Composition and orientation of the core region of novel HIV-1 entry inhibitors influences metabolic stability

Article abstract of DOI:10.3390/molecules25061430

Fostemsavir/temsavir is an investigational HIV-1 entry inhibitor currently in late-stage clinical trials. Although it holds promise to be a first-in-class Env-targeted entry inhibitor for the clinic, issues with bioavailability relegate its use to salvage

Full text of DOI:10.3390/molecules25061430

molecules
Communication
Composition and Orientation of the Core Region of
Novel HIV-1 Entry Inhibitors Influences
Metabolic Stability
Rama Karadsheh †, Megan E. Meuser † and Simon Cocklin *
Department of Biochemistry & Molecular Biology, Drexel University College of Medicine, Rooms 10307, 10309,
and 10315, 245 North 15th Street, Philadelphia, PA 19102, USA; rk885@drexel.edu (R.K.);
mem484@drexel.edu (M.E.M.)
* Correspondence: sc349@drexel.edu; Tel.: +1-215-762-7234 or +1-215-762-4979; Fax: 215-762-4452
† These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Antonio Carta
Received: 28 February 2020; Accepted: 19 March 2020; Published: 21 March 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Fostemsavir/temsavir is an investigational HIV-1 entry inhibitor currently in late-stage
clinical trials. Although it holds promise to be a first-in-class Env-targeted entry inhibitor for the clinic,
issues with bioavailability relegate its use to salvage therapies only. As such, the development of
a small molecule HIV-1 entry inhibitor that can be used in standard combination antiretroviral therapy
(cART) remains a longstanding goal for the field. We previously demonstrated the ability of extending
the chemotypes available to this class of inhibitor as the first step towards this overarching goal. In
addition to poor solubility, metabolic stability is a crucial determinant of bioavailability. Therefore,
in this short communication, we assess the metabolic stabilities of five of our novel chemotype
entry inhibitors. We found that changing the piperazine core region of temsavir alters the stability
of the compound in human liver microsome assays. Moreover, we identified an entry inhibitor
with more than twice the metabolic stability of temsavir and demonstrated that the orientation of
the core replacement is critical for this increase. This work further demonstrates the feasibility of
our long-term goal—to design an entry inhibitor with improved drug-like qualities—and warrants
expanded studies to achieve this.
Keywords: HIV-1 entry inhibitor; metabolic stability; docking; antiviral; surface plasmon resonance;
Cyp P450
1. Introduction
The HIV-1 Env glycoprotein complex on the surface of a virion orchestrates the two sequential
and specific binding events that allow for entry into susceptible cells. Env is a heterotrimer of gp41
and gp120 subunits that undergo the conformational changes, prompted by receptor engagement,
necessary for the fusion of viral and cellular membranes. As Env is the primary determinant of
viral infectivity, it is an attractive target for both pre- and post-exposure prophylaxis and is the focal
immunogen of the search for a viable vaccine. Two entry inhibitors are in use in the clinic (enfuvirtide
and maraviroc) but are only suitable for salvage therapies. Thus, a well-tolerated, orally bioavailable,
and virally-targeted entry inhibitor that can treat the majority of isolates of the virus has been actively
sought for many years.
The most promising entry inhibitor class has been developed by Bristol-Myers Squibb. The initial
compound, BMS-378806, and subsequent iterations, share a piperazine core chemotype and target the
HIV-1 Env gp120 subunit [
1]. The most promising analog from this class of inhibitors, BMS-626529
((ViiV Healthcare: Temsavir), is potent against many of the major subtypes of HIV-1, but unfortunately
Molecules 2020, 25, 1430; doi:10.3390/molecules25061430
www.mdpi.com/journal/molecules

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suffers from poor bioavailability due to its low solubility. However, BMS-663068 (ViiV Healthcare:
Fostemsavir), a phosphooxymethyl prodrug of temsavir, has performed favorably in clinical trials
and is slated for release late 2020 [2]. Unfortunately, due to suboptimal solubility after cleavage
of the prodrug, and breadth issues against specific subtypes of HIV-1, this entry inhibitor is only
recommended for treatment-experienced patients with limited therapeutic options.
Our group has been actively exploring the bioisosteric modification of the piperazine-based
entry inhibitors, as exemplified by temsavir/fostemsavir. This is part of an overarching effort to
improve upon their drug-like properties and breadth and to hopefully bring them closer to use in
standard combination antiretroviral therapy (cART) [3–5]. We have performed proof-of-concept studies
demonstrating that we can scaffold-hop from the piperazine chemotype while retaining nanomolar
inhibitory profiles and retaining target engagement. However, before this study, we were yet to
investigate whether these initial new chemotypes have any improved drug-like qualities. Therefore, in
this short communication, we investigate whether any of our novel chemotypes have any improved
ADME (absorption, distribution, metabolism, and excretion) properties using both computational and
experimental methods. Moreover, in the course of this study, we establish a computational workflow
that can be used to predict the metabolic stability of next-gen compounds, thus further streamlining
future design processes.
2. Results and Discussion
We have previously successfully used field-based three-dimensional similarity virtual screening
experiments, in combination with bioisosteric replacements, to identify novel scaffolds that could
function as entry inhibitors [3,4]. These studies resulted in the discovery of compounds SC11
(dipyrrolidine), SC12 (pyrrolo-pyrazole), SC15 (azetidine), and SC28 (azabicyclo-hexane). These
compounds display IC50 values in the 2–40 nM range against HIV-1B41 Env pseudotyped HIV-1
(Figure 1). In this study, we introduce two additional compounds, SC46 and SC54, which have
a reversed azabicyclo-hexane and a dimethyl-piperazine core, respectively. These new compounds also
have inhibitory effects on HIV-1B41 Env pseudotyped HIV-1 in the low nanomolar range (Figure 1).
Figure 1. Chemical structures of the compounds used in this study. Half maximal inhibitory
concentrations (IC50) against HIV-1B41 Env pseudotyped HIV-1 are given. Structural differences in the
core regions are highlighted in blue. The chemical structures were drawn with ChemAxon software
(Budapest, Hungary).

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Moreover, all of these compounds (SC11
,
SC12
,
SC15
,
SC28
,
SC46, and SC54), as well as
temsavir/BMS-626529, interact with the SOSIP recombinant Env trimer derived from the B41 isolate [
with different kinetics (Figure 2).
6]
Figure 2. Sensorgrams depicting the interaction of (A) SC46 and (B) SC54 with recombinant trimeric
HIV-1B41 SOSIP Env. Colored lines represent the raw data of the different concentrations of compound
used, and the black lines represent the fit that was derived using a 1:1 binding model. A table showing
the interaction parameters for all compounds used in this study is also provided.
Having demonstrated antiviral activity and target engagement for the previously published and
new compounds, we next analyzed the predicted ADME properties of the compounds and compared
them with those of the temsavir/BMS-626529. To accomplish this comparison, we performed in silico
prediction of drug-like metrics of the compounds as implemented in the oral non-central nervous
system (CNS) drug profile in StarDrop 6.6 (Optibrium, Ltd., Cambridge, UK) [
described this drug profile and component models in Tuyishime et al. [ ], and additional details
are online at the StarDrop FAQs (http://www.optibrium.com/community/faq/adme-qsar-models).
A probabilistic scoring algorithm [ ] is used to combine all of the component model predictions in the
7]. We have previously
3
8
oral non-CNS drug profile into an overall score, with scores range from 0 to 1 (0 suggesting extremely
non-drug-like, and 1 indicating the perfect drug). A plot of this metric versus aqueous solubility
(logS) is shown in Figure 3. As can be seen, none of the new chemotypes display improved solubility
compared to temsavir/BMS-626529. However, SC12 had an overall improved oral non-CNS drug
profile score, primarily due to improved scoring against the human intestinal absorption model.

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Figure 3. Plot showing the StarDrop 6.6 (Optibrium, Ltd., Cambridge, UK)-derived logS versus the
score from a multimetric oral non-CNS profile for the BMS and SC compounds used in this study.
As the bioavailability of temsavir/BMS-626529 is a primary reason for its use only in salvage
therapies and none of our first-generation chemotypes displayed improved predicted aqueous solubility,
a primary determinant of bioavailability, we chose to investigate further. Orally administered drugs
must pass through the intestinal wall and then the portal circulation to the liver, with both sites being
common regions of first-pass metabolism. As such, many drugs may be adversely metabolized before
adequate plasma concentrations are reached. Therefore, we next sought to computationally investigate
whether or not our compounds had improved predicted metabolic stability. We performed this analysis
using the P450 module in StarDrop 6.6 (Optibrium, Ltd., Cambridge, UK). We first predicted the
major metabolizing cytochrome P450 isoforms for each compound using the WhichP450
™ model [9].
The results of this analysis are shown in Figure 4.
Figure 4. Prediction of the major metabolizing CYP isoform for the compounds in this study. Prediction
was achieved using the StarDrop (version 6.6) P450 module. BMS-626529 was also predicted to be
predominantly metabolized via the 3A4 isoform.
All of the SC compounds are predicted to primarily be metabolized by CYP3A4. Temsavir/BMS-
626529 was also predicted to be metabolized by CYP3A4, which has been determined to be the primary

Molecules 2020, 25, 1430
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metabolizing isoform [10] As such, we next analyzed the predicted metabolic lability of each of the
compounds against CYP3A4 by comparing the overall composite site lability (CSL) score and the
number of labile sites [11]. The CSL score is a metric that reflects the overall efficiency of metabolism
of the molecule and is calculated by combining the labilities of individual sites within the compound.
Table 1 shows the CSL scores for CYP3A4 for all of the SC compounds and temsavir/BMS-626529. As
can be seen, the differences between the CSL scores of the compounds are minor; there is no significant
difference between the numbers of predicted labile sites. Such small changes in the CSL scores are not to
be expected to be correlated with appreciable differences in experimental CYP3A4 half-life or intrinsic
clearance [11]. This is because other factors that govern the rate of metabolism, such as reduction
rates and whether or not the compound binds to the enzyme. Therefore, a primary assumption
in this analysis is that all of the compounds bind to CYP3A4 with similar binding affinity, which
itself is influenced by compound properties such as size and lipophilicity. We, therefore, performed
predictive binding affinity calculations using the hydrogen bond and dehydration (HYDE) energy
scoring function in SeeSAR 9.2 (BioSolveIT Gmbh, Germany) [12]. For this analysis, we used the
structure of the human CYPA4 bound to an inhibitor (PDB ID 4D78) [13]. The HYDE scoring function
in SeeSAR provides a range of affinities, spanning an upper and lower limit. We, therefore, used the
lower limit as the affinity parameter to compare between molecules. Table 1 shows the affinity values
derived from this analysis. Unlike the previous analyses, the prediction of CYP3A4 binding resulted in
more considerable differences between compounds, with the affinities being predicted in the range of
0.8–800 mM. Combining the results from all of these predictions (CSL scores, labile sites, and predicted
CYP3A4 affinity), it would appear that compound SC28 and its core-reversed analog SC46 should
have greater metabolic stability than the other compounds. Additionally, based just upon the number
of labile sites, SC12 should have the lowest metabolic stability.
Table 1. Composite site lability (CSL) scores, number of labile sites, and predicted CYP3A4 affinities
of compounds used in this study. CSL values and the number of labile sites were determined using
StarDrop (version 6.6). Affinity to CYP3A4 was predicted using SeeSAR (version 9.2).
Compound
CSL 3A4
Labile Sites
Predicted 3A4 Affinity (M)
SC11
SC12
SC15
SC28
SC46
0.9514
0.9627
0.9564
0.9516
0.9474
0.9396
0.9416
3
4
3
2
3
3
3
0.004849324
0.083703353
0.003455543
0.767515588
0.166818977
0.006416669
0.000802
SC54
BMS-626529
Armed with our prediction that SC28 and SC46 may have increased metabolic stability compared
to temsavir/BMS-626529 and the other compounds, we next performed metabolic stability assays
using human liver microsomes. Metabolic stability analysis was performed as outlined in Lu et al. [14]
and as described explicitly in Materials and Methods. Compounds testosterone (low stability),
propranolol (medium stability), and warfarin (high stability) were included as in-line controls. Table 2
shows the metabolic stabilities of the compounds used in this study, along with derived predictive
pharmacokinetic (PK) parameters. As can be seen, the compounds have a range of stabilities, as
1
indicated by their respective half-lives (T /2). As predicted from the sheer number of labile sites,
1
SC12 had the shortest half-life of just over 6 /2 minutes. No clear correlation with any of the other
potentially predictive parameters could be gauged for compounds BMS-626529, SC11, SC15, and SC54.
However, rather pleasingly, compounds SC28 and SC46 had the greatest half-lives of 144.6 and
91.2 min, respectively, demonstrating a clear correlation with their predicted affinity to CYP3A4.

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Table 2. Metabolic stabilities of control and test compounds in a human liver microsome assay.
Predictive PK parameters were derived as outlined in Materials and Methods.
T1/2
Clint
Clapp
Clh
Eh
Compound
ID
k
(min)
(mL/min/mg) (mL/min/kg) (mL/min/kg)
(%)
Testosterone
Propranolol
Warfarin
SC11
0.05134
0.01305
0.00352
0.07253
0.105828
0.05268
0.00479
0.00760
0.022234
0.010075
13.5
53.1
196.6
9.6
6.548348
13.2
144.6
91.2
31.16817
68.78436
0.1027
0.0261
0.0070
99.005
25.170
6.797
16.639
11.145
5.073
83.19
55.72
25.37
87.49
91.0753
83.55
31.61
42.28
68.19353
49.27757
0.1451
139.887
204.0972779
101.588
9.245
14.657
42.8802911
19.43028912
17.498
SC12
SC15
SC28
SC46
0.211656436
0.1054
0.0096
0.0152
0.04446845
0.020149929
18.21506087
16.710
6.322
8.455
13.63870629
9.855514403
SC54
BMS-626529
3. Materials and Methods
3.1. Compounds
BMS-626529 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). SC11 was
synthesized following the procedures described in Tuyishime et al. [ ]. SC12 SC15, and SC28 were
]. Compounds SC46 and SC54 were synthesized de
3
,
synthesized as described in Tuyishime et al. [
novo, as outlined below.
4
3.1.1. Synthesis of SC46
General procedure for preparation of Compound 2
To a solution of Compound
(11.00 g, 132.39 mmol) at 110 ◦C was added Compound
27.38 mmol) (Scheme 1). The mixture was stirred at 140 ◦C for 16 hours under N2. TLC (petroleum
ether/ethyl acetate = 1:1, Rf = 0.3) indicated 30% of Reactant 1 remained, and one major new spot with
larger polarity was detected. The residue was purified by column chromatography (SiO2, petroleum
A
1 (5.00 g,
≈
ether/ethyl acetate = 2/1 to 1:1) to give Compound
2 (3.00 g, 13.09 mmol, 47.80% yield) as a white solid.
1H-NMR (ET5676-11-P1B, 400 MHz, CDCl3): 10.25 (br. s., 1 H), 9.08 (s, 1 H), 7.56 (s, 1 H), 7.38 (t, J =
2.8 Hz, 1 H), 6.71 (t, J = 2.6 Hz, 1 H), 4.04 (s, 3 H), 2.54 (s, 3 H).
Scheme 1. Preparation of Compound 2.
General Procedure for Preparation of Compound 3
A mixture of Compound
(Scheme 2). EtMgBr (3 M, 15.02 mL) was added dropwise at
2
(2.80 g, 12.21 mmol) in 2-MeTHF (1◦20.00 mL) was cooled to
−
10 ◦C
−
10 C, followed by the addition of
pyridine (500.00 mg, 6.32 mmol). The mixture was cooled to
51.28 mmol) was added. The mixture was stirred at 25 ◦C for 48 h under N2. TLC (petroleum ether/ethyl
−
45 ◦C, and then Compound
B
(7.00 g,
acetate = 1/1, Rf = 0.2) indicated
polarity was detected. The mixture was quenched with H2O (250 mL), and extracted with ethyl acetate
(200 mL 3). The organic was dried over Na2SO4 and concentrated to give the residue. The residue
≈
40% of Reactant 1 remained, and one major new spot with larger
×
was purified by column chromatography (SiO2, petroleum ether/ethyl acetate = 2:1 to 1:1) to give
1
Compound
3
(2.00 g, 6.07 mmol, 49.74% yield) as a yellow solid. H-NMR (ET5676-35-P1A, 400 MHz,

Molecules 2020, 25, 1430
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CDCl3): 10.93 (br. s., 1 H), 9.04 (s, 1 H), 8.24–8.31 (m, 1 H), 7.68 (s, 1 H), 4.35 (q, J = 7.0 Hz, 2 H),
3.94–4.00 (m, 3 H), 2.49 (s, 3 H), 1.34 (t, J = 7.2 Hz, 3 H).
Scheme 2. Preparation of Compound 3.
General Procedure for Preparation of Compound 3-1
A mixture of Compound
3
(1.00 g, 3.04 mmol) K2CO3 (1 M, 15.34 mL) in MeOH (15.00 mL) was
◦
stirred at 20 C for 1 h (Scheme 3). TLC (petroleum ether/ethyl acetate = 1/1,Rf = 0.01) indicated
Reactant 1 was consumed completely and one new spot formed. The reaction was clean, according to
TLC. The MeOH was concentrated and diluted with H2O (150 mL). The mixture was extracted with
ethyl acetate (100 mL
×
2). The aqueous phase was acidified with HCl to pH = 1, and the solid was
filtered and dried to give a residue. The residue was dried to give Compound
3
-1
(400.00 mg, crude) as
1
a yellow solid. H-NMR (ET5676-38-P1B, 400 MHz, DMSO-d6): 12.40 (br. s., 1 H), 9.20 (s, 1 H), 8.26 (d,
J = 3.6 Hz, 1 H), 7.84 (s, 1 H), 3.94 (s, 1 H), 3.13 (s, 1 H).
Scheme 3. Preparation of Compound 3-1.
General Procedure for Preparation of Compound 5
To a mixture of Compound
4 (1.00 g, 5.04 mmol) and TEA (1.00 g, 9.88 mmol) was added benzoyl
chloride (1.00 g, 7.11 mmol) dropwise at 0 ◦C (Scheme 4). The mixture was stirred at 25 ◦C for 16 h.
TLC (petroleum ether/ethyl acetate = 2/1, Rf = 0.3) indicated Reactant 1 was consumed completely,
and one new spot formed. The reaction was clean, according to TLC. The mixture was quenched
with H2O (50 mL), and extracted with ethyl acetate (50 Ml
and concentrated to give the residue. The residue was purified by column chromatography (SiO2,
petroleum ether/ethyl acetate = 2/1 to 1:1) to give Compound
×
3). The organic was dried over Na2SO4
1
5
(1.40 g, crude) as a white solid. H-NMR
(ET5676-28-P1A, 400 MHz, CDCl3): 8.17 (s, 1 H), 7.33–7.39 (m, 2 H), 7.28–7.32 (m, 3 H), 7.20–7.25 (m,
1 H), 6.99 (s, 1 H), 6.95 (s, 1 H), 6.82 (s, 1 H), 6.79 (s, 1 H), 5.42 (s, 2 H), 4.26 (s, 5 H), 3.78 (s, 3 H).
Scheme 4. Preparation of Compound 5.

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General Procedure for Preparation of Compound 6
A solution of Compound
(1.40 g, 4.63 mmol) in HCl/EtOAc (30.00 mL, 4M) was stirred at 25 ◦C
5
for 3 h (Scheme 5). The mixture was concentrated to give Compound
6 (900.00 mg, 3.77 mmol, 81.43%
1
yield, HCl) as a white solid. H-NMR (ET5676-37-P1A, 400 MHz, DMSO-d6): 9.73 (br. s., 1 H), 9.15
(br. s., 1 H), 8.60 (d, J = 4.0 Hz, 1 H), 7.80 (d, J = 7.2 Hz, 2 H), 7.47–7.54 (m, 1 H), 7.38–7.46 (m, 2 H),
3.25–3.39 (m, 4 H), 2.99 (d, J = 2.8 Hz, 1 H), 1.98 (br. s., 2 H).
Scheme 5. Preparation of Compound 6.
General Procedure for Preparation of Compound SC46
A mixture of Compound 3-1 (700.00 mg, 2.32 mmol), Compound
6 (610.00 mg, 2.56 mmol), DIEA
(900.00 mg, 6.96 mmol) and HATU (1.32 g, 3.48 mmol) in DMF (25.00 mL) was stirred at 28 ◦C for 15 h
under N2 atmosphere (Scheme 6). LC-MS showed Reactant 1 was consumed completely, and one peak
with desired MS was detected. The mixture was poured into H2O (150 mL), and extracted with DCM
(100 mL
×
3). The mixture was dried over Na2SO4 and concentrated to give a residue. The residue
was purified by prep-HPLC (basic condition) to give SC46 (30.00 mg, 2.65% yield) as a white solid.
1H-NMR (ET5676-52-P1B, 400 MHz, DMSO-d6): 12.38 (br. s., 1 H), 9.21 (s, 1 H), 8.56 (d, J = 4.0 Hz, 1 H),
8.22 (s, 1 H), 7.87 (s, 1 H), 7.79 (d, J = 7.2 Hz, 2 H), 7.47–7.53 (m, 1 H), 7.37–7.46 (m, 2 H), 3.99 (s, 3 H),
3.89 (d, J = 11.6 Hz, 1 H), 3.63–3.70 (m, 1 H), 3.59 (d, J = 10.8 Hz, 2 H), 2.64 (br. s., 1 H), 2.52 (d, J =
2.4 Hz, 3 H), 1.92 (br. s., 2 H).
Scheme 6. Preparation of Compound SC46.
3.1.2. Synthesis of SC54
General Procedure for the Preparation of Compound 7
To a mixture of 2,6-dimethylpiperazine (Compound C, 1.0 g, 8.8 mmol) in DCM (10 mL) was
added (Boc)2O (1.9 g, 8.8 mmol) at RT (Scheme 7). After stirred at RT overnight, the reaction solvents
were removed under reduced pressure to give crude tert-butyl 3,5-dimethylpiperazine-1-carboxylate
(Compound
7) as a yellow oil (1.8 g, Y = 96%), which was used in the next step without purification.
LC-MS (ESI): m/z (M + 1)+ = 215.10.

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Scheme 7. Preparation of Compound 7.
General Procedure for the Preparation of Compound 8
To a mixture of Compound (1.0 g, 4.7 mmol) in DCM (10 mL) was added benzoyl chloride
(787.0 mg, 5.6 mmol) and TEA (944.0 mg, 9.3 mmol) at RT (Scheme 8). After stirred at RT for
1 h, the reaction mixture was diluted with DCM (10 mL) and washed with brine (10 mL 3).
7
×
The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by silica gel column chromatography (PE/EA = 3:1, v/v) to give
tert-butyl 4-benzoyl-3,5-dimethylpiperazine-1-carboxylate (Compound 8) as brown solid (1.4 g, Y =
94.0%). LC-MS (ESI): m/z (M + 1)+ = 319.17.
Scheme 8. Preparation of Compound 8.
General Procedure for the Preparation of Compound 9
A mixture of Compound
at RT for 1h (Scheme 9). The reaction solvents were removed in vacuo to give crude (2,
6-dimethylpiperazin-1-yl)(phenyl) methanone (Compound ) as a brown solid (1.1 g), which was used
in the next step without purification. LC-MS (ESI): m/z (M + 1)+ = 219.39.
8 (1.0 g, 8.8 mmol) in HCl/dioxane (4 M, 10 mL) was stirred
9
H
N
Boc
N
HCl, dioxane (4 M)
RT, 1 h
N
9
N
8
O
O
Scheme 9. Preparation of Compound 9.
General Procedure for the Preparation of Compound SC54
To a solution of Compound 3-1 (28.0 mg, 0.09 mmol) in DMF (1 mL) was added
(2,6-dimethylpiperazin-1-yl)(phenyl) methanone (Compound
9, 24.0 mg, 0.11 mmol), HATU (42.4 mg,
0.11 mmol), and DIPEA (24.0 mg, 0.19 mmol) at 0 ◦C (Scheme 10). After the reaction mixture was

Molecules 2020, 25, 1430
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stirred at RT for 1 h, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with brine.
The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure.
The residue was purified by prep-HPLC (C18, 40–100% MeCN in H2O with 0.1% formic acid) to give
1-(4-benzoyl-3,5-dimethylpiperazin-1-yl)-2-(4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo
[2,3-c]pyridin-3-yl)ethane-1,2-dione as white solid (Compound SC54, 11.0 mg, Y = 24.0%). LC-MS
(ESI): m/z (M + 1)+ = 502.22; 1H-NMR (400 MHz, CDCl3)
δ 11.04 (br. s, 1H), 9.11 (s, 1H), 8.25 (d, J =
3.2 Hz, 1H), 7.77 (s, 1H), 7.44–7.42 (m, 3H), 7.38–7.36 (m, 2H), 4.49–4.46 (m, 3H), 4.06 (s, 3H), 3.78–3.75
(m, 1H), 3.41–3.37 (m, 1H), 3.09–3.05 (m, 1H), 2.56 (s, 3H), 1.42–1.39 (m, 6H).
Scheme 10. Preparation of Compound SC54.
3.2. Cells
HEK293T cells (gift from Dr. Irwin Chaiken, Drexel University, Philadelphia, PA, USA) were
cultured in Dulbecco’s Modified Eagle’s Medium (DMEM). The medium was supplemented with
10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM l-glutamine. Human astroglioma
U87 cells (obtained from Prof. Hongkui Deng, Peking University, and Prof. Dan Littman, New York
University, USA, through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID,
NIH) [15
10% FBS, 100 U/mL penicillin, 100
,
16] express CD4 and CCR5 receptors. U87 cells were cultured in DMEM, supplemented with
g/mL streptomycin, 2 mM l-glutamine, 300 g/mL G418 (Thermo
µ
µ
Scientific, Waltham, MA, USA), and 1 µg/mL Puromycin (Thermo Scientific). Both cell lines were
incubated continuously at 37 ◦C, ensuring a humidified 5% CO2 environment.
3.3. Proteins
The recombinant, truncated trimer B41 SOSIP.664 gp140 was expressed in HEK293F cells.
Post-expression, the soluble trimer was purified by mAb 2G12-affinity chromatography followed by
size-exclusion chromatography as described previously [6]. Through NIH AIDS Reagent Program,
Division of AIDS, NIAID, NIH, the recombinant human antibody IgG b12 anti-HIV-1 gp120 was
acquired. The monoclonal form of the anti-HIV-1 gp120 antibody, IgG1 b12, was obtained from
Dr. Dennis Burton and Carlos Barbas. As previously described, p24 was produced in-house [17].
A vector containing His-tagged HIV-1NL4-3CA on the C-terminus (a gift from Dr. Eric Barklis, Oregon
Health and Science University, Portland, OR, USA) was transformed into BL21-Codon Plus (DE3)-RIL
Competent Cells (Agilent Technologies, Wilmington, DE, USA). The vector expressed in autoinduction
ZYP-5052 medium at 30 ◦C, and remained shaking overnight at 250 rpm [18]. Bacterial cultures were
spun down at 8000 rpm. After discarding the supernatant, the remaining pellet was resuspended using
1
×
PBS. The resuspension was lysed by sonication and spun down at 45,000 rpm. The supernatant
was filtered through a 0.45 m filter, and promptly applied to a Talon cobalt resin affinity column
(Clonetech Laboratories, Mountain View, CA, USA). Elution of bound protein was accomplished
using 1 PBS with 250 mM imidazole. The elutions comprising of purified CA-H6 were merged and
µ
×

Molecules 2020, 25, 1430
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dialyzed overnight into 20 mM Tris-HCl pH 8.0. Subsequently, these were concentrated to 120 µM,
flash-frozen, and stored at −80 ◦C.
3.4. Production of Pseudotyped Viruses
HEK293T cells were plated in 6-well plates with density of 1
×
106 cells/well. After 24 h, the two
vectors (vector 1 and vector 2) were co-transfected in a 3:4 ratio, respectively [16]. Vector 1 is an HIV-1
pNL4-3-Luc+R-E plasmid with two main features. The vector contains a premature stop codon in the
Env gene and replaces HIV-1 Nef gene with the luciferase-reporter gene [19]. Vector 2 is a plasmid
expressing the HIV-1B41 gp160 Env [20]. Transient transfections of these vectors were carried out via
calcium phosphate (ProFection Mammalian Transfection System, Promega, Madison, WI) for 5 h in
DMEM without supplements. After the 5 h incubation period, the medium was replaced with fresh
culture media. Seventy-two hours post-transfection, virus was collected by filtering supernatants.
The result was single-round infectious virus with pseudotyped envelopes that contained a luciferase
reporter [16]. This was aliquoted and stored at −80 ◦C.
3.5. ELISA-Based Quantification of p24 Content
Mouse anti-p24 (Abcam, ab9071) coated an ELISA plate (50 ng/well) overnight at 4 ◦C. Following
the overnight incubation, the plate was blocked for 2 h with 3% (w/v) room temperature BSA 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 (expressed and purified as previously described) was used as a standard.
The next day, 0.5% PBST was used to wash the plate. Subsequently, rabbit anti-p24 (Abcam, ab63913)
was added in a 1:5000 dilution at room temperature. After 2 h, PBST was used to wash unbound
rabbit anti-p24. Next, goat anti-rabbit-HRP was added in a 1:5000 dilution at room temperature
for one hour. PBST was used to thoroughly wash the plate. Subsequently, a solution of 0.4 mg/mL
o-phenylenediamine in a phosphate-citrate buffer with sodium perborate (Sigma-Aldrich) was added.
This was incubated in the dark for 30 min. Using Multiskan™ GO Microplate Spectrophotometer
(Thermo Scientific), the optical densities were measured at 450 nm.
3.6. Single-Round Infection Assay
The single-round HIV-1 infection assay was performed as previously described [19
U87.CD4.CCR5 cells were seeded in Greiner Bio-one 96-well luminometer-compatible tissue culture
plates with a density of 1.2
104 cells/well. After 24 h, compounds underwent serial dilutions in DMEM.
DMSO was used as a vehicle control (Sigma). These dilutions were mixed with thawed pseudotyped
HIV-1 virus (normalized to p24 content) and added to the U87 cells 100 L/well. The plates were
incubated for 48 h at 37 ◦C. Post-incubation, the medium was aspirated from each well, and 50
L/well
of 1X luciferase lysis buffer (Promega) was added. The plates were placed at
80 ◦C for one freeze-thaw
cycle. After thawing, 50 L/well of the luciferase assay substrate (Promega) was added and the
,21,22].
×
µ
µ
−
µ
luciferase activity of each well was measured by a GloMax 96 microplate luminometer (Promega).
3.7. SPR Direct Interaction Analysis
3.7.1. Immobilization of Env Constructs
A ProteOn XPR36 SPR Protein Interaction Array System (Bio-Rad Laboratories, Hercules, CA,
USA) was used to analyze kinetic interactions of the compounds to the B41 SOSIP.664 gp140 at 25 ◦C.
First, the ProteOn GLH sensor chip was preconditioned. This was done with two 10 second pulses
of the following: 50 mM NaOH, 100 mM HCl, and 0.5% (w/v) sodium dodecyl sulfide. PBST buffer
(20 mM Na-phosphate, 150 mM NaCl, and 0.005% (v/v) polysorbate 20, pH 7.4) was then used to
equilibrate the system. The activation of the GLH sensor chip surface occurred by a 10 minute injection
with a 1:100 dilution of a 1:1 mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

Molecules 2020, 25, 1430
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(0.2 M) and sulfo-N-hydroxysuccinimide (0.05 M). Promptly following chip activation, 100 µg/mL
soluble gp140 B41.SOSIP.664 trimers in 10 mM sodium acetate (pH 4.5) were injected across ligand flow
channels for 15 min at a flow rate of 25 L/min. After immobilization of the soluble cleaved construct,
µ
excessive active ester groups were capped by a 5 min injection of 1 M ethanolamine HCl at a pH of 8.5.
This resulted in immobilized Env constructs at a density of 14,000 RUs (response unit, which is an
arbitrary unit that corresponds to 1 pg/mm2). As a control, IgG b12 in 10 mM sodium acetate (pH 4.5)
was used as a reference surface and matched the density of the immobilized SOSIP.
3.7.2. Direct Binding Analysis
The volume of compound stock solutions was increased to 30 µL with 100% DMSO. PBST pH
7.4 was added to the compound stocks to reach a final volume of 1 mL. This is required in order to
match the concentration of DMSO of the compound stocks to that of running buffer (3% DMSO). As
stated in the results, the starting concentrations of the serial dilutions were then prepared in running
buffer (PBS, 3% (v/v) DMSO, 0.005% (v/v) polysorbate 20, pH 7.4). These dilutions were injected to flow
across the surfaces at a rate of 100 µ
l min−1, for a 2-min association phase, followed by up to a 10-min
dissociation phase. The Proteon instrument’s "one shot kinetics" completed these phases with ease [23].
Data were analyzed using the ProteOn Manager Software version 3.0 (Bio-Rad). The responses of
a buffer injection and from the reference flow cell were subtracted to account for non-specific binding
and injection artifacts. Experimental data were fitted to a simple 1:1 binding model. The average
association [ka] and dissociation [kd] rates generated from 3–5 data sets were averaged and defined the
equilibrium dissociation constant (KD).
3.8. Metabolic Stability Evaluation of Compounds in Human Liver Microsomes.
Metabolic stability analysis was performed as outlined in Lu et al. [14].
Materials: Warfarin was purchased from Sigma (St. Louis, MO, USA). Testosterone was purchased
from Acros (Geel, Belgium). Propranolol was purchased from TRC (Toronto, Ont., Canada). NADPH
was purchased from Roche (Shanghai, China); all inorganic salts erre of analytical grade and were
purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). All organic solvents were of
HPLC grade and were purchased from Sigma-Aldrich, USA. Pooled human liver microsomes (HLM,
Batch # 452161) were purchased from BD Gentest Corporation (Woburn, MA, USA). Distilled water,
prepared from demineralized water, was used throughout the study.
Procedure:
Preparation of solutions. Test compound was weighed and dissolved in 100% DMSO to get
10 mM stock solution. The stock solution was diluted to 100 µM with mixture of acetonitrile and
H2O (1:1). The final concentrations of DMSO and acetonitrile were equal to or less than 0.1%. Stock
solutions of testosterone, warfarin, and propranolol were prepared to concentrations of 10 mM in 100%
DMSO, respectively. The stock solution for each compound was diluted into 100 µM with mixture
of acetonitrile and H2O (1:1). The final concentrations of DMSO and acetonitrile were equal or less
than 0.1%.
Microsomal incubation. Liver microsomes incubations were conducted in duplicate in 96-well
plates. Each well contained 40
0.625 mg/mL human liver microsomes, and test compound (1.25
preincubation at 37 ◦C, 10
L of 5.0 mM NADPH in 0.1M potassium phosphate buffer was added to
µL of 0.1M potassium phosphate buffer (pH 7.4), 4.125 mM MgCl2,
µM) or positive control. After a 5-min
µ
initiate the enzymatic reaction. The final component concentrations were 0.1 M potassium phosphate
buffer (pH 7.4); 1.0 mM NADPH; 3.3 mM MgCl2; 0.5 mg/mL human liver microsomes and test
compound (1.0
µM) or positive control (1.0
µM). Reactions were terminated at various time points (0, 5,
10, 20, 40 min) by adding 150
µL of ice-cold methanol and acetonitrile (1:1, v/v) containing the internal
standard. A parallel incubation was performed using 0.1 M potassium phosphate buffer (pH 7.4)
instead of NADPH as the negative control and reactions was terminated at 40 min after incubation
at 37 ◦C.

Molecules 2020, 25, 1430
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LC-MS/MS analysis: A Waters liquid chromatographic system was used. Chromatographic
separation of compounds was achieved using various ratios of buffer A (water, 0.1% formic acid)
and buffer B (acetonitrile, 0.1% formic acid) and an ACQUITY UPLC BEH C18 column (50
×
2.1 mm,
1.7
µ
m) at ambient temperature (25 ◦C). The flow rate was maintained at 0.6 mL/min. Detection was
performed on an API4000 Q-Trap mass spectrometer equipped with TurboIonSpray (ESI) Interface
(Applied Biosystems, Concord, Ontario, Canada). Analyst 1.5 software packages (Applied Biosystems)
were used to control the LC-MS/MS system, and for data acquisition and processing.
Data analysis: The peak area ratio of a test compound to internal standard was plotted as
a percentage of the relevant zero time point control (%remaining) for each reaction. The rate of
metabolism (k) is the slope of the linear regression from log percentage remaining versus incubation
1
time. The in vitro
T
/2 was calculated as
−
0.693/k. The calculated rate was used to extrapolate the
in vivo parameters: Intrinsic clearance (Clint) = k/c, where c is the microsomal protein concentration
(0.5 mg/mL for liver microsomes); for a human, apparent clearance (Clapp) = Clint b/d
(mL/min/kg), where a, b, and d are the scaling factors for normalizing Clint to human body weight.
The scaling factors a = 45 mg/g (microsomal protein/liver weight), b = 1500 g (liver weight), and d =
×
a
×
70 kg (body weight); hepatic clearance (Clh) = (Clapp
blood flow); hepatic extraction ratio (Eh) = (Clh/Q) × 100 (%).
×
Q)/Clapp + Q), where Q = 20 mL/min/kg (liver
4. Conclusions
In this short communication, we applied several computational analyses to ascertain whether or
not we could predict the metabolic stabilities of our first-generation scaffold-hopped entry inhibitors.
We computationally determined drug-like properties, predicted the major metabolizing cytochrome
P450 isoform, assesses metabolic liability, and performed affinity predictions of the compounds with
CYP3A4. This combined analysis suggested that compounds SC28 and SC46 may have increased
metabolic stability compared to the parental molecule temsavir/BMS-626529. This prediction was borne
out by experimental determination of the compound half-lives in human liver microsome stability
assays. Excitingly, SC28 had more than twice the metabolic stability of temsavir/BMS-626529, and its
increase even over SC46 demonstrates that the orientation of the core replacement is critical for this
increase, probably due to its effect on the affinity of the compound for binding to CYP3A4. Moreover,
both SC28 and SC46 had very low toxicities, with therapeutic indices (TI: CC50/IC50) of over 10,000
(see supplementary information). This work also establishes another addition to our computational
workflow that can be used to predict the metabolic stability of next-gen compounds, thus further
streamlining future design processes.
Supplementary Materials: The supplementary materials are available online.
Author Contributions: Conceptualization, R.K., M.E.M., and S.C.; methodology, R.K., M.E.M., and S.C.; formal
analysis, R.K., M.E.M., and S.C.; writing—original draft preparation, R.K., M.E.M., and S.C.; supervision, S.C.;
project administration, S.C.; funding acquisition, S.C. All authors have read and agreed to the published version
of the manuscript.
Funding: This work was supported by NIH/NIAID grant AI118415 (Cocklin, PI) and NIH/NIGMS grant
GM125396 (transitioning to R01AI150491) (Cocklin, PI). M.E.M. is also partially supported by NIH training
grant T32-MH079785.
Acknowledgments: We would like to thank Alexej Dick for help formatting the synthesis section and proofreading.
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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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).