Field-based affinity optimization of a novel azabicyclohexane scaffold HIV-1 entry inhibitor

Article abstract of DOI:10.3390/molecules24081581

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.

Full text of DOI:10.3390/molecules24081581

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; mem484@drexel.edu (M.E.M.);
aaa396@drexel.edu (A.A.R.); ad3474@drexel.edu (A.D.)
Department of Integrative Structural and Computational Biology, Collaboration for AIDS Vaccine Discovery,
the Scripps Research Institute, La Jolla, CA 92037, USA; gozorows@scripps.edu (G.O.);
andrew@scripps.edu (A.B.W.)
2
*
Correspondence: sc349@drexel.edu; Tel.: +1-215-762-7234 or +1-215-762-4979; Fax: +1-215-762-4452
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Stefano Aquaro
Received: 19 February 2019; Accepted: 20 April 2019; Published: 22 April 2019
ꢀꢁꢂꢃꢄꢅꢆ
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
Molecules 2019, 24, 1581; doi:10.3390/molecules24081581
www.mdpi.com/journal/molecules

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BMS-663068 prodrug entry inhibitor indicate that it would only be of utility as salvage therapy for
highly treatment-experienced patients.
Given the enormous potential of HIV-1 entry inhibitors as both pre- and post-exposure prophylactic
regimens, our group has been exploring the modification of piperazine scaffold entry inhibitors [12
We have successfully expanded, designed and tested a number of scaffolds other than piperazine,
developing compounds with nanomolar potency and specificity to HIV-1 Env [16 20 21]. Moreover,
–19].
,
,
we have recently demonstrated by using a novel surface plasmon resonance (SPR) interaction assay,
that the off-rate of the compounds for a soluble recombinant Env trimer is strongly correlated with the
potency of the compounds in the single round infection assay [20]. The results presented herein further
extends our work developing novel entry inhibitors by demonstrating the conformational effects of
previously disclosed entry inhibitors on the Env complex. Moreover, we demonstrate the affinity
optimization of one of these scaffolds, the azabicyclohexane scaffold, which has the most pronounced
effect on the conformation of the Env target. The results from this work have broad implications for
future HIV-1 entry inhibitor designs that capitalize on the malleability and metastability of the Env
protein complex.
2. Results and Discussion
To gain insight into optimization strategies and mechanisms of action of a selection of our
entry inhibitor compounds with differing core scaffolds, we wished to perform molecular docking
against the published structure of the HIV-1 SOSIP.664 gp140 trimer. First, however, we had to
confirm that the compounds actually do directly interact with the Env complex. We have previously
demonstrated the interaction of four of our HIV-1 entry inhibitors with the B41 SOSIP.664 gp140
trimer using surface plasmon resonance [20]. Therefore, as a precursor to molecular modeling,
we performed SPR interaction analysis upon SC11 (dipyrrolidine scaffold), SC15 (azetidine scaffold),
SC28 (azabicyclo-hexane scaffold), and SC45 (tetrahydropyridine scaffold) (Figure 1). Figure 2 shows
the representative sensograms for each compound interacting 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
in each compound are colored blue.

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Figure 2. Sensorgrams showing the interaction of small molecule compounds with B41 SOSIP.664
gp140 trimer immobilized on the sensor chip. ( SC11 (concentrations shown are 10 M and down in
a 1:2 dilution series), ( SC15 (2 M, 1:4 dilution series), ( SC28 (10 M, 1:2 dilution series), and (
SC45 (40 M, 1:3 dilution series). Colored lines represent actual data collected from the dilution series,
A
)
µ
B)
µ
C)
µ
D)
µ
whereas black lines 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
gp140 trimer.
Compound
ka (M−1s−1
)
kd (s−1
)
KD
3.83 ± 1.12 × 103
3.01 ± 0.188 × 105
1.39 ± 0.14 × 104
1.38 ± 0.045 × 104
5.02 ± 2.67 × 10−4
5.44 ± 0.677 × 10−3
6.99 ± 0.43 × 10−3
1.52 ± 0.0294 × 10−2
SC11
SC15
SC28
SC45
0.131 µM
0.0181 µM
0.511 µM
1.10 µM
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
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
co-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
structure, similar to the co-crystalized ligand BMS-386150 (Figure 3), without any need to perform
flexible docking. However, the azabicyclo-hexane compound SC28 did not successfully dock using the
previous rigid docking protocol, and instead, we had to use flexible protein-ligand docking in order to
achieve a plausible model of SC28 at the binding site. This need for flexibility in the docking protocol
was subsequently found to be due to the fact that the presence of the azabicyclo-hexane moiety, in order
to dock to the binding pocket, appears to induce changes in the orientations 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

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well documented that changes or interactions at one site can have profound effects upon the overall
structure and conformation of the Env complex. Moreover, it has been demonstrated recently that the
β20/21 loop is a regulator of conformational transitions in HIV-1 Env and it is possible that modulation
of this region by SC28 could induce gross changes in structure [23,24].
Figure 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
(analogue; amine-oxadiazole) highlighting the difference in the orientation of the W427 indole ring.
Based on the SPR and docking results, and specifically the participation of the β20/21 loop in the
binding of SC28, we decided to test the effects of four of the compounds on the overall structure of B41
SOSIP.664 using negative-stain electron microscopy (NS-EM). B41 SOSIP.664 has been reported to be a
strong immunogen like the “gold standard” BG505 SOSIP.664, with most broadly neutralizing epitopes
presented on its surface, yet its appearance by NS-EM suggests a much greater degree of movement of
gp120 subunits relative to the three-fold symmetry axis, a behavior sometimes referred to as trimer
“breathing” [25–27]. When no compound is included, a small population (about 20–25%) of imaged
B41 trimers adopted what we infer to be a more open conformation, while the remaining trimers
had a more tightly-packed, closed phenotype (Figure 5A). The inclusion of a 10-fold molar 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 assessment on

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the stabilizing effects of the small molecules on the trimer, we used differential scanning calorimetry
(DSC) to measure changes in thermostability upon the addition of small molecules (Figure 5B). All
four compounds had a positive impact on B41 SOSIP.664 trimer thermostability, with SC11 having the
largest effect (Figure 5B). The combined observations of a measured increase in thermostability (DSC)
and an inferred shift towards a more compact state (EM) suggested that the compounds are able to
halt the natural dynamics of an Env trimer (that are critical for the virus during receptor recognition
and host cell fusion), and are in agreement with the role of the β20/21 loop in stabilization of the ground
state of Env [23]. Because SC28 had both experimentally-determined stabilizing effects and in silico
suggestions of inducing conformational changes, the results prompted us to search for and design
analogues of the azabicyclohexane scaffold with improved affinity.
Figure 5. Biophysical characterization of B41 SOSIP.664 incubated with SC11, SC15, SC28 or SC45
) 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
samples were stained with 2% (w/v) uranyl formate. ( ) Differential scanning calorimetry curves (left)
and summary of melting temperatures (Tm) (right). Primary and secondary peaks are those with
.
(
A
B
the highest and second highest intensity (Cp, specific heat capacity), respectively.
change from the inhibitor-free control.
∆Tm is the relative
In the absence of any experimentally-derived structural information on the bioactive conformation
of our inhibitors, we used the docking model of SC28 as described above as an input into Spark
(Cresset, UK) to identify nonclassical bioisosteres of the selected region. From the literature available
on the piperazine-based entry inhibitors, it is clear that a primary determinant of potency is the
methyltriazole-azaindole head group of the compounds [28
–31]. We, therefore, focused on this region,
looking for changes suggested by Spark that may modulate either drug-like parameters or potency,
and were significantly different from the methyltriazole-azaindole head group of SC28. [16
From this analysis, we docked five compounds onto the B41 SOSIP Env structure (PDB code: 6MUG) [22
,
20
,
21
,
32
]
]
using Glide (XP-mode), and looked at the overall ligand-protein interaction energies. Table 2 shows
the calculated ligand-protein interaction energies for these compounds in comparison to the parental
compound SC28.

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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
SC49
SC50
SC52
SC55
SC56
−16.13700
−6.58188
−12.87666
−2.25288
−10.12901
−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 (Figure 4). 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. Figure 6 shows the representative sensorgrams for each compound interacting with the B41
SOSIP.664 gp140 trimer and Table 3 shows the kinetic parameters obtained after analysis.
As can be seen in Table 3, 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

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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.
The results of this analysis are shown in Figure S1 and S2 and the values are summarized in Table 4.
Figure 6. Sensorgrams showing the interaction of small molecule compounds with B41 SOSIP.664
gp140 trimer immobilized on the sensor chip. (
in a 1:2 dilution series), ( SC49 (100 M, 1:2 dilution series), (
SC52 (100 M, 1:2 dilution series), ( SC55 (100 M, 1:2 dilution series), and (F)
A
)
SC28 (concentrations shown are 10
SC50 (200 M, 1:2 dilution series),
SC56 (10 nM,
µM and down
B)
µ
C)
µ
(
D)
µ
E
)
µ
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 Table 2.

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Table 3. Kinetics and affinity of novel SC28 derivatives binding to soluble, cleaved recombinant Env.
N.D. = not determined.
Compound
SC28
ka (M−1s−1
)
kd (s−1
)
KD (µM)
0.504 µM
1.39 ± 0.14 × 104
9.49 ± 1.6 × 102
N.D.
6.99 ± 0.43 × 10−3
3.43 ± 0.36 × 10−2
N.D.
SC49
36.1 µM
SC50
114 ± 51 µM
23.7 ± 2.4 µM
11.4 ± 3.7 µM
0.526 nM
SC52
N.D.
N.D.
SC55
N.D.
N.D.
1.22 ± 0.067 × 107
6.39 ± 0.31 × 10−3
SC56
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
(Budapest, Hungary).
Therapeutic
Index
IC50 B41
(µM)
IC50 HxBc2
(µM)
IC50 JRCSF
(µM)
IC50 JRFL
(µM)
IC50 YU-2
(µM)
Median IC50
CC50
(µM)
Compound
(µM)
(CC50/IC50
)
SC28 (parental)
0.035 ±
0.021 ± 0.005
0.026 ± 0.009
0.17 ± 0.05
0.48 ± 0.05
57.2 ± 26.5
0.15 ± 0.17
190 ± 54
326 ± 81
1291.21
0.0062
SC49
SC50
26 ± 19
62 ± 1.4
NA
33.32 ± 11.56
45 ± 15
7.31
6.63
7.1 ± 4.2
6.9 ± 5.2
28.92 ± 5.62
24.28 ± 6.2
22.6 ± 4.2
18 ± 9
120 ± 18
SC52
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
SC56
2.02 ± 0.81
0.73 ± 0.56
0.45 ± 0.35
0.45 ± 0.33
1.15 ± 0.81
0.96 ± 0.59
73 ± 14
75.85
0.051 ± 0.008
0.011 ± 0.003
0.046 ± 0.009
0.13 ± 0.015
0.11 ± 0.049
0.0698 ± 0.045
94 ± 10
1344.71
As summarized in Table 4, SC28 and each of the analogues tested exhibited antiviral effects in 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 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 over that of

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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, San Diego, CA, USA); ProteOn XPR36 SPR Protein Interaction Array System, GLH sensorchip,
(Bio-Rad Laboratories, Hercules, CA, USA); MicroCal VP-Capillary differential scanning calorimeter,
Automated Origin 7.0 software (Malvern Panalytical, 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
under N2. A solution of 1 (30 g, 136 mMol) in THF (100 mL) was added dropwise slowly that the
temperature was kept below 40 to
60 ◦C. The reaction mixture was warmed to 50 ◦C and stirred
−
60 ◦C with vigorous stirring
−
−
−
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), dried over Na2SO4, filtered, and concentrated. To the residue CH2Cl2 (100 mL) was added
×
200 mL). The organic extracts were washed with brine
and the solid formed was collected by filtration and washed with CH2Cl2 (50 mL) to give
yield) as a brown solid. H-NMR (400 MHz CDCl3): δ 8.61–8.91 (m, 1 H) 7.93 (d, J = 1.6 Hz, 1 H) 7.43 (t,
J = 2.4 Hz, 1 H) 6.78 (t, J = 2.4 Hz, 1 H).
2
(8 g, 27%
1

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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):
J = 3.2 Hz, 1 H), 6.83 (d, J = 2.8 Hz, 1 H).
δ 12.75 (br. s., 1 H), 8.27 (d, J=1.2 Hz, 1 H), 7.86 (d,
General Procedure for the Preparation of 4
A mixture of
for 16 h. TLC (petroleum ether: ethyl acetate = 2:1, Rf = 0.01) showed that the reaction was complete.
3
(800 mg, 4.96 mMol) in MeOH (10 mL) and conc. HCl (10 mL) was stirred at 90 ◦C
The organic solvent was evaporated, and the precipitate was filtered off and dried to give
4
(500 mg,
1
56% yield) as a brown solid. H-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,
1
83% yield) as a white solid. H-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

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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-ium chloride (566 mg, 3.83 mMol). Then, 5a (523 mg, 3.83 mMol) was
◦
added slowly to the solution, and the mixture was stirred at 25 C for 15 h. TLC (ethyl acetate:
petroleum ether = 2:1, Rf = 0.01) showed that the conversion was more than 50% and LCMS showed the
desired mass of the product. Water was added (20 mL) slowly to the mixture at 0 ◦C. The precipitate
was filtered off, and dried to give 6 (200 mg, crude) 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
HATU (287 mg, 754 mMol). After stirred at 25 ◦C for 30 min, 6a (150 mg, 754 mMol) was added and the
mixture was stirred at 25 ◦C for 16 h. TLC (petroleum ether: ethyl acetate = 0:1, Rf = 0.3) showed that
the reaction was complete and LCMS showed the desired mass of the product. The mixture was diluted
with EtOAc (20 mL), washed with water (20 mL), brine (20 mL), dried over Na2SO4, and concentrated
to give 7 (300 mg, crude) as a yellow solid.
General Procedure for the Preparation of 8
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. H-NMR: ET6983-14-P1B (400 MHz
1
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. s.,
1 H), 1.75–2.00 (m, 2 H).

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3.2.2. The Synthesis of SC50
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
(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 h, then ethyl 2-chloro-2-oxo-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 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 with brine (30 mL
×
2mL), dried over Na2SO4, filtered and concentrated in vacuum to give
9
(3 g, 68% yield) as a brown
1
solid. H-NMR: ET6822-13-P1A (400 MHz CDCl3):
4.24–4.35 (m, 2 H), 1.23–1.36 (m, 3 H).
δ 8.56 (s, 1 H), 8.40 (s, 1 H), 7.88 (d, J = 2.4 Hz, 1 H),
General Procedure for the Preparation of 10
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
1
solid. H-NMR: ET6822-14-P1A (400 MHz DMSO-d6):
J = 2.4 Hz, 1 H).
δ 13.35 (br. s., 1 H), 8.47–8.65 (m, 1 H), 8.12 (d,
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 min,
then 6a (745 mg, 3.7 mMol) was added in portions and the mixture was stirred for 5 h. TLC (petroleum
ether:ethyl acetate = 1:1, Rf = 0.6.) showed one main spot was generated. The mixture was poured

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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 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 = 1:1) to
give 11 (2.0 g, 68% yield) as a yellow solid.
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
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.
General Procedure for the Preparation of 13
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%
1
yield) as a yellow solid. H-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 = 0.4)
showed reaction completion, LCMS showed the desired product was formed. After cooling to 25 ◦C,
ethyl acetate (30 mL) was added and combined and concentrated under reduce pressure. The residue

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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):
8.96 (d, J =1.6 Hz, 1 H), 8.91 (s, 1 H), 8.68 (d,
δ
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).
3.2.3. Synthesis of SC52
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-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 min. The reaction was
then 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
reduced pressure. The residue was purified by column 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
[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, and filtered
to give the desired product 17 (22 g, 62.9% yield) as a white solid. LC-MS (ESI): m/z [M + 1]+ = 192.33.

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Preparation of 6-Allyl-5,7-dichloropyrazolo[1,5-a]pyrimidine (18)
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%
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.
Preparation of 6-Allyl-5-chloro-N-(4-methoxybenzyl)pyrazolo[1,5-a] pyrimidin-7-amine (19)
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), 6.89–6.86 (m,
2H), 6.73 (d, J = 2.4 Hz, 1H), 6.71 (d, J = 3.6 Hz, 1H), 5.94 (s, 2H), 3.70 (s, 3H).

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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)
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.

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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)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 (2 mL) was added. The resulting mixture was stirred at room temperature for 1 h. The reaction
mixture was acidified to pH~5 with 1N aqueous HCI solution and then diluted with EtOAc (20 mL)
and H2O (20 mL), extracted with EtOAc (30 mL
×
2). The combined organic phases were dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by column
chromategraphy (silica gel, 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.
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,
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).
3.2.4. The Synthesis of SC55
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)

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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 add◦ed
40 C
dropwise to a solution of 1 M vinylmagnesium bromide in THF (182 mL, 182 mMol) at
−
(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 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 brine
(2 × 200 mL) and water (200 mL), dried (Na2SO4); concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (petroleum spirit/ethyl acetate = 3:1, v/v) to give
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)
To a mixture of 4-fluoro-1H-indole-7-carbonitrile (900 mg, 5.6 mMol) in DCM (15 mL) was
added methyl-2-chloro-2-oxoacetate (2.84 g, 8.76 mMol), then the reaction mixture was cooled to 0 ◦C,
AlCl3 (1.5 g, 11.2 mMol) was added in portions, then the reaction mixture was stirred at 0 ◦C for 30
min.Then reaction mixture was quenched with MeOH and extracted with ethyl acetate (3
×
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
spirit/ethyl acetate = 3:1, v/v) to give methyl 2-(7-cyano-4-fluoro-1H-indol-3-yl)-2-oxoacetate (660 mg,
48% yield). LC-MS (ESI): m/z [M + 1]+ = 247.44.

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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 h, 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)
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-hydroxy-carbamimidoyl)
-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
acetate = 3:1, v/v) to give 31 (315 mg, 55% yield). LC-MS (ESI): m/z [M + 1]+ = 450.63.

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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 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-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).
3.2.5. 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
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
−
40 ◦C. The reaction mixture was sealed
−
×
organic layers were washed with brine (50 mL) and H2O (50 mL), dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure. The residue was purified by silica gel column

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(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)
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
methyl oxalyl chloride (3.6 g, 19.7 mMol) in DCM (30 mL) slowly at 0 ◦C. The reaction mixture was
stirred for 30 h and then quenched with saturated NaHCO3 solution (10 mL), and extracted with DCM
(30 mL). The organic phase was washed with brine (10 mL) and H2O (10 mL), 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 36 as a yellow solid (2.2 g, Y = 43.1%). LC-MS
(ESI): m/z [M + 1]+ = 259.09.
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 (12.8 mL) and extracted with DCM (20 mL). The organic phase was concentrated under reduced
pressure to give 37 as a white solid (630 mg, yield = 30.3%). LC-MS (ESI): m/z [M + 1]+ = 245.09.

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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)
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
partitioned between DCM and water. The organic layer was separated, and the aqueous layer was
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)

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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 h, 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,
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
g/mL G418 (Thermo Scientific,
U/mL penicillin, 100 g/mL streptomycin and 2 mM l-glutamine, 300
µ
µ
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

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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
via sonication. The lysed sample was spun down at 45,000 rpm, the clarified supernatant was filtered
through a 0.45 m filter, and immediately applied to a Talon cobalt resin affinity column (Clonetech
×
PBS and lysed
µ
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
Vector 1 is an envelope-deficient HIV-1 pNL4-3-Luc+R-E plasmid which carries the luciferase-reporter
×
106 cells/well) [41].
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 L/well of luciferase assay substrate (Promega), a GloMax
µ
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
tissue culture plates (Olympus Plastics, San Diego, CA, USA). After 24 h, cells were treated with
×
104 cells/well) were seeded in 96-well

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compound (0.1–1000
µ
M) or DMSO (Sigma) for 48 h at 37 ◦C. Subsequently, 10
µ
L 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
sulfo-N-hydroxysuccinimide (0.05 M). Immediately after chip activation, 100
gp140 B41.SOSIP.664 trimers in 10 mM sodium acetate, pH 5.0 was injected across ligand flow channels
for 15 min at a flow rate of 25
L min−1. A 5-min injection of 1 M ethanolamine HCl (pH 8.5) was then
µ
g mL−1 soluble cleaved
µ
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
across the surfaces at a flow rate of 100 µ
L min−1, 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
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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
total dose of ~25 e−/Å2 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
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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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