Synthesis, enantiomeric resolution and biological evaluation of hiv capsid inhibition activity for racemic, (S)-and (r)-pf74

Article abstract of DOI:10.3390/molecules26133919

PF74 is a capsid-targeting inhibitor of HIV replication that effectively perturbs the highly sensitive viral uncoating process. A lack of information regarding the optical purity (enantiomeric excess) of the single stereogenic centre of PF74 has resulted in ambiguity as to the potency of different samples of this compound. Herein is described the synthesis of enantiomerically enriched (S)-and (R)-PF74 and further enrichment of the samples (≥98%) using chiral HPLC resolution. The biological activities of each enantiomer were then evaluated, which determined (S)-PF74 (IC₅₀ 1.5 μM) to be significantly more active than (R)-PF74 (IC₅₀ 19 μM). Computational docking studies were then conducted to rationalise this large discrepancy in activity, which indicated different binding conformations for each enantiomer. The binding energy of the conformation adopted by the more active (S)-PF74 (?G = ?73.8 kcal/mol) was calculated to be more favourable than the conformation adopted by the less active (R)-enantiomer (?G = ?55.8 kcal/mol) in agreement with experimental observations.

Full text of DOI:10.3390/molecules26133919

molecules
Article
Synthesis, Enantiomeric Resolution and Biological
Evaluation of HIV Capsid Inhibition Activity for
Racemic, (S)- and (R)-PF74
Stuart Ruddell 1 , Elena Sugrue 2 , Sarah Memarzadeh , Lorna Mae Hellam , Sam J. Wilson * and
David J. France *
1
1
2,
1
,
1
2
School of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK; stuart.ruddell@tum.de (S.R.);
s.memarzadeh.1@research.gla.ac.uk (S.M.); l.hellam.1@research.gla.ac.uk (L.M.H.)
MRC-University of Glasgow Centre for Virus Research, Institute of Infection, Immunity and Inflammation,
University of Glasgow, Glasgow G12 8TA, UK; elena.sugrue@glasgow.ac.uk
*
Correspondence: sam.wilson@glasgow.ac.uk (S.J.W.); david.france@glasgow.ac.uk (D.J.F.)
Abstract: PF74 is a capsid-targeting inhibitor of HIV replication that effectively perturbs the highly
sensitive viral uncoating process. A lack of information regarding the optical purity (enantiomeric excess)
of the single stereogenic centre of PF74 has resulted in ambiguity as to the potency of different samples
of this compound. Herein is described the synthesis of enantiomerically enriched (S)- and (R)-PF74 and
further enrichment of the samples (
each enantiomer were then evaluated, which determined (S)-PF74 (IC 1.5
≥
98%) using chiral HPLC resolution. The biological activities of
M) to be significantly more
M). Computational docking studies were then conducted to rationalise
this large discrepancy in activity, which indicated different binding conformations for each enantiomer.
The binding energy of the conformation adopted by the more active (S)-PF74 ( G = 73.8 kcal/mol)
was calculated to be more favourable than the conformation adopted by the less active (R)-enantiomer
∆G = −55.8 kcal/mol) in agreement with experimental observations.
µ
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
50
ꢀ
ꢁꢂꢃꢄꢅꢆ
active than (R)-PF74 (IC50 19
µ
Citation: Ruddell, S.; Sugrue, E.;
Memarzadeh, S.; Hellam, L.M.;
Wilson, S.J.; France, D.J. Synthesis,
Enantiomeric Resolution and
Biological Evaluation of HIV Capsid
Inhibition Activity for Racemic, (S)-
and (R)-PF74. Molecules 2021, 26, 3919.
https://doi.org/10.3390/
∆
−
(
Keywords: antiviral; stereochemistry; racemization; docking
molecules26133919
1
. Introduction
Academic Editors: Graeme Barker
and Simona Rapposelli
Human Immunodeficiency Virus (HIV) is a pathogenic retrovirus that infects cells of
the human immune system and, if left untreated, can result in Acquired Immunodeficiency
Syndrome (AIDS). An estimated 38 million people are infected worldwide and, since
the epidemic began in the early 1980s, approximately 33 million people have died from
Received: 31 May 2021
Accepted: 23 June 2021
Published: 26 June 2021
HIV-related deaths [1,2]. Combination antiretroviral therapy (cART) currently provides
effective viral suppression for most patients, but concerns including drug resistance and
adverse drug events provide a powerful motivation for investigation into new mechanisms
to treat HIV-1 infection [3].
The HIV-1 capsid protein (CA) multimerizes to form a fullerene-like shell that en-
capsidates the viral RNA genome and is essential for both early and late stages of the
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
viral replication cycle [4]. Small molecule ligands that target CA have been identified,
highlighted by lenacapavir (GS-6207, Figure 1), which is currently in phase 2/3 clinical
trials. The small molecule PF74 was the first to be co-crystallised with CA, and has been
Copyright: © 2021 by the authors. widely studied [5–7]. PF74 has been shown to both stabilise and destabilise the HIV capsid,
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This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
but the precise mechanism by which PF74 exerts its antiviral effect has not been fully
established. Regardless of whether the stabilising or destabilising effect is more important,
interfering with capsid stability perturbs the highly sensitive uncoating process, and thus
inhibits HIV infection [6,8–11].
4
.0/).
Molecules 2021, 26, 3919. https://doi.org/10.3390/molecules26133919
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3919
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Figure 1. Structures of lenacapavir and (S)-PF74.
PF74 is a non-natural derivative of phenylalanine, a sub-structure that has been
further optimised in lenacapavir. However, crucial information regarding the stereogenic
centre present in PF74 is scarce in published syntheses and studies on the biological
activities of this compound, and is not readily available from commercial suppliers of
PF74 [5,6,12–14]. As a derivative of phenylalanine, PF74 is prepared from enantioenriched
material from natural sources. Although there is a vast body of literature on strategies to
avoid epimerisation during peptide coupling [15], the inherent asymmetry of biological
systems necessitates that control of absolute stereochemical configuration of small molecule
inhibitors remains integral to their activity [14,16–18].
The scope of this project was to study stereochemical integrity in the synthesis of PF74,
and evaluate the biological activity of the two enantiomers in both cell assay and through
computational docking.
2. Results and Discussion
2.1. Synthesis and Characterisation
Our improved synthesis of PF74 was conducted based on a previously reported lit-
erature synthesis, with some optimisation [13]. Due to the likelihood of epimerisation in
this route, we chose to synthesise (S)-, (R)- and racemic PF74 using enantiomerically en-
riched starting materials, which allowed us to assess any epimerisation and to compare our
synthetic material with commercially available PF74. A PyBop-mediated amide coupling
between 2-methylindole-3-acetic acid (
methyl ester hydrochloride (2a 2b and 2c, respectively) was utilised to generate the amide
containing methyl ester (Scheme 1). Yields for these amidations were greater when using
PyBop than those obtained using HATU [13]. Following this amidation, saponification of
methyl ester to acid was conducted. Subsequent HATU-mediated amide coupling of
1) and either (S)-, (R)- or racemic phenylalanine
,
3
3
4
acid 4 (a, b and c) with N-methylaniline generated PF74 (5a, 5b and 5c).
Scheme 1. Synthesis of PF74 in three different enantiomer ratios.
While extensive literature precedent suggests that stereochemical integrity should
be maintained during the coupling and hydrolysis protocols to form
nucleophilicity of N-methylaniline posed a significant risk of epimerisation during the
formation of tertiary amide 19]. As a result of the sterically hindered and electronically
deactivated nature of this secondary aniline, the lifetime of the activated ester intermediate
4 [15], the poor
5
[

Molecules 2021, 26, 3919
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(
formed upon reaction of acid
4
with HATU) is extended and elevated temperatures were
◦
required (80 C). The combination of these two factors greatly increases the likelihood
of oxazolone-mediated epimerisation [20,21]. In order to investigate the occurrence of
epimerisation under our reaction conditions, analytical chiral HPLC was utilised (Figure 2).
This analysis confirmed that significant epimerisation was present in each of the syntheses
of PF74 (5a and 5b). Consequently, given the variable erosion of stereochemical integrity
we observed and the similarity between our route and the chosen precedent, this implied
that the previously reported synthesis also produced PF74 with ambiguous optical purity.
Figure 2. Chiral HPLC traces of: (A) 5a, (B) 5b, (C) 5c, (D) Enantiomerically enriched (S)-PF74.
To assess the integrity of the stereogenic carbon in commercial PF74 (as this informa-
tion is typically unavailable), two samples were purchased and analysed using chiral HPLC.
This revealed a high degree of variability, with one sample being highly enantioenriched
(er 99:1, Figure 2D), the other nearly racemic (S:R 51:49). With this observation, it seemed
plausible that the hitherto reported biological activities of PF74 used either racemic PF74 or
PF74 with ambiguous enantiomeric ratios. With the aim of testing the biological activity of
each enantiomer of PF74, enantiomeric resolution was conducted using chiral HPLC. Using
this approach, 1 mg each of highly enantioenriched (S)-PF74 (S:R 99.2:0.8) and (R)-PF74
(S:R 1.6:98.4) was generated.
2.2. Biological Evaluation
2.2.1. Antiviral Activity
Activity assays were conducted in the Human Embryonic Kidney cell line (HEK293T)
using a VSV G protein (VSV-G) pseudotyped single cycle HIV-1 reporter system (CSGW) [22].
Use of this reporter system enables quantification of infected cells using flow cytometry,
where the percentage of GFP positive cells is used as the metric of infection. By titrating
CSGW on HEK293T cells, the baseline infectivity of the virus (in the absence of PF74) was
established (e.g., 1
DMSO (vehicle, negative control) to the assay had no effect on the infectivity of the virus
Figure 3, green). Additionally, when the cells were treated with 10 M synthetic (racemic)
µL CSGW infected ca. 10% of cells) (Figure 3, red). The addition of
(
µ
PF74, effective inhibition of viral infectivity was observed, as indicated by decrease in the
percentage of cells infected for a given dose of CSGW (Figure 3, blue).

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Figure 3. CSGW infection of HEK 293T cells in the presence of no compound (red), DMSO vehicle
(green) and 10 µM racemic PF74 (blue).
When the cells were treated with the highly enantiomerically enriched enantiomers of
PF74, differential activities were observed (Figure 4). With (R)-PF74 no significant inhibition
of viral infectivity was observed above that of the baseline CSGW infectivity. With (S)-PF74
however, significant inhibition of viral infectivity was observed, indicating (S)-PF74 to be
considerably more active.
Figure 4. CSGW infection of HEK293T cells in the presence of DMSO vehicle (green), 10
purple) and 10 µM (S)-PF74 (orange).
µM (R)-PF74
(
To quantify this difference more conclusively, IC50 values were determined for each
of the enantiomers (Figure 5). For this dose response assay, a fixed amount of virus was
added (to produce approximately 15–20% cell infection) and a titration of each enantiomer
of PF74 was administered to the cells. The results were fitted to the Hill equation and
plotted. (S)-PF74 was determined to have an IC50 of 1.5
IC50 of 19 µM (Figure 5).
µM and the less active (R)-PF74 an

Molecules 2021, 26, 3919
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Figure 5. Dose response assays of (R)- and (S)-PF74 (black and red, respectively) for IC50 determina-
tion, plotted with Hill equation fit lines (grey).
2.2.2. Docking Studies
To rationalise the difference in efficacy between the two enantiomers, docking studies
were conducted using the Schrödinger suite. (S)-PF74 binding at the CA hexamer binding
site was shown to have seven potential favourable interactions. These include -cation
π
interactions between the indole and benzyl groups both to Lys70 as well as hydrogen
bonding interactions from the indole to Gln63, and between Asn57 and the phenylalanine
sub-structure embedded in PF74 (Figure 6A). Three locations of unfavourable solvent
exposure were also identified, all in the region of the N-methyl aniline of PF74. These
findings closely matched the literature reported binding interactions of the PF74-capsid
crystal structure, thus generating high confidence in the chosen method [
the binding energy of (S)-PF74 was calculated using a MMGBSA function and determined
to have a G of 73.8 kcal/mol. Docking of (R)-PF74 showed an alternative conformation
to that observed for (S)-PF74. This conformation indicated only four potential favourable
interactions comprised of three -cation interactions to Lys70 and a single water-mediated
6]. Additionally,
∆
−
π
hydrogen bonding interaction (Figure 6B). Three sites of unfavourable solvent exposure
for the R-enantiomer were also identified, two on the phenylalanine phenyl component of
PF74, and one on the indole methyl group. The binding energy of (R)-PF74 was calculated
using the same MMGBSA function and determined to have a ∆G of −55.8 kcal/mol.
Figure 6. View of CA residues with potential interactions with PF74 in the binding pocket, with hydrogen bonds shown as
dotted black lines. Numbers indicate bond lengths in ångströms. (A) (S)-PF74, (B) (R)-PF74.
The more favourable calculated binding conformation of (S)-PF74 corroborates the
experimental observation that (S)-PF74 was a more potent HIV inhibitor than (R)-PF74.

Molecules 2021, 26, 3919
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Therefore, the results of these computational studies constitute a reasonable explanation as
to why the efficacy of (S)-PF74 was approximately 15-fold greater than that of (R)-PF74.
3. Materials and Methods
3.1. General Chemical Methods
All reagents were purchased from commercial suppliers and used without further
purification unless otherwise stated. Reactions involving air-sensitive agents and dry
◦
solvents were performed in glassware that had been dried in an oven (150 C) or flame-
dried in vacuo and allowed to cool in vacuo before being flushed with argon. These
reactions were carried out with the exclusion of air using an argon atmosphere. Reactions
were monitored by thin layer chromatography (TLC, Merck, Darmstadt, Germany) on
silica gel 60 covered aluminium sheets. TLC plates were developed under UV-light and/or
with an acidic ethanolic anisaldehyde solution or a KMnO solution. NMR spectra (see
4
1
Supplementary Materials) were recorded on a DPX-400 spectrometer ( H NMR at 400 MHz,
1
3
1
C NMR at 100 MHz) (Bruker, Billerica, MA, USA) or a DPX-500 spectrometer ( H NMR
at 500 MHz and 13C NMR at 125 MHz) (Bruker, Billerica, MA, USA). Chemical shifts are
reported in ppm. H NMR spectra were recorded with CDCl or CD OD as the solvent
1
3
3
1
3
using residual CHCl3 (
NMR spectra the chemical shifts are reported relative to the central resonance of CDCl3
= 77.16) or CD OD ( = 49.00). Signals in NMR spectra are described as singlet (s),
δ
= 7.26) or CHD OD (
δ
= 3.31) as internal standard, and for
C
2
(
δ
δ
3
doublet (d), triplet (t), quartet (q), multiplet (m), broad (br) or a combination of these,
which refers to the spin–spin coupling pattern observed. Spin–spin coupling constants are
reported in Hertz (Hz) and are uncorrected. Two-dimensional NMR spectroscopy (COSY,
HSQC, HMBC, NOESY) and 13C DEPT NMR spectroscopy were used where appropriate
1
13
to assist the assignment of signals in the H and C NMR spectra. IR spectra were
obtained employing a FTIR-8400 instrument (Shimadzu, Kyoto, Japan) with a Golden
Gate™ attachment that uses a type IIa diamond as a single reflection element so that the IR
spectrum of the compound (solid or liquid) could be detected directly (thin layer). Melting
point ranges were collected using a Barnstead Electrothermal 9100 melting point apparatus
(Thermo Fisher Scientific, Waltham, MA, USA). High resolution mass spectra were recorded
under ESI or EI conditions by the analytical services at the University of Glasgow. Liquid
chromatography-mass spectrometry was conducted using a Dionex UltiMate 3000 LC
system (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a LCQ Fleet ion trap
mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). A Dr Maisch GmbH
Reprosil Gold 120 C18 3
at 214 nm. A linear gradient of 5–95% HPLC grade acetonitrile in ultrapure water with
µ
m 150
×
4 mm column was used with UV absorption detected
−
1
0
.1% trifluoroacetic acid over 10 or 40 min was utilised with a flow rate of 1 mL min
Chiral HPLC analysis was conducted using a LC-20AD prominence liquid chromatograph
Shimadzu, Kyoto, Japan) with a CBM-20A prominence communications bus module, a
.
(
DGU-20A5 prominence degasser and a SPD-M20A prominence diode array detector. A
Shimadzu CTO-20AC prominence column oven was utilised in combination with Diacel
Chemical Industries chiralcel OD-H 5
with an isocratic mobile phase of HPLC grade isopropyl alcohol in HPLC grade hexane.
µ
m 4.6
×
250 mm reverse phase analytical column
3.2. Compound Synthesis and Characterization
3.2.1. General Procedure to Methyl Ester (3)
Phenylalanine methyl ester hydrochloride 2 (1.05 equiv), PyBOP (1 equiv.) and N,N-
diisopropylethylamine (3 equiv.) were added to a solution of 2-methyl-3-indoleacetic acid
1
(1 equiv.) in N,N-dimethylformamide (0.4 M). The resulting solution was stirred at room
temperature for 18 h. The crude mixture was diluted with water and extracted with ethyl
acetate. The combined organic layers were washed with 5% aqueous lithium chloride,
dried over magnesium sulfate, filtered, and concentrated in vacuo. The product was
purified by column chromatography on silica gel using an eluent of 5–20% ethyl acetate in
dichloromethane.

Molecules 2021, 26, 3919
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Methyl (2-(2-Methyl-1H-indol-3-yl)acetyl)-L-phenylalaninate (3a)
Following the general procedure described above, 12.2 g (quantitative) of 3a was
obtained as a viscous oil.
1H NMR (400 MHz, CDCl3)
d, J = 7.9, Ar-H), 7.18 (1H, t, J = 7.5, Ar-H), 7.13 (1H, d, J = 7.8, Ar-H), 7.09 (1H, d, J = 7.9,
Ar-H), 7.00 (2H, t, J = 7.5, 2 Ar-H), 6.71 (2H, d, J = 7.5, 2 Ar-H), 5.99 (1H, d, J = 7.8,
CONH), 4.84 (1H, dt, J = 6.6, 6.6, -CH), 3.64 (2H, s, -COCH ),), 3.64 (3H, s, -CH ), 2.97 (1H,
δ 7.88 (1H, s, Ar-NH), 7.42 (1H, d, J = 7.8, Ar-H), 7.31 (1H,
×
×
-
2
3
dd, J = 13.9, 5.4, -CH ), 2.91 (1H, dd, J = 13.8, 5.5, -CH ), 2.29 (3H, s, -CH ).
2
2
3
13C NMR (101 MHz, CDCl3)
δ
171.9 (-CONH), 171.1 (-COOR), 135.6 (Ar-C), 135.5 (Ar-C),
Ar-CH), 128.4 (Ar-C), 127.0 (Ar-CH), 121.9 (Ar-
CH), 120.2 (Ar-CH), 118.0 (Ar-CH), 110.5 (Ar-CH), 104.7 (Ar-C), 52.8 (-CH), 52.3 (-OCH ),
133.4 (Ar-C), 129.2 (2
×
Ar-CH), 128.5 (2
×
3
3
7.7 (-CH ), 32.2 (-CH ), 11.6 (-CH ).
2 2 3
+
HRMS (ESI) exact mass calculated for C H N O Na [M + Na] m/z 373.1523, found
21
22
2
3
m/z 373.1514. IR (thin film) 2952, 1741, 1651.
Methyl (2-(2-Methyl-1H-indol-3-yl)acetyl)-D-phenylalaninate (3b)
Following the general procedure outlined above, 719 mg (78%) of compound 3b was
obtained as a viscous oil.
1H NMR (400 MHz, CDCl3)
J = 7.7, Ar-H), 7.18 (1H, t, J = 6.8, Ar-H), 7.13 (1H, d, J = 7.8, Ar-H), 7.09 (1H, t, J = 7.8, Ar-H),
δ 7.92 (1H, s, Ar-NH), 7.42 (1H, d, J = 7.7, Ar-H), 7.31 (1H, d,
6
.09 (2H, t, J = 6.7, 2
×
Ar-H), 6.70 (2H, d, J = 6.5, 2
×
Ar-H), 6.00 (1H, d, J = 8.3, -CONH),
4.90–4.76 (1H, m, -CH), 3.64 (2H, s, -CH ), 3.64 (3H, s, -CH ), 3.03–2.85 (2H, m, -CH ), 2.28
2
3
2
(
3H, s, -CH3).
13C NMR (101 MHz, CDCl3)
δ
171.9 (-CONH), 171.1 (-COOR), 135.6 (Ar-C), 135.5 (Ar-C),
Ar-CH), 128.4 (Ar-C), 127.0 (Ar-CH), 121.9 (Ar-
CH), 120.2 (Ar-CH), 118.0 (Ar-CH), 110.5 (Ar-CH), 104.6 (Ar-C), 52.8 (-CH), 52.3 (-OCH ),
133.4 (Ar-C), 129.1 (2
×
Ar-CH), 128.5 (2
×
3
3
7.7 (-CH ), 32.1 (-CH ), 11.6 (-CH ).
2 2 3
+
HRMS (ESI) exact mass calculated for C H N O Na [M + Na] m/z 373.1523, found
21
22
2
3
m/z 373.1514. IR (thin film) 2952, 1741, 1651.
Methyl (2-(2-Methyl-1H-indol-3-yl)acetyl)-DL-phenylalaninate (3c)
Following the general procedure described above, 925 mg (quantitative) of 3c was
obtained.
1H NMR (400 MHz, CDCl3)
J = 7.9, Ar-H), 7.18 (1H, t, J = 7.7, Ar-H), 7.15–7.11 (1H, m, Ar-H), 7.11–7.05 (1H, m, Ar-H),
δ
7.89 (1H, s, Ar-NH), 7.42 (1H, d, J = 7.8, Ar-H), 7.31 (1H, d,
7
.00 (2H, t, J = 7.7, 2
×
Ar-H), 6.70 (2H, d, J = 7.5, 2
×
Ar-H), 5.99 (1H, d, J = 8.3, -CONH),
4.88–4.80 (1H, m, -CH), 3.64 (2H, s, -CH ), 3.64 (3H, s, -CH ), 3.00–2.86 (2H, m, -CH ), 2.28
2
3
2
(
3H, s, -CH3).
13C NMR (101 MHz, CDCl3)
δ
171.9 (-CONH), 171.1 (-COOR), 135.6 (Ar-C), 135.5 (Ar-C),
Ar-CH), 128.4 (Ar-C), 127.0 (Ar-CH), 121.9 (Ar-
CH), 120.2 (Ar-CH), 118.0 (Ar-CH), 110.5 (Ar-CH), 104.7 (Ar-C), 52.8 (-CH), 52.3 (-OCH ),
133.4 (Ar-C), 129.2 (2
×
Ar-CH), 128.5 (2
×
3
3
7.7 (-CH ), 32.2 (-CH ), 11.6 (-CH ).
2 2 3
+
HRMS (ESI) exact mass calculated for C H N O Na [M + Na] m/z 373.1523, found
21
22
2
3
◦
m/z 373.1514. IR (thin film) 2952, 1741, 1651. Melting point 126–130 C.
3.2.2. General Procedure to Acid (4)
To a stirred solution of (1 equiv) in tetrahydrofuran (0.23 M) and water (0.34 M)
3
was added a solution of sodium hydroxide (1.5 equiv) in methanol (1 M NaOH) and the
resulting solution was stirred vigorously at room temperature for 18 h. The reaction mixture
was diluted with water and the crude biphasic mixture was washed with dichloromethane
to remove organic impurities. The aqueous phase was then acidified with 2 M aqueous
hydrochloric acid forming a thick white precipitate. The aqueous phase was extracted with

Molecules 2021, 26, 3919
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dichloromethane and the combined organic phases were dried with magnesium sulfate,
filtered, and concentrated in vacuo. The compound was used without further purification.
(
2-(2-Methyl-1H-indol-3-yl)acetyl)-D-phenylalanine (4a)
Following the general procedure described above, 989 mg (97%) of 4a was obtained.
1H NMR (400 MHz, CDCl3)
8.14 (1H, s, Ar-NH), 7.34 (1H, d, J = 7.8, Ar-CH), 7.26 (1H, d,
J = 7.0, Ar-CH), 7.16 (1H, t, J = 7.1, Ar-CH), 7.09 (1H, t, J = 7.7, Ar-CH), 7.09 (1H, t, J = 7.7
Ar-CH), 6.99 (2H, t, J = 7.6, 2 Ar-CH), 6.74 (2H, d, J = 7.1, 2 Ar-CH), 6.15 (1H, d, J = 7.9
δ
,
,
×
×
-
CONH), 4.76 (1H, dd, J = 13.1, 6.7, -CH), 3.61 (2H, s, -CH ), 3.00 (1H, dd, J = 13.9, 5.2,
2
-
CH ), 2.89 (1H, dd, J = 13.9, 6.7, -CH ), 2.13 (3H, s, -CH ).
2
2
3
13C NMR (101 MHz, CDCl3)
33.8 (Ar-C), 129.2 (2 Ar-CH), 128.6 (2
Ar-CH), 120.1 (Ar-CH), 117.8 (Ar-CH), 110.7 (Ar-CH), 103.7 (Ar-C), 53.2 (-CH), 37.0 (-CH ),
δ
= 174.0 (-COOH), 172.9 (-CONH), 135.5 (Ar-C), 135.4 (Ar-C),
Ar-CH), 128.2 (Ar-C), 127.0 (Ar-CH), 121.8
1
(
×
×
2
3
1.8 (-CH ), 11.4 (-CH ).
2 3
+
HRMS (ESI) exact mass calculated for C H N O Na [M + Na] m/z 359.1366, found
20
20
2
3
m/z 359.1355. IR (thin film) 2916, 1724, 1618.
2-(2-Methyl-1H-indol-3-yl)acetyl)-l-phenylalanine (4b)
Following the general procedure outlined above, 358 mg (74%) of 4b was obtained as
a solid.
(
1H NMR (400 MHz, CDCl3)
t, J = 7.4, Ar-CH), 7.13 (1H, t, J = 8.3, Ar-CH), 7.11 (1H, t, J = 8.3, Ar-CH), 7.07–7.00 (2H, m, 2
Ar-CH), 6.75 (2H, d, J = 7.5,2 Ar-CH), 5.99 (1H, br s, -CONH), 4.69 (1H, br s, -CH), 3.64
δ
7.89 (1H, s, Ar-NH), 7.36–7.29 (2H, m, 2
×
Ar-CH), 7.18 (1H,
×
×
(
2H, br s, -CH ), 3.04 (1H, br dd, J = 13.8, 5.5, -CH ), 2.94 (1H, br dd, J = 13.8, 5.5, -CH ),
2
2
2
2.20 (3H, s, -CH3).
13C NMR (101 MHz, CDCl3)
33.8 (Ar-C), 129.2 (2 Ar-CH), 128.6 (2
Ar-CH), 120.1 (Ar-CH), 117.8 (Ar-CH), 110.7 (Ar-CH), 103.7 (Ar-C), 53.2 (-CH), 37.0 (-CH ),
δ
= 174.0 (-COOH), 172.9 (-CONH), 135.5 (Ar-C), 135.4 (Ar-C),
Ar-CH), 128.2 (Ar-C), 127.0 (Ar-CH), 121.8
1
(
×
×
2
3
1.8 (-CH ), 11.4 (-CH ).
2 3
+
HRMS (ESI) exact mass calculated for C H N O Na [M + Na] m/z 359.1366, found
20
20
2
3
◦
m/z 359.1355. IR (thin film) 2916, 1724, 1618. Melting point 140–143 C.
2-(2-Methyl-1H-indol-3-yl)acetyl)-D-phenylalanine (4c)
Following the general procedure outlined above, 491 mg (quantitative) of 4c was
obtained.
(
Spectroscopic data matched that of acid 4a.
3.2.3. Preparation of PF74 (5)
PF74 was prepared according to a modified literature procedure [13]. To a stirred solution
of acid
49
4 (100 mg, 0.300 mmol) in N,N-dimethylformamide (0.3 mL) was added N-methylaniline
(
µ
L, 0.45 mmol), HATU (171 mg, 0.450 mmol) and N,N-diisopropylethylamine (0.11 mL,
◦
0.60 mmol) and the resulting solution was heated to 65 C and stirred for 48 h. To the reaction
mixture was added dichloromethane and half saturated aqueous sodium hydrogen carbonate
solution and the aqueous phase was extracted with dichloromethane. The combined organic
phases were washed with 5% aqueous lithium chloride solution, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The crude mixture was purified by column chromatography
on silica gel using an eluent of 1–5% ethanol in dichloromethane.
Natural (S)-PF74 (5a)
Following the general procedure described above, 63 mg (49%) of 5a was obtained.
1H NMR (400 MHz, CDCl3)
d, J = 8.0, Ar-CH), 7.18–7.04 (5H, m, 5
2H, m, 2 Ar-CH), 6.22 (1H, d, J = 8.3, -CONH), 4.81 (1H, ddd, J = 8.2, 6.9, 6.9, -CH),
.59 (1H, s, -COCH ), 3.58 (1H, s, -COCH ), 3.17 (3H, s, -NCH ), 2.74 (1H, dd, J = 13.3, 6.9,
δ
8.26 (1H, s, Ar-NH), 7.39–7.31 (4H, m, 4
Ar-CH), 6.97–6.89 (2H, m, 2
×
Ar-CH), 7.26 (1H,
Ar-CH), 6.71–6.65
×
×
(
3
×
2
2
3
-
CH ), 2.54 (1H, dd, J = 13.3, 7.0, -CH ), 2.26 (3H, s, -CH ).
2 2 3

Molecules 2021, 26, 3919
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13C NMR (101 MHz, CDCl3)
35.5 (2 Ar-C), 129.9 (2
Ar-CH), 127.5 (2 Ar-CH), 126.8 (Ar-CH), 121.6 (Ar-CH), 119.9 (Ar-CH), 117.9 (Ar-CH),
10.6 (Ar-CH), 104.6 (Ar-C), 51.2 (-CH), 38.9 (-CH ), 37.8 (-NCH ), 32.3 (-COCH ), 11.7
δ
171.4 (-CONR ), 170.9 (-CONH), 142.6 (Ar-C), 136.2 (Ar-C),
2
1
(
1
×
×
Ar-CH), 129.3 (2
×
Ar-CH), 128.4 (Ar-C, 2
×
Ar-CH), 128.2
×
2
3
2
(
-CH3).
+
HRMS (ESI) exact mass calculated for C H N O Na [M + Na] m/z 448.1995, found
27
27
3
2
m/z 448.1977. IR (thin film) 2924, 1638, 1595.
Chiral HPLC (OD-H, 8% i-PrOH in n-hexane, 1 mL min 1, λ 208 nm) tRnatural = 32.511
−
min (54.8%), tRunnatural = 40.831 min (45.2%). [α]1
9.8C
+1.40 (c = 1.00, CHCl ). Preparative
3
D
−
1
chiral HPLC purified (S)-PF74 (OD-H, 8% i-PrOH in n-hexane, 1 mL min
,
λ
208 nm)
tRnatural = 32.847 min (99.2%), tRunnatural = 41.995 min (0.8%). [α]1
9.5C
+ 520.0 (c = 1.00,
D
CHCl3).
Unnatural (R)-PF74 (5b)
Following the general procedure described above, 70 mg (55%) of 5b was obtained.
1H NMR (400 MHz, CDCl3)
7.92 (1H, s, Ar-NH), 7.40–7.27 (5H, m, 5 Ar-CH), 7.19–7.03
Ar-CH), 6.93 (2H, d, J = 7.0, 2 Ar-CH), 6.66 (2H, d, J = 7.3, 2 Ar-CH), 6.17
1H, d, J = 8.3, -CONH), 4.78 (1H, ddd, J = 7.1, 7.1, 7.1, -CH), 3.58 (1H, s, -COCH ), 3.55 (1H,
δ
×
(
(
5H, m, 5
×
×
×
2
s, -COCH ), 3.16 (3H, s, -NCH ), 2.72 (1H, dd, J = 13.3, 6.9, -CH ), 2.51 (1H, dd, J = 13.3, 7.0,
2
3
2
-
CH ), 2.33 (3H, s, -CH ).
2 3
13C NMR (101 MHz, CDCl3)
δ
171.4 (-CONR ), 170.9 (-CONH), 142.6 (Ar-C), 136.2 (Ar-C),
2
1
35.5 (Ar-C), 133.5 (Ar-C), 129.9 (2
Ar-CH), 128.2 (Ar-CH), 127.5 (2
17.9 (Ar-CH), 110.6 (Ar-CH), 104.6 (Ar-C), 51.2 (-CH), 38.9 (-CH ), 37.8 (-NCH ), 32.3
×
Ar-CH), 129.3 (2
Ar-CH), 126.8 (Ar-CH), 121.6 (Ar-CH), 119.9 (Ar-CH),
×
Ar-CH), 128.4 (Ar-C), 128.4 (2
×
×
1
2
3
(
-COCH ), 11.7 (-CH ).
2 3
+
HRMS (ESI) exact mass calculated for C H N O Na [M + Na] m/z 448.1995, found
27
27
3
2
m/z 448.1977. IR (thin film) 2924, 1638, 1595.
HPLC (OD-H, 8% i-PrOH in n-hexane, 1 mLmin 1,
−
λ
208 nm) tRnatural = 32.550 min
0.2C
(
35.0%), tRunnatural = 40.678 min (65.0%). [α]2
−5.57 (c = 1.00, CHCl3).
D
Racemic PF74 (5c)
Following the general procedure described above, 69 mg (54%) of 5c was obtained.
1H NMR (400 MHz, CDCl3)
7.96 (1H, s, Ar-NH), 7.41–7.27 (5H, m, 5 Ar-CH), 7.19–7.02
5H, m, 5 × Ar-CH), 6.93 (2H, d, J = 6.8, 2 × Ar-CH), 6.65 (2H, d, J = 6.9, 2 × Ar-CH), 6.18
1H, d, J = 8.3, -CONH), 4.78 (1H, ddd, J = 7.1, 7.1, 7.1, -CH), 3.58 (1H, s, -COCH ), 3.57 (1H,
δ
×
(
(
2
s, -COCH ), 3.16 (3H, s, -NCH ), 2.72 (1H, dd, J = 13.3, 6.9, -CH ), 2.51 (1H, dd, J = 13.3, 7.0,
2
3
2
-
CH ), 2.32 (3H, s, -CH ).
2 3
13C NMR (101 MHz, CDCl3)
δ
171.4 (-CONR ), 170.9 (-CONH), 142.6 (Ar-C), 136.2 (Ar-C),
2
1
35.5 (Ar-C), 133.5 (Ar-C), 129.9 (2
Ar-CH), 128.2 (Ar-CH), 127.5 (2
17.9 (Ar-CH), 110.6 (Ar-CH), 104.6 (Ar-C), 51.2 (-CH), 38.9 (-CH ), 37.8 (-NCH ), 32.3
×
Ar-CH), 129.3 (2
Ar-CH), 126.8 (Ar-CH), 121.6 (Ar-CH), 119.9 (Ar-CH),
×
Ar-CH), 128.4 (Ar-C), 128.4 (2
×
×
1
2
3
(
-COCH ), 11.7 (-CH ).
2 3
+
HRMS (ESI) exact mass calculated for C H N O Na [M + Na] m/z 448.1995, found
27
27
3
2
m/z 448.1977. IR (thin film) 2924, 1638, 1595.
HPLC (OD-H, 8% i-PrOH in n-hexane, 1 ML min 1,
−
λ
208 nm) tRnatural = 32.227 min
(
50.4%), tRunnatural = 40.120 min (49.6%). [α]1
9.2C
+ 0.85 (c = 1.00, CHCl3).
D
3
3
.3. Biological Assays—Materials and Methods
.3.1. Cells and Viruses
HEK293T suspension cells were obtained as a generous gift from Professor Massimo
Palmarini (Centre for Virus Research, University of Glasgow) and were cultured in DMEM
Dulbecco’s modified Eagle’s medium) with 10% FCS (fetal calf serum) and 10 g/mL
gentamicin. The VSV-G pseudotyped single-cycle HIV-1 reporter lentiviral vector CSGW
(
µ

Molecules 2021, 26, 3919
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is self-inactivating, and encodes GFP expressed from an internal spleen focus-forming
virus (SFFV) long terminal repeat (LTR) [22], packaged using GagPol derived from HIV-1
NL4-3 [23]. CSGW virus stocks were prepared as described previously [23], by transient
transfection of HEK 293T cells, the supernatant was harvested at 48 h post transfection and
clarified using a 0.45-µm-pore-size filter.
3.3.2. Virus Titrations
Cells were seeded in 96-well plates (50,000 cells/well) immediately prior to infection
or treatment. Briefly, suspension HEK293T cells were infected with a titrated challenge of
serially diluted CSGW-containing supernatant. At 48 h post infection, cells were trypsinized
◦
and incubated at 37 C to enable a single cell suspension and fixed (4% formaldehyde
saline). The fixed cells were incubated to allow complete virus inactivation prior to analysis.
Quantification of GFP positive cells (representative of GFP-reporter CSGW infection) was
carried out using flow cytometry with 10,000 events acquired per well (Guava EasyCyte
cytometer). From the titration performed with CSGW, the concentration of virus-containing
supernatant required to produce 15–20% infection of cells was calculated. This dilution
was then used in in fixed CSGW dose assays.
3.3.3. Compound Fixed Dose (Virus Titration) Assay
For fixed compound dose assays, a CSGW titration as described above was performed with
one exception. The final addition of 50
µ
L fresh media described above was substituted with
desired final concentration)
L 4% formaldehyde
the addition of 50 L of a 4% DMSO solution of compound (at 4
µ
×
in media. The plates were then incubated for 48 h before being fixed (100
saline) and analysed by flow cytometry.
µ
3
.3.4. Compound Dose Response (Fixed Virus Dose) Assay
For compound dose response assays, each compound was titrated across a 96-well
plate to give a 50 L solution with a maximum final concentration of 100 M to a minimum
final concentration of 95 pM ( 4 dilution series). In addition to the compound, each well
contained 100 L of a suspension of HEK293T cells in fresh media (50,000 cells/well) and
L of diluted CSGW (at the titre previously calculated to produce ~15–20% infection)
in fresh media with a final concentration of 1% DMSO per well. The plates were then
µ
µ
×
µ
50 µ
incubated for 48 hours before being fixed (100
by flow cytometry.
µL 4% formaldehyde saline) and analysed
3
.4. Molecular Docking Methods
All structure preparation and docking was performed using the Schrödinger suite
build 2019-1) The enantiomers of PF74 were drawn with ChemDraw and converted to a
D structure and energy minimized using the OPLS3e forcefield in LigPrep (Schrödinger
Release 2019-1: LigPrep, Schrödinger, LLC, New York, NY, USA 2019). The hexameric
quaternary structure of capsid PDB: 2XDE [ ] was generated in Pymol (The PyMOL
(
3
5
Molecular Graphics System, Version 2.0 Schrödinger, LLC, Schrödinger, Inc., New York,
NY, USA) and used for docking. Protein Prep Wizard (Schrödinger Release 2019-1: Protein
Preparation Wizard; Epik, Schrödinger, LLC, New York, NY, USA 2019) was used to prep
PDB structures for Prime (Schrödinger Release 2019-1: Prime, Schrödinger, LLC, New
York, NY, USA 2019) water molecules were kept and minimized. Molecular docking was
performed using Glide and the OPLS3e force field (Schrödinger Release 2019-1: Glide,
Schrödinger, LLC, New York, NY, USA 2021). Molecular Mechanics/Generalized Born
Surface Area (MMGBSA) protein–ligand interaction energies were calculated for the top
poses of both enantiomers in a rigid receptor with the VSGB solvent model and the OPLS3e
forcefield (Schrödinger Release 2019-1: Maestro, Schrödinger, LLC, New York, NY, USA
2019).

Molecules 2021, 26, 3919
11 of 12
4. Conclusions
To summarise, our improved syntheses of (S)-, (R)- and racemic PF74 were based on a
previously published procedure [13]. Chiral HPLC analyses of the synthesised materials
were conducted, which established a significant degree of epimerisation was present in each
batch. Preparative chiral HPLC was utilised to generate highly enantiomerically enriched
( 98%) samples of (S)- and (R)-PF74. Evaluation of the biological activities was conducted
≥
in HEK293T cells to assess the ability of each enantiomer to inhibit the infectivity of the
HIV viral vector CSGW-VSV-G. In broad agreement with recently published work [14], this
testing determined (S)-PF74 to be significantly more active (IC50 = 1.5
µM) than (R)-PF74
(
IC50 = 19 M). In an effort to rationalise this observation, docking studies were conducted
µ
which showed the two enantiomers adopted different conformations in the PF74 binding
site. The more active (S)-PF74 had additional favourable interactions compared to (R)-PF74,
which was reflected in the calculated binding energies of each conformation, the more
active (S)-PF74 (
∆
G of
−
73.8 kcal/mol) being significantly lower in energy than (R)-PF74
(
∆G of −55.8 kcal/mol).
Supplementary Materials: The following are available online: NMR spectra.
Author Contributions: Conceptualization, S.R., E.S., S.J.W. and D.J.F.; Data curation, S.R., E.S.,
S.M. and L.M.H.; Funding acquisition, S.J.W. and D.J.F.; Investigation, S.R., E.S., S.M. and L.M.H.;
Resources, S.J.W. and D.J.F.; Writing—original draft, S.R., E.S., S.M., L.M.H., S.J.W. and D.J.F. All
authors have read and agreed to the published version of the manuscript.
Funding: Financial support from the EPSRC (EP/K503058/1 to S.R., EP/R513222/1 to L.M.H. and
EP/V008404/1 to S.J.W. and D.J.F.) and University of Glasgow is gratefully acknowledged.
Data Availability Statement: Not applicable.
Acknowledgments: We would like to thank Massimo Palmarini (Centre for Virus Research, Univer-
sity of Glasgow) for supplying HEK293T suspension cells and Alistair Boyer (School of Chemistry,
University of Glasgow) for assistance with chiral HPLC.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of compounds are not available from the authors.
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