Diaryl ethers with carboxymethoxyphenacyl motif as potent HIV-1 reverse transcriptase inhibitors with improved solubility

Article abstract of DOI:10.1080/14756366.2017.1387542

In search of new non-nucleoside reverse transcriptase inhibitors (NNRTIs) with improved solubility, two series of novel diaryl ethers with phenacyl moiety were designed and evaluated for their HIV-1 reverse transcriptase inhibition potentials. All compounds exhibited good to excellent results with IC₅₀ at low micromolar to submicromolar concentrations. Two most active compounds (7e and 7 g) exhibit inhibitory potency comparable or even better than that of nevirapine and rilpivirine. Furthermore, SupT1 and CD4⁺ cell infectivity assays for the most promising (7e) have confirmed its strong antiviral potential while docking studies indicate a novel binding interactions responsible for high activity.

Full text of DOI:10.1080/14756366.2017.1387542

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Diaryl ethers with carboxymethoxyphenacyl motif
as potent HIV-1 reverse transcriptase inhibitors
with improved solubility
Tomasz Frączek, Rafał Kamiński, Agnieszka Krakowiak, Evelien Naessens,
Bruno Verhasselt & Piotr Paneth
To cite this article: Tomasz Frączek, Rafał Kamiński, Agnieszka Krakowiak, Evelien Naessens,
Bruno Verhasselt & Piotr Paneth (2018) Diaryl ethers with carboxymethoxyphenacyl motif as potent
HIV-1 reverse transcriptase inhibitors with improved solubility, Journal of Enzyme Inhibition and
Medicinal Chemistry, 33:1, 9-16, DOI: 10.1080/14756366.2017.1387542
To link to this article: http://dx.doi.org/10.1080/14756366.2017.1387542
©
2017 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
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Date: 04 November 2017, At: 12:43

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 33, NO. 1, 9–16
https://doi.org/10.1080/14756366.2017.1387542
RESEARCH PAPER
Diaryl ethers with carboxymethoxyphenacyl motif as potent HIV-1 reverse
transcriptase inhibitors with improved solubility
a
a
a,b
c
c
Tomasz Fr a˛ czek
Piotr Paneth
, Rafał Kami nꢀ ski , Agnieszka Krakowiak
, Evelien Naessens , Bruno Verhasselt
and
a
a
b
Institute of Applied Radiation Chemistry, Lodz University of Technology, Lodz, Poland; Department of Bioorganic Chemistry, Centre of
c
Molecular and Macromolecular Studies, Polish Academy of Sciences, Lodz, Poland; Department of Clinical Chemistry, Microbiology and
Immunology, Ghent University, Ghent University Hospital, Ghent, Belgium
ABSTRACT
ARTICLE HISTORY
In search of new non-nucleoside reverse transcriptase inhibitors (NNRTIs) with improved solubility, two ser-
ies of novel diaryl ethers with phenacyl moiety were designed and evaluated for their HIV-1 reverse tran-
scriptase inhibition potentials. All compounds exhibited good to excellent results with IC50 at low
micromolar to submicromolar concentrations. Two most active compounds (7e and 7 g) exhibit inhibitory
potency comparable or even better than that of nevirapine and rilpivirine. Furthermore, SupT1 and CD4
cell infectivity assays for the most promising (7e) have confirmed its strong antiviral potential while dock-
Received 4 July 2017
Revised 27 August 2017
Accepted 22 September 2017
þ
KEYWORDS
NNRTI; reverse transcriptase;
HIV; drug solubility
ing studies indicate a novel binding interactions responsible for high activity.
Introduction
mutation rate of the virus, and strains resistant to antiretroviral
drugs emerge. Consequently, the pharmacotherapy may become
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have pro-
ven their effectiveness as components of highly active antiretro-
ineffective, moreover, cross-resistance between NNRTIs is pos-
11–14
sible
. Another problem is that the NNRTIs binding site of RT
1
–3
viral therapy . Their relatively low toxicity, as compared to other
antiretroviral drugs, makes them a very attractive class of com-
favours non-polar compounds, which are usually poorly soluble in
water. This is especially the case in second-generation NNRTIs, as
both ETV and RPV are practically insoluble in water and require
4
–7
pounds used in treating HIV-1 infections . Currently, there are
five registered NNRTIs, first generation: nevirapine (NVP), efavirenz
1
5,16
special formulations
. For these reasons there is a need to
(
EFV), delavirdine, and second generation: etravirine (ETV) and ril- develop new NNRTIs with improved potency against resistant HIV
1
7–19
pivirine (RPV). Because HIV-1 reverse transcriptase (RT) has a low mutants and better pharmacokinetics
. First generation NNRTIs
fidelity – its error rate was reported to be in the range of like NVP and EFV are rigid molecules that bind well to the wild-
ꢀ
3
ꢀ5
8–10
1
0
–10
per nucleotide addition
– there is a very high type RT, but a single amino acid mutation in the binding site can
CONTACT Bruno Verhasselt
Hospital, Ghent, Belgium; Piotr Paneth
Supplemental data for this article can be accessed here.
Bruno.Verhasselt@UGent.be
Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent University
Institute of Applied Radiation Chemistry, Lodz University of Technology, Lodz, Poland
piotr.paneth@p.lodz.pl
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1
0
T. FRA˛CZEK ET AL.
significantly decrease their affinity to the enzyme. Second gener- materials. Diaryl ether parts (9a–f) of the new NNRTIs were syn-
ation NNRTIs have flexible structures which allows them to adapt thesised from phenols and aryl fluorides in N-methylpyrrolidone
2
0
34,35
to a modified binding site of mutant RT . Usually, second gener- (Figure 3) as described earlier
. In case of 9b Chan-Lam cou-
3
7
ation NNRTIs have 2–3 aromatic rings with an ether, thioether, pling was used . Hydroxyacetophenones were O-alkylated with
short alkyl or amino group located between the rings that acts as ethyl chloroacetate. Subsequent exchange of ethyl to methyl
a hinge that allows the inhibitors to bind in different conforma- afforded pure and solid methyl esters, which were selectively bro-
2
0,21
tions and overcome resistance mutations
. An excellent review minated with N-bromosuccinimide and p-toluenesulfonic acid in
1
8
38
on the chemical diversity of NNRTIs was written by Zhan et al. . chloroform (10a–d) (Figure 3) . Final deesterification was per-
Diaryl ethers are one of the classes of second generation NNRTIs. formed using potassium carbonate in a mixture of methylene
There are several interesting inhibitors belonging to this class, chloride, methanol and water (room temperature, 1–2 days).
including 1 – the most potent NNRTI reported to date (against Structures of obtained compounds are given in Table 1. Detailed
wild type RT) and doravirine (2), which is in phase III clinical trials synthetic procedures and characterisation data of reported com-
Figure 1)
As mentioned above, poor solubility in water results in reduced
bioavailability, and there is an increasing awareness of the need to
2
2–24
(
.
pounds can be found in the supplemental material.
design NNRTIs with improved pharmacokinetics. Several Molecular docking
approaches were used by different authors to achieve better solu-
39
The RCSB Protein Data Bank (PDB) contains over 150 crystallo-
graphic structures of HIV-1 reverse transcriptase-NNRTI complexes.
Basing on structural similarity features, four PDB entries were
selected for wild type RT receptor preparation: 3DRP, 4H4M, 3C6T
and 2YNG (the structures of corresponding reference ligands can
be found in the supplemental file, Fig. S1). RT mutant structures
were taken from 3DRS, 3MEG (K103N), 3DRR, 1JLC (Y181C) and
2
5,26
27,28
bility of NNRTIs: salt formation
, prodrug formation
, add-
2
9–31
23
ition of polar substituents
, modification of crystal structure
3
2
or reduced halogenations .
Our goal was to design second generation NNRTIs with
improved solubility and chemical stability. Building on common
3
3–35
substructures of several diaryl ether (3–5)
(
and azole NNRTIs
3
6
6) we designed two new scaffolds: 7a and 8a (Figure 2). The
4
RW4, 3BGR (K103N/Y181C). In addition, 4G1Q structure was used
as a wild type RT complex with RPV. Protein structures were pre-
new structures feature phenacyl moiety as an alternative to hydro-
lytically labile amide, found in some NNRTIs (Figure 2).
4
0
pared using Protein Preparation Wizard in Maestro . After a pre-
processing step with default settings, all waters, ions and small
molecules were removed, hydrogen bond network was optimised,
followed by global optimisation of all atoms. Receptor grids were
prepared with default box size, centred on a reference ligand from
Materials and methods
Synthesis
Compounds 7a–g (resorcinol type) and 8a–f (catechol type) were the crystallographic structure. To simulate a small-scale adaptation
synthesised in several steps from commercially available starting of the receptor upon binding of a ligand, van der Waals radii of
Figure 1. Structures of a catechol diether with the lowest EC50 reported to date (1) and doravirine (2).
Figure 2. Structures of several diaryl ether NNRTIs (3–5), RDEA806 (6), and our newly designed compounds (7a, 8a).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
11
ꢁ
ꢁ
Figure 3. Synthesis scheme (a) K
2
CO
3
, N-methylpyrrolidone, 120 C, 4 h (b) BBr
3
, CH
2
Cl
2
, 0–25 C, 5 days (c) ethyl chloroacetate, K
2
CO
3
, KI, acetone, reflux, 4 h (d) NaOH,
ꢁ
ꢁ
CH
2
Cl
2
– CH
3
OH (9:1), 25 C 1 h, then diluted HCl (e) CH
3
OH, p-toluenesulfonic acid, reflux, 4 h (f) N-bromosuccinimide, p-toluenesulfonic acid, CHCl
3
, 25 C, 12 h (g)
ꢁ
ꢁ
ꢁ
K CO
2 3
, acetone, 25 C, 4 h (h) K
2
CO
3
, CH
Cl
2 2
– CH
3
OH – H
2
O, 25 C, 1–2 days (i) Cu(CH
3
COO)
2
, pyridine, CH
Cl
2 2
, 25 C, 2–3 days. R1-R4 groups are as in Table 1.
Table 1. Structures of synthesised compounds.
R1
R2
R3
R4
7
7
7
7
7
a
b
c
d
e
CN
Cl
CN
CN
CN
2-Cl
2-Cl
3-Cl
H
H
H
H
H
H
CH
CH
CH
CH
2
COOK
2
COOK
2
COOK
2
COOK
2-Cl
7
7
8
8
8
f
CN
CN
CN
CN
CN
2-Cl
2-Cl
4-Cl
5-Cl
4-Cl
2-CH
3-CH
H
H
H
3
CH
CH
CH
CH
2
COOK
2
COOK
2
COOK
2
COOK
g
a
b
c
3
8d
8e
8f
CN
CN
CN
4-Cl
5-Cl
5-Cl
2-CH
3-CH
H
3
CH
CH
2
COOK
COOK
3
2

1
2
T. FRA˛CZEK ET AL.
atoms with partial charges less than 0.2 were scaled down by a maintained at maximum 500,000 cells/mL, in Iscove’s Modified
4
1
factor of 0.9 . Several docking protocols were used:
Dulbecco’s Medium (IMDM) supplemented with 2 mM L-glutamine
Life Technologies) and 10% (v/v) heat-inactivated foetal calf
serum (Hyclone, Thermo Fisher Scientific, Waltham, MA), as
(
ꢂ
ꢂ
Standard and extra precision (XP) Glide docking with default
4
7
4
2–45
described earlier .
settings
. Three best poses were stored. For XP docking the
Lyophilised compound was dissolved in dimethyl sulfoxide to a
threshold to reject a minimised pose was increased to 0.9 kcal/
mol.
concentration of 5 mM, and further diluted in cell culture medium
þ
(
SupT1: IMDM and CD4 T cells: RPMI). Cells were incubated 2 h
Custom protocol 1 (CP1): The following positional constraint
was added during receptor grid preparation – a 1 Å sphere
centred at the oxygen atom of diphenyl ether motif of a refer-
ence ligand (from the crystallographic structure) had to be
occupied by a neutral H-bond acceptor atom of a docked lig-
and. All ligand poses which did not comply with the constraint
were rejected.
Custom protocol 2: ligand poses obtained with the standard or
custom (CP1) protocol were examined, and one best pose was
manually selected (basing on the similarity to the reference lig-
and pose from crystallographic structure). All ligands were then
aligned to the selected pose using the Flexible Ligand
Alignment tool from Maestro, using a maximal common sub-
structure. Subsequently, ligands were docked using Glide XP
with the sampling mode set to “None (refine only)”
prior infection in appropriate medium as described above, now
also supplemented with diluted compound. Subsequently, cells
þ
(
SupT1: 50,000 per 96 well, CD4 T cells: 250,000 per 96 well)
were infected with HIV (HIV NL4–3-GFP-I, an infectious virus
expressing green fluorescent protein (GFP) from gfp-IRES-nef
4
8
mRNA expressed from the nef locus, as described earlier .
Medium was as described above, now also supplemented with
ꢂ
þ
diluted compound (For CD4 T cells, PHA was left out). Medium
was refreshed after 24 h, keeping compound and supplement con-
centrations constant. After 72 h, cells were harvested, measured by
flow cytometry for GFP expression and counted, using
a
MACSQuant flow cytometer (Miltenyi Biotec, Bergisch Gladbach,
Germany). Infection rate measured by GFP expression was
between 6 and 20%. Cell numbers were used to measure cytotox-
icity (cell numbers in non-infected cultures supplemented with
compound were compared to cell numbers obtained in parallel
cultures without compound added: a reduction with more than
Only the best scoring pose obtained with either protocol was
kept for each docked ligand. The complete results can be found in
the supplemental file.
10% was considered to indicate cytotoxicity).
Results and discussion
IC50 measurements
RT inhibitory activity
HIV RT inhibitory activity of the new compounds was measured
using the colorimetric Reverse Transcriptase Assay kit (Roche, 7a and 8a were tested for the inhibitory activity against HIV-1 RT.
Basel, Switzerland). The assay was performed according to the Both compounds were found to be NNRTIs with IC50 of
manufacturer’s instruction with the only modification that 2 ng 1.23 ± 0.05 lM and 23.4 ± 1.6 lM, respectively. Using 7a and 8a as
(instead of suggested 4–6 ng which caused the reaction to run too lead compounds several of their analogues were prepared and
fast) of enzyme supplied was used for the each reaction. Stock tested in vitro. Replacement of nitrile with chlorine in 7b was det-
solutions of examined and reference compounds (NVP (TCI, Tokyo, rimental to the inhibitory activity (Table 2). Changing the position
Japan) and RPV) containing 5% dimethyl sulfoxide (Merck, or removing the chlorine atom in the central ring of 7a also
Darmstadt, Germany) were used to prepare dilutions ranging from resulted in increased IC50 values (7c and 7d, Table 2), but in case
0
.1 to 50 mM. Averaged results from at least two measurements of catechol ethers moving the position of chlorine from 4 in 8a to
were used to obtain an inhibition curve. IC50 values were obtained 5 in 8b was beneficial for activity (about seven-fold decrease the
by interpolating the curve using non-linear regression.
IC50 value, Table 2). Modifications introduced to the phenacyl moi-
ety of the new inhibitors resulted in several interesting findings.
7
e was found to be the most active NNRTI in this study with IC50
Solubility
0.36 ± 0.01 mM,
more
potent
and nearly
than
as
the
drug
as
NVP
RPV
(IC50 ¼ 0.75 ± 0.02 mM)
potent
Substances were dissolved in ultrapure deionised water at room
temperature up to concentrations of 50 g/L. Solutions were centri-
fuged and their aliquots were transferred into weighed glass vials Table 2. IC50 values, solubility and docking scores of examined compounds.
(
d ¼ 0.01 mg). The samples were dried under reduced pressure and Compound
IC50 [mM]
Solubility [g/L]
Mean docking score
the vials were weighed again. The solubility was calculated from 7a
1.23 ± 0.05
6.75 ± 0.50
8.30 ± 1.3
9.78 ± 0.67
0.36 ± 0.01
4.71 ± 0.43
0.65 ± 0.03
23.4 ± 1.6
3.40 ± 0.30
1.90 ± 0.11
4.70 ± 0.70
3.07 ± 0.22
1.27 ± 0.05
0.75 ± 0.02
0.32 ± 0.04
13.1
4.3
>50.0
>50.0
3.2
8.9
3.8
>50.0
>70.0
>50.0
14.4
–14.94
–14.56
–14.76
–14.39
–17.07
–14.97
–15.14
–14.85
–14.73
–16.60
–14.89
–15.30
–16.59
–
7
7
7
7
7
7g
8
b
c
d
e
f
mass difference divided by sample volume.
Infection assays and cytotoxicity measurements
þ
CD4 T cells were harvested from normal donors and cultured in
a
b
Roswell Park Memorial Institute medium (RPMI) supplemented
8
with 2 mM L-glutamine (Life Technologies, Merelbeke, Belgium), 8c
1
0% (v/v) heat-inactivated foetal calf serum (Hyclone, Thermo 8d
8e
f
39.9
Fisher Scientific, Waltham, MA), 100 U/mL penicillin and 100 mg/mL
streptomycin (Life Technologies), 20 ng/mL interleukin-2 (IL-2; spe-
8
NVP
>50.0
0.17
a
b
cific activity 10 U/ng, Peprotech, London, UK), and with 1 mg/mL RPV
phytohemagglutinin (PHA) mitogen (Thermo Fisher Scientific), 72 h
0.00002
–
a
49
Morelock et al.
15
4
6
b
prior to infection, as described earlier . SupT1 cells were
Janssen et al. .

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
13
(
IC50 ¼ 0.32 ± 0.04 mM). Analogous modification in catechol series
resulted in 8c, more potent that its parent compound 8a (IC50 against mutated forms of the enzyme we have performed docking
.9 ± 0.11 mM). Methylation of 7a in the phenacyl ring yielded 7f studies of 7e and RPV – clinically used drug of the second gener-
less potent) and 7 g, which was found to be another potent ation that is active against these mutations. For wild-type, K103N,
inhibitor of HIV-1 RT, slightly more active than NVP (IC50 and Y181C forms 3DRP, 3DRS and 3DRR structures were chosen,
.65 ± 0.03 mM). Catechol analogues 8d-f were found to possess
In order to assess expected activity of our best compound
1
(
0
comparable, low micromolar activity (Table 2).
Molecular docking analysis
Using one of the three docking protocols, a good pose for every
inhibitor could be found for all four receptors. Averaged docking
scores are given in Table 2 (individual scores are in Table S4 of
supplemental material). Interestingly, the obtained mean docking
scores show quite good qualitative correlation with IC50 values
(
Table 2). For example, for compounds 7a–g the docking scores
almost correctly rank the inhibitors by their activity: 7e, 7 g, 7f,
a, 7c, 7 b, 7d, with only 7f being swapped with 7a, as well as 7c
7
with 7b. For 8a–f the docking scores also were able to identify
the three most active inhibitors. The results show that the molecu-
lar docking may be a valuable tool in further development of
NNRTIs from this chemical class.
The docking analysis was also useful in gaining some insight
into the binding interactions of the new inhibitors with RT. The
diaryl ether part of examined compounds binds in the hydropho-
bic cavity formed by non-polar aromatic amino acid residues of
Tyr181, Tyr188, Phe227, Trp229 and Tyr318 (Figure 4(a)). The 3-
chloro-5-cyanophenyl ring forms a p–p stacking interactions with
Trp229 and Tyr188. The chlorine atom in the central aromatic ring
points towards the carbonyl oxygen of Tyr188, forming a halogen
bond. The carbonyl group of phenacyl moiety forms a hydrogen
bond with the backbone oxygen of Lys103. Finally, the carboxyl
Figure 5. Comparison of docking scores of 7e and RPV to selected RT mutants.
Table 3. Threshold toxic concentrations and antiviral activity at 10-fold lower
concentration of compounds examined.
Compound
Threshold toxicity [mM]
Antiviral activity
group of three-ringed inhibitors locates itself at the solvent 7a
5
50
50
50
50
>50
50
50
5
50
50
50
5
–
þ
–
7
7
7
7
b
c
exposed entrance to the binding site, forming a hydrogen bond
with Val106. In case of compounds 7e, 8c and 8f, the additional
phenyl ring between phenacyl and glycolic acid synthons is pre-
d
e
–
þ
þ
þ
–
dicted to bind in an unexplored pocket adjacent to the entrance 7f
7
8
8
8
g
to the NNRTIs binding site (Figure 4(b)). This pocket may be an
attractive target for the further optimisation of the new inhibitors.
The predicted binding modes of 7e, 8c and 8f suggest the exist-
ence of a strong ionic interaction between the inhibitors carboxyl 8d
and guanidine of Arg199, which explains the unusually high-dock- 8e
a
b
c
–
þ
–
þ
–
8f
ing scores for these three compounds (e.g. –19.62 for 7e in 3C6T
receptor, see Table S3), and may also be the cause of 7e potency.
NVP
RPV
50
0.5
þ
þ
Figure 4. (a) Predicted binding mode of 7a (PDB: 3C6T). Turquoise dashed lines – p–p stacking, red dashed lines – hydrogen bonds, purple dashed line – halogen
bond. (b) Predicted binding mode of 7e (PDB: 3C6T) viewed from the entrance to the NNRTIs binding site. Some residues removed for clarity.

1
4
T. FRA˛CZEK ET AL.
Figure 6. Cells were incubated with dilutions of 7e and infected with HIV-1. Plots indicate concentration (in mM) vs. infection rate (normalised to level without inhibi-
þ
tor) for (a) SupT1 and (b) CD4 T cells.
respectively, in case of 7e, because they share the same reference whether antiviral activity was observed at 10-fold lower concentra-
ligand. For K103N/Y181C double mutant 4RW4 structure was used. tion than this threshold toxic concentration are given in Table 3.
RPV was docked to 4G1Q (WT), 3MEG (K103N), 3JLC (Y181C) and
BGR (K103N/Y181C), which all have RPV as the native ligand in antiviral activity at less than 10% of toxic concentration (Table 3).
Several compounds tested in infectivity assay did not show
3
crystal structures, with the exception of 3JLC, as there is no crys- Nonetheless, of those who did, 7e inhibited infection most clearly
tallographic data in PDB for RPV – Y181C complex. The results below concentrations which affected cellular viability. Therefore,
show a small decrease of docking scores for RPV in K103N and this compound was tested more extensively in SupT1 cells, as well
þ
K103/Y181C, and a significant drop for Y181C mutant (in this case as in peripheral blood CD4 cells. As shown in Figure 6, both in
the decrease may be overestimated since this is not the native SupT1 cells as in primary T cells, IC50 was around 0.25 mM (toxicity
crystal structure, Figure 5). Interestingly, 7e shows practically the was only apparent above 20 mM). The measurements were run in
parallel with NVP as a reference, and it showed IC50 of 0.04 mM, in
line with literature .
same scores for WT, Y181C and K103N/Y181C forms, and an
increased score for K103N (Figure 5). This suggests that 7e is not
sensitive to these mutations.
5
0
Conclusions
Solubility
Two novel scaffolds of diaryl ether NNRTIs with phenacyl moiety
were designed in this study. With the aid of molecular modelling
several modifications of the core structures were prepared. Using
molecular docking to several crystallographic structures and thor-
ough conformational search of ligands, it was possible to obtain
quite accurate predictions of structure–activity relationship. All
synthesised compounds showed inhibitory activity against wild-
type HIV-1 RT. In general, resorcinol-based compounds possessed
better activities than catechol-based compounds, and are more
promising candidates for further development. One of the com-
pounds, 7e, was found to be a very potent NNRTI in enzymatic
assay, and is predicted to display novel interactions with the RT.
The presented compounds were designed to possess a good
aqueous solubility, which was achieved in all cases. Given those
encouraging results, the inhibitors were subjected to biological
evaluation of their efficacy against HIV infection in vitro. 7e proved
The aqueous solubility (at pH ¼7) of synthesised compounds is pre-
sented in Table 2. Many of the compounds show a good solubility,
exceeding 50g/L. Exact measurements of the maximum solubility
proved to be infeasible due to the formation of micelles, aggregates
and ultimately gels for increasing concentrations of the compounds.
This behaviour results from the amphiphilic character of the exam-
ined compounds and elongated shapes of their molecules, which
gives them surfactant-like properties. The general trend observed
for all inhibitors is that catechol-based ethers (8a–f) are better sol-
uble than their resorcinol counterparts (7a–g). Interestingly, remov-
ing or replacing the chlorine atom in two-position of resorcinol-
based ethers like in case of 7c and 7d significantly increased their
solubility. However, as discussed above, the two-position of the
chlorine atom is optimal for the inhibitory activity. Methyl substitu-
tion in the phenacyl ring was also detrimental for the solubility. The
most active compound 7e shows relatively low solubility, but it is
still ca. 100,000 times greater than that of RPV. Calculated octanol-
water partition coefficients (logD) for examined compounds range
from 0.97 to 3.05, which is close to that of NVP (2.49), and signifi-
cantly lower than that of RPV (5.47, Table S5).
þ
to be a potent anti-viral in SupT1 and CD4 T cell infectivity
assays. These results show our design could deliver highly water-
soluble NNRTIs, at least one compound displays potent antiviral
activity in infection assays in vitro.
Disclosure statement
The authors report no conflicts of interest.
Infection assays
First, toxicity of the compounds was determined in SupT1 cells.
Since even minor toxicity affects the support of viral replication by
Funding
the cell, compounds should be active well below threshold toxic The reported studies were supported by the grant 2011/02/A/ST4/
concentration (i.e. concentration at which toxicity is observed in 00246 (2012–2017) from the Polish National Science Centre (NCN)
1
0-fold titration series). The threshold toxic concentration, and to PP, and by Ghent University grant BOF17/GOA/013, HIVERA

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
15
IRIFCURE and grants from the Research Foundation Flanders
FWO) to BV. BV is a Senior Clinical Investigator of the FWO.
pyrimidinyl]amino]benzonitrile (R278474, rilpivirine). J Med
Chem 2005;48:1901–9.
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ORCID
Tomasz Fr a˛ czek
Agnieszka Krakowiak
Bruno Verhasselt
http://orcid.org/0000-0002-2496-9508
http://orcid.org/0000-0002-0273-2972
http://orcid.org/0000-0002-4645-1144
Piotr Paneth
http://orcid.org/0000-0002-3091-8387
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