Field-Based Affinity Optimization of a Novel Azabicyclohexane Scaffold

molecules

Article Field-Based Aﬃnity Optimization of a Novel Azabicyclohexane Scaﬀold HIV-1 Entry Inhibitor

Megan E. Meuser 1, Adel A. Rashad 1, Gabriel Ozorowski 2, Alexej Dick 1, Andrew B. Ward 2 and Simon Cocklin 1,*

1 Department of Biochemistry & Molecular Biology, Drexel University College of Medicine, Rooms 10307, 10309, and 10315, 245 North 15th Street, Philadelphia, PA 19102, USA; [email protected] (M.E.M.); [email protected] (A.A.R.); [email protected] (A.D.) 2 Department of Integrative Structural and Computational Biology, Collaboration for AIDS Vaccine Discovery, the Scripps Research Institute, La Jolla, CA 92037, USA; [email protected] (G.O.); [email protected] (A.B.W.) * Correspondence: [email protected]; Tel.: +1-215-762-7234 or +1-215-762-4979; Fax: +1-215-762-4452

Academic Editor: Stefano Aquaro Received: 19 February 2019; Accepted: 20 April 2019; Published: 22 April 2019

Abstract: Small-molecule HIV-1 entry inhibitors are an extremely attractive therapeutic modality. We have previously demonstrated that the entry inhibitor class can be optimized by using computational means to identify and extend the chemotypes available. Here we demonstrate unique and differential effects of previously published antiviral compounds on the gross structure of the HIV-1 Env complex, with an azabicyclohexane scaffolded inhibitor having a positive effect on glycoprotein thermostability. We demonstrate that modification of the methyltriazole-azaindole headgroup of these entry inhibitors directly effects the potency of the compounds, and substitution of the methyltriazole with an amine-oxadiazole increases the affinity of the compound 1000-fold over parental by improving the on-rate kinetic parameter. These findings support the continuing exploration of compounds that shift the conformational equilibrium of HIV-1 Env as a novel strategy to improve future inhibitor and vaccine design efforts.

Keywords: bioisosteres; HIV-1 Env; antiviral; surface plasmon resonance; computer-aided drug design

1. Introduction The HIV-1 Env complex, the sole viral protein on the outer surface of the virion, is the main focus of antigen design for antibody-based vaccines and small molecule entry inhibitors [1]. The Env complex itself is a trimer of heterodimeric subunits, gp120 and gp41, and orchestrates the series of events that allow deposition of the viral contents into the host cell, making it a primary determinant of viral infectivity. Over recent years, several structures of recombinant Env trimers have been determined by cryo-electron microscopy and x-ray crystallography, including membrane-extracted trimers and engineered soluble trimer mimics [2–11]. These structures have revealed many insights about the structural features that govern sensitivity and resistance to neutralization of HIV-1 by antibodies and small molecules. A number of academic groups and pharmaceutical companies are investing time and eﬀort into the development of small molecule inhibitors of the HIV-1 entry process. Encouraging results have been obtained in one entry inhibitor group, originally developed by Bristol-Myers Squibb, and currently being further developed by ViiV Healthcare [12]. Phase III trials of BMS-663068 (or fostemsavir as it has been dubbed) are ongoing. However, all results from these studies on

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BMS-663068 prodrug entry inhibitor indicate that it would only be of utility as salvage therapy for highlyMolecules treatment-experienced 2018, 23, x patients. 2 of 30 Given the enormous potential of HIV-1 entry inhibitors as both pre- and post-exposure prophylactic Given the enormous potential of HIV-1 entry inhibitors as both pre- and post-exposure regimens, our group has been exploring the modification of piperazine scaffold entry inhibitors [12–19]. prophylactic regimens, our group has been exploring the modification of piperazine scaffold entry We have successfully expanded, designed and tested a number of scaffolds other than piperazine, inhibitors [12–19]. We have successfully expanded, designed and tested a number of scaffolds other developing compounds with nanomolar potency and specificity to HIV-1 Env [16,20,21]. Moreover, than piperazine, developing compounds with nanomolar potency and specificity to HIV-1 Env we have recently demonstrated by using a novel surface plasmon resonance (SPR) interaction assay, [16,20,21]. Moreover, we have recently demonstrated by using a novel surface plasmon resonance that the off-rate of the compounds for a soluble recombinant Env trimer is strongly correlated with the (SPR) interaction assay, that the off-rate of the compounds for a soluble recombinant Env trimer is potency of the compounds in the single round infection assay [20]. The results presented herein further strongly correlated with the potency of the compounds in the single round infection assay [20]. The extends our work developing novel entry inhibitors by demonstrating the conformational effects of results presented herein further extends our work developing novel entry inhibitors by previously disclosed entry inhibitors on the Env complex. Moreover, we demonstrate the affinity demonstrating the conformational effects of previously disclosed entry inhibitors on the Env optimization of one of these scaffolds, the azabicyclohexane scaffold, which has the most pronounced complex. Moreover, we demonstrate the affinity optimization of one of these scaffolds, the effect on the conformation of the Env target. The results from this work have broad implications for azabicyclohexane scaffold, which has the most pronounced effect on the conformation of the Env future HIV-1 entry inhibitor designs that capitalize on the malleability and metastability of the Env target. The results from this work have broad implications for future HIV-1 entry inhibitor designs protein complex. that capitalize on the malleability and metastability of the Env protein complex. 2. Results and Discussion 2. Results and Discussion To gain insight into optimization strategies and mechanisms of action of a selection of our entryTo inhibitor gain insight compounds into optimization with differing strategies core scaandff olds,mechanisms we wished of action to perform of a selection molecular of our docking entry againstinhibitor the compounds published with structure differing of thecore HIV-1 scaffolds, SOSIP.664 we wished gp140 to trimer.perform First,molecular however, docking we against had to confirmthe published that the structure compounds of the actually HIV-1 SOSIP.664 do directly gp140 interact trimer. with First, the Env however, complex. we Wehad have to confirm previously that demonstratedthe compounds the actually interaction do directly of four interact of our with HIV-1 the Env entry complex. inhibitors We withhave thepreviously B41 SOSIP.664 demonstrated gp140 trimerthe interaction using surface of four plasmon of our HIV-1 resonance entry [20 inhibi]. Therefore,tors with asthe a B41 precursor SOSIP.664 to molecular gp140 trimer modeling, using wesurface performed plasmon SPR resonance.[20] interaction analysis Therefore, upon as SC11a precursor(dipyrrolidine to molecular scaffold), modeling,SC15 (azetidinewe performed scaff old),SPR SC28interaction(azabicyclo-hexane analysis upon scaSC11ffold), (dipyrrolidine and SC45 (tetrahydropyridine scaffold), SC15 (azetidine scaffold) scaffold), (Figure1 SC28). Figure (azabicyclo-2 shows thehexane representative scaffold), sensogramsand SC45 for(tetrahydropyridine each compound interacting scaffold) with(Figure the B411). SOSIP.664Figure 2 gp140shows trimer the andrepresentative Table1 shows sensograms the kinetic for parameters each compound obtained inte afterracting analysis. with the B41 SOSIP.664 gp140 trimer and Table 1 shows the kinetic parameters obtained after analysis.

Figure 1. Chemical structures of compounds SC11, SC15, SC28, and SC45. The diﬀerent core scaﬀolds Figure 1. Chemical structures of compounds SC11, SC15, SC28, and SC45. The different core scaffolds in each compound are colored blue. in each compound are colored blue.

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Figure 2. Sensorgrams showing the interaction of small molecule compounds with B41 SOSIP.664 gp140Figure trimer 2. Sensorgrams immobilized showing on the sensorthe interaction chip. (A) ofSC11 small(concentrations molecule compounds shown are with 10 µ MB41 and SOSIP.664 down in agp140 1:2 dilution trimer series),immobilized (B) SC15 on the(2 µ sensorM, 1:4 dilutionchip. (A series),) SC11 (concentrations (C) SC28 (10 µM, shown 1:2 dilution are 10 series),µM and and down (D) SC45in a 1:2(40 dilutionµM, 1:3 series), dilution (B series).) SC15 (2 Colored µM, 1:4 lines dilution represent series), actual (C) dataSC28 collected (10 µM, from1:2 dilution the dilution series), series, and whereas(D) SC45 black (40 µM, lines 1:3 signify dilution the series). fits to a Colored 1:1 binding lines model. represent Interaction actual data parameters collected derived from the from dilution 5 sets ofseries, data whereas are given black in Table lines1 .signify the fits to a 1:1 binding model. Interaction parameters derived from 5 sets of data are given in Table 1. Table 1. Kinetics and affinity of compounds SC11, SC15, SC28, and SC45 binding to B41 SOSIP.664 gp140Table trimer.1. Kinetics and affinity of compounds SC11, SC15, SC28, and SC45 binding to B41 SOSIP.664 gp140 trimer. 1 1 1 Compound ka (M− s− ) kd (s− ) KD SC11 Compound 3.83 k1.12a (M−1s10−1)3 5.02kd (s−2.671) 10 4 KD 0.131 µM ± × ± × − SC15 SC11 3.013.830.188 ± 1.12 ×10 105 3 5.025.44 ± 2.670.677 × 10−4 10 30.131 µM0.0181 µM ± × ± × − SC28 SC15 1.393.01 0.14± 0.18810 × 4105 5.446.99 ± 0.6770.43 × 10−103 30.0181 µM0.511 µM ± × ± × − SC45 SC28 1.381.390.045 ± 0.14 ×10 104 4 6.991.52 ± 0.430.0294 × 10−3 10 20.511 µM 1.10 µM ± × ± × − SC45 1.38 ± 0.045 × 104 1.52 ± 0.0294 × 10−2 1.10 µM As predicted, each of the compounds interacted robustly with the B41 SOSIP.664 gp140 trimer, As predicted, each of the compounds interacted robustly with the B41 SOSIP.664 gp140 trimer, albeit with differing kinetics, providing a clear basis for computational docking of the inhibitors into albeit with differing kinetics, providing a clear basis for computational docking of the inhibitors into the structure of soluble Env trimer. Therefore, we docked all of the compounds onto the recently the structure of soluble Env trimer. Therefore, we docked all of the compounds onto the recently published B41 SOSIP Env structure in complex with BMS-386150, using the binding pocket of the published B41 SOSIP Env structure in complex with BMS-386150, using the binding pocket of the co- co-crystalized ligand (PDB code: 6MUG) [22] to examine potential differences in binding orientation. crystalized ligand (PDB code: 6MUG) [22] to examine potential differences in binding orientation. Interestingly, compounds SC11, SC15 and SC45 were easily docked onto the binding site of B41.SOSIP Interestingly, compounds SC11, SC15 and SC45 were easily docked onto the binding site of structure, similar to the co-crystalized ligand BMS-386150 (Figure3), without any need to perform B41.SOSIP structure, similar to the co-crystalized ligand BMS-386150 (Figure 3), without any need to flexible docking. However, the azabicyclo-hexane compound SC28 did not successfully dock using the perform flexible docking. However, the azabicyclo-hexane compound SC28 did not successfully dock previous rigid docking protocol, and instead, we had to use flexible protein-ligand docking in order to using the previous rigid docking protocol, and instead, we had to use flexible protein-ligand docking achieve a plausible model of SC28 at the binding site. This need for flexibility in the docking protocol in order to achieve a plausible model of SC28 at the binding site. This need for flexibility in the was subsequently found to be due to the fact that the presence of the azabicyclo-hexane moiety, in order docking protocol was subsequently found to be due to the fact that the presence of the azabicyclo- to dock to the binding pocket, appears to induce changes in the orientations of certain pocket side hexane moiety, in order to dock to the binding pocket, appears to induce changes in the orientations chains, especially in the β20/21 loop residue W427, and the α1 helix residue W112 (Figures3 and4). It is of certain pocket side chains, especially in the β20/21 loop residue W427, and the α1 helix residue W112 (Figures 3 and 4). It is well documented that changes or interactions at one site can have profound

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Moleculeseffects 2018upon, 23 ,the x overall structure and conformation of the Env complex. Moreover, it has4 ofbeen 30 welldemonstrated documented recently that changes that the or β20/21 interactions loop is a atregulator one site of can conformational have profound transitions eﬀects upon in theHIV-1 overall Env effectsand it uponis possible the overall that modulation structure andof this conformati region byon SC28of the could Env inducecomplex. gross Moreover, changes itin has structure been structure and conformation of the Env complex. Moreover, it has been demonstrated recently that the demonstrated[23,24]. recently that the β20/21 loop is a regulator of conformational transitions in HIV-1 Env β loop is a regulator of conformational transitions in HIV-1 Env and it is possible that modulation and20/ 21it is possible that modulation of this region by SC28 could induce gross changes in structure of this region by SC28 could induce gross changes in structure [23,24]. [23,24].

Figure 3. Docking models of compounds SC11, SC15, SC28 and SC45 with B41 SOSIP structure PDB Figure6MUG. 3. Docking models of compounds SC11, SC15, SC28 and SC45 with B41 SOSIP structure FigurePDB 6MUG. 3. Docking models of compounds SC11, SC15, SC28 and SC45 with B41 SOSIP structure PDB 6MUG.

Figure 4. Comparison of the docking models of compounds SC28 (parental; methyltriazole) and SC56 Figure 4. Comparison of the docking models of compounds SC28 (parental; methyltriazole) and SC56 (analogue; amine-oxadiazole) highlighting the diﬀerence in the orientation of the W427 indole ring. (analogue; amine-oxadiazole) highlighting the difference in the orientation of the W427 indole ring.

FigureBased 4. on Comparison the SPR and of the docking docking results, models and of compounds speciﬁcally SC28 the (parental; participation methyltriazole) of the β20 and/21 loop SC56 in the binding(analogue;Based of SC28 on amine-oxadiazole) the, we SPR decided and docking to testhighlighting the results, eﬀects the an of differend four specifically ofce the in the compounds the orientation participation on of thethe overallW427of the indole β structure20/21 ring.loop ofin B41the SOSIP.664binding of using SC28 negative-stain, we decided to electron test the microscopy effects of four (NS-EM). of theB41 compounds SOSIP.664 on has the been overall reported structure to be of a strongB41 BasedSOSIP.664 immunogen on the using SPR like negative-stain and the “golddocking standard” results, electron BG505an micrd specificallyoscopy SOSIP.664, (NS-EM). the with participation most B41 broadlySOSIP.664 of neutralizingthe has β20/21 been loop epitopesreported in the bindingpresentedto be a strong of onSC28 its immunogen, surface, we decided yet like its to appearancethe test “gold the effects standard” by NS-EM of four BG505 suggests of the SOSIP.664, compounds a much with greater on most the degree broadlyoverall ofmovement structureneutralizing of of B41gp120epitopes SOSIP.664 subunits presented using relative onnegative-stain its to surfac the three-folde, yetelectron its symmetry appearance microscopy axis, by (NS-EM). aNS-EM behavior suggests B41 sometimes SOSIP.664 a much referred has greater been to reporteddegree as trimer of to“breathing”movement be a strong of [ 25immunogen gp120–27]. subunits When like no relativethe compound “gold to standard”the is thre included,e-fold BG505 asymmetry small SOSIP.664, population axis, with a be most (abouthavior broadly 20–25%)sometimes neutralizing of referred imaged epitopesB41to as trimers trimer presented “breathing” adopted on what its [25–27]. surfac we infere, When yet to its no be appearance compou a more opennd by is included, conformation,NS-EM suggests a small while poa muchpulation the remaininggreater (about degree 20–25%) trimers of movementhadof imaged a more ofB41 tightly-packed, gp120 trimers subunits adopted closed relative what phenotype to we the infer thre (Figuretoe-fold be a 5 symmetrymoreA). The open inclusion axis, conformation, a be ofhavior a 10-fold whilesometimes molar the remaining excess referred of toanytrimers as oftrimer the had four“breathing” a more compounds tightly-packed, [25–27]. appears When closed to no drive compou phenotyp thend equilibrium ise (Figureincluded, from5A). a small The open inclusionpo topulation closed of trimers,(abouta 10-fold 20–25%) at molar least ofqualitatively.excess imaged of anyB41 Statistics oftrimers the four adopted of compounds NS-EM what analysis weappears infer are summarized to drivbe ae more the inequilibrium open Table conformation, S1. Tofrom make open a while better to closed the assessment remaining trimers, on at trimersleast qualitatively. had a more tightly-packed,Statistics of NS-EM closed analysis phenotyp aree (Figuresummarized 5A). The in inclusionTable S1. ofTo a 10-foldmake a molarbetter excess of any of the four compounds appears to drive the equilibrium from open to closed trimers, at least qualitatively. Statistics of NS-EM analysis are summarized in Table S1. To make a better

Molecules 2019, 24, 1581 5 of 30 Molecules 2018, 23, x 5 of 30 assessment on the stabilizing effects of the small molecules on the trimer, we used differential the stabilizing effects of the small molecules on the trimer, we used differential scanning calorimetry scanning calorimetry (DSC) to measure changes in thermostability upon the addition of small (DSC) to measure changes in thermostability upon the addition of small molecules (Figure5B). All molecules (Figure 5B). All four compounds had a positive impact on B41 SOSIP.664 trimer four compounds had a positive impact on B41 SOSIP.664 trimer thermostability, with SC11 having the thermostability, with SC11 having the largest effect (Figure 5B). The combined observations of a largest effect (Figure5B). The combined observations of a measured increase in thermostability (DSC) measured increase in thermostability (DSC) and an inferred shift towards a more compact state (EM) and an inferred shift towards a more compact state (EM) suggested that the compounds are able to suggested that the compounds are able to halt the natural dynamics of an Env trimer (that are critical halt the natural dynamics of an Env trimer (that are critical for the virus during receptor recognition for the virus during receptor recognition and host cell fusion), and are in agreement with the role of and host cell fusion), and are in agreement with the role of the β20/21 loop in stabilization of the ground the β20/21 loop in stabilization of the ground state of Env [23]. Because SC28 had both experimentally- state of Env [23]. Because SC28 had both experimentally-determined stabilizing effects and in silico determined stabilizing effects and in silico suggestions of inducing conformational changes, the suggestions of inducing conformational changes, the results prompted us to search for and design results prompted us to search for and design analogues of the azabicyclohexane scaffold with analogues of the azabicyclohexane scaffold with improved affinity. improved affinity.

Figure 5. Biophysical characterization of B41 SOSIP.664 incubated with SC11, SC15, SC28 or SC45. Figure 5. Biophysical characterization of B41 SOSIP.664 incubated with SC11, SC15, SC28 or SC45. (A) Representative negative-stain EM 2D class averages. The control sample contained no compound. (A) Representative negative-stain EM 2D class averages. The control sample contained no compound. Averaged particles with phenotypes of “breathing” trimers are highlighted with yellow boxes. All Averaged particles with phenotypes of “breathing” trimers are highlighted with yellow boxes. All samples were stained with 2% (w/v) uranyl formate. (B) Differential scanning calorimetry curves (left) samples were stained with 2% (w/v) uranyl formate. (B) Differential scanning calorimetry curves (left) and summary of melting temperatures (Tm) (right). Primary and secondary peaks are those with and summary of melting temperatures (Tm) (right). Primary and secondary peaks are those with the the highest and second highest intensity (Cp, specific heat capacity), respectively. ∆Tm is the relative highest and second highest intensity (Cp, specific heat capacity), respectively. ΔTm is the relative change from the inhibitor-free control. change from the inhibitor-free control. In the absence of any experimentally-derived structural information on the bioactive conformation In the absence of any experimentally-derived structural information on the bioactive of our inhibitors, we used the docking model of SC28 as described above as an input into Spark conformation of our inhibitors, we used the docking model of SC28 as described above as an input (Cresset, UK) to identify nonclassical bioisosteres of the selected region. From the literature available into Spark (Cresset, UK) to identify nonclassical bioisosteres of the selected region. From the literature on the piperazine-based entry inhibitors, it is clear that a primary determinant of potency is the available on the piperazine-based entry inhibitors, it is clear that a primary determinant of potency methyltriazole-azaindole head group of the compounds [28–31]. We, therefore, focused on this region, is the methyltriazole-azaindole head group of the compounds.[28–31] We, therefore, focused on this looking for changes suggested by Spark that may modulate either drug-like parameters or potency, region, looking for changes suggested by Spark that may modulate either drug-like parameters or and were significantly different from the methyltriazole-azaindole head group of SC28.[16,20,21,32] potency, and were significantly different from the methyltriazole-azaindole head group of SC28. From this analysis, we docked five compounds onto the B41 SOSIP Env structure (PDB code: 6MUG) [22] [16,20,21,32] From this analysis, we docked five compounds onto the B41 SOSIP Env structure (PDB using Glide (XP-mode), and looked at the overall ligand-protein interaction energies. Table2 shows code: 6MUG)[22] using Glide (XP-mode), and looked at the overall ligand-protein interaction the calculated ligand-protein interaction energies for these compounds in comparison to the parental energies. Table 2 shows the calculated ligand-protein interaction energies for these compounds in compound SC28. comparison to the parental compound SC28. Interestingly, all of the azabicyclohexane analogues produced broadly similar overall poses with the distortion of the β20/21 loop region but a range of calculated interaction energies. The two compounds with the greatest interaction energies in this analysis were SC28 and SC56, which differ only in the replacement of the methyltriazole with an amine-oxadiazole. This change only appeared

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Table 2. Calculated Overall Ligand-Protein interaction energies for the novel azabicyclohexane analogues.

Calculated Overall Ligand-Protein Interaction Energy Compound Kcal/Mol SC28 16.13700 − SC49 6.58188 − SC50 12.87666 − SC52 2.25288 − SC55 10.12901 − SC56 18.37654 −

Interestingly, all of the azabicyclohexane analogues produced broadly similar overall poses with the distortion of the β20/21 loop region but a range of calculated interaction energies. The two compounds with the greatest interaction energies in this analysis were SC28 and SC56, which differ only in the replacement of the methyltriazole with an amine-oxadiazole. This change only appeared to have one large effect upon the binding pocket, with the orientation of the W427 side chain being completely different between the two binding models (Figure4). This analysis suggests that SC56 should have greater affinity for the HIV-1 Env than the parental compound, whereas the other modifications run a range of having potentially lower or equal affinities to SC28. We therefore chose to synthesize these compounds in order to test the predictions from the design and docking analyses. The docking of the new compounds (designated SC49, SC50, SC52, SC55, and SC56) onto the B41 SOSIP Env structure implies a rank order of affinities. Therefore, after synthesis, and prior to antiviral testing, we sought to establish that the new compounds retained the target specificity of the parental SC28 compound and whether this rank order held true. We again chose to demonstrate this via SPR. Figure6 shows the representative sensorgrams for each compound interacting with the B41 SOSIP.664 gp140 trimer and Table3 shows the kinetic parameters obtained after analysis. As can be seen in Table3, all analogues retained target specificity and interacted with the B41 SOSIP.664 gp140 trimer. A range of affinities were observed between the analogues with a general agreement with the docking results, i.e., SC49 and SC52 having lower affinities relative to SC28. Rather satisfyingly, however, was the finding that SC56 indeed had a greater affinity for the B41 SOSIP.664 gp140 trimer than SC28 with a 1000-fold difference improvement in the KD of SC56 (0.5 nM) in comparison to the parental SC28 (0.5 µM) and similar overall binding responses to SC28 but over a concentration series 1000-fold more dilute. The dissociation rates of SC56 and SC28 are nearly identical indicating that the contributing factor in the kinetics to this increase in affinity is the association rate with the ka parameter of SC56 increasing by a factor of 1000 in comparison to SC28. After demonstrating that each of the five SC28 derivatives and SC28 interact with the B41 SOSIP.664 gp140 trimer, and that we had greatly improved the affinity in one of the analogues, SC56, we then tested them for potency against HIV-1 using the HIV-1 single round infection assay. In this + system, 293T cells are co-transfected with the envelope-deficient HIV-1 NL4-3 vector (pNL4-3-LucR E−; a gift of N. R. Landau, New York University), [33] which carries the luciferase-reporter gene; and the envelope expressing vector from the B41 [26], HxBc2 [34], JR-CSF [35,36], JR-FL [37], or YU-2 [38,39] HIV-1 isolates. This co-transfection yields recombinant single-round infectious envelope-pseudotyped luciferase-reporter HIV-1 viruses. The B41, JR-CSF, JR-FL, and YU-2 envelopes were all originally isolated by directly cloning samples from HIV-1 infected patients and therefore were never subjected to the potential selection imposed by passage of the virus in tissue culture. B41, JR-CSF, JR-FL, and YU-2 are all relatively resistant to neutralization by soluble CD4 and antibodies directed against the HIV-1 envelope glycoproteins and are classified as tier 2 isolates that utilize the CCR5-receptor for entry. Therefore they are representative of the clinically most abundant viruses. The use of the HxBc2 reference strain Env, along with the Envs from four primary R5-tropic viruses allows an assessment of the generality of results obtained. The pseudotyped viruses are then used to infect U87.CD4.CCR5 (B41, JR-CSF, JR-FL, and YU-2) or U87.CD4.CXCR4 (HxBc2) target cells in the presence and absence Molecules 2019, 24, 1581 7 of 30

of compounds and infectivity is quantiﬁed by measuring luciferase levels in cell lysates (Luciferase Assay System, Promega, Fitchburg, WI, USA) using a microplate luminometer (GloMax, Promega). The toxicity of the compounds was also assessed in parallel as outlined in Materials & Methods. MoleculesThe results2018, 23 of, x this analysis are shown in Figure S1 and S2 and the values are summarized in Table47. of 30

FigureFigure 6. Sensorgrams 6. Sensorgrams showing showing the the interaction interaction of small moleculemolecule compounds compounds with with B41 B41 SOSIP.664 SOSIP.664 gp140gp140 trimer trimer immobilized immobilized on on the the sensor sensor chip. chip. ( (AA)) SC28 (concentrations(concentrations shown shown are are 10 10µM µM and and down down in ain 1:2 a 1:2dilution dilution series), series), (B ()B SC49) SC49 (100(100 µM,µM, 1:2 1:2 dilution dilution series), ((CC))SC50 SC50(200 (200µ M,µM, 1:2 1:2 dilution dilution series), series), (D) SC52 (100 µM, 1:2 dilution series), (E) SC55 (100 µM, 1:2 dilution series), and (F) SC56 (10 nM, (D) SC52 (100 µM, 1:2 dilution series), (E) SC55 (100 µM, 1:2 dilution series), and (F) SC56 (10 nM, 1:2 dilution series). Colored lines represent actual data, whereas black lines indicate the ﬁts to a 1:1 1:2 dilution series). Colored lines represent actual data, whereas black lines indicate the fits to a 1:1 interaction model. Interaction parameters derived from ﬁve sets of data are given in Table2. interaction model. Interaction parameters derived from five sets of data are given in Table 2.

Table 3. Kinetics and affinity of novel SC28 derivatives binding to soluble, cleaved recombinant Env. N.D. = not determined.

Compound ka (M−1s−1) kd (s−1) KD (µM) SC28 1.39 ± 0.14 × 104 6.99 ± 0.43 × 10−3 0.504 µM SC49 9.49 ± 1.6 × 102 3.43 ± 0.36 × 10−2 36.1 µM SC50 N.D. N.D. 114 ± 51 µM SC52 N.D. N.D. 23.7 ± 2.4 µM SC55 N.D. N.D. 11.4 ± 3.7 µM SC56 1.22 ± 0.067 × 107 6.39 ± 0.31 × 10−3 0.526 nM

After demonstrating that each of the five SC28 derivatives and SC28 interact with the B41 SOSIP.664 gp140 trimer, and that we had greatly improved the affinity in one of the analogues, SC56,

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we then tested them for potency against HIV-1 using the HIV-1 single round infection assay. In this system, 293T cells are co-transfected with the envelope-deficient HIV-1 NL4-3 vector (pNL4-3- LucR+E−; a gift of N. R. Landau, New York University), [33] which carries the luciferase-reporter gene; LucR+E−; a gift of N. R. Landau, New York University), [33] which carries the luciferase-reporter gene; and the envelope expressing vector from the B41 [26], HxBc2 [34], JR-CSF [35,36], JR-FL [37], or YU- 2 [38,39] HIV-1 isolates. This co-transfection yields recombinant single-round infectious envelope- pseudotyped luciferase-reporter HIV-1 viruses. The B41, JR-CSF, JR-FL, and YU-2 envelopes were all originally isolated by directly cloning samples from HIV-1 infected patients and therefore were never Moleculessubjected to2019 the potential, 24, 1581 selection imposed by passage of the virus in tissue culture. B41, JR-CSF, JR- 8 of 30 FL, and YU-2 are all relatively resistant to neutralization by soluble CD4 and antibodies directed against the HIV-1 envelope glycoproteins and are classified as tier 2 isolates that utilize the CCR5- receptor for entry. Therefore they are representative of the clinically most abundant viruses. The use receptorTable for entry. 3. Kinetics Therefore they and are aﬃ representativenity of novel of theSC28 clinicallyderivatives most abundant binding viruses. to The soluble, use cleaved recombinant Env. of the HxBc2 reference strain Env, along with the Envs from four primary R5-tropic viruses allows an assessmentN.D. = ofnot the generality determined. of results obtained. The pseudotyped viruses are then used to infect U87.CD4.CCR5 (B41, JR-CSF, JR-FL, and YU-2) or U87.CD4.CXCR4 (HxBc2) target cells in the presence and absence of compounds and infectivity is1 quantified1 by measuring luciferase levels1 in presence andCompound absence of compounds and infectivityka (M −is quantifieds− ) by measuring luciferase kd (s −levels) in KD (µM) cell lysates (Luciferase Assay System, Promega, Fitchburg, WI, USA) using a microplate luminometer (GloMax, Promega). The toxicity of the compounds was also4 assessed in parallel as outlined in 3 (GloMax, Promega).SC28 The toxicity of the compounds1.39 0.14 was also10 assessed in parallel6.99 as0.43 outlined10 in− 0.504 µM Materials & Methods. The results of this analysis± are shown× in Figure S1 and S2 and± the values× are Materials & Methods. The results of this analysis are shown in2 Figure S1 and S2 and the values are 2 summarized in TableSC49 4. 9.49 1.6 10 3.43 0.36 10− 36.1 µM As summarized in Table 4, SC28 and each of± the analogues× tested exhibited antiviral± effects× in As summarizedSC50 in Table 4, SC28 and each of N.D.the analogues tested exhibited antiviral N.D. effects in 114 51 µM the single-round infection assay utilizing viruses pseudotyped with HIV-1 Env from either the B41, ± HxBc2, JR-CSF, JR-FL, or YU-2 isolates. The analogues exhibited a range of potencies, largely in HxBc2, JR-CSF, SC52JR-FL, or YU-2 isolates. The analoguesN.D. exhibited a range of potencies, N.D. largely in 23.7 2.4 µM correlation with the docking and the SPR results. However, we found that SC56 despite having a ± much higher affinity for the B41 SOSIP.664 gp140 trimer than SC28, had a median IC50 only two-fold much higher affinitySC55 for the B41 SOSIP.664 gp140 trimerN.D. than SC28, had a median IC50 only N.D. two-fold 11.4 3.7 µM over that of SC28. We have previously demonstrated that the potency of our entry inhibitors has a ± direct correlationSC56 with the kinetic off-rate1.22 parameter.[20]0.067 Comparison107 of the 6.39kinetics 0.31of SC28 10and 3 0.526 nM direct correlation with the kinetic off-rate parameter.[20]± × Comparison of the kinetics± of SC28× and− SC56 shows that the off-rates for their interaction with the B41 SOSIP.664 gp140 trimer are almost identical, providing a rationale for their similar potencies, and further corroborating our previous study. Table 4. Potency and toxicity of compounds against HIV-1B41, HIV-1HxBc2, HIV-1JRCSF, HIV-1JRFL, and HIV-1YU-2 Env pseudotyped HIV-1. The chemical structures were drawn with ChemAxon software Table 4. Potency and toxicity of compounds against HIV-1B41, HIV-1HxBc2, HIV-1JRCSF, HIV-1JRFL, and Table 4. Potency and toxicity of compounds against HIV-1B41, HIV-1HxBc2, HIV-1JRCSF, HIV-1JRFL, and HIV-1(Budapest,YU-2 Env pseudotyped Hungary). HIV-1. The chemical structures were drawn with ChemAxon software HIV-1YU-2 Env pseudotyped HIV-1. The chemical structures were drawn with ChemAxon software (Budapest, Hungary). (Budapest, Hungary). Therapeutic ICIC50 50 B41IC50 ICIC5050HxBc2 IC50 IC50ICJRCSF50 Median IC50 JRFL TherapeuticIC50 YU-2 Median IC50 CC50 Compound IC50 IC50 IC50 IC50 IC50 Median CC50 Therapeutic Index Compound B41( µM)HxBc2 JRCSF(µM) JRFL YU-2(µM) IC50 (µCCM)50 Index(µ M) (µM) (µM) Compound B41 HxBc2 JRCSF JRFL YU-2 IC50 (µM) Index (CC50/IC50) (µM) (µM) (µM) (µM) (µM) (µM) (µM) (CC50/IC50) (µM) (µM) (µM) (µM) (µM) (µM) (CC50/IC50) SC28SC28 (parental)(parental) SC28 (parental)

0.0350.035 ± 0.021 ± 0.026 ± 0.17 ± 0.48 ± 0.15 ± 190 ± 0.035 ± ±0.021 ± 0.0210.026 ± 0.0050.17 ± 0.0260.48 ± 0.0090.15 ± 0.17 1900.05 ± 1291.21 0.48 0.05 0.15 0.17 190 54 1291.21 0.00620.0062 0.005 0.009± 0.05 0.05± 0.17 ±54 1291.21± ± ± Molecules 2018, 23, x 0.0062 0.005 0.009 0.05 0.05 0.17 54 9 of 30

Molecules 2018, 23, x 9 of 30 Table 4. Cont.

SC49 SC49 Table 4. Cont. Molecules 2018SC50, 23, x 7.1 ± 6.9 ± 28.92 ± 24.28 ± 22.6 ± 18 ± 9 120 ± 6.63 9 of 30 Molecules 2018, 23, x 62 ± 33.32 ± 57.2 ± 326 ± 9 of 30 264.2 ± 19 625.2 ± 5.62NA 33.326.2 ± 57.24.2 ± 45 ± 15 32618 ± 7.31 26 ±26 19 191.4 62NA 1.411.56 26.5 NA 45 ± 15 33.32 8111.56 7.31 57.2 26.5 45 15 326 81 7.31 SC50 7.1 ± ± 6.91.4 ± 28.92Table± ± 4. Cont.24.2811.56 ± 22.626.5 ± 18 ± 9 120±81 ± 6.63 ± ± ± 4.2 5.2 Table5.62 4. Cont.6.2 4.2 18

SC50 7.1 ± 6.9 ± 28.92 ± 24.28 ± 22.6 ± 18 ± 9 120 ± 6.63 SC50 7.1 ± 6.9 ± 28.92 ± 24.28 ± 22.6 ± 18 ± 9 120 ± 6.63 SC50 4.2 5.2 5.62 6.2 4.2 18 4.2 5.2 5.62 6.2 4.2 18

SC52 0.437.1 ± 4.20.44 ± 2.02 6.9 ± 5.2NA 28.921.35 ± 5.621.06 ± 24.289.2 6.2± 8.65 22.6 4.2 18 9 120 18 6.63 ± ± ± ± ± ± ± 0.1 0.17 0.25 0.44 0.668 1.1

SC52 0.43 ± 0.44 ± 2.02 ± NA 1.35 ± 1.06 ± 9.2 ± 8.65 0.1 0.17 0.25 0.44 0.668 1.1

SC52 0.43 ± 0.44 ± 2.02 ± NA 1.35 ± 1.06 ± 9.2 ± 8.65 SC52 SC52 0.430.1 ± 0.440.17 ± 2.020.25 ± NA 1.350.44 ± 1.060.668 ± 9.21.1 ± 8.65 0.1 0.17 0.25 0.44 0.668 1.1

0.43 0.1 0.44 0.17 2.02 0.25 NA 1.35 0.44 1.06 0.668 9.2 1.1 8.65 ± ± ± ± ± ±

SC55 2.02 ± 0.73 ± 0.45 ± 0.45 ± 1.15 ± 0.96 ± 73 ± 14 75.85 0.81 0.56 0.35 0.33 0.81 0.59 SC55 2.02 ± 0.73 ± 0.45 ± 0.45 ± 1.15 ± 0.96 ± 73 ± 14 75.85 SC55 0.81 0.56 0.35 0.33 0.81 0.59

SC55 2.02 ± 0.73 ± 0.45 ± 0.45 ± 1.15 ± 0.96 ± 73 ± 14 75.85 SC55 2.02 ± 0.73 ± 0.45 ± 0.45 ± 1.15 ± 0.96 ± 73 ± 14 75.85 0.812.02 0.810.56 0.730.35 0.560.33 0.450.81 0.350.59 0.45 0.33 1.15 0.81 0.96 0.59 73 14 75.85 0.81 ± 0.56 0.35± 0.33 0.81± 0.59 ± ± ± ±

SC56 0.051 ± 0.011 ± 0.046 ± 0.13 ± 0.11 ± 0.0698 ± 94 ± 10 1344.71 0.008 0.003 0.009 0.015 0.049 0.045 SC56 0.051 ± 0.011 ± 0.046 ± 0.13 ± 0.11 ± 0.0698 ± 94 ± 10 1344.71

0.0510.008 0.0080.003 0.0110.009 0.0030.015 0.0460.049 0.0090.045 0.13 0.015 0.11 0.049 0.0698 0.045 94 10 1344.71 ± ± ± ± ± ± ± SC56 0.051 ± 0.011 ± 0.046 ± 0.13 ± 0.11 ± 0.0698 ± 94 ± 10 1344.71 SC56 0.0510.008 ± 0.0110.003 ± 0.0460.009 ± 0.130.015 ± 0.110.049 ± 0.06980.045 ± 94 ± 10 1344.71 0.008 0.003 0.009 0.015 0.049 0.045

3. Material and Methods

3. MaterialAs summarizedand Methods in Table4, SC28 and each of the analogues tested exhibited antiviral eﬀects in the 3.1. General Information single-round infection assay utilizing viruses pseudotyped with HIV-1 Env from either the B41, HxBc2, DMEM, FBS, penicillin, streptomycin and L-glutamine, G418, puromycin, Multiskan™ GO 3.3.1. Material General andInformation Methods JR-CSF,3.Microplate Material JR-FL,andSpectrophotometer, Methods or YU-2 FEI isolates. Talos Arctica The electron analogues microscope, exhibited FEI Ceta 16M a CMOS range camera of potencies, largely in correlation (ThermoDMEM, Scientific, FBS, penicillin,Waltham, streptomycinMA, USA); BL21-Codon and L-glutamine, Plus (DE3)-RIL G418, puromycin, Competent Multiskan™ Cells (Agilent GO with3.1.Microplate General the InformationSpectrophotometer, docking and theFEI Talos SPR Arctica results. electron However, microscope, FEI we Ceta found 16M CMOS that cameraSC56 despite having a much higher 3.1.Technologies, General Information Wilmington, DE, USA); Talon cobalt resin affinity column (Clonetech Laboratories, aMountain(ThermoﬃnityDMEM, Scientific, View, for FBS, the CA, penicillin,Waltham, B41USA); SOSIP.664calcium streptomycinMA, phosphate,USA); gp140BL21-Codon and 5X L-glutamine, luciferase trimer Plus lysis (DE3)-RIL thanG418, buffer, puromycin,SC28 Competentluciferase, had Multiskan™ assay Cells a median substrate, (Agilent GO IC50 only two-fold over that of DMEM, FBS, penicillin, streptomycin and L-glutamine, G418, puromycin, Multiskan™ GO MicroplateGloMaxTechnologies, 96 microplateSpectrophotometer, Wilmington, luminomete DE, FEI USA);r Talos(Promega, Talon Arctica cobalt Madison, electron resin WI, microscope,affinity USA); column mouse FEI anti-p24Ceta(Clonetech 16M (ab9071, CMOS Laboratories, Abcam,camera Microplate Spectrophotometer, FEI Talos Arctica electron microscope, FEI Ceta 16M CMOS camera (ThermoCambridge,Mountain Scientific, View, MA, CA, USA); Waltham, USA); Triton calcium MA, X-100, USA);phosphate, O-phenylened BL21-Codon 5X luciferaseiamine, Plus phosphate-citratelysis(DE3)-RIL buffer, Competentluciferase buffer assay Cells with substrate, (Agilentsodium (Thermo Scientific, Waltham, MA, USA); BL21-Codon Plus (DE3)-RIL Competent Cells (Agilent Technologies,perborate,GloMax 96 DMSOmicroplate Wilmington, (Sigma-Aldrich, luminomete DE, USA);r (Promega,St. TalonLouis, cobalt Madison,MO, USA);resin WI, affinity 96-well USA); column mouseluminometer-compatible anti-p24(Clonetech (ab9071, Laboratories, Abcam, tissue Technologies, Wilmington, DE, USA); Talon cobalt resin affinity column (Clonetech Laboratories, MountaincultureCambridge, plates View, MA, (Greiner CA, USA); USA); Bio-one,Triton calcium X-100, Monroe, phosphate, O-phenylened NC, 5XUSA); luciferaseiamine, Cell Countinglysisphosphate-citrate buffer, Kit-8 luciferase cell buffer proliferation assay with substrate, sodium and Mountain View, CA, USA); calcium phosphate, 5X luciferase lysis buffer, luciferase assay substrate, GloMaxcytotoxicityperborate, 96 microplateDMSO assay (Dojindo(Sigma-Aldrich, luminomete Molecularr (Promega,St. Louis,Technologies, Madison,MO, USA); Rockville, WI, 96-well USA); MD, mouseluminometer-compatible USA); anti-p24 96-well (ab9071, tissue Abcam,culture tissue GloMax 96 microplate luminometer (Promega, Madison, WI, USA); mouse anti-p24 (ab9071, Abcam, Cambridge,platesculture (Olympus plates MA, (Greiner USA);Plastics, TritonBio-one, San X-100,Diego, Monroe, O-phenylenedCA, NC,USA) USA);; ProteOniamine, Cell XPR36Countingphosphate-citrate SPR Kit-8 Protein cell buffer Interactionproliferation with sodium Array and Cambridge, MA, USA); Triton X-100, O-phenylenediamine, phosphate-citrate buffer with sodium perborate,System,cytotoxicity GLH DMSO assay sensorchip, (Dojindo(Sigma-Aldrich, (Bio-RadMolecular St. Laboratories, Louis,Technologies, MO, USA);Hercules, Rockville, 96-well CA, MD, luminometer-compatibleUSA); USA); MicroCal 96-well tissueVP-Capillary culture tissue perborate, DMSO (Sigma-Aldrich, St. Louis, MO, USA); 96-well luminometer-compatible tissue culturedifferentialplates (Olympusplates scanning (Greiner Plastics, calorimeter,Bio-one, San Diego, Monroe, AutomatedCA, NC,USA) USA);; ProteOnOrigin Cell 7.0XPR36Counting software SPR Kit-8 Protein (Malverncell Interactionproliferation Panalytical, Array and cytotoxicitycultureSystem, platesGLH assay (Greinersensorchip, (Dojindo Bio-one, (Bio-RadMolecular Monroe, Laboratories, Technologies, NC, USA); Hercules, Rockville, Cell Counting CA, MD, USA); USA);Kit-8 MicroCal 96-wellcell proliferation tissueVP-Capillary culture and

platescytotoxicitydifferential (Olympus assayscanning Plastics, (Dojindo calorimeter, San Molecular Diego, Automated CA,Technologies, USA); ProteOnOrigin Rockville, XPR367.0 MD,software SPR USA); Protein (Malvern96-well Interaction tissue Panalytical, culture Array plates (Olympus Plastics, San Diego, CA, USA); ProteOn XPR36 SPR Protein Interaction Array System, GLH sensorchip, (Bio-Rad Laboratories, Hercules, CA, USA); MicroCal VP-Capillary differentialSystem, GLH scanning sensorchip, calorimeter, (Bio-Rad Laboratories,Automated OriginHercules, 7.0 CA, software USA); MicroCal(Malvern VP-CapillaryPanalytical, differential scanning calorimeter, Automated Origin 7.0 software (Malvern Panalytical,

Molecules 2019, 24, 1581 9 of 30

SC28. We have previously demonstrated that the potency of our entry inhibitors has a direct correlation with the kinetic oﬀ-rate parameter [20]. Comparison of the kinetics of SC28 and SC56 shows that the oﬀ-rates for their interaction with the B41 SOSIP.664 gp140 trimer are almost identical, providing a rationale for their similar potencies, and further corroborating our previous study.

3. Material and Methods

3.1. General Information DMEM, FBS, penicillin, streptomycin and L-glutamine, G418, puromycin, Multiskan™ GO Microplate Spectrophotometer, FEI Talos Arctica electron microscope, FEI Ceta 16M CMOS camera (Thermo Scientific, Waltham, MA, USA); BL21-Codon Plus (DE3)-RIL Competent Cells (Agilent Technologies, Wilmington, DE, USA); Talon cobalt resin affinity column (Clonetech Laboratories, Mountain View, CA, USA); calcium phosphate, 5X luciferase lysis buffer, luciferase assay substrate, GloMax 96 microplate luminometer (Promega, Madison, WI, USA); mouse anti-p24 (ab9071, Abcam, Cambridge, MA, USA); Triton X-100, O-phenylenediamine, phosphate-citrate buffer with sodium perborate, DMSO (Sigma-Aldrich, St. Louis, MO, USA); 96-well luminometer-compatible tissue culture plates (Greiner Bio-one, Monroe, NC, USA); Cell Counting Kit-8 cell proliferation and cytotoxicity assay (Dojindo Molecular Technologies, Rockville, MD, USA); 96-well tissue culture plates (Olympus Plastics,Molecules 2018 San, 23 Diego,, x CA, USA); ProteOn XPR36 SPR Protein Interaction Array System, GLH sensorchip,10 of 30 (Bio-Rad Laboratories, Hercules, CA, USA); MicroCal VP-Capillary differential scanning calorimeter, AutomatedWestborough, Origin MA, 7.0 USA); software Protein (Malvern Preparation Panalytical, Wiza Westborough,rd implemented MA, with USA); Maestro Protein (Schrödinger Preparation WizardMaestro implemented Version 11.5.011, with MaestroNew York, (Schrödinger NY, USA, MaestroMM share Version Version 11.5.011, 4.1.011, New New York, York, NY, NY, USA, USA, mM shareRelease Version 2018-1, 4.1.011, Platform New Darwin-x86_64). York, NY, USA, SC11 Release was synthesized 2018-1, Platform as outlined Darwin-x86_64). in Tuyishime SC11et al. [21].was synthesizedSC15, SC28, asand outlined SC45 were in Tuyishime synthesized et al. as [ 21outlined]. SC15 in, SC28 Tuyishime, and SC45 et al.were [16]. synthesized as outlined in Tuyishime et al. [16]. 3.2. Chemistry 3.2. Chemistry 3.2.1. Synthesis of SC49 3.2.1. Synthesis of SC49 General procedure for the preparation of 2 General Procedure for the Preparation of 2

Vinylmagnesium bromide 1a (1(1 M,M, 543.04543.04 mL)mL) waswas cooledcooled belowbelow −60 °CC with vigorous stirring − ◦ under N2.. A A solution solution of of 1 1 (30 (30 g, g, 136 136 mmol) mMol) in in THF THF ( (100100 mL) was added dropwise slowly that the temperature was keptkept belowbelow −60 °C.C. The reaction mixture waswas warmedwarmed toto −40 to −50 °CC and stirred − ◦ − − ◦ for an additionaladditional 11 h.h. TLC (petroleum(petroleum ether:ether: ethylethyl acetateacetate == 2:1, Rf = 0.47)0.47) show show that the reaction was complete. Saturated aqueous NH 4ClCl (200 (200 mL) mL) was was added slowly. The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 200 mL mL).). The organic extracts were washed with brine 2 4 × 2 2 (200 mL),mL), drieddried overover NaNa2SO4,, filtered, ﬁltered, and concentrated. To thethe residueresidue CHCH2Cl2 (100 mL) was added 2 2 and the solid formed was collected by ﬁltrationfiltration andand washedwashed withwith CHCH2Cl2 (50(50 mL) mL) to to give give 2 (8(8 g, g, 27% 11 3): yield) asas aa brownbrown solid.solid. H-NMRH-NMR (400(400 MHz MHz CDCl CDCl3): δ δ8.61–8.91 8.61–8.91 (m, (m, 1 1 H) H) 7.93 7.93 (d, (d,J =J =1.6 1.6 Hz, Hz, 1 H)1 H) 7.43 7.43 (t, J(t,= J 2.4= 2.4 Hz, Hz, 1 H)1 H) 6.78 6.78 (t, (t,J = J 2.4= 2.4 Hz, Hz, 1 H).1 H). General Procedure for the Preparation of 3

A mixture of 2 (4 g, 18.6 mmol) and CuCN (3.3 g, 37.2 mmol) in DMF (30 mL) was stirred at 150 °C for 1 h, after which TLC (petroleum ether: ethyl acetate = 2: 1, Rf = 0.51) showed reaction completion. The mixture was diluted with EtOAc (100 mL), washed with water (100 mL), brine (100 mL), and concentrated. The residue was purified by column chromatography on silica gel and eluted with petroleum ether: ethyl acetate = 4: 1 to give 3 (900 mg, 30% yield) as a yellow solid. 1H-NMR: ET6983-5-P1A (400 MHz DMSO-d6): δ 12.75 (br. s., 1 H), 8.27 (d, J=1.2 Hz, 1 H), 7.86 (d, J = 3.2 Hz, 1 H), 6.83 (d, J = 2.8 Hz, 1 H). General Procedure for the Preparation of 4

A mixture of 3 (800 mg, 4.96 mmol) in MeOH (10 mL) and conc. HCl (10 mL) was stirred at 90 °C for 16 h. TLC (petroleum ether: ethyl acetate = 2:1, Rf = 0.01) showed that the reaction was complete. The organic solvent was evaporated, and the precipitate was filtered off and dried to give

Molecules 2018,, 23,, xx 10 of 30

Westborough, MA, USA); Protein Preparation Wizard implemented with Maestro (Schrödinger Maestro Version 11.5.011, New York, NY, USA, MM share Version 4.1.011, New York, NY, USA, Release 2018-1, Platform Darwin-x86_64). SC11 was synthesized as outlined in Tuyishime et al. [21]. SC15, SC28, and SC45 were synthesized as outlined in Tuyishime et al. [16].

3.2. Chemistry

3.2.1. Synthesis of SC49

General procedure for the preparation of 2

Vinylmagnesium bromide 1a (1 M, 543.04 mL) was cooled below −60 °C with vigorous stirring under N2. A solution of 1 (30 g, 136 mmol) in THF (100 mL) was added dropwise slowly that the temperature was kept below −60 °C. The reaction mixture was warmed to −40 to −50 °C and stirred for an additional 1 h. TLC (petroleum ether: ethyl acetate = 2:1, Rf = 0.47) show that the reaction was complete. Saturated aqueous NH4Cl (200 mL) was added slowly. The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 200 mL). The organic extracts were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated. To the residue CH2Cl2 (100 mL) was added and the solid formed was collected by filtration and washed with CH2Cl2 (50 mL) to give 2 (8 g, 27% 1 yield) as a brown solid. 1H-NMR (400 MHz CDCl3): δ 8.61–8.91 (m, 1 H) 7.93 (d, J = 1.6 Hz, 1 H) 7.43 Molecules 2019, 24, 1581 10 of 30 (t, J = 2.4 Hz, 1 H) 6.78 (t, J = 2.4 Hz, 1 H). General Procedure for the Preparation of 3 General Procedure for the Preparation of 3

A mixture of of 22 (4(4 g, g, 18.6 18.6 mmol) mMol) and and CuCN CuCN (3.3 (3.3 g, g,37.2 37.2 mmol) mMol) in DMF in DMF (30 (30mL) mL) was was stirred stirred at 150 at °C for 1 h, after which TLC (petroleum ether: ethyl acetate = 2: 1, Rf = 0.51) showed reaction 150°C for◦C for1 h, 1 after h, after which which TLC TLC (petroleum (petroleum ether: ether: et ethylhyl acetate acetate = =2:2: 1, 1, Rf Rf == 0.51)0.51) showed reaction completion. The The mixture mixture was was diluted diluted with with EtOAc EtOAc (100 (100 mL), mL), washed washed with with water water (100 (100mL), mL), brine brine (100 (100mL), mL),and concentrated. and concentrated. The residue The residue was purified was puriﬁed by column by column chromatography chromatography on silica on gel silica and gel eluted and 1 witheluted petroleum with petroleum ether: ethyl ether: acetate ethyl = acetate 4: 1 to =give4: 13 to(900 give mg,3 30%(900 yield) mg, 30% as a yield) yellow as solid. a yellow 1H-NMR: solid. 1ET6983-5-P1A (400 MHz DMSO-d6): δ 12.75 (br. s., 1 H), 8.27 (d, J=1.2 Hz, 1 H), 7.86 (d, J = 3.2 Hz, 1 H), ET6983-5-P1AH-NMR: ET6983-5-P1A (400 MHz DMSO- (400 MHzd δ DMSO- 12.75 (br.d6): s.,δ 112.75 H), 8.27 (br. (d, s., J 1=1.2 H), Hz, 8.27 1 (d,H), J7.86=1.2 (d, Hz, J = 13.2 H), Hz, 7.86 1 H), (d, 6.83J = 3.2 (d, Hz, J = 2.8 1 H), Hz, 6.83 1 H). (d, J = 2.8 Hz, 1 H). General Procedure for the Preparation of 4 General Procedure for the Preparation of 4

Molecules 2018, 23, x 11 of 30 A mixture ofof 33 (800(800mg, mg, 4.96 4.96 mMol) mmol) in in MeOH MeOH (10 (10 mL) mL) and and conc. conc. HCl HCl (10 (10 mL) mL) was was stirred stirred at 90at ◦90C °CforMolecules 16for h. 16 2018 TLC h., 23TLC (petroleum, x (petroleum ether: ether: ethyl acetateethyl acetate= 2:1, Rf= 2:1,= 0.01) Rf showed= 0.01) showed that the reactionthat the wasreaction complete.11 wasof 30 1 Thecomplete.4 (500 organic mg, The56% solvent organic yield) was as solvent a evaporated, brown was solid. evaporated, and H-NMR the precipitate and (400 th MHze precipitate was MeOD): ﬁltered wasδ o8.39ﬀ filteredand (d, driedJ = off4.0 toandHz, give dried1 H),4 (500 8.23to give mg, (d, 56%J4 = (500 2.8 yield) Hz,mg, 1 as56% H), a brown yield)7.15 (d, as solid. J a= brown 3.21H-NMR Hz, solid. 1 H). (400 1H-NMR MHz MeOD):(400 MHzδ 8.39 MeOD): (d, J =δ 8.394.0 Hz, (d, 1J = H), 4.0 8.23 Hz, (d, 1 H),J = 8.232.8 Hz,(d, 1GeneralJ = H), 2.8 7.15 Hz, Procedure (d, 1 H),J = 7.153.2 for Hz, (d, the J 1 = H). Preparation3.2 Hz, 1 H). of 5

General Procedure for the Preparation of 5 General Procedure for the Preparation of 5

A solution of 4 (450 mg, 2.50 mmol), DIEA (967 mg, 7.49 mmol), and HATU (1.04 g, 2.75 mmol) in THF (10 mL) was stirred at 25 °C for 0.5 h. Then methylamine (675 mg, 9.99 mmol, HCl salt) was A solutionA solution of 4 (450 of 4 mg, (450 2.50 mg, mMol), 2.50 mmol), DIEA (967DIEA mg, (967 7.49 mg, mMol), 7.49 mmol), and HATU and HATU (1.04 g, (1.04 2.75 mMol)g, 2.75 inmmol) THF added,in THF and(10 mL)the mixturewas stirred was at stirred 25 °C atfor 25 0.5 °C h. for Then 16 h.methylamine TLC (dichloromethane: (675 mg, 9.99 methanol mmol, HCl = 20: salt) 1, Rfwas = (10 mL) was stirred at 25 ◦C for 0.5 h. Then methylamine (675 mg, 9.99 mMol, HCl salt) was added, 0.65)added, showed and the that mixture the reaction was stirred was complete. at 25 °C forThe 16 mixture h. TLC was (dichloromethane: diluted with EtOAc methanol (20 mL), = 20: washed 1, Rf = and the mixture was stirred at 25 ◦C for 16 h. TLC (dichloromethane: methanol = 20: 1, Rf = 0.65) with water (20 mL), brine (20 mL) and dried over Na2SO4, and concentrated. The residue was purified showed0.65) showed that the that reaction the reaction was complete. was complete. The mixture The mixture was diluted was diluted with EtOAcwith EtOAc (20 mL), (20 washedmL), washed with bywith column water chromatography(20 mL), brine (20 on mL) silica and gel dried and over elut Naed 2withSO4, andpetroleum concentrated. ether: EtOAc The residue = 5:1 to was give purified 5 (400 water (20 mL), brine (20 mL) and dried over Na2SO4, and concentrated. The residue was puriﬁed by mg, 83% yield) as a white solid. 1H-NMR: ET6983-9-P1A (400 MHz, CDCl3): δ 10.50 (br. s., 1 H), 8.01 columnby column chromatography chromatography on silicaon silica gel andgel and eluted elut withed with petroleum petroleum ether: ether: EtOAc EtOAc= 5:1 = to5:1 give to give5 (400 5 (400 mg, (d, J = 1.2 Hz, 1 H), 7.92 (br. s., 1 H),1 7.47 (br. s., 1 H), 7.26 (s, 1 H), 6.69 (br. s., 2 H), 3.07 (d, J = 4.8 Hz, 83%mg, 83% yield) yield) as a whiteas a white solid. solid.1H-NMR: H-NMR: ET6983-9-P1A ET6983-9-P1A (400 (400 MHz, MHz, CDCl CDCl): δ3):10.50 δ 10.50 (br. (br. s., 1s., H), 1 H), 8.01 8.01 (d, 1 H). 3 J(d,= 1.2J = 1.2 Hz, Hz, 1 H), 1 H), 7.92 7.92 (br. (br. s., 1 s., H), 1 7.47H), 7.47 (br. s.,(br. 1 s., H), 1 7.26H), 7.26 (s, 1 H),(s, 1 6.69 H), (br.6.69 s., (br. 2 H),s., 2 3.07 H), (d,3.07J = (d,4.8 J = Hz, 4.8 1 Hz, H). General1 H). Procedure for the Preparation of 6 General Procedure for the Preparation of 6 General Procedure for the Preparation of 6

Compound 5 (370 mg, 1.92 mmol) was added to a mixture of AlCl3 (1.53 g, 11.5 mmol) and 1- ethyl-3-methylimidazol-3-iumCompound 5 (370 mg, 1.92 chloride mmol) (566 was mg, added 3.83 tommol). a mixture Then, of 5a AlCl (5233 mg,(1.53 3.83 g, 11.5mmol) mmol) was addedand 1- slowlyethyl-3-methylimidazol-3-ium to the solution, and the chloride mixture (566was mg, stirred 3.83 at mmol). 25 °C Then,for 15 5a h. (523 TLC mg, (ethyl 3.83 acetate: mmol) waspetroleum added etherslowly = 2:1,to the Rf solution,= 0.01) showed and the that mixture the conversion was stirred was at more 25 °C than for 50%15 h. and TLC LCMS (ethyl showed acetate: the petroleum desired massether of= 2:1, the Rf product. = 0.01) showedWater was that added the conversion (20 mL) slowwas lymore to thethan mixture 50% and at LCMS0 °C. The showed precipitate the desired was filteredmass of off, the and product. dried Waterto give was 6 (200 added mg, crude)(20 mL) as slow a yellowly to solid.the mixture at 0 °C. The precipitate was Generalfiltered off,Procedure and dried for tothe give Preparation 6 (200 mg, of crude) 7 as a yellow solid. General Procedure for the Preparation of 7

To a solution of 6 (200 mg, 754 mmol and DIEA (292 mg, 2.26 mmol) in DMF (5 mL) was added HATUTo (287 a solution mg, 754 of mmol). 6 (200 mg, After 754 stirred mmol at and 25 °CDIEA for (29230 min, mg, 6a 2.26 (150 mmol) mg, 754in DMF mmol) (5 mL)was addedwas added and theHATU mixture (287 wasmg, 754stirred mmol). at 25 After °C for stirred 16 h. TLCat 25 (petroleum°C for 30 min, ether: 6a (150ethyl mg, acetate 754 mmol)= 0:1, Rf was = 0.3) added showed and thatthe mixturethe reaction was wasstirred complete at 25 °C and for LCMS 16 h. TLC showed (petroleum the desired ether: mass ethyl of acetatethe product. = 0:1, TheRf = mixture0.3) showed was dilutedthat the withreaction EtOAc was (20complete mL), washed and LCMS with showed water the(20 desiredmL), brine mass (20 of mL),the product. dried over The Namixture2SO4, andwas concentrateddiluted with toEtOAc give 7(20 (300 mL), mg, washed crude) aswith a yellow water solid. (20 mL), brine (20 mL), dried over Na2SO4, and Generalconcentrated Procedure to give for 7 (300the Preparation mg, crude) ofas 8a yellow solid. General Procedure for the Preparation of 8

Molecules 2018, 23, x 11 of 30

4 (500 mg, 56% yield) as a brown solid. 1H-NMR (400 MHz MeOD): δ 8.39 (d, J = 4.0 Hz, 1 H), 8.23 (d, J = 2.8 Hz, 1 H), 7.15 (d, J = 3.2 Hz, 1 H). General Procedure for the Preparation of 5

A solution of 4 (450 mg, 2.50 mmol), DIEA (967 mg, 7.49 mmol), and HATU (1.04 g, 2.75 mmol) in THF (10 mL) was stirred at 25 °C for 0.5 h. Then methylamine (675 mg, 9.99 mmol, HCl salt) was added, and the mixture was stirred at 25 °C for 16 h. TLC (dichloromethane: methanol = 20: 1, Rf = 0.65) showed that the reaction was complete. The mixture was diluted with EtOAc (20 mL), washed with water (20 mL), brine (20 mL) and dried over Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel and eluted with petroleum ether: EtOAc = 5:1 to give 5 (400 mg, 83% yield) as a white solid. 1H-NMR: ET6983-9-P1A (400 MHz, CDCl3): δ 10.50 (br. s., 1 H), 8.01 (d, J = 1.2 Hz, 1 H), 7.92 (br. s., 1 H), 7.47 (br. s., 1 H), 7.26 (s, 1 H), 6.69 (br. s., 2 H), 3.07 (d, J = 4.8 Hz, 1 H). General Procedure for the Preparation of 6

Molecules 2019, 24, 1581 11 of 30

Compound 5 (370 mg, 1.92 mmol) was added to a mixture of AlCl3 (1.53 g, 11.5 mmol) and 1- Compound 5 (370 mg, 1.92 mMol) was added to a mixture of AlCl (1.53 g, 11.5 mMol) and ethyl-3-methylimidazol-3-ium chloride (566 mg, 3.83 mmol). Then, 5a (523 mg,3 3.83 mmol) was added 1-ethyl-3-methylimidazol-3-ium chloride (566 mg, 3.83 mMol). Then, 5a (523 mg, 3.83 mMol) was slowly to the solution, and the mixture was stirred at 25 °C for 15 h. TLC (ethyl acetate: petroleum added slowly to the solution, and the mixture was stirred at 25 C for 15 h. TLC (ethyl acetate: ether = 2:1, Rf = 0.01) showed that the conversion was more than 50%◦ and LCMS showed the desired petroleum ether = 2:1, Rf = 0.01) showed that the conversion was more than 50% and LCMS showed the mass of the product. Water was added (20 mL) slowly to the mixture at 0 °C. The precipitate was desired mass of the product. Water was added (20 mL) slowly to the mixture at 0 C. The precipitate filtered off, and dried to give 6 (200 mg, crude) as a yellow solid. ◦ was ﬁltered oﬀ, and dried to give 6 (200 mg, crude) as a yellow solid. General Procedure for the Preparation of 7 General Procedure for the Preparation of 7

To aa solutionsolution ofof 66 (200(200 mg,mg, 754754 mMolmmol and DIEA (292(292 mg,mg, 2.262.26 mMol)mmol) in DMF (5 mL) was added HATU (287(287 mg,mg, 754 754 mMol). mmol). After After stirred stirred at at 25 25◦C °C for for 30 30 min, min, 6a 6a (150 (150 mg, mg, 754 754 mMol) mmol) was was added added and and the mixturethe mixture was was stirred stirred at 25 at◦ C25 for °C 16 for h. 16 TLC h. TLC (petroleum (petroleum ether: ether: ethyl ethyl acetate acetate= 0:1, = Rf0:1,= Rf0.3) = showed0.3) showed that thethat reaction the reaction was completewas complete and LCMS and LCMS showed showed the desired the desired mass of mass the product. of the product. The mixture The mixture was diluted was withdiluted EtOAc with (20 EtOAc mL), washed(20 mL), with washed water with (20 mL),water brine (20 (20mL), mL), brine dried (20 over mL), Na dried2SO4 ,over and concentratedNa2SO4, and toconcentrated give 7 (300 to mg, give crude) 7 (300 as mg, a yellow crude) solid. as a yellow solid. Molecules 2018, 23, x 12 of 30 Molecules 2018, 23, x 12 of 30 General Procedure for the Preparation ofof 88

A solution of 7 (300 mg, 674 mmol) in TFA (4 mL) was stirred at 25 °C for 16 h. LCMS showed A solution of 7 (300(300 mg,mg, 674674 mMol)mmol) in TFA (4(4 mL)mL) waswas stirredstirred atat 2525 ◦°CC forfor 1616 h.h. LCMSLCMS showedshowed that the reaction was complete. The mixture was concentrated to give the crude product 8 TFA salt that the reactionreaction waswas complete.complete. The mixture was co concentratedncentrated to give the crude product 8 TFA salt (200 mg, crude) as a yellow solid. (200(200 mg,mg, crude)crude) asas aa yellowyellow solid.solid. General Procedure for the Preparation of SC49 General Procedure forfor thethe PreparationPreparation ofof SC49SC49

To a solution of 8 (200 mg, 579 mmol) and DIEA (150 mg, 1.16 mmol) in DCM (5 mL) was added To aa solutionsolution ofof 88 (200(200 mg,mg, 579579 mMol)mmol) andand DIEADIEA (150(150 mg,mg, 1.161.16 mMol)mmol) in DCM (5 mL) was added 8a (163 mg, 1.16 mmol). Then the mixture was stirred at 25 °C for 16 h. LCMS showed that the desired 8a (163 mg, mg, 1.16 1.16 mmol). mMol). Then Then the the mixture mixture was was stirred stirred at 25 at °C 25 for◦C 16 for h. LCMS 16 h. LCMSshowed showed that the thatdesired the product was produced. The mixture was concentrated. The residue was purified by neutral prep- desiredproduct productwas produced. was produced. The mixture The mixture was concentrat was concentrated.ed. The residue The residue was purified was puriﬁed by neutral by neutral prep- HPLC to give SC49 (22 mg, 8% yield) as a light yellow solid. 1H-NMR: ET6983-14-P1B (400 MHz prep-HPLCHPLC to give to giveSC49SC49 (22 (22mg, mg, 8% 8%yield) yield) as asa light a light yellow yellow solid. solid. 1H-NMR:1H-NMR: ET6983-14-P1B ET6983-14-P1B (400 (400 MHz CDCl3): δ 11.25 (br. s., 1 H), 9.26 (s, 1 H), 8.20 (d, J = 2.4 Hz, 1 H), 7.92 (d, J = 4.4 Hz, 1 H), 7.52 (br. s., 1 CDCl33): δδ 11.2511.25 (br. s., 1 H), 9.26 (s, 1 H), 8.20 (d,(d, JJ = 2.4 Hz, 1 H), 7.92 (d, J = 4.44.4 Hz, Hz, 1 H), 7.52 (br. s., 1 H), 7.36–7.48 (m, 4 H), 4.34 (d, J = 12.0 Hz, 1 H), 3.46–3.90 (m, 3 H), 3.08 (d, J = 4.8 Hz, 3 H), 2.61 (br. H), 7.36–7.487.36–7.48 (m,(m, 44 H),H), 4.344.34 (d, (d,J J= =12.0 12.0 Hz, Hz, 1 1 H), H), 3.46–3.90 3.46–3.90 (m, (m, 3 3 H), H), 3.08 3.08 (d, (d,J = J 4.8= 4.8 Hz, Hz, 3 H),3 H), 2.61 2.61 (br. (br. s., s., 1 H), 1.75–2.00 (m, 2 H). 1s., H), 1 H), 1.75–2.00 1.75–2.00 (m, (m, 2 H). 2 H).

3.2.2. The Synthesis of SC50 3.2.2. The Synthesis of SC50 General Procedure for the Preparation of 9 General Procedure for the Preparation of 9

To a mixture of 1-ethyl-3-methylimidazol-3-ium chloride (4.1 g, 27.9 mmol) was added AlCl3 To a mixture of 1-ethyl-3-methylimidazol-3-ium chloride (4.1 g, 27.9 mmol) was added AlCl3 (11.2 g, 83.7 mmol) in portions at 0 °C and stirred for 30 min, then 2 (3.0 g, 13.9 mmol) was added in (11.2 g, 83.7 mmol) in portions at 0 °C and stirred for 30 min, then 2 (3.0 g, 13.9 mmol) was added in portions at 0 °C under N2. The mixture was stirred at 0 °C for 0.5 hour, then ethyl 2-chloro-2-oxo- portions at 0 °C under N2. The mixture was stirred at 0 °C for 0.5 hour, then ethyl 2-chloro-2-oxoacetate (3.8 g, 27.9 mmol) was added dropwise, then the mixture was stirred at 25 °C for 2 h. TLC acetate (3.8 g, 27.9 mmol) was added dropwise, then the mixture was stirred at 25 °C for 2 h. TLC (petroleum ether: ethyl acetate = 3: 1, Rf = 0.2) showed the reaction was occurred and one new spot (petroleum ether: ethyl acetate = 3: 1, Rf = 0.2) showed the reaction was occurred and one new spot was generated. The mixture was poured onto water (50 mL) and stirred for 10 min. The aqueous was generated. The mixture was poured onto water (50 mL) and stirred for 10 min. The aqueous phase was extracted with ethyl acetate (50 mL × 3 mL). The combined organic phase was washed phase was extracted with ethyl acetate (50 mL × 3 mL). The combined organic phase was washed with brine (30 mL × 2mL), dried over Na2SO4, filtered and concentrated in vacuum to give 9 (3 g, 68% with brine (30 mL × 2mL), dried over Na2SO4, filtered and concentrated in vacuum to give 9 (3 g, 68% yield) as a brown solid. 1H-NMR: ET6822-13-P1A (400 MHz CDCl3): δ 8.56 (s, 1 H), 8.40 (s, 1 H), 7.88 yield) as a brown solid. 1H-NMR: ET6822-13-P1A (400 MHz CDCl3): δ 8.56 (s, 1 H), 8.40 (s, 1 H), 7.88 (d, J = 2.4 Hz, 1 H), 4.24–4.35 (m, 2 H), 1.23–1.36 (m, 3 H). (d, J = 2.4 Hz, 1 H), 4.24–4.35 (m, 2 H), 1.23–1.36 (m, 3 H). General Procedure for the Preparation of 10 General Procedure for the Preparation of 10

Molecules 2018, 23, x 12 of 30

A solution of 7 (300 mg, 674 mmol) in TFA (4 mL) was stirred at 25 °C for 16 h. LCMS showed that the reaction was complete. The mixture was concentrated to give the crude product 8 TFA salt (200 mg, crude) as a yellow solid.

General Procedure for the Preparation of SC49

To a solution of 8 (200 mg, 579 mmol) and DIEA (150 mg, 1.16 mmol) in DCM (5 mL) was added 8a (163 mg, 1.16 mmol). Then the mixture was stirred at 25 °C for 16 h. LCMS showed that the desired product was produced. The mixture was concentrated. The residue was purified by neutral prep- HPLC to give SC49 (22 mg, 8% yield) as a light yellow solid. 1H-NMR: ET6983-14-P1B (400 MHz CDCl3): δ 11.25 (br. s., 1 H), 9.26 (s, 1 H), 8.20 (d, J = 2.4 Hz, 1 H), 7.92 (d, J = 4.4 Hz, 1 H), 7.52 (br. s., 1 H), 7.36–7.48 (m, 4 H), 4.34 (d, J = 12.0 Hz, 1 H), 3.46–3.90 (m, 3 H), 3.08 (d, J = 4.8 Hz, 3 H), 2.61 (br. Molecules 2019, 24, 1581 12 of 30 s., 1 H), 1.75–2.00 (m, 2 H).

3.2.2. The Synthesis of SC50

General Procedure for the Preparation ofof 99

To aa mixturemixture ofof 1-ethyl-3-methylimidazol-3-ium1-ethyl-3-methylimidazol-3-ium chloride (4.1 g,g, 27.927.9 mMol)mmol) waswas addedadded AlClAlCl33 (11.2 g, 83.7 mMol)mmol) in portions at 0 ◦°CC and stirred for 30 min, then 2 (3.0 g, 13.9 mMol)mmol) was added in portions atat 00 ◦°CC underunder N N2.2. TheThe mixture mixture was was stirred stirred at at 0 ◦0C °C for for 0.5 0.5 h, thenhour, ethyl then 2-chloro-2-oxo-acetateethyl 2-chloro-2-oxo- (3.8acetate g, 27.9 (3.8 mMol) g, 27.9 was mmol) added was dropwise, added dropwise, then the mixturethen the was mixture stirred was at 25 stirred◦C for at 2 25 h. TLC°C for (petroleum 2 h. TLC ether:(petroleum ethyl acetateether: ethyl= 3: 1,acetate Rf = 0.2) = 3: showed 1, Rf = 0.2) the reactionshowedwas the occurredreaction was and oneoccurred new spot and wasone generated.new spot Thewas mixturegenerated. was The poured mixture onto was water po (50ured mL) onto and water stirred (50 for mL) 10 min. and Thestirred aqueous for 10 phase min. wasThe extractedaqueous with ethyl acetate (50 mL 3 mL). The combined organic phase was washed with brine (30 mL phase was extracted with ×ethyl acetate (50 mL × 3 mL). The combined organic phase was washed× 2mL),with brine dried (30 over mL Na× 2mL),2SO4, dried ﬁltered over and Na concentrated2SO4, filtered in and vacuum concentrated to give in9 (3 vacuum g, 68% to yield) give as9 (3 a browng, 68% 1 solid.yield) asH-NMR: a brown ET6822-13-P1A solid. 1H-NMR: (400 ET6822-13-P1A MHz CDCl3): (400δ 8.56 MHz (s, 1 CDCl H), 8.403): δ (s, 8.56 1 H), (s, 7.881 H), (d, 8.40J = (s,2.4 1 Hz, H), 17.88 H), 4.24–4.35(d, J = 2.4 (m,Hz, 21 H),H), 1.23–1.364.24–4.35 (m,(m, 32 H).H), 1.23–1.36 (m, 3 H). Molecules 2018, 23, x 13 of 30 Molecules 2018, 23, x 13 of 30 General Procedure for the Preparation ofof 1010

To a mixture of 9 (3 g, 9.5 mmol) in MeOH (30 mL) and H2O (10 mL) was added K2CO3 (2.6 g, 19 To a mixture of 9 (3(3 g, g, 9.5 9.5 mmol) mMol) in in MeOH MeOH (30 (30 mL) mL) and and H H2O2O (10 (10 mL) mL) was was added added K2 KCO2CO3 (2.63 (2.6 g, 19 g, mmol) in one portion at 25 °C under N2. The mixture was stirred at 25 °C for 5 h. TLC (petroleum 19mmol) mMol) in inone one portion portion at at25 25 °C◦ Cunder under N N2. 2The. The mixture mixture was was stirred stirred at at 25 25 °C◦C for for 5 5 h. h. TLC TLC (petroleum ether:ethyl acetate = 1: 1, Rf = 0.03) showed the reaction was completed. The mixture was concentrated ether:ethyl acetate == 1: 1, 1, R Rf == 0.03)0.03) showed showed the the reaction reaction was was comp completed.leted. The The mixture mixture was was concentrated concentrated in reduced pressure at 45 °C.f The residue was poured onto water (50 mL) and stirred for 10 min. The in reduced reduced pressure pressure at at 45 45 °C.C. The The residue residue was was pour poureded onto onto water water (50 (50mL) mL) and andstirred stirred for 10 for min. 10 min.The mixture was acidified with◦ HCl (1 M) to pH ~4, the aqueous phase was extracted with ethyl acetate Themixture mixture was acidified was acidiﬁed with withHCl (1 HCl M) (1to M)pH to~4, pH the ~4, aqueous the aqueous phase was phase extracted was extracted with ethyl with acetate ethyl (50 mL × 3mL). The combined organic phase was washed with brine (30 mL × 2mL), dried with (50acetate mL (50× 3mL). mL The3mL). combined The combined organic organic phase phasewas washed was washed with brine with brine(30 mL (30 × mL2mL),2mL), dried driedwith anhydrous Na2SO× 4, filtered and concentrated in vacuum to give 10 (2.0 g, 73% yield) as a× yellow solid. withanhydrous anhydrous Na2SO Na4, 2filteredSO4, ﬁltered and concentrated and concentrated in vacuum in vacuum to give to 10 give (2.010 g,(2.0 73% g, yield) 73% yield)as a yellow as a yellow solid. 1H-NMR: ET6822-14-P1A (400 MHz DMSO-d6): δ 13.35 (br. s., 1 H), 8.47–8.65 (m, 1 H), 8.12 (d, J = 2.4 1 1 solid.H-NMR:H-NMR: ET6822-14-P1A ET6822-14-P1A (400 MHz (400 DMSO- MHz DMSO-d6): δ 13.35d ): δ(br.13.35 s., 1 (br. H), s., 8.47–8.65 1 H), 8.47–8.65 (m, 1 H), (m, 8.12 1 H), (d, 8.12 J = 2.4 (d, Hz, 1 H). 6 Hz,J = 2.4 1 H). Hz, 1 H). General Procedure for the Preparation of 11 General Procedure for the Preparation ofof 1111

To a mixture of 10 (900 mg, 3.1 mmol) in DMF (30 mL) was added HATU (1.55 g, 4.08 mmol) To aa mixturemixture ofof10 10(900 (900 mg, mg, 3.1 3.1 mMol) mmol) in in DMF DMF (30 (30 mL) mL) was was added added HATU HATU (1.55 (1.55 g, 4.08 g, 4.08 mMol) mmol) and DIEAand DIEA (1.22 (1.22 g, 9.4 g, mMol) 9.4 mmol) in one in portionone portion at 25 atC 25 under °C under N . TheN2. mixtureThe mixture was stirredwas stirred at 25 atC 25 for °C 30 for min, 30 and DIEA (1.22 g, 9.4 mmol) in one portion at◦ 25 °C under2 N2. The mixture was stirred at◦ 25 °C for 30 min, then 6a (745 mg, 3.7 mmol) was added in portions and the mixture was stirred for 5 h. TLC min,then 6athen (745 6a mg, (745 3.7 mg, mMol) 3.7 mmol) was added was inadded portions in portions and the mixtureand the was mixture stirred was for stirred 5 h. TLC for (petroleum 5 h. TLC ether:ethyl(petroleum acetateether:ethyl= 1:1, acetate R = 0.6.)= 1:1, showed Rf = 0.6.) one showed main one spot main was spot generated. was generated. The mixture The mixture was poured was (petroleum ether:ethyl acetatef = 1:1, Rf = 0.6.) showed one main spot was generated. The mixture was poured onto water (50 mL) and stirred for 10 min. The aqueous phase was extracted with ethyl acetate poured onto water (50 mL) and stirred for 10 min. The aqueous phase was extracted with ethyl acetate (30 mL × 2). The combined organic phase was washed with brine (20 mL × 2), dried with anhydrous (30 mL × 2). The combined organic phase was washed with brine (20 mL × 2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100–200 mesh silica gel, petroleum ether:ethyl acetate = (column height: 250 mm, diameter: 100 mm, 100–200 mesh silica gel, petroleum ether:ethyl acetate = 1:1) to give 11 (2.0 g, 68% yield) as a yellow solid. 1:1) to give 11 (2.0 g, 68% yield) as a yellow solid. General Procedure for the Preparation of 12 General Procedure for the Preparation of 12

A mixture of 11 (2.0 g, 4.28 mmol) in HCl/EtOAc (20 mL) was stirred at 25 °C for 5 h. LCMS A mixture of 11 (2.0 g, 4.28 mmol) in HCl/EtOAc (20 mL) was stirred at 25 °C for 5 h. LCMS showed the reaction was completed, and the desired product was generated. The mixture was filtered showed the reaction was completed, and the desired product was generated. The mixture was filtered and washed with EtOAc, and concentrated in vacuum to give 12 (1.2 g, 76% yield) as a yellow solid. and washed with EtOAc, and concentrated in vacuum to give 12 (1.2 g, 76% yield) as a yellow solid. General Procedure for the Preparation of 13 General Procedure for the Preparation of 13

Molecules 2018, 23, x 13 of 30

To a mixture of 9 (3 g, 9.5 mmol) in MeOH (30 mL) and H2O (10 mL) was added K2CO3 (2.6 g, 19 mmol) in one portion at 25 °C under N2. The mixture was stirred at 25 °C for 5 h. TLC (petroleum ether:ethyl acetate = 1: 1, Rf = 0.03) showed the reaction was completed. The mixture was concentrated in reduced pressure at 45 °C. The residue was poured onto water (50 mL) and stirred for 10 min. The mixture was acidified with HCl (1 M) to pH ~4, the aqueous phase was extracted with ethyl acetate (50 mL × 3mL). The combined organic phase was washed with brine (30 mL × 2mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give 10 (2.0 g, 73% yield) as a yellow solid. 1H-NMR: ET6822-14-P1A (400 MHz DMSO-d6): δ 13.35 (br. s., 1 H), 8.47–8.65 (m, 1 H), 8.12 (d, J = 2.4 Hz, 1 H).

General Procedure for the Preparation of 11

To a mixture of 10 (900 mg, 3.1 mmol) in DMF (30 mL) was added HATU (1.55 g, 4.08 mmol) and DIEA (1.22 g, 9.4 mmol) in one portion at 25 °C under N2. The mixture was stirred at 25 °C for 30 Moleculesmin, then2019 6a, 24 (745, 1581 mg, 3.7 mmol) was added in portions and the mixture was stirred for 5 h.13 TLC of 30 (petroleum ether:ethyl acetate = 1:1, Rf = 0.6.) showed one main spot was generated. The mixture was poured onto water (50 mL) and stirred for 10 min. The aqueous phase was extracted with ethyl acetate onto(30 mL water × 2). (50 The mL) combined and stirred organic for 10 phase min. was The aqueouswashed with phase brine was (20 extracted mL × 2), with dried ethyl with acetate anhydrous (30 mL 2). The combined organic phase was washed with brine (20 mL 2), dried with anhydrous Na SO , ×Na2SO4, filtered and concentrated in vacuum. The residue was purified× by silica gel chromatography2 4 ﬁltered(column and height: concentrated 250 mm, indiameter: vacuum. 100 The mm, residue 100–200 was me puriﬁedsh silica by gel, silica petroleum gel chromatography ether:ethyl acetate (column = height:1:1) to give 250 11 mM, (2.0 diameter: g, 68% yield) 100 mM,as a yellow 100–200 solid. mesh silica gel, petroleum ether:ethyl acetate = 1:1) to give 11 (2.0 g, 68% yield) as a yellow solid. General Procedure for the Preparation of 12 General Procedure for the Preparation of 12

A mixture of 11 (2.0(2.0 g,g, 4.284.28 mMol)mmol) inin HClHCl/EtOAc/EtOAc (20 mL)mL) waswas stirredstirred atat 2525 ◦°CC forfor 55 h.h. LCMS showed the reaction was completed, and the desired product was generated. The mixture was filtered ﬁltered and washed with EtOAc, and concentrated in vacuum toto givegive 1212 (1.2(1.2 g,g, 76%76% yield)yield) asas aa yellowyellow solid.solid. Molecules 2018, 23, x 14 of 30 Molecules 2018, 23, x 14 of 30 General Procedure for the Preparation ofof 1313

To a mixture of 12 (1.5 g, 4.09 mmol) and benzoyl chloride (1.15 g, 8.17 mmol) in DCM (30 mL) To aa mixturemixture ofof 1212 (1.5(1.5 g,g, 4.094.09 mMol)mmol) andand benzoylbenzoyl chloridechloride (1.15 g, 8.17 mMol)mmol) in DCM (30 mL) was added TEA (413 mg, 4.09 mmol) in one portion at 25 °C under N2. The mixture was stirred at 25 was addedadded TEATEA (413(413 mg,mg, 4.094.09 mMol)mmol) inin oneone portionportion atat 2525 ◦°CC underunder NN22. The The mixture was stirred at 25 °C for 2 h. LC-MS showed the reaction was completed. The mixture was concentrated in reduced ◦°CC for 2 h.h. LC-MS showed the reactionreaction waswas completed.completed. The mixture was concentrated in reduced pressure at 45 °C. The residue was purified by prep-HPLC (acid conditions) to give 13 (600 mg, 31% pressure at 45 ◦°C.C. The residue was puriﬁedpurified by prep-HPLC (acid conditions) to give 13 (600 mg, 31% 1 6 yield) as a yellow solid. H-NMR11 (400 MHz DMSO-d ): δ 13.22 (br. s., 1 H), 9.04 (d, J = 4.8 Hz, 1 H), yield) as aa yellowyellow solid.solid. H-NMR (400 MHz DMSO- d6):): δδ 13.2213.22 (br. (br. s., s., 1 1 H), 9.04 (d, J = 4.84.8 Hz, Hz, 1 H), 8.80 (d, J = 2.4 Hz, 1 H) , 8.10 (d, J = 2.8 Hz, 1 H), 7.22–7.56 (m, 5 H), 4.01 (d, J = 12.4 Hz, 1 H), 3.67 (d, 8.80 (d, JJ == 2.4 Hz, 1 H),H) ,8.10 8.10 (d, (d,J J= = 2.82.8 Hz,Hz,1 1H), H), 7.22–7.56 7.22–7.56 (m, (m, 5 5 H), H), 4.01 4.01 (d, (d,J =J =12.4 12.4 Hz, Hz, 1 1 H), H), 3.67 3.67 (d, (d,J J =7.2 Hz, 1 H), 3.42 (dd, J = 18.4, 11.2 Hz, 2 H), 2.51–2.57 (m, 1 H), 1.88 (d, J = 7.6 Hz, 2 H). =J =7.27.2 Hz, Hz, 1 1 H), H), 3.42 3.42 (dd, (dd,J J= =18.4, 18.4,11.2 11.2Hz, Hz,2 2 H), H),2.51–2.57 2.51–2.57 (m, (m, 1 1 H), H), 1.88 1.88 (d, (d,J J= = 7.67.6 Hz,Hz, 22 H).H). General Procedure for the Preparation of SC50 General Procedure forfor thethe PreparationPreparation ofof SC50SC50

To a solution of 13 (100 mg, 0.212 mmol), 3-pyridylboronic acid (39 mg, 0.318 mmol) in dioxane To aa solutionsolution ofof 1313 (100(100 mg,mg, 0.2120.212 mMol),mmol), 3-pyridylboronic3-pyridylboronic acidacid (39(39 mg,mg, 0.3180.318 mMol)mmol) in dioxane (5 mL) and H2O (1 mL) was added Pd(dppf)Cl2 (15.5 mg, 21 mmol) and K2CO3 (87.9 mg, 637 mmol) (5(5 mL)mL) andand HH22O (1 mL) waswas addedadded Pd(dppf)ClPd(dppf)Cl22 (15.5(15.5 mg,mg, 2121 mMol)mmol) andand KK22CO3 (87.9(87.9 mg, 637 mMol)mmol) in one portion at 25 °C then heated at 110 °C for 12 h. TLC (dichloromethane:methanol = 10:1, Rf = in oneone portionportion at at 25 25 C°C then then heated heated at at 110 110C °C for for 12 h.12 TLCh. TLC (dichloromethane:methanol (dichloromethane:methanol= 10:1, = 10:1, R = R0.4)f = 0.4) showed reaction completion,◦ LCMS showed◦ the desired product was formed. After cooling tof 25 showed0.4) showed reaction reaction completion, completion, LCMS LCMS showed showed the desiredthe desired product product was was formed. formed. After After cooling cooling to 25 to 25C, °C, ethyl acetate (30 mL) was added and combined and concentrated under reduce pressure. The◦ ethyl°C, ethyl acetate acetate (30 mL) (30 wasmL) addedwas added and combined and combined and concentrated and concentrated under reduceunder pressure.reduce pressure. The residue The residue was purified by prep-TLC (dichloromethane:methanol = 10:1, Rf = 0.4) to give SC50 (90 mg, residue was purified by prep-TLC (dichloromethane:methanol = 10:1, Rf = 0.4) to give SC50 (90 mg, 91% yield) as an off-white solid. 1H-NMR (400 MHz MeOD): δ 8.96 (d, J =1.6 Hz, 1 H), 8.91 (s, 1 H), 91% yield) as an off-white solid. 1H-NMR (400 MHz MeOD): δ 8.96 (d, J =1.6 Hz, 1 H), 8.91 (s, 1 H), 8.68 (d, J = 4.8 Hz, 1 H), 8.19–8.32 (m, 2 H), 7.64 (dd, J = 8.0, 4.8 Hz, 1 H), 7.41–7.51 (m, 5 H), 4.56 (s, 1 8.68 (d, J = 4.8 Hz, 1 H), 8.19–8.32 (m, 2 H), 7.64 (dd, J = 8.0, 4.8 Hz, 1 H), 7.41–7.51 (m, 5 H), 4.56 (s, 1 H), 4.21 (d, J = 12.4 Hz, 1 H), 3.72–3.80 (m, 1 H), 3.57–3.71 (m, 2 H), 2.54 (s, 1 H), 1.88–2.05 (m, 2 H). H), 4.21 (d, J = 12.4 Hz, 1 H), 3.72–3.80 (m, 1 H), 3.57–3.71 (m, 2 H), 2.54 (s, 1 H), 1.88–2.05 (m, 2 H). 3.2.2. Synthesis of SC52 3.2.2. Synthesis of SC52 Preparation of tert-Butyl (3-benzoyl-3-azabicyclo [3.1.0]hexan-6-yl) Carbamate (14) Preparation of tert-Butyl (3-benzoyl-3-azabicyclo [3.1.0]hexan-6-yl) Carbamate (14)

At 0 °C, benzoyl chloride (97.4 mg, 0.70 mmol) was added to a mixture of tert-butyl (3- At 0 °C, benzoyl chloride (97.4 mg, 0.70 mmol) was added to a mixture of tert-butyl (3- azabicyclo[3.1.0]hexan-6-yl)carbamate (125.0 mg, 0.63 mmol) and TEA (127.0 mg, 1.26 mmol) in DCM azabicyclo[3.1.0]hexan-6-yl)carbamate (125.0 mg, 0.63 mmol) and TEA (127.0 mg, 1.26 mmol) in DCM (5 mL) dropwise. The resulting mixture was stirred at 0 °C for 30 mins. The reaction was then (5 mL) dropwise. The resulting mixture was stirred at 0 °C for 30 mins. The reaction was then quenched with H2O (20 mL). The mixture was extracted with DCM (50 mL × 2). The combined organic quenched with H2O (20 mL). The mixture was extracted with DCM (50 mL × 2). The combined organic layers was washed with brine (100 mL), dried over anhydrous Na2SO4 and concentrated under layers was washed with brine (100 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0–100% EtOAc in reduced pressure. The residue was purified by column chromatography (silica gel, 0–100% EtOAc in

Molecules 2018, 23, x 14 of 30

To a mixture of 12 (1.5 g, 4.09 mmol) and benzoyl chloride (1.15 g, 8.17 mmol) in DCM (30 mL) was added TEA (413 mg, 4.09 mmol) in one portion at 25 °C under N2. The mixture was stirred at 25 °C for 2 h. LC-MS showed the reaction was completed. The mixture was concentrated in reduced pressure at 45 °C. The residue was purified by prep-HPLC (acid conditions) to give 13 (600 mg, 31% yield) as a yellow solid. 1H-NMR (400 MHz DMSO-d6): δ 13.22 (br. s., 1 H), 9.04 (d, J = 4.8 Hz, 1 H), 8.80 (d, J = 2.4 Hz, 1 H) , 8.10 (d, J = 2.8 Hz, 1 H), 7.22–7.56 (m, 5 H), 4.01 (d, J = 12.4 Hz, 1 H), 3.67 (d, J =7.2 Hz, 1 H), 3.42 (dd, J = 18.4, 11.2 Hz, 2 H), 2.51–2.57 (m, 1 H), 1.88 (d, J = 7.6 Hz, 2 H).

General Procedure for the Preparation of SC50

To a solution of 13 (100 mg, 0.212 mmol), 3-pyridylboronic acid (39 mg, 0.318 mmol) in dioxane (5 mL) and H2O (1 mL) was added Pd(dppf)Cl2 (15.5 mg, 21 mmol) and K2CO3 (87.9 mg, 637 mmol) in one portion at 25 °C then heated at 110 °C for 12 h. TLC (dichloromethane:methanol = 10:1, Rf = Molecules0.4) showed2019, 24reaction, 1581 completion, LCMS showed the desired product was formed. After cooling14 to of 25 30 °C, ethyl acetate (30 mL) was added and combined and concentrated under reduce pressure. The residue was purified by prep-TLC (dichloromethane:methanol = 10:1, Rf = 0.4) to give SC50 (90 mg, was puriﬁed by prep-TLC (dichloromethane:methanol = 10:1, Rf = 0.4) to give SC50 (90 mg, 91% yield) 91% yield) as an off-white solid. 1H-NMR (400 MHz MeOD): δ 8.96 (d, J =1.6 Hz, 1 H), 8.91 (s, 1 H), as an oﬀ-white solid. 1H-NMR (400 MHz MeOD): δ 8.96 (d, J =1.6 Hz, 1 H), 8.91 (s, 1 H), 8.68 (d, 8.68 (d, J = 4.8 Hz, 1 H), 8.19–8.32 (m, 2 H), 7.64 (dd, J = 8.0, 4.8 Hz, 1 H), 7.41–7.51 (m, 5 H), 4.56 (s, 1 J = 4.8 Hz, 1 H), 8.19–8.32 (m, 2 H), 7.64 (dd, J = 8.0, 4.8 Hz, 1 H), 7.41–7.51 (m, 5 H), 4.56 (s, 1 H), 4.21 H), 4.21 (d, J = 12.4 Hz, 1 H), 3.72–3.80 (m, 1 H), 3.57–3.71 (m, 2 H), 2.54 (s, 1 H), 1.88–2.05 (m, 2 H). (d, J = 12.4 Hz, 1 H), 3.72–3.80 (m, 1 H), 3.57–3.71 (m, 2 H), 2.54 (s, 1 H), 1.88–2.05 (m, 2 H).

3.2.3.3.2.2. Synthesis of SC52

Preparation of tert-Butyl (3-benzoyl-3-azabicyclo [3.1.0]hexan-6-yl)[3.1.0]hexan-6-yl) CarbamateCarbamate ((1414))

At 0 °C, benzoyl chloride (97.4 mg, 0.70 mmol) was added to a mixture of tert-butyl (3- At 0 ◦C, benzoyl chloride (97.4 mg, 0.70 mMol) was added to a mixture of tert-butyl (3-azabicyclo[3.1.0]hexan-6-yl)carbamateazabicyclo[3.1.0]hexan-6-yl)carbamate (125.0 (125.0 mg, mg,0.63 mmol) 0.63 mMol) and TEA and (127.0 TEA mg, (127.0 1.26 mg, mmol) 1.26 in mMol) DCM (5 mL) dropwise. The resulting mixture was stirred at 0 °C for 30 mins. The reaction was then in DCM (5 mL) dropwise. The resulting mixture was stirred at 0 ◦C for 30 min. The reaction was thenquenched quenched with H with2O (20 H OmL). (20 The mL). mixture The mixture was extracted was extracted with DCM with (50 DCM mL (50× 2). mL The combined2). The combined organic Molecules 2018, 23, x 2 15 of 30 Moleculeslayers was 2018 , washed23, x with brine (100 mL), dried over anhydrous Na2SO4 and concentrated× 15under of 30 organic layers was washed with brine (100 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified puriﬁed by colu columnmn chromatography (silica gel, 0–100% EtOAc in petroleum ether) to give the title product (150.0 mg, 78.7% yield) as colorless oil. LC-MS (ESI): m//z petroleum+ ether) to give the title product (150.0 mg, 78.7% yield) as colorless oil. LC-MS (ESI): m/z [M + 1]+ = 303.14. [M + 1] + = 303.14. [M + 1] = 303.14. Preparation of (6-Amino-3-azabicyclo[3.1.0]hexan-3-yl)(phenyl) Methanone (15) Preparation of (6-Amino-3-azabicyclo[3.1.0]hexan-3-yl)(phenyl) Methanone ((1515))

To aa solutionsolution of 1414 (150(150 mg,mg, 0.4960.496 mMol)mmol) in DCM (5 mL), HCI-dioxane (2 (2 M, M, 3 3 mL) mL) was was added. added. The resulting mixture was stirred at roomroom temperaturetemperature forfor 88 h.h. After After completion completion of of the reaction indicatedindicated bybyby LCMSLCMSLCMS and andand TLC, TLC,TLC, the thethe mixture mixturemixture was waswas concentrated concentratedconcentrated under underunder reduced reducedreduced pressure pressurepressure to aﬀ ordtoto affordafford the crude thethe crudeamine amine15 (120 15 mg, (120(120 100% mg,mg, yield) 100%100% asyield)yield) colorless asas colorlesscolorless oil, which oil,oil, which waswhich used waswas in usedused the next inin thethe step nextnext directly stepstep directlydirectly without withoutwithout further + furtherpuriﬁcation. purification. LC-MS LC-MS (ESI): m (ESI):/z [M m+//1]z +[M[M= ++203.10. 1]1]+ == 203.10.203.10.

Preparation of 6-Allylpyrazolo[1,5-a]pyrimidine-5,7-diol (17))

To a solution of 1H-pyrazol-3-amine 16 (15.2(15.2 g,g, 182.9182.9 mmol)mmol) inin EtOHEtOH (200(200 mL)mL) werewere addedadded To a solution of 1H-pyrazol-3-amine 16 (15.2 g, 182.9 mMol) in EtOH (200 mL) were added NaOEt NaOEt (24.9 g, 365.8 mmol), followed by diethyl 2-allylmalonate (36.6 g, 182.9 mmol). The resulting (24.9 g, 365.8 mMol), followed by diethyl 2-allylmalonate (36.6 g, 182.9 mMol). The resulting mixture mixture was stirred at 100 °C overnight. After cooled down to RT, the precipitateipitate waswas collectedcollected byby was stirred at 100 ◦C overnight.2 After cooled down to RT, the precipitate was collected by filtration filtration and dissolved in H2O (500 mL). The resulted solution was acidified to pH~2 with 1N HCI, and filtered dissolved to ingive H2 theO (500 desired mL). product The resulted 17 (22 solution g, 62.9% was yield) acidified as a white to pH~2 solid. with LC-MS 1N HCI, (ESI): and m/ filteredz [M + and filtered to give the desired product 17 (22 g, 62.9% yield) as a white solid. LC-MS (ESI):+ m/z [M + to+ give the desired product 17 (22 g, 62.9% yield) as a white solid. LC-MS (ESI): m/z [M + 1] = 192.33. 1]+ == 192.33.192.33.

Preparation of 6-Allyl-5,7-dichloropyrazolo[1,5-a]pyrimidineloropyrazolo[1,5-a]pyrimidine ((18)

3 A solution of 17 (16.5(16.5 g,g, 86.486.4 mmol)mmol) inin POCIPOCI3 (100 mL) was stirred at 100 °C overnight. After 3 cooled down to RT, excess POCl3 waswas removedremoved underunder reducedreduced pressurepressure toto affordafford thethe 18 (17.2(17.2 g,g, 87.3%87.3% yield) as brown oil, which was used in the next step directly without further purification. LC-MS (ESI): + m//z [M[M ++ 1]1]+ =228.14,=228.14, 230.14.230.14.

Preparation of 6-Allyl-5-chloro-N-(4-methoxybenzyl)pyrazolo[1,5-a] pyrimidin-7-amine (19)

Molecules 2018, 23, x 15 of 30 petroleum ether) to give the title product (150.0 mg, 78.7% yield) as colorless oil. LC-MS (ESI): m/z + [M + 1]+ = 303.14.

Preparation of (6-Amino-3-azabicyclo[3.1.0]hexan-3-yl)(phenyl) Methanone (15)

To a solution of 14 (150 mg, 0.496 mmol) in DCM (5 mL), HCI-dioxane (2 M, 3 mL) was added. The resulting mixture was stirred at room temperature for 8 h. After completion of the reaction indicated by LCMS and TLC, the mixture was concentrated under reduced pressure to afford the crude amine 15 (120 mg, 100% yield) as colorless oil, which was used in the next step directly without + further purification. LC-MS (ESI): m/z [M + 1]+ = 203.10.

Preparation of 6-Allylpyrazolo[1,5-a]pyrimidine-5,7-diol (17)

To a solution of 1H-pyrazol-3-amine 16 (15.2 g, 182.9 mmol) in EtOH (200 mL) were added NaOEt (24.9 g, 365.8 mmol), followed by diethyl 2-allylmalonate (36.6 g, 182.9 mmol). The resulting mixture was stirred at 100 °C overnight. After cooled down to RT, the precipitate was collected by filtration and dissolved in H2O (500 mL). The resulted solution was acidified to pH~2 with 1N HCI, filtration and dissolved in H2O (500 mL). The resulted solution was acidified to pH~2 with 1N HCI, and filtered to give the desired product 17 (22 g, 62.9% yield) as a white solid. LC-MS (ESI): m/z [M + andMolecules filtered2019, to24 ,give 1581 the desired product 17 (22 g, 62.9% yield) as a white solid. LC-MS (ESI): m/z15 [M of 30+ + 1]+ = 192.33.

Preparation of 6-Allyl-5,7-dich 6-Allyl-5,7-dichloropyrazolo[1,5-a]pyrimidineloropyrazolo[1,5-a]pyrimidine (1818))

A solution of 17 (16.5 g, 86.4 mmol) in POCI3 (100 mL) was stirred at 100 °C overnight. After A solution of 17 (16.5 g, 86.4 mmol) in POCI3 (100 mL) was stirred at 100 °C overnight. After A solution of 17 (16.5 g, 86.4 mMol) in POCI3 (100 mL) was stirred at 100 ◦C overnight. After cooled down to RT, excess POCl3 was removed under reduced pressure to afford the 18 (17.2 g, 87.3% cooled down to RT, excess POCl3 was removed under reduced pressure to afford the 18 (17.2 g, 87.3% cooled down to RT, excess POCl3 was removed under reduced pressure to aﬀord the 18 (17.2 g, 87.3% yield) as brown oil, which was used in the next step directly without further purification. LC-MS (ESI): yield) as brown+ oil, which was used in the next step directly without further puriﬁcation. LC-MS (ESI): m/z [M + 1]+ =228.14, 230.14. m/z [M + 1]1]+ =228.14,=228.14, 230.14. 230.14. Preparation of 6-Allyl-5-chloro-N-(4-methoxybenzyl)pyrazolo[1,5-a] pyrimidin-7-amine (19) Preparation of 6-Allyl-5-chloro-N-(4-methoxybenzyl)pyrazolo[1,5-a] pyrimidin-7-aminepyrimidin-7-amine ((1919))

Molecules 2018, 23, x 16 of 30

To a solution ofof 18 (17.2(17.2 g,g, 75.4 mMol)mmol) and TEA (15.2 g, 150.8 mMol)mmol) in DCM (250 mL), PMBNH22 (10.3 g, 75.4 mMol)mmol) was added. The The mixture mixture was was stirred at room temperature for 24 h. The reaction solution was diluted with HH2O (250 mL), and extracted with DCM (250(250 mLmL × 2). The The combined combined organic organic 2 × layers were washed with brine (300(300 mL),mL), drieddried overover anhydrousanhydrous NaNa22SO4,, filtered ﬁltered and concentratedconcentrated under reduced pressure. The The residue residue was was purified puriﬁed by by column chromatography (silica (silica gel, 0–50% EtOAc in in petroleum petroleum ether) ether) to to give give the the 1919 (17.4(17.4 g, g,70.2% 70.2% yield) yield) as a as white a white solid. solid. LC-MS LC-MS (ESI): (ESI): m/z [Mm /+z + + 1 1 1][M =+ 329.09,1] = 329.09 331.04;, 331.04; H-NMRH-NMR (400 MHz, (400 MHz,DMSO- DMSO-d6): δ 8.12d6): δ(d,8.12 J = (d,2.4J Hz,= 2.4 1H), Hz, 8.01 1H), (t, 8.01 J = (t,6.8J =Hz,6.8 1H), Hz, 1H),7.21–7.17 7.21–7.17 (m, 2H), (m, 2H),6.89–6.84 6.89–6.84 (m, 2H), (m, 2H),6.41 (d, 6.41 J = (d, 2.0J =Hz,2.0 1H), Hz, 6.00–5.90 1H), 6.00–5.90 (m, 1H), (m, 5.12–5.08 1H), 5.12–5.08 (m, 1H), (m, 4.99 1H), (d,4.99 J (d,= 6.8J =Hz,6.8 2H), Hz, 4.90–4.88 2H), 4.90–4.88 (m, 1H), (m, 3.71 1H), (s, 3.71 3H), (s, 3H),3.47–3.45 3.47–3.45 (m, 2H). (m, 2H).

Preparation of 5-Chloro-8-(4-methoxybenzyl)-8H-pyrazolo[1,5-a]pyrrolo[3,2-e]pyrimidine ((2020))

A mixturemixture ofof19 19(2.2 (2.2 g, g, 6.69 6.69 mMol), mmol), K OsO K2OsOH2O4·H2O (234.8 (234.8 mg, mg, 0.67 mMol)0.67 mmol) and NaIO and NaIO(5 g,4 23.4 (5 g, mMol) 23.4 2 4· 4 mmol)in THF in/H THF/H2O (252 mL,O (25 4:1, mLv/ ,v 4:1,) was v/v stirred) was stirred at 0 ◦C at for 0 °C 5 h, for and 5 h, then and thethen PTSA the PTSA (1.15 (1.15 g, 6.69 g, mMol)6.69 mmol) was wasadded. added. The The mixture mixture was was stirred stirred at room at room temperature temperature for 2 for h. The2 h. reactionThe reacti solutionon solution was dilutedwas diluted with withH O (50H2O mL), (50 andmL), extracted and extracted with ethyl with acetateethyl acetate (50 mL (502). mL The × 2). combined The combined organic organic layers were layers washed were 2 × withwashed brine with (100 brine mL), (100 dried mL), over dried anhydrous over anhydrous Na2SO4, Na ﬁltered2SO4, and filtered concentrated and concentrated under reduced under pressure.reduced pressure.The residue The was residue puriﬁed was bypurified column by chromatographycolumn chromatography (silica gel, (silica 0–30% gel, EtOAc0–30% EtOAc in petroleum in petroleum ether) ether)to give to20 give(1.1 20 g, 52.6%(1.1 g, yield)52.6% asyield) a white as a solid.white LC-MSsolid. LC-MS (ESI): m (ESI):/z [M m/z+ 1] [M+ = +313.04, 1]+ = 313.04, 315.04; 315.04;1H-NMR 1H- (400NMR MHz, (400 DMSO-MHz, DMSO-d6): δ 8.23d6): (d,δ 8.23J = (d,2.4 J Hz, = 2.4 1H), Hz, 7.41 1H), (d, 7.41J = 3.2(d, Hz,J = 3.2 1H), Hz, 7.32–7.30 1H), 7.32–7.30 (m, 2H), (m, 6.89–6.86 2H), 6.89– (m, 6.862H), (m, 6.73 2H), (d, J 6.73= 2.4 (d, Hz, J = 1H),2.4 Hz, 6.71 1H), (d, J6.71= 3.6 (d, Hz, J = 1H),3.6 Hz, 5.94 1H), (s, 2H),5.94 (s, 3.70 2H), (s, 3H).3.70 (s, 3H).

Preparation of 8-(4-Methoxybenzyl)-5-(3-methyl-1H-1,2,4-triazol-1-yl)-8H-pyrazolo[1,5-a]- pyrrolo[3,2-e]pyrimidine (21)

A mixture of 20 (1.1 g, 3.5 mmol) and 3-methyl-1H-1,2,4-triazole (1.4 g, 17.5 mmol) was stirred at 170 °C for 2 h. After cooled down to room temperature, the reaction mixture was diluted with H2O (30 mL) and extracted with DCM (30 mL × 2). The combined organic phases were washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0–100% EtOAc in petroleum ether) to give 21 (732 mg, 58.2% yield) as a yellow solid. LC-MS (ESI): m/z [M + 1]+ = 360.35.

Preparation of 5-(3-Methyl-1H-1,2,4-triazol-1-yl)-8H-pyrazolo[1,5-a] pyrrolo[3,2-e]-pyrimidine (22)

Molecules 2018, 23, x 16 of 30

To a solution of 18 (17.2 g, 75.4 mmol) and TEA (15.2 g, 150.8 mmol) in DCM (250 mL), PMBNH2 (10.3 g, 75.4 mmol) was added. The mixture was stirred at room temperature for 24 h. The reaction solution was diluted with H2O (250 mL), and extracted with DCM (250 mL × 2). The combined organic layers were washed with brine (300 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0–50% EtOAc in petroleum ether) to give the 19 (17.4 g, 70.2% yield) as a white solid. LC-MS (ESI): m/z [M + 1]+ = 329.09, 331.04; 1H-NMR (400 MHz, DMSO-d6): δ 8.12 (d, J = 2.4 Hz, 1H), 8.01 (t, J = 6.8 Hz, 1H), 7.21–7.17 (m, 2H), 6.89–6.84 (m, 2H), 6.41 (d, J = 2.0 Hz, 1H), 6.00–5.90 (m, 1H), 5.12–5.08 (m, 1H), 4.99 (d, J = 6.8 Hz, 2H), 4.90–4.88 (m, 1H), 3.71 (s, 3H), 3.47–3.45 (m, 2H).

Preparation of 5-Chloro-8-(4-methoxybenzyl)-8H-pyrazolo[1,5-a]pyrrolo[3,2-e]pyrimidine (20)

A mixture of 19 (2.2 g, 6.69 mmol), K2OsO4·H2O (234.8 mg, 0.67 mmol) and NaIO4 (5 g, 23.4 mmol) in THF/H2O (25 mL , 4:1, v/v) was stirred at 0 °C for 5 h, and then the PTSA (1.15 g, 6.69 mmol) was added. The mixture was stirred at room temperature for 2 h. The reaction solution was diluted with H2O (50 mL), and extracted with ethyl acetate (50 mL × 2). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0–30% EtOAc in petroleum ether) to give 20 (1.1 g, 52.6% yield) as a white solid. LC-MS (ESI): m/z [M + 1]+ = 313.04, 315.04; 1H- NMR (400 MHz, DMSO-d6): δ 8.23 (d, J = 2.4 Hz, 1H), 7.41 (d, J = 3.2 Hz, 1H), 7.32–7.30 (m, 2H), 6.89– 6.86Molecules (m, 20192H),, 24 6.73, 1581 (d, J = 2.4 Hz, 1H), 6.71 (d, J = 3.6 Hz, 1H), 5.94 (s, 2H), 3.70 (s, 3H). 16 of 30

Preparation of 8-(4-Methoxybenzyl)-5-(3-methyl-1H-1,2,4-triazol-1-yl)-8H-pyrazolo[1,5-a]- pyrrolo[3,2-e]pyrimidinePreparation of 8-(4-Methoxybenzyl)-5-(3-methyl-1 (21) H-1,2,4-triazol-1-yl)-8H-pyrazolo[1,5-a]- pyrrolo[3,2-e]pyrimidine (21)

A mixture ofof 2020 (1.1(1.1 g,g,3.5 3.5 mMol) mmol) and and 3-methyl-1 3-methyl-1HH-1,2,4-triazole-1,2,4-triazole (1.4 (1.4 g, g, 17.5 17.5 mMol) mmol) was was stirred stirred at 170at 170◦C °C for for 2 h.2 h. After After cooled cooled down down to to room room temperature, temperature, the the reaction reaction mixture mixture waswas diluteddiluted withwith HH22O (30 mL) and extracted with DCM (30 mLmL × 2).2). The The co combinedmbined organic organic phases phases were washed with brine × (30 mL),mL), drieddried over over anhydrous anhydrous Na 2NaSO24SO, ﬁltered4, filtered and and concentrated concentrated under under reduced reduced pressure. pressure. The residue The wasresidue puriﬁed was purified by column by column chromatography chromatography (silica gel, (silica 0–100% gel, 0–100% EtOAc inEtOAc petroleum in petroleum ether) to ether) give 21to (732give mg,21 (732 58.2% mg, yield) 58.2% as yield) a yellow as a solid.yellow LC-MS solid. LC-MS (ESI): m (ESI):/z [M +m/1]z +[M= +360.35. 1]+ = 360.35. Molecules 2018, 23, x 17 of 30 PreparationMolecules 2018, of23, 5-(3-Methyl-1x H-1,2,4-triazol-1-yl)-8H-pyrazolo[1,5-a] pyrrolo[3,2-e]-pyrimidine 17 ((2222 of)) 30

A solution of 21 (732 mg, 2.04 mmol) in TfOH/TFA (7 mL, 2:5, v/v) was stirred at room temperatureA solutionsolution forof 1h.of21 The21(732 (732 reaction mg, mg, 2.04 mixture2.04 mMol) mmol) inwas TfOH concin /TfOH/TFATFAentrated (7 mL, under 2:5,(7 mL,v reduced/v) was2:5, stirred pressure.v/v) was at room Thestirred temperatureresidue at room was purifiedfortemperature 1h. The by reactionprep-HPLC for 1h. The mixture (C18,reaction was 30–100% mixture concentrated acetonitrile was conc underentrated in H reduced2O with under pressure. 0.1% reduced formic The pressure. acid) residue to giveThe was residue22 puriﬁed (166 wasmg, by 34%purified yield) by as prep-HPLC a white solid. (C18, LC-MS 30–100% (ESI): acetonitrile m/z [M + 1] in+ =H 240.19;2O with 1H-NMR 0.1% formic (400 MHz,acid) to DMSO- give 22d6 ):(166 δ 13.37 mg, prep-HPLC (C18, 30–100% acetonitrile in H2O with 0.1% formic acid) to give 22 (166 mg, 34% yield) as (br.34% s, yield) 1H), 9.41as a white(s, 1H), solid. 8.21 LC-MS(d, J = 2.4 (ESI): Hz,+ m 1H),/z [M 7.30 + 1]1 (d,+ = J240.19; = 3.2 Hz, 1H-NMR 2H), 7.21 (400 (d, MHz, J = 3.2 DMSO- Hz, 1H),d6): 6.69 δ 13.37 (d, a white solid. LC-MS (ESI): m/z [M + 1] = 240.19; H-NMR (400 MHz, DMSO-d6): δ 13.37 (br. s, 1H), J9.41(br. = 2.4 s, (s, 1H),Hz, 1H), 1H), 9.41 8.21 2.47(s, (d, 1H), J(s,= 3H).2.48.21 Hz, (d, 1H),J = 2.4 7.30 Hz, (d, 1H),J = 7.303.2 Hz, (d, 2H),J = 3.2 7.21 Hz, (d, 2H),J = 7.213.2Hz, (d, J 1H), = 3.2 6.69 Hz, (d, 1H),J = 6.692.4 Hz,(d, 1H),J = 2.4 2.47 Hz, (s, 1H), 3H). 2.47 (s, 3H). Preparation of Ethyl 2-(5-(3-methyl-1H-1,2,4-triazol-1-yl)-8H-pyrazolo [1,5-a]pyrrolo[3,2-e]- pyrimidin-8-yl)acrylatePreparation of Ethyl 2-(5-(3-methyl-12-(5-(3-methyl-1 (23) HH-1,2,4-triazol-1-yl)-8-1,2,4-triazol-1-yl)-8HH-pyrazolo-pyrazolo [1,5-a]pyrrolo[3,2-e]-pyrimidin [1,5-a]pyrrolo[3,2-e]- -8-yl)acrylatepyrimidin-8-yl)acrylate (23) (23)

To a stirred solution of 22 (166(166 mg,mg, 0.6930.693 mMol)mmol) and ethyl propiolate (68 mg, 0.693 mMol)mmol) in DCMTo (5 a mL)mL) stirred waswas addedsolutionadded thethe of solution solution22 (166 of mg,of PPh PPh 0.6933 3(181.8 (181.8 mmol) mg, mg, and 0.693 0.693 ethyl mMol) mmol) propiolate in in DCM DCM (3(68 (3 mL) mg, mL) dropwise 0.693 dropwise mmol) at 0at ◦in C.0 °C.TheDCM The reaction (5 reactionmL) mixturewas mixtureadded was the stirredwas solution stirred at room of at PPh temperatureroom3 (181.8 temperature mg, for 0.693 4 h forand mmol) 4 thenh andin concentrated DCM then (3 concentrated mL) under dropwise reduced under at 0 reducedpressure.°C. The pressure.reaction The residue mixtureThe was residue puriﬁedwas was stirred bypurified column at room by chromatography colu temperaturemn chromatography for (silica 4 h gel, and (silica 0–100% then gel, concentrated EtOAc 0–100% in petroleum EtOAc under in petroleumspirit)reduced to pressure. give spirit)23 (85 to The give mg, residue 36.4%23 (85 was yield)mg, purified36.4% as a whiteyield) by colu solid.as amn white LC-MS chromatography solid. (ESI): LC-MSm/z [M (silica(ESI):+ 1] mgel,+/=z [M0–100%338.10. + 1]+ EtOAc= 338.10. in petroleum spirit) to give 23 (85 mg, 36.4% yield) as a white solid. LC-MS (ESI): m/z [M + 1]+ = 338.10. Preparation of 2-(5-(3-Methyl-1H-1,2,4-triazol-1-yl)-8H-pyrazolo [1,5-a]pyrrolo[3,2-e]-pyrimidin-8- yl)acrylicPreparation acid of (2-(5-(3-Methyl-124) H-1,2,4-triazol-1-yl)-8H-pyrazolo [1,5-a]pyrrolo[3,2-e]-pyrimidin-8- yl)acrylic acid (24)

To a solution of 23 (85 mg, 0.252 mmol) in THF(5 mL), LiOH (12.1 mg, 0.504 mmol) dissolved in H2O To(2 mL) a solution was added. of 23 (85The mg, resulting 0.252 mmol) mixture in wasTHF(5 sti rredmL), at Li roomOH (12.1 temperature mg, 0.504 for mmol) 1 h. Thedissolved reaction in mixtureH2O (2 mL) was was acidified added. to ThepH~5 resulting with 1N mixture aqueous was HCI sti solutionrred at room and thentemperature diluted withfor 1 EtOAch. The reaction(20 mL) andmixture H2O was (20 mL),acidified extracted to pH~5 with with EtOAc 1N aqueous (30 mL ×HCI 2). Thesolution combined and then organic diluted phases with were EtOAc dried (20 overmL) anhydrousand H2O (20 Na mL),2SO 4extracted and concentrated with EtOAc under (30 reducedmL × 2). pressureThe combined. The residue organic was phases purified were bydried column over chromategraphyanhydrous Na2SO (silica4 and gel, concentrated 0–10% MeOH under in DCM)reduced to pressuregive 24 (35. The mg, residue 45% yield) was aspurified a white by solid. column LC- MSchromategraphy (ESI): m/z [M (silica+ 1]+ = gel,310.09. 0–10% MeOH in DCM) to give 24 (35 mg, 45% yield) as a white solid. LC- MS (ESI): m/z [M + 1]+ = 310.09.

Molecules 2018, 23, x 17 of 30

A solution of 21 (732 mg, 2.04 mmol) in TfOH/TFA (7 mL, 2:5, v/v) was stirred at room temperature for 1h. The reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (C18, 30–100% acetonitrile in H2O with 0.1% formic acid) to give 22 (166 mg, 34% yield) as a white solid. LC-MS (ESI): m/z [M + 1]+ = 240.19; 1H-NMR (400 MHz, DMSO-d6): δ 13.37 (br. s, 1H), 9.41 (s, 1H), 8.21 (d, J = 2.4 Hz, 1H), 7.30 (d, J = 3.2 Hz, 2H), 7.21 (d, J = 3.2 Hz, 1H), 6.69 (d, J = 2.4 Hz, 1H), 2.47 (s, 3H).

Preparation of Ethyl 2-(5-(3-methyl-1H-1,2,4-triazol-1-yl)-8H-pyrazolo [1,5-a]pyrrolo[3,2-e]- pyrimidin-8-yl)acrylate (23)

To a stirred solution of 22 (166 mg, 0.693 mmol) and ethyl propiolate (68 mg, 0.693 mmol) in DCM (5 mL) was added the solution of PPh3 (181.8 mg, 0.693 mmol) in DCM (3 mL) dropwise at 0 °C. The reaction mixture was stirred at room temperature for 4 h and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 0–100% EtOAc in petroleum spirit) to give 23 (85 mg, 36.4% yield) as a white solid. LC-MS (ESI): m/z [M + 1]+ = 338.10. Molecules 2019, 24, 1581 17 of 30

Preparation of 2-(5-(3-Methyl-1H-1,2,4-triazol-1-yl)-8H-pyrazolo [1,5-a]pyrrolo[3,2-e]-pyrimidin-8- Preparationyl)acrylic acid of ( 2-(5-(3-Methyl-124) H-1,2,4-triazol-1-yl)-8H-pyrazolo [1,5-a]pyrrolo[3,2-e]-pyrimidin -8-yl)acrylic acid (24)

To aa solutionsolution ofof 2323 (85(85 mg,mg, 0.2520.252 mMol)mmol) inin THF(5THF(5 mL),mL), LiOHLiOH (12.1(12.1 mg,mg, 0.5040.504 mMol)mmol) dissolved in H22O (2 mL) was added. The resulting mixture was stirredstirred at room temperaturetemperature forfor 11 h.h. The reaction mixture waswas acidiﬁedacidified toto pH~5pH~5 with 1N aqueous HCI solution and then diluted with EtOAc (20 mL) and HH22O (20 mL), extracted with EtOAc (30(30 mLmL × 2). The combined organic phases were dried over × anhydrous NaNa22SO4 andand concentrated under reduced pressure pressure.. The residue was puriﬁedpurified by columncolumn Moleculeschromategraphychromategraphy 2018, 23, x (silica gel, gel, 0 0–10%–10% MeOH MeOH in in DCM) DCM) to togive give 24 24(35(35 mg, mg, 45% 45% yield) yield) as aas white a white solid.18 solid. ofLC- 30 LC-MSMS (ESI): (ESI): m/zm [M/z [M+ 1]++ =1] 310.09.+ = 310.09. Preparation of N-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(5-(3- methyl-1H-1,2,4-triazol-1-yl)-8H- pyrazolo[1,5-a]pyrrolo[3,2-e]pyrimidin-8-yl)acrylamidePreparation of N-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(5-(3- (SC52) methyl-1 H-1,2,4-triazol-1-yl)-8H-pyrazolo [1,5-a]pyrrolo[3,2-e]pyrimidin-8-yl)acrylamide (SC52)

To a a mixture mixture of of24 24(35 (35mg, mg,0.113 0.113mmol), mMol), DIPEA DIPEA(29.2 mg, (29.2 0.226 mg, mmol) 0.226 and mMol)(6-amino-3- and (6-amino-3-azabicyclo[3.1.0]hexan-3-yl)(phenyl)methanoneazabicyclo[3.1.0]hexan-3-yl)(phenyl)methanone (22.9 mg, (22.90.113 mg,mmol) 0.113 in mMol) DCM in(5 DCMmL) was (5 mL) added was HATUadded HATU(51.7 mg, (51.7 0.136 mg, mmol) 0.136 mMol) slowly slowly at RT. atThe RT. reaction The reaction mixture mixture was wasstirred stirred at room at room temperature temperature for 2 30for min. 30 min. After After reaction reaction completion, completion, the mixture the mixture was wasdiluted diluted with with H O H(102O mL), (10 mL),and then and extracted then extracted with withDCM DCM (20 mL (20 × mL 2). The2). Thecombined combined organic organic layers layers were were washed washed with with brine, brine, dried dried over over anhydrous 2 4 × Na2SOSO4 andand concentrated concentrated under under reduced reduced pressure. The The residue residue was was purified puriﬁed by prep-HPLC (C18 2 2 column, 30%~100% MeCN MeCN in in H H2O,O, with with 0.1% 0.1% formic formic acid acid in in H H2O)O) to to give give the the title title product product (8.5 (8.5 mg, mg, ++ 11 15.2% yield) asas aa whitewhite power.power. LC-MSLC-MS (ESI):(ESI):m m/z/z[M [M+ +1] 1] == 494.09; H-NMR (400 MHz, DMSO-DMSO-dd66): δ 9.43 (s, 1H), 8.63 (d, JJ = 3.23.2 Hz, 1H), 8.12 (d, J = 2.42.4 Hz, Hz, 1H), 1H), 7.47–7.36 7.47–7.36 (m, (m, 4H), 4H), 7.34–7.28 (m, 2H), 6.68 (d, J = 2.4 Hz, 1H), 6.30 (d, J = 2.02.0 Hz, 1H), 6.06 (d, J = 2.02.0 Hz, 1H), 3.95 (d, J = 12.412.4 Hz, 1H), 3.69–3.65 (m, 1H), 3.47–3.43 (m, 1H), 3.35 (d, J = 11.211.2 Hz, Hz, 1H), 1H), 2.48 2.48 (s, (s, 3H), 3H), 2.35– 2.35–2.322.32 (m, (m, 1H), 1H), 2.03–1.97 2.03–1.97 (m, (m, 1H), 1H), 1.82–1.75 (m, 2H).

3.2.3.3.2.4. The The Synthesis of SC55 Synthesis of 2-(7-(5-amino-1,2,4-oxadiazol-3-yl)-4-fluoro-1of 2-(7-(5-amino-1,2,4-oxadiazol-3-yl)-4-fluoro-1H-indol-3-yl)-H-indol-3-yl)-N-(3-benzoyl-3-azabicycloN-(3-benzoyl-3- azabicyclo[3.1.0]hexan-6-yl)-2-oxoacetamide[3.1.0]hexan-6-yl)-2-oxoacetamide (SC55) (SC55) Preparation of 7-Bromo-4-fluoro-1H-indole (26)

A solution of 1-bromo-4-fluoro-2-nitrobenzene 25 (10 g, 45.5 mmol) in THF (200 mL) was added dropwise to a solution of 1 M vinylmagnesium bromide in THF (182 mL, 182 mmol) at −40 °C (bath temp). The reaction was stirred at −40 °C for 3 h, and saturated aqueous NH4Cl was added. The layers were separated, and the organic layer was evaporated. The crude product was purified flash chromatography, giving 4.2 g (43%) of 26 (43% yield). LC-MS (ESI): m/z [M + 1]+ = 215.15. Preparation of 4-Fluoro-1H-indole-7-carbonitrile (27)

A mixture of 26 (2.2 g, 10.3 mmol) and CuCN (4.6 g, 51.4 mmol) in DMF (20 mL) was refluxed for 16 h. After cooling to room temperature, the reaction mixture was poured onto a solution of ammonia in MeOH (100 mL, sat.) and the solid was removed by filtration. The filtrate was added to

Molecules 2018, 23, x 18 of 30

Preparation of N-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(5-(3- methyl-1H-1,2,4-triazol-1-yl)-8H- pyrazolo[1,5-a]pyrrolo[3,2-e]pyrimidin-8-yl)acrylamide (SC52)

To a mixture of 24 (35 mg, 0.113 mmol), DIPEA (29.2 mg, 0.226 mmol) and (6-amino-3- azabicyclo[3.1.0]hexan-3-yl)(phenyl)methanone (22.9 mg, 0.113 mmol) in DCM (5 mL) was added HATU (51.7 mg, 0.136 mmol) slowly at RT. The reaction mixture was stirred at room temperature for 30 min. After reaction completion, the mixture was diluted with H2O (10 mL), and then extracted with DCM (20 mL × 2). The combined organic layers were washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by prep-HPLC (C18 column, 30%~100% MeCN in H2O, with 0.1% formic acid in H2O) to give the title product (8.5 mg, + 1 15.2% yield) as a white power. LC-MS (ESI): m/z [M + 1]+ = 494.09; 1H-NMR (400 MHz, DMSO-d6): δ 9.43 (s, 1H), 8.63 (d, J = 3.2 Hz, 1H), 8.12 (d, J = 2.4 Hz, 1H), 7.47–7.36 (m, 4H), 7.34–7.28 (m, 2H), 6.68 (d, J = 2.4 Hz, 1H), 6.30 (d, J = 2.0 Hz, 1H), 6.06 (d, J = 2.0 Hz, 1H), 3.95 (d, J = 12.4 Hz, 1H), 3.69–3.65 (m, 1H), 3.47–3.43 (m, 1H), 3.35 (d, J = 11.2 Hz, 1H), 2.48 (s, 3H), 2.35–2.32 (m, 1H), 2.03–1.97 (m, 1H), 1.82–1.75 (m, 2H).

3.2.3. The Synthesis of SC55 Molecules 2019, 24, 1581 18 of 30 Synthesis of 2-(7-(5-amino-1,2,4-oxadiazol-3-yl)-4-fluoro-1H-indol-3-yl)-N-(3-benzoyl-3- azabicyclo[3.1.0]hexan-6-yl)-2-oxoacetamide (SC55) Preparation of 7-Bromo-4-ﬂuoro-1H-indole (26) Preparation of 7-Bromo-4-fluoro-1H-indole (26)

A solution of 1-bromo-4-fluoro-2-nitrobenzene 25 (10 g, 45.5 mmol) in THF (200 mL) was added A solution of 1-bromo-4-fluoro-2-nitrobenzene 25 (10 g, 45.5 mMol) in THF (200 mL) was added dropwise to a solution of 1 M vinylmagnesium bromide in THF (182 mL, 182 mmol) at −40 °C (bath dropwise to a solution of 1 M vinylmagnesium bromide in THF (182 mL, 182 mMol) at 40 ◦C temp). The reaction was stirred at −40 °C for 3 h, and saturated aqueous NH4Cl was added. The− layers (bath temp). The reaction was stirred at 40 C for 3 h, and saturated aqueous NH Cl was added. were separated, and the organic layer −was ◦evaporated. The crude product was 4purified flash The layers were separated, and the organic layer was evaporated. The crude product was purified chromatography, giving 4.2 g (43%) of 26 (43% yield). LC-MS (ESI): m/z [M + 1]+ = 215.15. flash chromatography, giving 4.2 g (43%) of 26 (43% yield). LC-MS (ESI): m/z [M + 1]+ = 215.15. Preparation of 4-Fluoro-1H-indole-7-carbonitrile (27) Preparation of 4-Fluoro-1H-indole-7-carbonitrile (27)

A mixture ofof 2626 (2.2(2.2 g, g, 10.3 10.3 mMol) mmol) and and CuCN CuCN (4.6 (4.6 g, g, 51.4 51.4 mMol) mmol) in in DMF DMF (20 (20 mL) mL) was was refluxed refluxed for Molecules 2018, 23, x 19 of 30 16forh. 16 After h. After cooling cooling to room to room temperature, temperature, the reaction the reaction mixture mixture was poured was ontopoured a solution onto a ofsolution ammonia of ammoniain MeOH (100in MeOH mL, sat.) (100 and mL, the sat.) solid and was the removed solid was by removed filtration. by The filtration. filtrate wasThe addedfiltrate towas a mixture added to of a mixture of water (50 mL)/ammonia (60 mL, sat. aq.) and extracted with EtOAc/Ether (1/1) until TLC water (50 mL)/ammonia (60 mL, sat. aq.) and extracted with EtOAc/Ether (1/1) until TLC analysis analysis showed no product in the aqueous phase. The combined organic extracts were washed with showed no product in the aqueous phase. The combined organic extracts were washed with brine brine (2 × 200 mL) and water (200 mL), dried (Na2SO4); concentrated under reduced pressure. The (2 200 mL) and water (200 mL), dried (Na SO ); concentrated under reduced pressure. The residue residue× was purified by silica gel column chromatography2 4 (petroleum spirit/ethyl acetate = 3:1, v/v) was purified by silica gel column chromatography (petroleum spirit/ethyl acetate = 3:1, v/v) to give to give 4-fluoro-7-cyanoindole 27 as a tan yellow solid, yield 55%), LC-MS (ESI): m/z [M + 1]+ = 161.23. 4-fluoro-7-cyanoindole 27 as a tan yellow solid, yield 55%), LC-MS (ESI): m/z [M + 1]+ = 161.23. Preparation of Methyl 2-(7-cyano-4-fluoro-1H-indol-3-yl)-2-oxoacetate (28) Preparation of Methyl 2-(7-cyano-4-fluoro-1H-indol-3-yl)-2-oxoacetate (28)

To a a mixture mixture of of 4-fluoro-1H-indole-7-carbonitrile 4-fluoro-1H-indole-7-carbonitrile (900 (900 mg, mg, 5.6 mmol) 5.6 mMol) in DCM in DCM(15 mL) (15 was mL) added was methyl-2-chloro-2-oxoacetateadded methyl-2-chloro-2-oxoacetate (2.84 g,(2.84 8.76 mmol), g, 8.76 mMol), then the then reaction the reaction mixture mixture was cooled was to cooled 0 °C， toAlCl 0 ◦C,3 AlCl（1.53 g,(1.5 11.2 g, 11.2mmol mMol)）was was added added in portions in portions,，then then the the reaction reaction mixture mixture was was stirred stirred at at 0 0 °C◦C for for 30 min.Then reactionreaction mixturemixture was was quenched quenched with with MeOH MeOH and and extracted extracted with with ethyl ethyl acetate acetate (3 (330 × mL) 30 mL) and × washedand washed with brine.with Thebrine. organic The organic layer was layer dried was over dried anhydrous over Naanhydrous2SO4, filtered Na2SO and4, concentratedfiltered and concentratedunder reduced under pressure. reduced The pressure. residue wasThe purifiedresidue wa bys silicapurified gel by column silica chromatographygel column chromatography (petroleum (petroleumspirit/ethyl acetatespirit/ethyl= 3:1, vacetate/v) to give = 3:1, methyl v/v) 2-(7-cyano-4-fluoro-1 to give methyl 2-(7-cyano-4-fluoro-1H-indol-3-yl)-2-oxoacetateH-indol-3-yl)-2- (660 mg, + oxoacetate48% yield). (660 LC-MS mg, (ESI):48% yield).m/z [M LC-MS+ 1] = (ESI):247.44. m/z [M + 1]+ = 247.44. Preparation of 2-(7-Cyano-4-fluoro-1H-indol-3-yl)-2-oxoacetic Acid (29)

To a mixture of methyl 2-(7-cyano-4-fluoro-1H-indol-3-yl)-2-oxoacetate (28, 660 mg, 2.68 mmol) in THF (10 mL) and water (2.5 mL), LiOH (257.4 mg, 10.7 mmol) was added, this mixture was stirred at room temperature for 4 hr, the reaction was monitored as by LC-MS, the solvent was concentrated and acidified with 2N HCl, extracted with ethyl acetate (25 ml × 2).The combined organic layers were washed with brine and dried over Na2SO4,the mixture was concentrated to provide 2-(7-cyano-4- fluoro-1H-indol-3-yl)-2-oxoacetic acid (29, 560 mg, 90% yield) as a yellow solid. LC-MS (ESI): m/z [M + 1]+ = 233.28.

Preparation of N-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(7-cyano-4-fluoro-1H-indol-3-yl)-2- oxoacetamide (30)

Molecules 20182018,, 2323,, xx 1919 ofof 3030 a mixture of water (50 mL)/ammonia (60 mL, sat. aq.) and extracted with EtOAc/Ether (1/1) until TLC analysis showed no product in the aqueous phase. The combined organic extracts were washed with 2 4 brine (2 × 200 mL) and water (200 mL), dried (Na2SO4);); concentratedconcentrated underunder reducedreduced pressure.pressure. TheThe residueresidue waswas purifiedpurified byby silicasilica gelgel columncolumn chromatographychromatography (petroleum(petroleum spirit/ethylspirit/ethyl acetateacetate == 3:1,3:1, v/v)) + toto givegive 4-fluoro-7-cyanoindole4-fluoro-7-cyanoindole 27 asas aa tantan yellowyellow solid,solid, yieldyield 55%),55%), LC-MSLC-MS (ESI):(ESI): m/z [M[M ++ 1]1]+ == 161.23.161.23. Preparation of Methyl 2-(7-cyano-4-fluoro-1H-indol-3-yl)-2-oxoacetate-indol-3-yl)-2-oxoacetate ((28))

To a mixture of 4-fluoro-1H-indole-7-carbonitrile (900 mg, 5.6 mmol) in DCM (15 mL) was added ， 3 methyl-2-chloro-2-oxoacetate (2.84(2.84 g,g, 8.768.76 mmol),mmol), thenthen thethe reactionreaction mixturemixture waswas cooledcooled toto 00 °C°C，AlCl3 （1.5 g, 11.2 mmol）was added in portions，thenthen thethe reactionreaction mixturemixture waswas stirredstirred atat 00 °C°C forfor 3030 min.Then reaction mixture was quenched with MeOH and extracted with ethyl acetate (3 × 30 mL) 2 4 and washed with brine. The organic layer was dried over anhydrous Na2SO4,, filteredfiltered andand concentrated under reduced pressure. The residue was purified by silica gell columncolumn chromatographychromatography (petroleum spirit/ethyl acetate = 3:1, v/v) to give methyl 2-(7-cyano-4-fluoro-1H-indol-3-yl)-2- (petroleumMolecules 2019 spirit/ethyl, 24, 1581 acetate = 3:1, v/v) to give methyl 2-(7-cyano-4-fluoro-1H-indol-3-yl)-2-19 of 30 + oxoacetate (660 mg, 48% yield). LC-MS (ESI): m//z [M[M ++ 1]1]+ = 247.44. Preparation of 2-(7-Cyano-4-fluoro-1H-indol-3-yl)-2-oxoacetic Acid (29) PreparationPreparation of of2-(7-Cyano-4-fluoro-1 2-(7-Cyano-4-ﬂuoro-1H-indol-3-yl)-2-oxoaceticH-indol-3-yl)-2-oxoacetic Acid Acid (29 (29) )

To a mixture of methyl 2-(7-cyano-4-fluoro-1H-indol-3-yl)-2-oxoacetate-indol-3-yl)-2-oxoacetate ((28,, 660660 mg,mg, 2.682.68 mmol)mmol) To a mixture of methyl 2-(7-cyano-4-fluoro-1H-indol-3-yl)-2-oxoacetate (28, 660 mg, 2.68 mMol) inin THFTHF (10(10 mL)mL) andand waterwater (2.5(2.5 mL),mL), LiOHLiOH (257.4(257.4 mg,mg, 10.710.7 mmol)mmol) waswas added,added, thisthis mixturemixture waswas stirredstirred in THF (10 mL) and water (2.5 mL), LiOH (257.4 mg, 10.7 mMol) was added, this mixture was at room temperature for 4 hr, the reaction was monitored as by LC-MS, the solvent was concentrated stirred at room temperature for 4 h, the reaction was monitored as by LC-MS, the solvent was and acidified with 2N HCl, extracted with ethyl acetate (25 ml × 2).The combined organic layers were concentrated and acidified with 2N HCl, extracted with ethyl acetate (25 mL 2).The combined organic 2 4 washed with brine and dried over Na2SO4,the,the mixturemixture waswas concentratedconcentrated toto× provideprovide 2-(7-cyano-4-2-(7-cyano-4- layers were washed with brine and dried over Na2SO4,the mixture was concentrated to provide fluoro-1fluoro-1H-indol-3-yl)-2-oxoacetic-indol-3-yl)-2-oxoacetic acidacid ((29,, 560560 mg,mg, 90%90% yield)yield) asas aa yellowyellow solid.solid. LC-MSLC-MS (ESI):(ESI): m//z [M[M 2-(7-cyano-4-fluoro-1+ H-indol-3-yl)-2-oxoacetic acid (29, 560 mg, 90% yield) as a yellow solid. LC-MS + 1]+ == 233.28.233.28. (ESI): m/z [M + 1]+ = 233.28. Preparation of N-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(7-cyano-4-fluoro-1H-indol-3-yl)-2- PreparationPreparation of of NN-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(7-cyano-4-fluoro-1-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(7-cyano-4-fluoro-1H-indol-3-yl)-2H-indol-3-yl)-2- oxoacetamide-oxoacetamide (30 ())30 )

Molecules 2018, 23, x 20 of 30

At rt, to a mixture of 2-(7-cyano-4-fluoro-1H-indol-3-yl)-2-oxoacetic acid (29, 560 mg, 2.41 mmol) in DMFAt rt,(5 mL) to a mixturewas added of 2-(7-cyano-4-ﬂuoro-1(6-amino-3-azabicyclo[3.1.0]hexan-3-yl)(phenyl)methanoneH-indol-3-yl)-2-oxoacetic acid (29, 560 mg, (488.2 2.41 mg, mMol) 2.41

inmmol), DMF HATU (5 mL) (1.37 was g, added 3.6 mmol) (6-amino-3-azabicyclo[3.1.0]hexan-3-yl)(phenyl)methanone and TEA (731.0 mg, 7.23 mmol), then the reaction mixture (488.2 was mg, 2.41stirred mMol), at RT HATUfor 2 h. (1.37 Then g, reaction 3.6 mMol) mixture and TEA was (731.0filtered mg, through 7.23 mMol), Celite.then The thefiltrate reaction was concentrated mixture was stirredunder reduced at RT for pressure. 2 h. Then The reaction residue mixture was purified was filtered by silica through gel column Celite. Thechromatography filtrate was concentrated (petroleum underether/ethyl reduced acetate pressure. = 3:1, v/v The) to residue give 30 was (534 purified mg, 53% by yield). silica gelLC-MS column (ESI): chromatography m/z [M + 1]+ = 417.51. (petroleum ether/ethyl acetate = 3:1, v/v) to give 30 (534 mg, 53% yield). LC-MS (ESI): m/z [M + 1]+ = 417.51. Preparation of N-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(4-fluoro-7-(N-hydroxy- Preparation of N-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(4-fluoro-7-(N-hydroxy-carbamimidoyl) -1carbamimidoyl)-1H-indol-3-yl)-2-oxoacetamideH-indol-3-yl)-2-oxoacetamide (31). (31).

At rt, to a mixture of 30 (534 mg, 1.28 mmol) in EtOH (8 mL) was added hydroxylamine At rt, to a mixture of 30 (534 mg, 1.28 mMol) in EtOH (8 mL) was added hydroxylamine hydrochloride (178.4 mg, 2.56 mmol), and TEA (388.56 mg, 3.84 mmol), then the reaction mixture was hydrochloride (178.4 mg, 2.56 mMol), and TEA (388.56 mg, 3.84 mMol), then the reaction mixture was stirred at RT for 3 h. Then reaction mixture was diluted with ethyl acetate (30 mL) and washed with stirred at RT for 3 h. Then reaction mixture was diluted with ethyl acetate (30 mL) and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced brine. The organic layer was dried over anhydrous Na2SO4, ﬁltered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether/ethyl pressure. The residue was puriﬁed by silica gel column chromatography (petroleum ether/ethyl acetate = 3:1, v/v) to give 31 (315 mg, 55% yield). LC-MS (ESI): m/z [M + 1]+ = 450.63. acetate = 3:1, v/v) to give 31 (315 mg, 55% yield). LC-MS (ESI): m/z [M + 1]+ = 450.63. Preparation of N-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(4-fluoro-7-(5-(trichloro-methyl)-1,2,4- oxadiazol-3-yl)-1H-indol-3-yl)-2-oxoacetamide (32)

To a mixture of 31 (315 mg, 0.7 mmol) in 2,2,2-trichloroacetic anhydride (3 mL) then the reaction mixture was stirred at 80 °C for 10 h. The reaction mixture was cooled to rt, petroleum ether (10 mL) was added and the mixture was filtered to give 32 (61 mg, 15% yield) as a brown solid. LC-MS (ESI): m/z [M + 1]+ = 577.82. Preparation of 2-(7-Nitro-1H-indazol-3-yl)acetic acid SC55

To a solution of 32 (61 mg, 0.11 mmol) in DMF (1 mL) was added a solution of ammonia in MeOH (7 M, 2 mL) and the resulting mixture stirred at r.t. for 16 h. The reaction mixture was

Molecules 2018, 23, x 20 of 30

At rt, to a mixture of 2-(7-cyano-4-fluoro-1H-indol-3-yl)-2-oxoacetic acid (29, 560 mg, 2.41 mmol) in DMF (5 mL) was added (6-amino-3-azabicyclo[3.1.0]hexan-3-yl)(phenyl)methanone (488.2 mg, 2.41 mmol), HATU (1.37 g, 3.6 mmol) and TEA (731.0 mg, 7.23 mmol), then the reaction mixture was stirred at RT for 2 h. Then reaction mixture was filtered through Celite. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 3:1, v/v) to give 30 (534 mg, 53% yield). LC-MS (ESI): m/z [M + 1]+ = 417.51.

Preparation of N-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(4-fluoro-7-(N-hydroxycarbamimidoyl)-1H-indol-3-yl)-2-oxoacetamide (31).

At rt, to a mixture of 30 (534 mg, 1.28 mmol) in EtOH (8 mL) was added hydroxylamine hydrochloride (178.4 mg, 2.56 mmol), and TEA (388.56 mg, 3.84 mmol), then the reaction mixture was stirred at RT for 3 h. Then reaction mixture was diluted with ethyl acetate (30 mL) and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetateMolecules =2019 3:1,, 24 v/v, 1581) to give 31 (315 mg, 55% yield). LC-MS (ESI): m/z [M + 1]+ = 450.63. 20 of 30

Preparation of N-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(4-fluoro-7-(5-(trichloro-methyl)-1,2,4- Preparation of N-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(4-ﬂuoro-7-(5-(trichloro-methyl)- oxadiazol-3-yl)-1H-indol-3-yl)-2-oxoacetamide (32) 1,2,4-oxadiazol-3-yl)-1H-indol-3-yl)-2-oxoacetamide (32)

To a mixture of 31 (315 mg, 0.7 mmol) in 2,2,2-trichloroacetic anhydride (3 mL) then the reaction To a mixture of 31 (315 mg, 0.7 mMol)mmol) in 2,2,2-trichloroacetic 2,2,2-trichloroacetic anhydride (3 mL) then the reaction mixture was stirred at 80 °C for 10 h. The reaction mixture was cooled to rt, petroleum ether (10 mL) mixture was stirred at 80 ◦C for 10 h. The reaction mixture was cooled to rt, petroleum ether (10 mL) was added and the mixture was filtered to give 32 (61 mg, 15% yield) as a brown solid. LC-MS (ESI): was added andand thethe mixturemixture was was ﬁltered filtered to to give give32 32(61 (61 mg, mg, 15% 15% yield) yield) as aas brown a brown solid. solid. LC-MS LC-MS (ESI): (ESI):m/z m/z [M + 1]+ = 577.82. m/z[M +[M1] + 1]= 577.82.= 577.82. Preparation of 2-(7-Nitro-1H-indazol-3-yl)acetic acid SC55 Preparation of 2-(7-Nitro-1H-indazol-3-yl)acetic acid SC55

MoleculesTo a2018a solutionsolution, 23, x of of32 32(61 (61 mg, mg, 0.11 0.11 mMol) mmol) in DMFin DMF (1 mL) (1 mL) was addedwas added a solution a solution of ammonia of ammonia in MeOH21 of in30 (7MeOH M, 2 mL)(7 M, and 2 mL) the resulting and the mixtureresulting stirred mixture at r.t. stirred For 16 at h. r.t. The for reaction 16 h. The mixture reaction was concentrated,mixture was concentrated, the residue was purified by prep. HPLC (C18, 0~90 acetonitrile in H2O with 0.1% formic the residue was puriﬁed by prep. HPLC (C18, 0~90 acetonitrile in H2O with 0.1% formic acid) to + 1 acid)provide to provideSC55 (6.1 SC55 mg, (6.1 12.2% mg, yield) 12.2% as yield) a white as a solid. white LC-MS solid. LC-MS (ESI): m (ESI):/z [M m/z+ 1] [M+ = +475.41, 1] = 475.41,1H-NMR H- NMR (400 MHz, CDCl3): δ 10.59 (s, 1H), 9.07 (d, J = 3.2 Hz, 1H), 7.87 (dd, J = 8.3,4.4 Hz, 1H), 7.60–7.47 (400 MHz, CDCl3): δ 10.59 (s, 1H), 9.07 (d, J = 3.2 Hz, 1H), 7.87 (dd, J = 8.3,4.4 Hz, 1H), 7.60–7.47 (m, 1H),(m, 1H), 7.42–7.26 7.42–7.26 (m, 4H),(m, 4H), 7.00 (dd,7.00 J(dd,= 10.4,8.4 J = 10.4,8.4 Hz, 1H), Hz, 5.451H), (s, 5.45 2H), (s, 4.28 2H), (d, 4.28J = 12.7(d, J Hz,= 12.7 1H), Hz, 3.74–3.50 1H), 3.74– (m, 3.502H)2.60 (m, (s,2H)2.60 1H), 2.21–1.91(s, 1H), 2.21–1.91 (m, 1H), (m, 1.89–1.73 1H), 1.89–1.73 (m, 2H). (m, 2H).

3.2.4.3.2.5. Synthesis Synthesis of SC56

Preparation of 7-Bromo-4-methoxy-1H-indole (34)

To a solutionsolution of 1-bromo-4-methoxy-2-nitrobenzene1-bromo-4-methoxy-2-nitrobenzene (33, 10.0 g, 43.1 mmol) mMol) in in THF THF (100 (100 mL) were added vinylmagnesium bromide (110 mL) dropwise at −40 °C. The reaction mixture was sealed were added vinylmagnesium bromide (110 mL) dropwise at 40 ◦C. The reaction mixture was sealed and stirred at −40 °C for 1.5 hour. After warmed up to room −temperature, the resulting solution was and stirred at 40 ◦C for 1.5 h. After warmed up to room temperature, the resulting solution was quenched with− sat. NH4Cl (120 mL) and extracted with ethyl acetate (2 × 150 mL). The combined quenched with sat. NH4Cl (120 mL) and extracted with ethyl acetate (2 150 mL). The combined organic layers were washed with brine (50 mL) and H2O (50 mL), dried× over anhydrous Na2SO4, organic layers were washed with brine (50 mL) and H2O (50 mL), dried over anhydrous Na2SO4, filteredﬁltered and concentrated under reduced pressure. The The residue residue was was purified puriﬁed by silica gel column (petroleum ether/ethyl acetate = 2:1, v/v) to give 34 as a yellow solid (6.8 g, yield = 70.1%). LC-MS (ESI): m/z [M/M + 1]+ = 226.04/228.04.

Preparation of 4-Methoxy-1H-indole-7-carbonitrile (35)

An oven-dried screw cap test tube was charged with a magnetic stir bar, CuCN (152.4 mg, 0.8 mmol), then 34 (6.8 g, Y = 70.1%) in DMF (75 mL) was added into the tube. The reaction mixture was stirred at 140 °C overnight. After cooled down to room temperature, the resulting solution was filtered through Celite. The filtrate was diluted with ethyl acetate (100 mL) and washed with saturated NaHCO3 solution (30 mL), water (30 mL) and brine (30 mL). The organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column (petroleum ether/ethyl acetate = 1:1, v/v) to give 35 as a yellow solid (3.4 g, yield = 65.8%). LC-MS (ESI): m/z [M + 1]+ = 173.10.

Preparation of Methyl 2-(7-cyano-4-methoxy-1H-indol-3-yl)-2-oxoacetate (36)

Molecules 2018, 23, x 21 of 30 Molecules 2018, 23, x 21 of 30 concentrated, the residue was purified by prep. HPLC (C18, 0~90 acetonitrile in H2O with 0.1% formic concentrated, the residue was purified by prep. HPLC (C18, 0~90 acetonitrile in H2O with 0.1% formic acid) to provide SC55 (6.1 mg, 12.2% yield) as a white solid. LC-MS (ESI): m/z [M + 1]+ = 475.41, 1H- acid) to provide SC55 (6.1 mg, 12.2% yield) as a white solid. LC-MS (ESI): m/z [M + 1]+ = 475.41, 1H- NMR (400 MHz, CDCl3): δ 10.59 (s, 1H), 9.07 (d, J = 3.2 Hz, 1H), 7.87 (dd, J = 8.3,4.4 Hz, 1H), 7.60–7.47 NMR (400 MHz, CDCl3): δ 10.59 (s, 1H), 9.07 (d, J = 3.2 Hz, 1H), 7.87 (dd, J = 8.3,4.4 Hz, 1H), 7.60–7.47 (m, 1H), 7.42–7.26 (m, 4H), 7.00 (dd, J = 10.4,8.4 Hz, 1H), 5.45 (s, 2H), 4.28 (d, J = 12.7 Hz, 1H), 3.74– 3.50 (m, 2H)2.60 (s, 1H), 2.21–1.91 (m, 1H), 1.89–1.73 (m, 2H).

3.2.4. Synthesis of SC56

Preparation of 7-Bromo-4-methoxy-1H-indole (34)

To a solution of 1-bromo-4-methoxy-2-nitrobenzene (33, 10.0 g, 43.1 mmol) in THF (100 mL) were added vinylmagnesium bromide (110 mL) dropwise at −40 °C. The reaction mixture was sealed and stirred at −40 °C for 1.5 hour. After warmed up to room temperature, the resulting solution was quenched with sat. NH4Cl (120 mL) and extracted with ethyl acetate (2 × 150 mL). The combined quenched with sat. NH4Cl (120 mL) and extracted with ethyl acetate (2 × 150 mL). The combined organic layers were washed with brine (50 mL) and H2O (50 mL), dried over anhydrous Na2SO4, organicMolecules 2019layers, 24, 1581were washed with brine (50 mL) and H2O (50 mL), dried over anhydrous Na212SO of 304, filtered and concentrated under reduced pressure. The residue was purified by silica gel column (petroleum ether/ethyl acetate = 2:1, v/v) to give 34 as a yellow solid (6.8 g, yield = 70.1%). LC-MS (ESI):(petroleum m/z [M/M ether +/ ethyl1]+ = 226.04/228.04. acetate = 2:1, v/v) to give 34 as a yellow solid (6.8 g, yield = 70.1%). LC-MS (ESI): m/z [M/M + 1]+ = 226.04/228.04. (ESI): m/z [M/M + 1]+ = 226.04/228.04. Preparation of 4-Methoxy-1H-indole-7-carbonitrile (35) Preparation of 4-Methoxy-1H-indole-7-carbonitrile (35)

An oven-dried screw cap test tube was charged with a magnetic stir bar, CuCN (152.4 mg, 0.8 An oven-dried screw cap test tube was charged with a magnetic stir bar, CuCN (152.4 mg, mmol), then 34 (6.8 g, Y = 70.1%) in DMF (75 mL) was added into the tube. The reaction mixture was 0.8 mMol), then 34 (6.8 g, Y = 70.1%) in DMF (75 mL) was added into the tube. The reaction mixture stirred at 140 °C overnight. After cooled down to room temperature, the resulting solution was was stirred at 140 ◦C overnight. After cooled down to room temperature, the resulting solution filtered through Celite. The filtrate was diluted with ethyl acetate (100 mL) and washed with saturatedwas filtered NaHCO through3 solution Celite. (30 The mL), filtrate water was (30 diluted mL) and with brine ethyl (30 acetate mL). The (100 organic mL) and phase washed was dried with saturated NaHCO3 solution (30 mL), water (30 mL) and brine (30 mL). The organic phase was dried oversaturated anhydrous NaHCO Na32solutionSO4, filtered (30 mL),and concentrated water (30 mL) under and brinereduced (30 pressure. mL). The The organic residue phase was was purified dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column (petroleum ether/ethyl acetate = 1:1, v/v) to give 35 as a yellow solid (3.4 g, yield =by 65.8%). silica LC-MS gel column (ESI): (petroleum m/z [M + 1] ether+ = 173.10./ethyl acetate = 1:1, v/v) to give 35 as a yellow solid (3.4 g, = 65.8%). LC-MS (ESI): m/z [M + 1]+ = 173.10. yield = 65.8%). LC-MS (ESI): m/z [M + 1]+ = 173.10. Preparation of Methyl 2-(7-cyano-4-methoxy-1H-indol-3-yl)-2-oxoacetate (36) Preparation of Methyl 2-(7-cyano-4-methoxy-1 H-indol-3-yl)-2-oxoacetate (36)

Molecules 2018, 23, x 22 of 30

To a mixture of 35 (3.4 g, 19.8 mmol) and AlCl3 (13.2 g, 98.8 mmol) in DCM (80 mL) was added To a mixture of 35 (3.4 g, 19.8 mMol) and AlCl (13.2 g, 98.8 mMol) in DCM (80 mL) was added methyl oxalyl chloride (3.6 g, 19.7 mmol) in DCM (303 mL) slowly at 0 °C. The reaction mixture was methyl oxalyl chloride (3.6 g, 19.7 mMol) in DCM (30 mL) slowly at 0 ◦C. The reaction mixture was stirred for 30 hour and then quenched with saturated NaHCO3 solution (10 mL), and extracted with stirred for 30 h and then quenched with saturated NaHCO3 solution (10 mL), and extracted with DCM DCM (30 mL). The organic phase was washed with brine (10 mL) and H2O (10 mL), dried over (30 mL). The organic phase was washed with brine (10 mL) and H2O (10 mL), dried over anhydrous anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by Na SO , ﬁltered and concentrated under reduced pressure. The residue was puriﬁed by silica gel silica2 gel4 column (petroleum ether/ethyl acetate = 1:1, v/v) to give 36 as a yellow solid (2.2 g, Y = 43.1%). column (petroleum ether/ethyl acetate = 1:1, v/v) to give 36 as a yellow solid (2.2 g, Y = 43.1%). LC-MS LC-MS (ESI): m/z [M + 1]+ = 259.09. (ESI): m/z [M + 1]+ = 259.09. Preparation of 2-(7-Cyano-4-methoxy-1H-indol-3-yl)-2-oxoacetic Acid (37) Preparation of 2-(7-Cyano-4-methoxy-1H-indol-3-yl)-2-oxoacetic Acid (37)

A mixture of 36 (2.2 g, 8.5 mmol) and LiOH (306.0 mg, 12.8 mmol) in THF/H2O (18 mL, 5:1, V/V) A mixture of 36 (2.2 g, 8.5 mMol) and LiOH (306.0 mg, 12.8 mMol) in THF/H2O (18 mL, 5:1, v/v) was stirred at at room room temperature temperature for for 2.5 2.5 hr. hr. The The reaction reaction mixture mixture was was adjusted adjusted to pH to pH= 6 with= 6 with 1 N HCl 1 N HCl(12.8 (12.8mL) mL)and andextracted extracted with with DCM DCM (20 (20 mL). mL). The The organic organic phase phase was was concentrated concentrated under under reduced + pressure toto givegive 3737 asas aa whitewhite solidsolid (630(630 mg,mg, yieldyield == 30.3%). LC-MS (ESI): m/z [M + 1]1]+ == 245.09.245.09. Preparation of N-(3-Benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(7-cyano-4-methoxy-1H-indol-3-yl)-2- oxoacetamide (38)

At room temperature, to a stirred solution of 37 (500 mg, 2.05 mmol) in DCM (30 mL) were added (6-amino-3-azabicyclo[3.1.0]hexan-3-yl)(phenyl)methanone HCl salt (540 mg, 2.26 mmol) and DIPEA (1 mL, 6.15 mmol), followed by HATU (935 mg, 2.46 mmol) in portions. The resulting mixture was stirred at room temperature for 30 min, and then diluted with DCM (10 mL) and water (20 mL). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0~20% MeOH in DCM) to afford the title compound 38 (300 mg, yield = 34%). LC-MS (ESI): m/z [M + 1]+ = 429.21. Preparation of N-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(7-(N-hydroxycarbamimidoyl)-4- methoxy-1H-indol-3-yl)-2-oxoacetamide (39)

Molecules 2018, 23, x 22 of 30

MoleculesTo 2018a mixture, 23, x of 35 (3.4 g, 19.8 mmol) and AlCl3 (13.2 g, 98.8 mmol) in DCM (80 mL) was 22added of 30 methyl oxalyl chloride (3.6 g, 19.7 mmol) in DCM (30 mL) slowly at 0 °C. The reaction mixture was To a mixture of 35 (3.4 g, 19.8 mmol) and AlCl3 (13.2 g, 98.8 mmol) in DCM (80 mL) was added stirred for 30 hour and then quenched with saturated NaHCO3 solution (10 mL), and extracted with methyl oxalyl chloride (3.6 g, 19.7 mmol) in DCM (30 mL) slowly at 0 °C. The reaction mixture was DCM (30 mL). The organic phase was washed with brine (10 mL) and H2O (10 mL), dried over stirred for 30 hour and then quenched with saturated NaHCO3 solution (10 mL), and extracted with anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by DCMsilica gel(30 column mL). The (petroleum organic ether/ethylphase was acetatewashed = 1:1,with v/v brine) to give (10 36mL) as aand yellow H2O solid (10 (2.2mL), g, driedY = 43.1%). over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by LC-MS (ESI): m/z [M + 1]+ = 259.09. silica gel column (petroleum ether/ethyl acetate = 1:1, v/v) to give 36 as a yellow solid (2.2 g, Y = 43.1%). + LC-MSPreparation (ESI): of m 2-(7-Cyano-4-methoxy-1/z [M + 1] = 259.09. H-indol-3-yl)-2-oxoacetic Acid (37)

Preparation of 2-(7-Cyano-4-methoxy-1H-indol-3-yl)-2-oxoacetic Acid (37)

A mixture of 36 (2.2 g, 8.5 mmol) and LiOH (306.0 mg, 12.8 mmol) in THF/H2O (18 mL, 5:1, V/V) was stirred at room temperature for 2.5 hr. The reaction mixture was adjusted to pH = 6 with 1 N HCl (12.8A mL) mixture and extractedof 36 (2.2 g,with 8.5 mmol)DCM (20and mL). LiOH The (306.0 organic mg, 12.8phase mmol) was inconcentrated THF/H2O (18 under mL, 5:1,reduced V/V) Molecules 2019, 24, 1581 22 of 30 waspressure stirred to atgive room 37 astemperature a white solid for (6302.5 hr. mg, The yield reaction = 30.3%). mixture LC-MS was (ESI): adjusted m/z to[M pH + 1] =+ 6 = with 245.09. 1 N HCl (12.8 mL) and extracted with DCM (20 mL). The organic phase was concentrated under reduced pressurePreparation to give of N37-(3-Benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(7-cyano-4-methoxy- as a white solid (630 mg, yield = 30.3%). LC-MS (ESI): m/z [M + 1]1H+ =-indol-3-yl)-2- 245.09. Preparationoxoacetamide of (N38-(3-Benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(7-cyano-4-methoxy-) 1H-indol-3-yl)-2 -oxoacetamidePreparation of (38N)-(3-Benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(7-cyano-4-methoxy-1H-indol-3-yl)-2- oxoacetamide (38)

At room temperature, temperature, to to a astirred stirred solution solution of 37 of (50037 (500 mg, mg, 2.05 2.05mmol) mMol) in DCM in DCM(30 mL) (30 were mL) added were (6-amino-3-azabicyclo[3.1.0]hexan-3-yl)(phenyl)methanone HCl salt (540 mg, 2.26 mmol) and DIPEA addedAt (6-amino-3-azabicyclo[3.1.0]hexan-3-yl)(phenyl)methanone room temperature, to a stirred solution of 37 (500 mg, 2.05 mmol) HCl salt in DCM (540 mg,(30 mL) 2.26 were mMol) added and DIPEA(1 mL, 6.15 (1 mL, mmol), 6.15 mMol), followed followed by HATU by HATU (935 mg, (935 2.46 mg, mmol) 2.46 mMol) in portions. in portions. The resulting The resulting mixture mixture was (6-amino-3-azabicyclo[3.1.0]hexan-3-yl)(phenyl)methanone HCl salt (540 mg, 2.26 mmol) and DIPEA wasstirred stirred at room at room temperature temperature for 30 for min, 30 min, and andthen thendiluted diluted with with DCM DCM (10 mL) (10 mL)and water and water (20 mL). (20mL). The (1 mL, 6.15 mmol), followed by HATU (935 mg, 2.46 mmol) in portions. The resulting mixture was Theorganic organic layer layer was was washed washed with with brine, brine, dried dried over over Na Na2SOSO4, filtered, filtered and and concentrated concentrated under under reduced stirred at room temperature for 30 min, and then diluted2 with4 DCM (10 mL) and water (20 mL). The pressure.pressure. The residue was purifiedpurified by flashflash chromatographychromatography (silica(silica gel,gel, 0~20%0~20% MeOH in DCM) toto organic layer was washed with brine, dried over Na2SO4, filtered and concentrated+ under reduced afford the title compound 3838 (300 mg, yield = 34%). LC-MS (ESI): m/z [M + 1] + = 429.21. pressure.afford the The title residue compound was purified(300 mg, by yield flash= chromatography34%). LC-MS (ESI): (silic/a gel,[M +0~20%1] = 429.21.MeOH in DCM) to Preparation of N-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(7-(N-hydroxycarbamimidoyl)-4-+ affordPreparation the title of compound 38 (300 mg, yield = 34%). LC-MS (ESI): m/z [M + 1] = 429.21. methoxy-1H-indol-3-yl)-2-oxoacetamide (39) PreparationN-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(7-( of N-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(7-(N-hydroxycarbamimidoyl)-4-methoxy-N-hydroxycarbamimidoyl)-4- 1H-indol-3-yl)-2-oxoacetamide (39) methoxy-1H-indol-3-yl)-2-oxoacetamide (39)

At room temperature, to a solution of 38 (300 mg, 0.7 mmol) in EtOH (10 mL) was added NH2OH (3 mL, 50 wt.% in water). After stirred at room temperature for 30 min, the reaction mixture was At room temperature, to a solution of 38 (300 mg, 0.7 mmol) in EtOH (10 mL) was added NH2OH partitionedAt room between temperature, DCM toand a solution water. The of 38 organic(300 mg, layer 0.7 mMol)was separated, in EtOH (10and mL) the wasaqueous added layer NH2 wasOH (3 mL, 50 wt.% in water). After stirred at room temperature for 30 min, the reaction mixture was (3 mL, 50 wt.% in water). After stirred at room temperature for 30 min, the reaction mixture was partitionedMolecules 2018 ,between 23, x DCM and water. The organic layer was separated, and the aqueous layer23 wasof 30 partitioned between DCM and water. The organic layer was separated, and the aqueous layer was

extracted with DCM. The combined organic layerslayers werewere drieddried overover NaNa22SO4,, filtered ﬁltered and and concentrated concentrated under reduced pressure to givegive the crude title product 3939,, which was used in the next step without + further puriﬁcationpurification (323.0(323.0 mg,mg, yieldyield == 100.0%). LC-MS LC-MS (ESI): (ESI): mm//z [M[M + 1]1]+ == 462.17.462.17.

Preparation ofofN -(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(4-methoxy-7-(5-(trichloro-methyl)-1,2,4N-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(4-methoxy-7-(5-(trichloro-methyl)- -oxadiazol-3-yl)-11,2,4-oxadiazol-3-yl)-1H-indol-3-yl)-2-oxoacetamideH-indol-3-yl)-2-oxoacetamide (40) (40)

At room temperature, to a solution of 39 (323.0 mg, 0.7 mmol) in anhydrous THF (10 mL) was added 2,2,2-trichloroacetic anhydride (648.0 mg, 2.1 mmol) dropwise. The resulting mixture was stirred at room temperature overnight. After completion of the reaction as indicated by LC-MS, the reaction was quenched with ice water. The resulting solution was extracted with DCM. The combined organic layers were washed with water, dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude title product 40, which was used in the next step without further purification. LC-MS (ESI): m/z [M/M + 1]+ = 588.00/590.00. Preparation of 2-(7-(5-Amino-1,2,4-oxadiazol-3-yl)-4-methoxy-1H-indol-3-yl)-N-(3-benzoyl-3- azabicyclo[3.1.0]hexan-6-yl)-2-oxoacetamide (SC56)

At room temperature, to a solution of 40 (411.0 mg, 0.7 mmol) in THF (10 mL) was added ammonia solution (5 mL). After stirred at room temperature for 1 hour, the reaction mixture was partioned between DCM and water. The organic layer was separated, and the aqueous layer was extracted with DCM. The combined organic layers were washed water, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (Gilson, C18, 10%~100% MeCN in water with 0.1% formic acid) to afford SC56 (4.3 mg, yield = 1%) as a white solid. LC-MS (ESI): m/z [M + 1]+ = 487.10; 1H-NMR (400 MHz, CDCl3): δ 10.55 (s, 1H), 8.94 (d, J = 3.2 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.56 (br. s, 1H), 7.46–7.38 (m, 5H), 6.81 (d, J = 8.4 Hz, 1H), 5.38 (s, 2H), 4.36– 4.32 (m, 1H), 4.04 (s, 3H), 3.75-3.66 (m, 3H), 2.64 (s, 1H), 1.91-1.83 (m, 2H).

3.3. Cells HEK293T cells (a gift from Dr. Irwin Chaiken, Drexel University, Philadelphia, PA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin and 2mM L-glutamine. Human astroglioma U87 cells stably expressing CD4/CCR5 or CD4/CXCR4 (obtained from Prof. Hongkui Deng, Peking University, China and Prof. Dan Littman,

Molecules 2018, 23, x 23 of 30 extracted with DCM. The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude title product 39, which was used in the next step without further purification (323.0 mg, yield = 100.0%). LC-MS (ESI): m/z [M + 1]+ = 462.17. Preparation of N-(3-benzoyl-3-azabicyclo[3.1.0]hexan-6-yl)-2-(4-methoxy-7-(5-(trichloro-methyl)- 1,2,4-oxadiazol-3-yl)-1H-indol-3-yl)-2-oxoacetamide (40)

Molecules 2019, 24, 1581 23 of 30

At room temperature, to a solution of 39 (323.0 mg, 0.7 mMol) in anhydrous THF (10 mL) was addedAt 2,2,2-trichloroacetic room temperature, anhydride to a solution (648.0 of mg,39 (323.0 2.1 mMol) mg, dropwise.0.7 mmol) Thein anhydrous resulting mixture THF (10 was mL) stirred was atadded room 2,2,2-trichloroacetic temperature overnight. anhy Afterdride completion(648.0 mg, of2.1 the mmol) reaction dropwise. as indicated The byresulting LC-MS, mixture the reaction was wasstirred quenched at room with temperature ice water. overnight. The resulting After solution comple wastion extracted of the reaction with DCM. as indicated The combined by LC-MS, organic the layersreaction were was washed quenched with with water, ice water. dried over The resultin Na2SO4g, ﬁlteredsolution and was concentrated extracted with under DCM. reduced The combined pressure to give the crude title product 40, which was used in the next step without further puriﬁcation. LC-MS organic layers were washed with water, dried over Na2SO4, filtered and concentrated under reduced m z + (ESI):pressure/ to[M /giveM + 1]the =crude588.00 title/590.00. product 40, which was used in the next step without further + Preparationpurification. ofLC-MS (ESI): m/z [M/M + 1] = 588.00/590.00. 2-(7-(5-Amino-1,2,4-oxadiazol-3-yl)-4-methoxy-1Preparation of 2-(7-(5-Amino-1,2,4-oxadiazol-3-yl)-4-methoxy-1H-indol-3-yl)-N-(3-benzoyl-3-azabicycloH-indol-3-yl)-N-(3-benzoyl-3- [3.1.0]hexan-6-yl)-2-oxoacetamide (SC56) azabicyclo[3.1.0]hexan-6-yl)-2-oxoacetamide (SC56)

At roomroom temperature, temperature, to to a solutiona solution of 40 of(411.0 40 (411.0 mg, 0.7mg, mMol) 0.7 mmol) in THF in (10 THF mL) was(10 mL) added was ammonia added solutionammonia (5 solution mL). After (5 stirredmL). After at room stirred temperature at room fortemperature 1 h, the reaction for 1 hour, mixture the was reaction partioned mixture between was DCMpartioned and water.between The DCM organic and layerwater. was The separated, organic la andyer thewas aqueous separated, layer and was the extracted aqueous with layer DCM. was extracted with DCM. The combined organic layers were washed water, dried over Na2SO4, filtered The combined organic layers were washed water, dried over Na2SO4, filtered and concentrated under reducedand concentrated pressure. under The residue reduced was pressure. purified byThe prep-HPLC residue was (Gilson, purified C18, by 10%~100% prep-HPLC MeCN (Gilson, in water C18, with10%~100% 0.1% formicMeCN acid)in water to a withfford 0.1%SC56 formic(4.3 mg, acid) yield to afford= 1%) SC56 as a (4.3 white mg, solid. yield LC-MS = 1%) as (ESI): a whitem/z solid.[M + LC-MS+ (ESI): 1m/z [M + 1]+ = 487.10; 1H-NMR (400 MHz, CDCl3): δ 10.55 (s, 1H), 8.94 (d, J = 3.2 Hz, 1H), 1] = 487.10; H-NMR (400 MHz, CDCl3): δ 10.55 (s, 1H), 8.94 (d, J = 3.2 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H),7.93 (d, 7.56 J (br.= 8.4 s, Hz, 1H), 1H), 7.46–7.38 7.56 (br. (m, s,5H), 1H), 6.81 7.46–7.38 (d, J = (m,8.4 5H), Hz, 1H),6.81 5.38(d, J (s,= 8.4 2H), Hz, 4.36–4.32 1H), 5.38 (m, (s, 1H), 2H), 4.04 4.36– (s, 3H),4.32 (m, 3.75-3.66 1H), 4.04 (m, (s, 3H), 3H), 2.64 3.75-3.66 (s, 1H), (m 1.91-1.83, 3H), 2.64 (m, 2H).(s, 1H), 1.91-1.83 (m, 2H).

3.3. Cells HEK293T cells (a gift from Dr. Irwin Chaiken, Drexel University, Philadelphia, PA, USA) were cultured in Dulbecco’s Modified Modified Eagle’s Medium (DMEM), 10% FBS, 100 U/mL U/mL penicillin, 100 µg/mLµg/mL streptomycin and 2mM lL-glutamine. Human astroglioma U87 cells stably expressing CD4/CCR5 CD4/CCR5 or CD4CD4/CXCR4/CXCR4 (obtained from Prof. Hongkui Deng, Peking University, China and Prof. Dan Littman, New York University, New Yory, NY, USA, through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH) [40,41] were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin and 2 mM l-glutamine, 300 µg/mL G418 (Thermo Scientific, Waltham, MA, USA) and 1µg/mL puromycin (Thermo Scientific, Waltham, MA, USA). Cells were incubated continuously in a humidified 5% CO2/95% air environment at 37 ◦C.

3.4. Proteins HEK293F cells were used to express the soluble cleaved trimer B41 SOSIP.664 gp140. The recombinant trimer was subsequently purified by mAb 2G12-affinity chromatography followed by size-exclusion chromatography as described previously (PMID: 25589637); IgG b12 anti HIV-1 gp120 was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Anti-HIV-1 gp120 Monoclonal (IgG1 b12) from Dr. Dennis Burton and Carlos Barbas); p24 was produced in-house as previously described [42]. Briefly, a vector containing C-terminally His-tagged HIV-1NL4-3CA (a gift from Dr. Eric Molecules 2019, 24, 1581 24 of 30

Barklis, Oregon Health and Science University,Portland, OR, USA) was transformed into BL21-Codon Plus (DE3)-RIL Competent Cells (Agilent Technologies, Wilmington, DE, USA) and expressed in autoinduction ZYP-5052 medium at 30 ◦C while shaking at 250 rpm overnight [43]. Bacterial cultures were spun down at 8000 rpm, and the supernatant was discarded. Cell pellets were resuspended in 1 PBS and lysed × via sonication. The lysed sample was spun down at 45,000 rpm, the clarified supernatant was filtered through a 0.45 µm filter, and immediately applied to a Talon cobalt resin affinity column (Clonetech Laboratories, Mountain View, CA, USA). Bound protein was eluted using 1 PBS with 250 mM imidazole. × Elutions containing purified CA-H6 were pooled, dialyzed overnight into 20 mM Tris-HCl pH 8.0 at 4 ◦C, concentrated to 120 µM, flash frozen, and stored at 80 C. − ◦ 3.5. Production of Pseudotyped Viruses Single-round infectious envelope-pseudotyped luciferase-reporter viruses were produced by a co-transfection of two vectors (3:4 ratio of vector 1:2) in 6-well plated 293T cells (1 106 cells/well) [41]. × Vector 1 is an envelope-deficient HIV-1 pNL4-3-Luc+R-E plasmid which carries the luciferase-reporter gene [33]. Vector 2 is a plasmid expressing the HIV-1 gp160 Env from the various isolates tested (B41, Hxbc2, YU2, JRCSF, and JRFL) [26,35–39,44–46]. Transient transfections of these vectors were carried out via calcium phosphate (ProFection Mammalian Transfection System, Promega, Madison, WI, USA) for 5 h. The DNA-containing medium was replaced with fresh culture media after the 5 h transfection incubation. Supernatants containing pseudovirus were collected 72 h post transfection, clarified, filtered, aliquoted and stored at 80 C. − ◦ 3.6. ELISA-Based Quantification of p24 Content ELISA plate was coated with 50 ng/well of mouse anti-p24 (ab9071, Abcam, Cambridge, MA, USA) overnight at 4 ◦C. Following the overnight incubation, the plate was blocked with 3% (w/v) BSA at room temperature for 2 h and washed with 0.5% (v/v) Tween in PBS. Pseudoviral stocks were lysed using 0.1% (v/v) Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) at 37 ◦C for 1 h and added to the plate overnight at 4 ◦C. Simultaneously, p24 protein (produced and purified as previously described) was used as a standard. The following day, the plate was washed with 0.5% PBST and a 1:5000 dilution of rabbit anti-p24 (ab63913, Abcam) was added for 2 h at room temperature. After washing with PBST to remove the unbound rabbit anti-p24 off the plate, goat anti-rabbit-HRP at a 1:5000 dilution was added for 1 h at room temperature. The plate was then extensively washed with PBST. Subsequently, a solution of 0.4 mg/mL O-phenylenediamine in a phosphate-citrate buffer with sodium perborate (Sigma-Aldrich) was added and incubated for 30 min in the dark. Optical densities were then obtained at 450 nm in a Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific).

3.7. Single-Round Infection Assay The single-round HIV-1 infection assay was performed as previously described [33,47,48]. Brieﬂy, U87.CD4.CCR5/CXCR4 (1.2 104 cells/well) target cells were seeded in 96-well luminometer-compatible × tissue culture plates (Greiner Bio-one, Monroe, NC, USA). After 24 h, compound or DMSO (vehicle control for compounds, Sigma) were mixed with pseudotyped viruses (normalized to p24 content), added to the target cells, and incubated for 48 h at 37 ◦C. Following the 48 h incubation, the media was removed from each well, and the cells were lysed using 50 µL/well of 1X luciferase lysis buﬀer (Promega) and one freeze-thaw cycle. After adding 50 µL/well of luciferase assay substrate (Promega), a GloMax 96 microplate luminometer (Promega) was used to measure the luciferase activity of each well.

3.8. Cellular Toxicity The viability of the U87.CD4.CCR5/CXCR4 cells was determined using the Cell Counting Kit-8 cell proliferation and cytotoxicity assay (Dojindo Molecular Technologies, Rockville, MD, USA) per the manufacturer’s instructions. U87.CD4.CCR5/CXCR4 cells (1.2 104 cells/well) were seeded in 96-well × tissue culture plates (Olympus Plastics, San Diego, CA, USA). After 24 h, cells were treated with Molecules 2019, 24, 1581 25 of 30

compound (0.1–1000 µM) or DMSO (Sigma) for 48 h at 37 ◦C. Subsequently, 10 µL of the CCK-8 solution was added to each well and incubated for 4 h at 37 ◦C. Following the 4 h incubation, the absorbance at 450 nm was measured in a Multiskan™ GO Microplate Spectrophotometer (Thermo Scientiﬁc). Untreated cells were used as a background control and 0.1% SDS treated cells were used as a positive control. The 50% cytotoxicity (CC50) value was deﬁned as the concentration of the compounds that reduced the viability (observed as a decrease in absorbance) of treated cells by 50% as compared with control cells.

3.9. SPR Direct Interaction Analysis

Immobilization of Env Constructs Interaction analyses were performed on a ProteOn XPR36 SPR Protein Interaction Array System (Bio-Rad Laboratories, Hercules, CA, USA) at 25 ◦C for all kinetic analyses. ProteOn GLH sensor chips were preconditioned with two short pulses each (10 s) of 50 mM NaOH, 100 mM HCl, and 0.5% (w/v) sodium dodecyl sulfide. Then the system was equilibrated with PBST buffer (20 mM Na-phosphate, 150 mM NaCl, and 0.005% [v/v] polysorbate 20, pH 7.4). The surface of a GLH sensorchip was activated by a 10 min injection with a 1:100 dilution of a 1:1 mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.2 M) and 1 sulfo-N-hydroxysuccinimide (0.05 M). Immediately after chip activation, 100 µg mL− soluble cleaved gp140 B41.SOSIP.664 trimers in 10 mM sodium acetate, pH 5.0 was injected across ligand flow channels 1 for 15 min at a flow rate of 25 µL min− . A 5-min injection of 1 M ethanolamine HCl (pH 8.5) was then performed to cap excess active ester groups on the sensor surface.This resulted in the immobilization of Env constructs at a density of 14,000 RUs (response unit, which is an arbitrary unit that corresponds to 1 pg/mm2). A reference surface was similarly created by immobilizing a non-specific protein (IgG b12) in 10 mM sodium acetate, pH 5.0 to match the density of immobilized HIV-1 proteins.

3.10. Direct Binding Analysis To prepare the compounds for analysis, the compound stock solutions were brought up to 30 µL in 100% DMSO and this was made to a final volume of 1 mL by addition of sample preparation buffer (PBS, pH 7.4) to ensure that the concentration of DMSO was matched with that of running buffer (3% DMSO). Serial dilutions from a starting concentrating as indicated in the result section were then prepared in running buffer (PBS, 3% [v/v] DMSO, 0.005% [v/v] polysorbate 20, pH 7.4) and injected 1 across the surfaces at a flow rate of 100 µL min− , for a 2.6 min association phase, followed by up to a 10 min dissociation phase using the “one shot kinetics” capability of the Proteon instrument [49]. Data were analyzed using the ProteOn Manager Software version 3.0 (Bio-Rad). To account for nonspecific binding and injection artifacts, the responses of a buffer injection and responses from the reference flow cell were subtracted. Experimental data were fitted to a simple 1:1 binding model (where applied). The average kinetic parameters (association [ka] and dissociation [kd] rates) generated from five data sets were used to define the equilibrium dissociation constant (KD).

3.11. Negative Stain Electron Microscopy (EM) SC compounds were dissolved in 100% methanol to a final concentration of 0.2 mg/mL and 10 µL aliquots (total of 2 µg compound) were evaporated using a SpeedVac. 400 µL of B41 SOSIP.664 (at 0.25 mg/mL in Tris-buffered saline [TBS]) were added to tubes containing lyophilized compound. The small molecules were resuspended by pipetting up and down and left to incubate at room temperature for 1 h. Final concentrations of trimer (approximate MW of peptide 215,000 Da) and small molecule (MW 513.5 Da) were estimated to be 0.93 µM and 9.73 µM, respectively. A control tube containing only B41 SOSIP.664 (at 0.25 mg/mL Tris-buffered saline) was also incubated under the same conditions. Following incubation, each sample was diluted to about 0.02 mg/mL protein concentration in TBS and a 3 µL aliquot was adsorbed onto a glow-discharged, carbon-coated Cu400 copper mesh Molecules 2019, 24, 1581 26 of 30 grid. The drop was blotted using Whatman #1 filter paper, a 3 µL drop of 2% (w/v) uranyl formate was added to the sample-treated surface of the grid, and the sample stained for 45 s before blotting. Samples were imaged using an FEI Talos Arctica electron microscope (Thermo Fisher) operating at 200 kV and an FEI Ceta 16M CMOS camera (Thermo Fisher). Single frame exposures were collected with a 2 total dose of ~25 e−/Å at a magnification of 73,000, resulting in a pixel size of 1.98 Å at the specimen plane. Data processing and analysis has been described previously [26,50].

3.12. Differential Scanning Aalorimetry (DSC) Experiments were performed using a MicroCal VP-Capillary differential scanning calorimeter (Malvern Panalytical, Westborough, MA, USA). Before the experiments were carried out, B41 SOSIP.664 was dialyzed against phosphate-buffered saline (PBS). The protein concentration was subsequently adjusted to 0.25 mg/mL. Similar to the EM methods above, 0.5 mL of B41 SOSIP.664 were added to tubes containing 2 µg lyophilized compound SC11, SC15, SC28 or SC45, mixed by pipetting, and allowed to incubate for 1 h at room temperature. The samples, along with a control containing protein alone, were into the instrument cell and thermal denaturation was probed at a scan rate of 90 ◦C/h, with PBS in the reference cell. Buffer correction, normalization, and baseline subtraction procedures were performed using the Automated Origin 7.0 software (Malvern Panalytical, Westborough, MA, USA). The data were fitted using a non-two-state model.

3.13. Molecular Modeling

3.13.1. Docking of Compounds SC11, SC15 and SC45 The Env protein (pdb code: 6MUG) was prepared by the Protein Preparation Wizard implemented with Maestro (Schrödinger Maestro Version 11.5.011, New York, NY, USA, mM share Version 4.1.011, New York, NY, USA, Release 2018-1, Platform Darwin-x86_64). The Grid box was centered on the co-crystalized BMS-386150. For validating the glide docking protocol, the original ligand BMS-386150 was built using the LigPrep tool (Schrödinger Maestro Version 11.5.011, New York, NY, USA) and docked using glide-XP mode. The predicted docked pose matched the original co-ordinates of the co-crystalized BMS-386150 with an RMS value of 0.1 Å. SC11, SC15 and SC45 was docked using the same glide-XP mode and the top ranked pose was selected. The docked protein-ligand complexes were then refined using Prime (VSGB solvation model and OPLS3e forcefield, entire protein refinement).

3.13.2. Docking of Compounds SC28, SC49, SC50, SC52, SC55 and SC56 Glide induced-Fit (extended sampling settings) was used to flexibly sample the ligands and the protein pocket. The top ranked poses were then refined using Prime (VSGB solvation model and OPLS3e forcefield, entire protein refinement).

4. Conclusions In this study, we demonstrated that four entry inhibitors with different core scaffolds still interact with the B41 SOSIP.664 gp140 trimer via SPR but with different kinetics. After examining the effects of these four compounds on the overall structure of B41 SOSIP.664 trimer using NS-EM and DSC, we discovered that all of them, including SC28 (azabicyclohexane core), were able to stabilize the SOSIP and the EM results suggested a shift in the conformational equilibrium of the SOSIP from a heterogeneous population of open and closed trimers to a more homogenous pool of closed trimers. The findings suggest that a molecule like SC28 is able to stabilize the SOSIP and halt or severly impair the ability of receptor-mediated conformational dynamics of Env, making it a potential allosteric fusion inhibitor. Using computational field- and structure-based design methods, we chose five analogues of SC28 that had a modified methyltriazole-azaindole head group and retained the azabicyclo-hexane core region. These five SC28 derivatives all retained target specificity to B41 SOSIP.664 gp140 trimers and exhibited antiviral activity. Most striking was the difference between SC28 and SC56, both Molecules 2019, 24, 1581 27 of 30 compounds had nearly identical potencies, but had a 1000-fold difference in affinity. We have previously demonstrated for this class of compounds that the dissociation rate is correlated to the potency and when looking at the contributing factor to this increase in affinity, we observed that both compounds had similar dissociation rates, but that the association rates also differed by a factor of 1000. This further confirms that by decreasing the dissociation rate we can improve the potency for future analogues of our entry inhibitors to help bring this class of inhibitors closer towards clinical utility. By continuing to explore compounds that shift the conformational equilibrium of Env trimers, this class of entry inhibitors will have profound implications for future inhibitor and vaccine design efforts.

Supplementary Materials: The following are available online at, Figure S1: Single round infection assay graphs, Figure S2: Toxicity of compounds, Figure S3: Sensorgram of SC56, Table S1: NS-EM statistics. Author Contributions: Conceptualization, S.C. and M.E.M.; Methodology, S.C., M.E.M, A.A.R., G.O., A.B.W., A.D.; Formal Analysis, S.C., M.E.M, A.A.R., G.O., A.B.W., A.D.; Investigation, M.E.M, A.A.R., G.O., A.D.; Writing-Original Draft Preparation, S.C., M.E.M, A.A.R., G.O.; Writing-Review & Editing, S.C., M.E.M, A.A.R., G.O., A.B.W., A.D.; Supervision, S.C.; Funding Acquisition, S.C., G.O., A.B.W. Funding: This work was supported in part by NIH grants AI118415 (Cocklin, PI) and GM125396 (Cocklin, PI). G.O. is supported by amfAR Mathilde Krim Fellowship in Basic Biomedical Research grant #109718-63-RKNT. A.B.W. acknowledges support from the Collaboration for AIDS Vaccine Discovery OPP1115782 (Bill and Melinda Gates Foundation). Electron microscopy datasets were collected at The Scripps Research Institute. Acknowledgments: We’d like to thank Cresset (UK; http://www.cresset-group.com/) for access to their software via an academic license. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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