substituents targeted at interaction with the Gly48.

Figure 2. X-ray crystal structure of PI 4b.

Based on molecular modeling studies, the acetamide substituents (OCH2CONHR) were envisaged to serve as hydrogen bond acceptor of the Gly48 backbone NH via its carbonyl group, while the acetamide NH would be able to interact with structural water molecules as a HB donor. However, the X-ray crystal data of PI 4f (PDB ID: 5AHB) revealed a slightly different arrangement (Figure 3). The acetamide NH points toward the carbonyl oxygen of Gly48, forming a close hydrogen bonding contact (d = 2.7 Å). As the electron density maps indicate only one clear position for the Gly48 carbonyl oxygen as well as the acetamide heteroatoms, this arrangement appears to be the sole conformation of this substituent. A similar interaction has been previously described by Ghosh et al. for a urethane substituent on the cyclopentylTHF (Cp-THF) moiety.26 Additionally, the water-mediated contact from the exo-oxygen on the bis-THF moiety to Gly48 (NH) can be observed too. However, the increased polarity of the acetamide analogues 4e−g is likely to contribute in the decreased activity of these analogues. Hence, we then directed our attention to a less polar amide isostere such as a fluoroalkene group in order to enhance cell permeability in comparison to these analogues, leading to the evaluation of 4h. As shown above, this analogue possesses an impressive absolute potency. The fluoroalkene isostere mimics the electron distribution around the amide oxygen, but the H-bond donating capacity is lost. In addition, the fluorine atom is an inferior hydrogen bond acceptor compared to a carbonyl oxygen.27 At the time of synthesis, the acetamide crystal structure, showing the amide NH interaction with the Gly48 backbone (see above), was not yet available, and also in this context it is interesting to observe the much increased potency of the fluoroalkene 4h compared to that of the 4033



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Figure 4. X-ray crystal structure of PI 4h.

Figure 3. X-ray crystal structure of PI 4f.

acetamide analogues. The crystal structure of the 4h (PDB ID: 5AGZ) bound to the protease shows at least two possible orientations of the fluoroalkene substituent. Interestingly, the fluoroalkene disorder can be qualitatively explained by a competing interaction of the Gly48 CO bond, and of the Arg108 guanidinium group, for the fluorine atom. Diederich has described the attractive interactions between a C−F group and electropositive regions in a protein (“fluorophilic residues”), including amide carbonyl groups, and guanidinium groups.28 For the amide interaction, the α1 bond angle (117.9°) is indicative of a favorable interaction, though the C F···CO distance (4.2 Å), and the α2 bond angle (82°) are suggestive of a weak interaction (Figure 4). Dalvit and Vulpetti have suggested that deshielded fluorine atoms (as measured by fluorine chemical shift) are superior for interactions with carbonyl groups.29 While the fluorine atom in a fluoroalkene is already deshielded (δF −105 ppm for 4h), those in a trifluoromethyl group are even more so. Hence, the trifluoropropyloxy substituted PI 4i (δF −65 ppm) was synthesized. In addition, it was also envisaged as a possible isostere of the methoxyethoxy group of PI 4b. Gratifyingly, analogue 4i possessed subnanomolar activity. Indeed, the X-ray crystal structure of PI 4i (PDB ID: 5AH8) reveals possible interactions between the CF3-group and the carbonyl-carbon of Gly48 (Figure 5). With distances of 3.8−4.1 Å, CF···C angles of 90°/103°, and F···CO angles of 125°/126°, the observed CF···CO contacts fit well within the reported ranges. This interaction appears to be more favorable to the water-mediated hydrogen bonding from Gly48 (NH) to the exo-oxygen on the bis-THF-B-ring, as this contact is lost with the trifluoropropyl substituent.

Figure 5. X-ray crystal structure of PI 4i, showing the possible interaction of the trifluoromethyl group with the carbonyl group.

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showed a better resistance profile against the mutant strains compared to the hydrophobic analogues, though increased polarity appeared to impair the permeability of these compounds. Replacing the polar groups with halogenated isosteric structures led to a marked increase in activity. A series of crystal structures of the PIs in complex with HIV PR provided structural insight into the geometry and type of interactions involved, providing a better understanding for future design and modeling studies.

Finally, the excellent activity of the chloride-substituted PIs 3j and 4j inspired further investigations. The crystal structures of these analogues bound to the protein were very similar (3j shown in Figure 6, PDB ID: 5AH7). Interestingly, a structural

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EXPERIMENTAL SECTION

General Methods. All chemical reagents were obtained from commercial sources and used without further purification. THF was distilled from Na/benzophenone immediately prior to use. CH2Cl2 and Et3N were dried over CaH2. Extra dry DMF and DMSO (H2O < 50 ppm) were purchased from commercial sources. All glassware was flame-dried under vacuum and cooled under N2 prior to use. Water- or air-sensitive reactions were performed under inert atmosphere, using dry solvents. Reactions were monitored by TLC (Merck Kieselgel 60 F254, aluminum sheet) and spots were visualized by UV and/or by exposure to an acidic solution of p-anisaldehyde, followed by brief heating. Flash column chromatography was performed on silica gel (Merck silica gel 60, particle size 40−63 μm). All reported solvent mixtures are volume measures. NMR spectra were recorded on a Bruker AV300 [300.13 MHz (1H NMR) and 75.47 MHz (13C NMR)] or a Bruker DPX400 [400.13 MHz (1H NMR) and 100.61 MHz (13C NMR)], respectively. The chemical shift (δ) is given in ppm using the residual solvent peak as an internal standard. The coupling constants (J) are given in Hertz (Hz). The multiplicity of the 1H NMR signals was designated as follows: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, m = multiplet, and br = broad. IR spectra were recorded on a Nicolet 380 FT-IR as a film and absorption peaks are given in cm−1. Optical rotation was measured on an Optical Activity POLAAR 2001 at 589 nm. Melting points were measured on a Gallenkamp melting point apparatus and are uncorrected. Lowresolution electrospray mass spectra were recorded with a Waters ZMD single quadrupole system. HRMS were measured on a Bruker APEX III FT-ICR MS system. The purity of all final compounds was determined by analytical reversed-phase high performance liquid chromatography (HPLC) using two different chromatographic systems and was found to be ≥95 in most cases. Any lower purity is specified at the end of the experimental description of this particular compound. Analytical HPLC was performed on a Waters Separation module 2795 system or a Acquity UPLC system equipped with a photodiode array detector. The first system consisted of the following: column, Waters XTerra MS C18 (3.5 μm, 4.6 mm × 100 mm); mobile phase A, 0.1% formic acid in H2O/MeOH (95:5); mobile phase B, MeOH. At a flow rate of 1.5 mL/min and a column temperature of 40 °C, gradient elution was performed from 0% B to 95% B over 12 min. The second system consisted of the following: column, Aquity UPLC BEH C18 (1.7 μm, 2.1 mm × 50 mm); mobile phase A, MeOH; mobile phase B, 10 mM NH4OAc in H2O/MeCN (90:10). At a flow rate of 0.7 mL/min and a column temperature of 70 °C, gradient elution was performed as follows: 0 min, 95% B; 1.30 min, 5% B; 1.5 min, 5% B; 1.70 min, 95% B. HPLC retention times and purity data for each target compound are provided in the Supporting Information. Typical Procedure for the Synthesis of Protease Inhibitors. 2-{(1S,4R,5R,6R)-6-(4-Methoxybenzyloxy)-2,8-dioxa-bicyclo[3.3.0]-octane-4-yloxy} acetic acid (15). A flask was charged with NaH (81 mg, 2.02 mmol, 60% in mineral oil). The solid was rinsed with hexane three times, dried under vacuum, diluted with THF (3 mL), and cooled to 0 °C. A solution of bromoacetic acid (267 mg, 1.92 mmol) in THF (5 mL) was added dropwise and the mixture was stirred for 10 min. A second flask was charged with NaH (54 mg, 1.34 mmol, 60% in mineral oil) and the solid was treated as described above. A solution of alcohol 8 (255 mg, 0.96 mmol) in THF (5 mL) was added and the mixture was stirred for 10 min. The content of the first flask was then transferred to the second flask via cannula, the

Figure 6. X-ray crystal structure of PI 3j.

water molecule was observed, with hydrogen bonding to the chlorine atom, and there are two distinct positions of the Gly48. Significantly, the observed distances between the “close” Gly48 oxygen with the chlorine atom (dO−Cl = 2.4−2.5 A), and the corresponding small CCl···O bond angles (Θ1 77−78°), clearly show that there is no halogen bond30 operating. Equally, the distances of the “distant” Gly48 oxygen with the chlorine atom (dO−Cl = 3.7−3.9 A) are too large, and the corresponding CCl···O bond angles (Θ1 67−69°) are too small. Instead we believe that the two Gly48 positions reflect the orientation of the PI within the crystal, with the “close” position being associated with the benzoxazole/aniline part of the PI, and the “distant” position with the bis-THF part. In this case, this would reflect a repulsive interaction between the Cl and the Gly48 carbonyl group, with the Cl being in contact with the structural water molecule. In this case, the chlorine would mimic an OH group of 3a and the increased lipophilicity could then be responsible for the increased activity. A similar observation was made by Ghosh and co-workers, with X-ray studies on a C4-difluorinated darunavir analogue with an equally potent antiviral activity against HIV-1 wild type and mutant viruses.31 In conclusion, we have reported the synthesis of a new series of substituted darunavir analogues having a side chain on the rigid bis-tetrahydrofuran moiety. The majority of the PI analogues synthesized inhibit HIV-1IIIB and a number of mutant strains at low nanomolar or even subnanomolar concentrations. Analogues with polar substituents generally 4035



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ppm; HRMS (ESI+) for C9H15NO5Na (M + Na)+ calcd 240.0842, found 240.0845. Protease Inhibitor 5f. To a solution of alcohol 6f (174 mg, 0.80 mmol) and Et3N (220 μL, 1.60 mmol) in CH2Cl2 (12 mL) was added N,N′-disuccinimidyl carbonate (DSC, 307 mg, 1.20 mmol) and the resulting mixture was stirred at rt for 18 h. After completion CH2Cl2 (10 mL) and sat. aq. NaHCO3 (5 mL) were added. The phases were separated and the aqueous phase was extracted with CH2Cl2 (3 × 10 mL/mmol). The combined organic phases were dried over anhydrous Na2SO4, filtered, and the solvent removed in vacuo. The crude product was then purified by column chromatography (CH2Cl2/MeOH 98:2 to 94:6) to afford the corresponding mixed carbonate 23f as a pale yellow oil (183 mg, 0.51 mmol, 64%). To a solution of this carbonate and Et3N (140 μL, 1.00 mmol) in CH2Cl2 (10 mL) was added amine 7c (200 mg, 0.49 mmol) and the resulting mixture was stirred at rt for 25 h. After completion CH2Cl2 (10 mL) and sat. aq. NaHCO3 (2.5 mL) were added. The phases were separated and the aqueous layer is extracted with CH2Cl2 (3 × 5 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered, and the solvent removed in vacuo. The crude product was purified by column chromatography (CH2Cl2/MeOH 97:3 to 95:5) and by HPLC (CH2Cl2/MeOH 97:3) to afford the PI 5f as a white solid (260 mg, 82%). Formula C31H43N3O10S; Mw 649.75; Rf 0.36 (CH2Cl2/MeOH 95:5); Mp 92− 94 °C; [α]D +15.9 (c 1.38, CHCl3, 24 °C); IR (film) 3361 (w), 2960 (w), 1716 (m), 1662 (m), 1258 (s), 1150 (vs), 1091 (s), 1023 (s), 730 (vs) cm−1; 1H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 9.0 Hz, 2 H), 7.32−7.18 (m, 5 H), 7.00 (d, J = 9.0 Hz, 2 H), 6.42 (br. s, 1 H), 5.77 (d, J = 5.3 Hz, 1 H), 5.14 (d, J = 8.8 Hz, 1 H), 5.08 (dt, J = 8.8, 6.0 Hz, 1 H), 3.99 (dd, J = 10.0, 6.3 Hz, 1 H), 3.96−3.85 (m, 3 H), 3.89 (s, 3 H), 3.83−3.75 (m, 2 H), 3.69 (dd, J = 10.0, 5.9 Hz, 1 H), 3.67−3.62 (m, 2 H), 3.49 (d, J = 3.2 Hz, 1 H), 3.18 (dd, J = 15.2, 8.7 Hz, 1 H), 3.06 (dd, J = 13.7, 4.0 Hz, 1 H), 3.03−2.95 (m, 2 H), 2.91 (dd, J = 8.5, 5.2 Hz, 1 H), 2.86 (s, 3 H), 2.85−2.74 (m, 2 H), 1.85 (m, 1 H), 0.94 (d, J = 6.6 Hz, 3 H), 0.90 (d, J = 6.7 Hz, 3 H) ppm; 13C NMR + DEPT (100 MHz, CDCl3) δ 169.5 (C), 163.1 (Car), 155.1 (C), 137.7 (Car), 129.7 (Car), 129.5 (2 × CHar), 129.3 (2 × CHar), 128.5 (2 × CHar), 126.6 (CHar), 114.4 (2 × CHar), 108.8 (CH), 80.4 (CH), 73.8 (CH2), 72.9 (CH), 72.2 (CH), 71.0 (CH2), 68.2 (CH2), 58.9 (CH2), 55.7 (CH3), 55.5 (CH), 53.7 (CH2), 51.4 (CH), 35.4 (CH2), 27.3 (CH), 25.6 (CH3), 20.2 (CH3), 19.9 (CH3) ppm; HRMS (ESI+) for C31H43N3O10SNa (M + Na)+ calcd 672.2561, found 672.2536. Antiviral Assays. The antiviral activity has been determined with a cell-based replication assay.32 This assay directly measures the ongoing replication of virus in MT4 cells via the specific interaction of HIV-tat with LTR sequences coupled to GFP. In the toxicity assay, a reduced expression of the GFP reporter protein serves as a marker for cellular toxicity of a compound. Briefly, various concentrations of the test compounds are brought into a 384-well microtiter plate. Subsequently, MT4 cells and HIV-1/LAI (wild type) are added to the plate at a concentration of 150 000 cells/mL and 200 cell culture infectious doses 50% (CCID50). To determine the toxicity of the test compound, mock-infected cell cultures containing an identical compound concentration range are incubated for 3 days (37 °C, 5% CO2) in parallel with the HIV-infected cell cultures. On the basis of the calculated percent inhibition for each compound concentration, dose response curves are plotted and EC50, pEC50, CC50, and pCC50 values are calculated. Protein Crystallography. Expression and Purification of HIV-1 Protease. The procedure has been published earlier with minor modifications.33 In short, the protease gene as a gift from Prof. R. Gallo was isolated using PCR and inserted to a pET11a expression vector. The Escherichia coli strains HB101 and XL-1 were used as hosts for cloning and the strain BL21 were used for protein expression. Cells were grown in LB medium to an OD of 1.0 before induction with 0.5 mM isopropyl-thio-β-D-1-galactoside. Cells were harvested after 3 h of induction and lysed using a French press in a lysis buffer containing 50 mM sodium phosphate, 0.3 M sodium chloride and 10 mM DTT (dithiothreitol) adjusted to pH 8.0. The lysate was collected in 15 min at 23 000 g and inclusion bodies were collected and washed by repeated precipitation and dissolving of the pellet using centrifugation

cooling was remove, and the resulting mixture was heated to reflux for 15 h. After completion, the reaction mixture was cooled to rt, diluted with THF (10 mL) and H2O (10 mL), acidified with 2 M HCl (5 mL), and extracted with EtOAc (3 × 25 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered, and the solvent removed in vacuo to give the desired product in sufficient purity. For characterization, the crude product was further purified by column chromatography (CH2Cl2/MeOH 97:3 to 90:10) to afford the title compound 15 as a pale yellow oil (292 mg, 93%). Formula C16H20O7; Mw 324.33; Rf 0.20 (CH2Cl2/MeOH 90:10); [α]D +17.9 (c 0.99, CHCl3, 25 °C); IR (film) 2937 (br. w), 1733 (m), 1514 (s), 1247 (s), 1123 (vs), 1026 (vs) cm−1; 1H NMR (400 MHz, CDCl3) δ 7.24 (d, J = 8.7 Hz, 2 H), 6.90 (d, J = 8.8 Hz, 2 H), 5.81 (d, J = 5.3 Hz, 1 H), 4.53 (d, J = 11.4 Hz, 1 H), 4.49 (d, J = 11.4 Hz, 1 H), 4.48 (d, J = 3.8 Hz, 1 H), 4.24 (ddd, J = 8.8, 8.0, 6.7 Hz, 1 H), 4.13 (d, J = 16.8 Hz, 1 H), 4.08 (dt, J = 10.3, 1.3 Hz, 1 H), 4.07 (d, J = 16.8 Hz, 1 H), 4.00 (dd, J = 10.3, 3.9 Hz, 1 H), 3.96 (dd, J = 9.2, 6.6 Hz, 1 H), 3.81 (s, 3 H), 3.58 (dd, J = 9.2, 8.0 Hz, 1 H), 3.00 (dd, J = 9.0, 5.2 Hz, 1 H) ppm; 13C NMR + DEPT (100 MHz, CDCl3) δ 173.8 (C), 159.5 (C), 129.4 (C), 129.3 (2 × CHar), 114.0 (2 × CHar), 108.7 (CH), 80.5 (CH), 76.3 (CH), 73.6 (CH2), 72.4 (CH2), 70.8 (CH2), 65.9 (CH2), 55.3 (CH3), 51.9 (CH) ppm; HRMS (ESI+) for C16H20O7Na (M + Na)+ calcd 347.1101, found 347.1096. 2-{(1S,4R,5R,6R)-6-Hydroxy-2,8-dioxa-bicyclo[3.3.0]-octane4-yloxy} N-methylacetamide (6f). To a solution of methylamine hydrochloride (275 mg, 4.20 mmol), N-(3-(dimethylamino)propyl)N′-ethylcarbodiimide hydrochloride (EDCI, 391 mg, 2.04 mmol) and Et3N (543 μL, 6.80 mmol) in CH2Cl2 (20 mL) at 0 °C were added hydroxybenzotriazole (HOBt, 275 mg 2.04 mmol) and a solution of acid 15 in CH2Cl2 (5 mL). The cooling was removed and the resulting mixture was stirred at rt for 15 h. After completion, the reaction mixture is poured into aq. sat. NaHCO3 (10 mL), the phases were separated, and the aqueous phase was extracted with CH2Cl2 (2 × 10 mL/mmol). The combined organic phases were washed with aq. HCl (10 mL/mmol, 1 M), dried over anhydrous Na2SO4, filtered, and the solvent removed in vacuo. The crude product was purified by column chromatography (CH2Cl2/MeOH 97:3 to 95:5) to afford the intermediate protected acetamide as a colorless oil (417 mg, 91%; Formula C17H23NO6; Mw 337.37; Rf 0.34 (CH2Cl2/MeOH 95:5); IR (film) 3369 (br. w), 2940 (w), 1663 (s), 1514 (s), 1247 (s), 1119 (s), 1027 (vs) cm−1; 1H NMR (400 MHz, CDCl3) δ 7.24 (d, J = 8.8 Hz, 2 H), 6.90 (d, J = 8.7 Hz, 2 H), 6.49 (br. s, 1 H), 5.80 (d, J = 5.2 Hz, 1 H), 4.51 (d, J = 11.7 Hz, 1 H), 4.48 (d, J = 11.7 Hz, 1 H), 4.40 (dt, J = 3.6, 1.1 Hz, 1 H), 4.24 (ddd, J = 9.0, 7.9, 6.6 Hz, 1 H), 4.04 (dt, J = 10.5, 1.1 Hz, 1 H), 3.98 (dd, J = 10.5, 3.6 Hz, 1 H), 3.97 (d, J = 14.9 Hz, 1 H), 3.96 (dd, J = 9.2, 6.6 Hz, 1 H), 3.91 (d, J = 14.9 Hz, 1 H), 3.82 (s, 3 H), 3.58 (dd, J = 9.2, 7.9 Hz, 1 H), 2.91 (ddt, J = 8.9, 5.1, 1.0 Hz, 1 H), 2.84 (d, J = 5.1 Hz, 3 H) ppm; 13C NMR + DEPT (100 MHz, CDCl3) δ 169.6 (C), 159.6 (Car), 129.4 (2 × CHar), 129.3 (Car), 114.0 (2 × CHar), 108.7 (CH), 80.6 (CH), 76.4 (CH), 73.9 (CH2), 72.6 (CH2), 71.0 (CH2), 68.4 (CH2), 55.3 (CH3), 51.8 (CH), 25.5 (CH3) ppm; LRMS (ESI+) m/z 360.2 (M + Na)+). To a solution of this intermediate PMB-ether (580 mg, 1.72 mmol) in MeOH (20 mL) under N2 was added Pd/C (145 mg, 25 w%, 10% Pd). The flask was then put under H2-atmosphere and the reaction was stirred at rt for 18 h. After completion the reaction mixture was filtered over Celite and the filter cake was washed with MeOH. The solvent was removed in vacuo to afford the title compound 6f as a white solid (421 mg, 99%) in sufficient purity. Formula C9H15NO5; Mw 217.22; Rf 0.30 (CH2Cl2/MeOH 93:7); Mp 128 °C; [α]D +31.8 (c 0.48, CHCl3, 26 °C); IR (film) 3401 (m), 3343 (m), 2965 (w), 2882 (w), 1652 (vs), 1118 (vs), 1020 (s), 1003 (s), 953 (s) cm−1; 1H NMR (400 MHz, CDCl3) δ 6.56 (br. s, 1 H, disappears after D2O-exchange), 5.82 (d, J = 5.1 Hz, 1 H), 4.59 (dtd, J = 8.7, 6.8, 4.7 Hz, 1 H, simplifies to dt, J = 8.7, 6.7 Hz, after D2O-exchange), 4.48 (m, 1 H), 4.06−4.00 (m, 4 H), 3.96 (d, J = 15.1 Hz, 1 H), 3.60 (dd, J = 9.4, 7.2 Hz, 1 H), 2.86 (d, J = 5.0 Hz, 3 H, simplifies to s after D2O-exchange), 2.87 (m, 1 H), 2.73 (d, J = 4.8 Hz, 1 H, disappears after D2O-exchange) ppm; 13C NMR + DEPT (100 MHz, CDCl3) δ 170.0 (C), 109.1 (CH), 80.2 (CH), 74.1 (CH2), 73.2 (CH2), 69.7 (CH), 68.3 (CH2), 53.1 (CH), 25.6 (CH3) 4036



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at 16 900 g. The inclusion bodies were dissolved in 8 M urea for 15 min at room temperature and passed through a Q-sepharose column and refolding was performed in dialysis against 20 mM sodium phosphate pH 6.5, 10 mM DTT, and 1 mM EDTA. After application to a S-Sepharose column of 1.5 mL per liter cell culture equilibrated with 50 mM MES (4-Morpholineethanesulfonic acid) pH 6.5, 5 mM β-mercaptoethanol, and 1 mM EDTA, the protease was eluted with 0.34 M sodium chloride. Concentration was done using Vivaspin to 4 mg/mL protein. Crystallization. The protein was cocrystallized with 40 mM of the inhibitors by adding a stock solution of the inhibitor dissolved in dimethyl sulfoxide. Crystals were prepared using the hanging drop method using 1 μL protein solution in 50 mM MES pH 5.5, 1 mM DTT, and 1 mM EDTA was mixed with a 1.2 M sodium chloride solution in the same buffer and drops were immediately micro-seeded with protease/inhibitor crystals. Crystals appearing after 2 days grew to a size of about 0.2 × 0.2 × 0.02 mm3 in a couple of weeks. Structure Determination. X-ray diffraction data was collected at end station I911−3 at MAX IV laboratory using MARCCD detectors. Data was processed and scaled with XDS and the CCP4 suite and the structure was visualized using Coot. The interpretation of the electron map and to some extent the refinement of the compound had in some areas extra levels of complexity, since the inhibitor bind in two alternate orientations with respect to the asymmetric unit. The major features of the compound could nevertheless be interpreted infallibly.

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Kottler, H.; Tessmer, U.; Hohenberg, H.; Krausslich, H.-G. Sequential steps in human immunodeficiency virus particle maturation revealed by alterations of individual gag polyprotein cleavage sites. J. Virol. 1998, 72, 2846−2854. (4) Kohl, N. E.; Emini, E. A.; Schleif, W. A.; Davis, L. J.; Heimbach, J. C.; Dixon, R. A. F.; Scolnick, E. M.; Sigal, I. S. Active human immundeficiency virus protease is required for viral infectivity. Proc. Natl. Acad. Sci. U. S. A. 1988, 85, 4686−4690. (5) Wensing, A. M. J.; van Maarseveen, N. M.; Nijhuis, M. Fifteen years of HIV protease inhibitors: raising the barrier to resistance. Antiviral Res. 2010, 85, 59−74. (6) (a) Roberts, J. D.; Bebenek, K.; Kunkel, T. A. The accuracy of reverse transcriptase from HIV-1. Science 1988, 242, 1171−1173. (b) Wensing, A. M.; Calvez, V.; Günthard, H. F.; Johnson, V. A.; Paredes, R.; Pillay, D.; Shafer, R. W.; Richman, D. D. 2014 Update of the drug resistance mutations in HIV-1. Top HIV Med. 2014, 22, 138− 45. (7) (a) Review: Ghosh, A. K.; Anderson, D. D.; Weber, I. T.; Mitsuya, H. Enhancing protein backbone binding − A fruitful concept for combating drug resistant HIV. Angew. Chem., Int. Ed. 2012, 51, 1778−1802. (b) Ghosh, A. K.; Thompson, W. J.; Fitzgerald, P. M.; Culberson, J. C.; Axel, M. G.; McKee, S. P.; Huff, J. R.; Anderson, P. S. Structure-based design of HIV-1 protease inhibitors: replacement of two amides and a 10 pi-aromatic system by a fused bis-tetrahydrofuran. J. Med. Chem. 1994, 37, 2506−2508. (8) (a) Ghosh, A. K.; Kincaid, J. F.; Cho, W.; Walters, D. E.; Krishnan, K.; Hussain, K. A.; Koo, Y.; Cho, H.; Rudall, C.; Holland, L.; Buthod, J. Potent HIV protease inhibitors incorporating high-affinity P2-ligands and (R)-(hydroxyethylamino)sulfonamide isostere. Bioorg. Med. Chem. Lett. 1998, 8, 687−690. (b) Surleraux, D. L. N. G.; Tahri, A.; Verschueren, W. G.; Pille, G. M. E.; deKock, H. A.; Jonckers, T. H. M.; Peeters, A.; DeMeyer, S.; Azijn, H.; Pauwels, R.; deBethune, M. P.; King, N. M.; Prabu-Jeyabalan, M.; Schiffer, C. A.; Wigerinck, P. B. T. P. Discovery and selection of TMC114, a next generation HIV-1 protease inhibitor. J. Med. Chem. 2005, 48, 1813−1822. (9) (a) Koh, Y.; Nakata, H.; Maeda, K.; Ogata, H.; Bilcer, G.; Devasamudram, T.; Kincaid, J. F.; Boross, P.; Wang, Y. F.; Ties, Y. F.; Volarath, P.; Gaddis, L.; Harrison, R. W.; Weber, I. T.; Ghosh, A. K.; Mitsuya, H. Novel bis-tetrahydrofuranylurethane-containing nonpeptidic protease inhibitor (PI) UIC-94017 (TMC114) with potent activity against multi-PI-resistant human immunodeficiency virus in vitro. Antimicrob. Agents Chemother. 2003, 47, 3123−3129. (b) De Meyer, S.; Azijn, H.; Surleraux, D.; Jochmans, D.; Tahri, A.; Pauwels, R.; Wigerinck, P.; de Bethune, M.-P. TMC114, a novel human immunodeficiency virus type 1 protease inhibitor active against protease inhibitor-resistant viruses, including a broad range of clinical isolates. Antimicrob. Agents Chemother. 2005, 49, 2314−2321. (10) Tie, Y.; Boross, P. I.; Wang, Y.-F.; Gaddis, L.; Hussain, A. K.; Leshchenko, S.; Ghosh, A. K.; Louis, J. M.; Harrison, R. W.; Weber, I. T. High resolution crystal structures of HIV-1 protease with a potent non-peptide inhibitor (UIC-94017) active against multi-drug-resistant clinical strains. J. Mol. Biol. 2004, 338, 341−352. (11) (a) Surleraux, D. L. N. G.; deKock, H. A.; Verschueren, W. G.; Pille, G. M. E.; Maes, L. J. R.; Peeters, A.; Vendeville, S.; DeMeyer, S.; Azijn, H.; Pauwels, R.; deBethune, M. P.; King, N. M.; PrabuJeyabalan, M.; Schiffer, C. A.; Wigerinck, P. B. T. P. Design of HIV-1 protease inhibitors active on multidrug-resistant virus. J. Med. Chem. 2005, 48, 1965−1973. (b) Miller, J. F.; Andrews, C. W.; Brieger, M.; Furfine, E. S.; Hale, M. R.; Hanlon, M. H.; Hazen, R. J.; Kaldor, I.; McLean, E. W.; Reynolds, D.; Sammond, D. M.; Spaltenstein, A.; Tung, R.; Turner, E. M.; Xu, R. X.; Sherrill, R. G. Ultra-potent P1 modified arylsulfonamide HIV protease inhibitors: The discovery of GW0385. Bioorg. Med. Chem. Lett. 2006, 16, 1788−1794. (c) Sherrill, R. G.; Furfine, E. S.; Hazen, R. J.; Miller, J. F.; Reynolds, D. J.; Sammond, D. M.; Spaltenstein, A.; Wheelan, P.; Wright, L. L. Synthesis and antiviral activities of novel N-alkoxy-arylsulfonamidebased HIV protease inhibitors. Bioorg. Med. Chem. Lett. 2005, 15, 3560−3564.

ASSOCIATED CONTENT

S Supporting Information *

Experimental procedure and spectral data for intermediates and additional target compounds. HPLC purity data for all tested compounds, and crystallographic data. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00358. Accession Codes

5AGZ, 5AH6, 5AH7, 5AH8, 5AH9, 5AHA, 5AHB, and 5AHC.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +44 (0)23 8059 3816. Fax: +44 (0)23 8059 3781. *E-mail: [email protected]. Tel.: +46 (0)46 222 4491. Fax: +46 (0)46 222 4710. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS K.H. thanks Janssen Pharmaceutica NV for funding. We thank Janssen Pharmaceutica NV for antiviral activity determination. REFERENCES

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Disubstituted Bis-THF Moieties as New P2 Ligands in Nonpeptidal HIV

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