Effect of stereo and regiochemistry towards wild and multidrug resistant HIV-1 virus: Viral potency of chiral PETT derivatives

Article abstract of DOI:10.1016/j.bcp.2004.01.019

Chiral derivatives of several substituted halopyridyl and thiazolyl PETT compounds were synthesized as non-nucleoside inhibitors of the reverse transcriptase (RT) enzyme of the human immunodeficiency virus (HIV-1). Molecular modeling studies indicated that because of the asymmetric geometry of the non-nucleoside inhibitors (NNRTI) binding pocket, the 'R' stereoisomers would fit the NNRTI binding pocket of the HIV-1 RT much better than the corresponding 'S' stereoisomers, as reflected by their 10⁴-fold lower K_i values. The 'R' stereoisomers of several PETT derivatives inhibited the recombinant RT in vitro with lower IC₅₀ values than their enantiomers. The active compounds were further evaluated for their ability to inhibit HIV-1 replication in human peripheral blood mononuclear cells (PBMCs). All the 'R' isomers again showed potent anti-HIV activity and inhibited the replication of the HIV-1 strains HTLV_IIIB in PBMCs at nanomolar concentrations whereas their enantiomers were less potent. The lead compounds for the respective groups were further tested against A17 (NNRTI-resistant, Y181C mutant RT), and A17Var (NNI-resistant Y181C±K103N mutant RT) as well as multidrug resistant viral strains. The results indicated that the lead compounds were several logs more potent than the standard NNRTI drug nevirapine. Structure-activity relationship among the derivatives showed preference of pyridyl unit with halo substitutions primarily at 5-position demonstrating the importance of both the stereochemistry as well as regiochemistry. Our data provides experimental evidence that the stereochemistry and the regiochemistry of non-nucleoside inhibitors can profoundly affect their anti-HIV activity.

Full text of DOI:10.1016/j.bcp.2004.01.019

Biochemical Pharmacology 67 (2004) 1933–1946
Effect of stereo and regiochemistry towards wild and multidrug resistant
$
HIV-1 virus: viral potency of chiral PETT derivatives
*
Taracad K. Venkatachalam, Chen Mao, Fatih M. Uckun
Department of Chemistry, Structural Biology and Virology, Parker Hughes Institute, 2657 Patton Road, St. Paul, MN 55113, USA
Received 28 August 2003; accepted 16 January 2004
Abstract
Chiral derivatives of several substituted halopyridyl and thiazolyl PETT compounds were synthesized as non-nucleoside inhibitors of
the reverse transcriptase (RT) enzyme of the human immunodeficiency virus (HIV-1). Molecular modeling studies indicated that because
of the asymmetric geometry of the non-nucleoside inhibitors (NNRTI) binding pocket, the ‘R’ stereoisomers would fit the NNRTI binding
4
pocket of the HIV-1 RT much better than the corresponding ‘S’ stereoisomers, as reflected by their 10 -fold lower K values. The ‘R’
i
stereoisomers of several PETT derivatives inhibited the recombinant RT in vitro with lower IC50 values than their enantiomers. The active
compounds were further evaluated for their ability to inhibit HIV-1 replication in human peripheral blood mononuclear cells (PBMCs). All
the ‘R’ isomers again showed potent anti-HIVactivity and inhibited the replication of the HIV-1 strains HTLVIIIB in PBMCs at nanomolar
concentrations whereas their enantiomers were less potent. The lead compounds for the respective groups were further tested against A17
(
NNRTI-resistant, Y181C mutant RT), and A17Var (NNI-resistant Y181C ꢀ K103N mutant RT) as well as multidrug resistant viral
strains. The results indicated that the lead compounds were several logs more potent than the standard NNRTI drug nevirapine. Structure–
activity relationship among the derivatives showed preference of pyridyl unit with halo substitutions primarily at 5-position demonstrating
the importance of both the stereochemistry as well as regiochemistry. Our data provides experimental evidence that the stereochemistry
and the regiochemistry of non-nucleoside inhibitors can profoundly affect their anti-HIV activity.
#
2004 Elsevier Inc. All rights reserved.
Keywords: HIV; Multidrug resistance; Non-nucleoside inhibitors; Regiochemistry; Stereochemistry; Thiourea
1
. Introduction
from AIDS [7]. The most commonly used combinations
include two nucleoside analogs, with or without a protease
inhibitor [8]. In recent years, structure-based design has
played an increasingly important role in the development
of useful drugs, as demonstrated by the success of HIV-
protease inhibitor design [9]. Nevirapine is currently the
only non-nucleoside inhibitor compound that has been
used in combination with AZT and/or protease inhibitors
for the treatment of HIV-1. A new series of effective drug
combinations most likely will involve other non-nucleo-
side inhibitors in combination with nucleoside and pro-
tease inhibitors as a triple-action regimen to combat the
growing problem encountered during therapy due to the
emergence of resistant mutant viral forms. NNRTIs inhibit
HIV RT by altering either of the conformation or mobility
of RT by binding to a specific allosteric site near the
polymerase site, thereby resulting in non-competitive inhi-
bition of the enzyme [10–12]. In a systematic search for
potent anti-AIDS drugs, Bell et al. and Cantrell et al.
[13,14] discovered the phenethylthiazolylthiourea (PETT)
Human immunodeficiency virus type-1 (HIV-1) is the
causative agent for the transmission and development of
the acquired immunodeficiency syndrome (AIDS) [1].
Current therapies target intervention in the replication
cycle of the virus. The main targets in contemporary drug
discovery efforts against HIV-1 is reverse transcriptase
(RT), a vital enzyme that is responsible for the reverse
transcription of retroviral RNA to proviral DNA [2–4].
Reverse transcriptase inhibitors constitute two types:
nucleoside inhibitors such as AZT, 3TC and DDI, and
non-nucleoside inhibitors (NNRTIs) such as nevirapine,
delavirdine and efavirenz [5,6]. Combination therapies
may be significant factor in the dramatic decrease of deaths
$
Supplementary data associated with this article can be found, in the
online version, at doi:10.1016/j.bcp.2004.01.019
*
Corresponding author. Tel.: þ1-651-204-3659; fax: þ1-651-204-3689.
E-mail address: fatih_uckun@ih.org (F.M. Uckun).
0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2004.01.019

1934
T.K. Venkatachalam et al. / Biochemical Pharmacology 67 (2004) 1933–1946
class of compounds as potent inhibitors of HIV-1 and
disclosed the structure–activity relationship among various
substituents in their structure. However, there was no report
on the role of stereochemistry or regiochemistry towards
anti-HIV activity of PETT derivatives. Following this lead
and using rational drug design we have identified several
structurally distinct thiourea compounds as potent NNRTIs
for HIV-RT [15–21] and examined their structure–activity
relationship. In order to examine the effect of stereochem-
istry of these PETT derivatives on HIV-activity we synthe-
sized several chiral thiourea derivatives and examined their
anti-HIVactivity against the HIV-1 strain as well as multi-
ple mutated strains. The following report gives the results
obtained during the course of that study. Additionally, this
study demonstrates for the first time that substitution on the
ethyl linker unit of these PETT derivatives plays a sig-
nificant role in their antiviral profile. It had been previously
hypothesized that substitution on the aromatic and hetero-
cyclic rings of the thiourea moiety imparted the biological
activity, and no attention had been given to substitutions on
the ethyl linker of the molecule.
depending on the compound and included either one or a
combination of the following: ethyl acetate, methanol,
chloroform, hexane, methylene chloride, THF and ether.
Condensing the respective chiral amines and thiocarbai-
midazole derivatives of 5 - and 6 -substituted amino pyr-
idines in anhydrous dimethylformamide made compounds.
The chiral amines were purchased from Aldrich chemical
company and were used without further purification. Thia-
zolyl and benzothiazolyl substituted thioureas were pre-
pared in a similar fashion using thiocarbaimidazole
derivatives of either substituted amino thiazoles or ben-
zothiazoles, respectively.
A general synthesis for the compounds is as follows:
thiocarbonyl di-imidazole and 2-amino-5-halo pyridine was
added to 100 ml of dry acetonitrile under nitrogen atmo-
sphere, and then stirred at room temperature for 12–15 h.
The precipitate obtained was filtered, washed with cold
acetonitrile, and dried thoroughly under vacuum to yield the
thiocarbonyl intermediate. In the subsequent step, these
intermediates were taken up in a dry flask under nitrogen,
50 ml of anhydrous dimethylformamide was added, and
the contents were stirred for 30 min at room temperature.
The corresponding chiral amine dissolved in 10 ml of dry
dimethylformamide was added to this solution and the
reaction mixture was heated to 110 8C for 15 h. The reac-
tion mixture was cooled to room temperature, then poured
into a crushed ice/water mixture and the contents were
stirred for an additional hour. The precipitate was filtered,
washed with cold water several times and dried under
vacuum. The dried precipitate was then dissolved in chloro-
form and washed with brine, water and finally the separated
chloroform layer was dried over anhydrous magnesium
sulfate. Filtration and evaporation of the solvent yielded
the target thiourea compound. Compounds were further
purified using column chromatography on silica gel, and
finally recrystallized using ethanol. A similar procedure
was followed for other substituted compounds using either
thiazolyl or benzothiazolyl precursors.
0
0
2
. Materials and methods
2
.1. General synthesis of thiourea compounds
All chemicals were purchased from Aldrich (Milwau-
kee, WI) and were used without further purification. Unless
otherwise noted, each reaction vessel was secured with
rubber septa, and the reaction was performed under a
nitrogen atmosphere. H, 13C NMR spectra were obtained
on a Varian Mercury 300 instrument at ambient tempera-
1
ture in DMSO-d . Chemical shifts are reported as d values
6
in parts per million (ppm) downfield from tetramethylsi-
lane (d ¼ 0:0 ppm) as an internal standard or from the
1
residual dimethylsulfoxide signal (d ¼ 2:49 ppm for H
1
3
NMR or d ¼ 39:7 ppm for C NMR). Splitting patterns
are designated as follows: s, singlet; d, doublet; t, triplet; m,
multiplet; br, broad peak. FT-IR spectra were recorded on a
Nicolet Prot e´ g e´ 460 spectrometer. Mass spectra were
performed on a Hewlett-Packard MALDI-TOF spectro-
meter (Model G2025A LD-TOF). Melting points were
determined using a Melt John’s apparatus and are uncor-
rected. HPLCs were done using a Hewlett-Packard 1100
series instrument consisting of an automatic sampler, an
electronic degasser, a thermostatic control unit, and a diode
array detector in conjunction with a Chemstation software
assembly. The column used was an analytical RP-18
Lichrospher column, 5 mm (4:6 mm  250 mm) and eluent
was 35:65 water (0.1% acetic acid):acetonitrile. The flow
rate was maintained at 1.0 ml/min and the detection wave-
length was set at 275 nm. The column was maintained at
room temperature throughout the analysis. Column chro-
matography was performed using silica gel obtained from
the Baker Company. The solvents used for elution varied
2.2. Stock HTLVIIIB virus
The HIV-1 strain HTLVIIIB, which was propagated in
CCRF-CEM, cells, was used in in-vitro assays of the anti-
HIV-1 activity of the synthesized thiourea derivatives. Cell
free supernatants of HTLVIIIB-infected CCRF-CEM cells
were harvested, dispensed into 1 ml aliquots, and frozen at
À70 8C. Periodic titration of stock virus was performed by
examining its cytopathic effects in MT-2 cells [22,23].
2.3. In vitro assays of anti-HIV-1 activity
Normal human peripheral blood mononuclear cells
(PBMCs) from HIV-negative donors were cultured 72 h
in RPMI 1640 supplemented with 20% (v/v) heat-inacti-
vated fetal bovine serum (FBS), 3% interleukin-2, 2 mM
L-glutamine, 25 mM HEPES, 2 g/l NaHCO , 50 mg/ml
3

T.K. Venkatachalam et al. / Biochemical Pharmacology 67 (2004) 1933–1946
1935
gentamicin, and 4 mg/ml phytohemagglutinin prior to
exposure to HIV-1 at a multiplicity of infection (MOI)
of 0.1 during a 1 h adsorption period at 37 8C in a humi-
by an extra methyl group, both ‘R’ and ‘S’stereoisomers, in
affecting their biological activities towards reverse tran-
scriptase.
dified 5% CO atmosphere. Subsequently, cells were cul-
6
Among the ten b-methyl phenylethyl pyridyl thiourea
compounds, the ‘R’ stereoisomers of compounds with
halogen (Br, Cl) or methyl substitutions respectively at
5-position, compounds 1, 3 and 5, were the most potent
(Table 1). These compounds displayed nanomolar IC50
values against recombinant RT in vitro. In contrast the ‘S’
stereoisomers showed lower potency with IC50 values in
the range of 1–8 mM concentrations. Similarly, only the ‘R’
stereoisomers of the b-methyl phenylethyl thiazolyl thiour-
eas (11 and 15) exhibited anti-HIV activity (Table 1).
Overall, the ‘R’ stereoisomers of all compounds inhibited
the recombinant RT in vitro with lower IC50 values than
their enantiomers.
Of the methyl phenyl thiourea (MPT) compounds (1–19)
whose ‘R’ stereoisomers exhibited nanomolar IC50 values
against recombinant RT, five compounds (1, 3, 5, 7, and 9)
were further evaluated for their ability to inhibit HIV-1
replication in human peripheral blood mononuclear cells.
These ‘R’ stereoisomers were active anti-HIV agents and
inhibited the replication of the HIV-1 strains HTLVIIIB
2
tured in 96-well microtiter plates (100 mm; 2 Â 10 cells/
ml) in the presence of various concentrations of thiourea
analogues or nevirapine, trovirdine or AZT and aliquots of
culture supernatants were removed from the wells on the
7
th day after infection for p24 antigen assays, as previously
described [22,23]. The applied p24 enzyme immunoassay
EIA) was the unmodified kinetic assay commercially
available from Coulter Corporation/Immunotech, Inc.
Westbrooke, ME), which utilizes a murine mAb to HIV
(
(
core protein coated onto microwell strips to which the
antigen present in the test culture supernatant samples
binds. Percent viral inhibition was calculated by compar-
ing the p24 values from untreated infected cells (i.e. virus
controls). In addition, the IC50 values measuring the activ-
ity of compounds against recombinant HIV-1 RT (rRT)
were determined using the Quan-RT assay system (Amer-
sham, Arlington Heights, IL, USA), which utilizes the
scintillation proximity assay principle [22,23].
(
NNRTI-sensitive) at nanomolar concentrations. When
3
. Results and discussion
examined for their ability to inhibit the replication of
the HIV-1 strain, A17 (NNRTI-resistant, Y181C mutant
RT) and A17Var (NNI-resistant, Y181C ꢀ K103N
mutant), majority of the ‘R’ isomers showed activity from
nanomolar to micromolar concentration range. The ‘R’
stereoisomers of MPT compounds were relatively less
potent against NNRTI-resistant HIV-strains than they were
against HTLVIIIB. Compound 3, the 5-chloropyridyl iso-
mer, was 380 times more active than nevirapine, 190 times
more than delavirdine, and two times more active than
trovirdine. We also examined the activity shown by these
compounds against A17 variant HIV-1 strain and found the
For the synthesis of thiourea compounds we followed
the general procedure shown in Scheme 1 as previously
reported [14–16].
In our previous studies [15,16], we modified the struc-
ture of phenylethyl thiourea compounds by introducing
various groups on the phenyl ring and evaluated their
activity towards RT. In this study, we focused on the role
of the chiral center of the phenyl ethyl thioureas for their
NNRTI activity. Accordingly, we prepared the first group
of chiral thiourea compounds to understand the role played
Scheme 1.

1936
T.K. Venkatachalam et al. / Biochemical Pharmacology 67 (2004) 1933–1946
Table 1
Effect of stereochemistry on anti-HIV activity of methyl phenylethyl substituted thiourea compounds
X
S
S
N
Y
S
N
Z
N
N
N
N
N
S
N
N
S
H
H
H
H
H
H
Compound
Isomer
X
Y
rRT (mM)
RT (mM) HTLV III
B
A17 (mM)
A17V (mM)
1
2
3
4
5
6
7
8
9
R
S
5-Br
5-Br
–
–
–
–
–
–
–
–
–
–
0.42
1.90
0.10
1.39
0.52
7.70
16.71
>100
8.40
3.50
5.05
>100
>100
>100
10.02
>100
>100
>100
38.75
23
0.003
0.030
<0.001
0.038
0.003
–
0.67
1.18
0.26
0.54
1.08
–
1.08
1.23
R
S
5-Cl
0.53
5-Cl
5-Me
>100
1.84
–
R
S
5-Me
R
S
6-Me
0.04
–
1.40
–
1.19
–
6-Me
H
H
–
R
S
0.028
–
2.34
–
0.54
–
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
9
R
S
4-Me
4-Me
4-Methylenecarbo
0.055
–
6.85
–
1.10
–
–
R
S
–
–
–
–
–
4-Methylenecarbo
–
–
–
R
S
–
H
–
–
–
–
H
4-Methylbenzo
–
–
–
R
R
R
–
–
–
–
–
–
Benzothiazolyl
–
–
–
–
4,6-Me
–
–
–
Nevirapine
Trovirdine
Delavirdine
–
–
–
–
–
–
–
0.034
0.007
0.009
0.0015–
0.005
0.002
100
0.50
50
–
100
100
100
1.5
–
–
–
0.8
–
–
1.5
Efavirenz [27]
Capravirin [28]
–
–
–
–
–
–
–
TMC-125 [29–31]
–
–
–
–
–
The anti-HIV activity was measured by determining the inhibition of the HIV-1 strain HTLVIIIB in human PBMCs as previously described in detail [21,22].
The cell-free RT inhibition assays using recombinant RT(rRT) and the Quan-RT assay kit (Amersham, Arlington Heights, IL) were performed as reported
[21,22].
entire series of these derivatives indicated activity in
nanomolar concentrations whereas the standard drugs,
namely nevirapine, trovirdine and delaviridine, were less
potent. Based on the above experimental result we con-
clude that these b-methyl phenylethyl thiourea compounds
may be useful candidates for further development espe-
cially since they showed remarkable in vitro activity
against mutant strains of HIV-1. Table 2 shows the activity
observed for b-methyl phenylethyl thioureas against RT
MDR. It is evident from the table values that the halo
pyridyl compounds exhibited potent activity at nanomolar
concentrations. The b-methyl phenyl ethyl thiourea com-
pounds were ten times more active compared to AZT, 65
times more potent than nevirapine and 19 times active than
delavirdine indicating the role played by the structure of
these compounds towards the mutant strains.
Our molecular modeling studies indicated that the ‘R’
stereoisomers of chiral halopyridyl and thiazolyl thiourea
compounds would fit the target NNRTI binding pocket on
HIV-RT better than their enantiomers. Unfavorable inter-
actions with the NNRTI binding pocket near the Y181 side
chain would impair the binding of the ‘S’ stereoisomer in
its lower energy, ‘‘staggered’’ conformation. This steric
hindrance could be relieved if the ‘S’ stereoisomer adopted
an energetically unfavorable ‘‘eclipsed’’ conformation.
In either case, the estimated binding energies would be
significantly higher for the ‘S’ compounds compared to
the ‘R’ isomers. Fig. 1 shows a representative picture of
Table 2
Activity of b-methyl phenylethylthioureas against multidrug resistance
strain of HIV-1
X
S
N
N
N
H
H
Compound
Isomer
X
RTMDR (mM)
1
R
S
5-Br
5-Br
5-Cl
5-Cl
5-Me
6-Me
H
0.02
0.17
0.02
0.16
0.05
0.10
0.19
0.15
1.40
0.40
2
3
R
S
4
5
R
R
R
–
7
9
AZT
–
Nevirapine
Delavirdine
–
–
–
–
The anti-HIV activity was measured by determining the inhibition of the
replication of the strain in human PBMC [21,22].

T.K. Venkatachalam et al. / Biochemical Pharmacology 67 (2004) 1933–1946
1937
well as the benzothiazolyl compounds. Additionally, it was
observed that halo-substitutions at the 5-position of the
pyridyl ring enhanced the activity of these thioureas.
Although a methyl substitution at the 6-position of the
pyridyl ring was tolerated, once again the 5-position was
preferred compared to the 6-position. This demonstrates
the importance of substituent regiospecificity in addition to
stereospecificity in these compounds.
3.2. The activity of chiral a-methyl benzyl thioureas
The synthesis followed the general procedure shown in
Scheme 1 using the appropriate chiral amines and thio-
carbonylimidazole derived from heterocyclic amines.
Table 3 gives the anti-HIV activity observed for all the
isomers of chiral a-methyl benzyl thiourea derivatives
towards recombinant reverse transcriptase.
Fig. 1. Molecular model of a phenethyl pyridyl thiourea molecule that is
docked into the non-nucleoside inhibitor reverse transcriptase binding
pocket. The phenyl group is attached through a two-carbon linker marked
as ‘B’ and ‘A’ in the figure. The chiral center in the molecule is labeled as
The a-methyl as well as a-ethyl groups were maintained
constant and the pyridyl group was replaced by thiozolyl
and benzothiazole rings. Additionally, we introduced sev-
eral substituents in those rings in order to extend our study
and have some understanding about structure–activity
relationship among these derivatives. From Table 3 it is
evident that all the ‘R’ isomers were active whereas their
enantiomers (‘S’) were inactive. This is in accordance
with the results obtained from chiral PETT derivatives
of b-methyl phenylethyl thiourea compounds as discussed
previously (Table 1). Substitution on the pyridyl ring at
5-position was essential to retain the activity of the com-
pounds. Among the substituents at 5-position, halo as well
as alkyl substitutions yielded active derivatives. Among the
methyl-substituted derivatives, the 5-methyl compound
was more active than the 6-methyl substituted compound
for these assays. Changing the pyridyl ring to thiazole ring
and methyl substitution at 4-position of the thiazolyl ring
did not measurably affect the activity of the compound.
When we extended the study towards benzothiazolyl
compounds, the activity of the compound decreased con-
siderably indicating that this substitution is not beneficial
for the PETT derivatives used in this study. Further intro-
duction of groups on the benzothiazolyl functionality
(compounds 38 and 43) also did not improve biological
activity.
The next aspect examined was the alteration of the chiral
methyl group of these a-methyl benzyl thioureas using
ethyl group instead. Unfortunately, we were able to only
use a racemic mixture for this purpose due to non-avail-
ability of the individual isomeric amines. The thiazolyl
compound (20) indicated an activity of 1.1 mM whereas the
pyridyl compound (28) was 15 times less potent against
recombinant reverse transcriptase. Interestingly, the
bromo- and chloro-substituted pyridyl compounds (25
and 26) showed activity in the range of 3–5 mM, which
was better than the unsubstituted pyridyl compound (28)
(13.1 mM). Methyl substitution at 4-position of the thia-
zolyl compound indicated only moderate activity, however,
‘B’. The pyridyl group is represented on the left hand side of the figure and
is connected through thiourea unit.
phenylethyl pyridylthiourea molecule docked into the
NNRTI binding site using previously described method
[
6,12].
Substitutions at the ‘R’ and ‘S’ positions would lead to
different conformations of the phenethyl group in the Wing
region. The ‘R’ stereoisomer is more energetically
2
favored than the ‘S’ stereoisomer, consistent with the
predicted better-fit, and thus stronger binding, with the
NNRTI binding pocket. However, the two-carbon linker
between the phenyl group and the thiourea group is
adjustable and more forgiving. The energetic difference
is minimized by adopting an alternative conformation to
maintain the tight fit with the binding pocket for both ‘R’
and ‘S’ stereoisomers, in comparison with those of the
compounds with only a one-carbon spacing. The com-
pounds with a one-carbon spacing are more rigid and their
‘
S’ stereoisomers have to overcome greater energetic
barrier to accommodate the binding pocket. Based on this
rationale we would expect that the ‘R’ stereoisomers of
b-methyl phenylethyl halopyridylthioureas may show
greater potency compared to the ‘S’ stereoisomers against
reverse transcriptase, which is consistent with our experi-
mental results. It is interesting to note that aza analogs of
chiral ((2-phosphonomethoxy)propyl) guanines of a ‘R’
stereoisomeric configuration were found to show potent
anti-HIV activity as compared to the ‘S’ isomers [24],
another result that is consistent with our experimental data
on chiral PETT derivatives.
3
.1. Structure–activity relationship among chiral
b-methyl phenethyl thioureas
Among the chiral derivatives studied in the previous
class of compounds it is evident that pyridyl compounds
exhibit more potent activity compared to the thiazolyl as

1938
T.K. Venkatachalam et al. / Biochemical Pharmacology 67 (2004) 1933–1946
Table 3
Effect of stereochemistry on anti-HIV activity of a-methyl benzyl substituted thiourea compounds
R
S
X
N
N
H
H
Compound
R
Isomer
X
rRT (mM)
RT (mM) HTLVIIIB
N
2
0
1
Ethyl
R/S
1.1
<0.001
S
N
Br
Cl
2
Methyl
Methyl
Methyl
Methyl
Ethyl
R
1.6
1.2
<0.01
<0.01
>1
22
23
24
25
26
27
R
N
N
Br
Cl
S
>100
>100
5.4
S
–
N
N
Br
Cl
R/S
R/S
R
0.001
0.001
<0.001
Ethyl
3.4
N
CH3
N
Methyl
0.8
S
N
28
29
30
Ethyl
R/S
S
13.1
>100
17.5
–
CH3
N
Methyl
Ethyl
–
S
O
C2H5
R/S
N
0.05
O
S
S
N
CH3
N
3
1
Ethyl
R/S
5.8
0.02
CH3
3
3
2
3
Ethyl
Ethyl
R/S
R/S
0.8
0.005
N
>100
–
S
CH3
34
Ethyl
R/S
>100
–
N
CH3

T.K. Venkatachalam et al. / Biochemical Pharmacology 67 (2004) 1933–1946
1939
Table 3 (Continued )
Compound
R
Isomer
X
rRT (mM)
RT (mM) HTLVIIIB
35
36
37
Methyl
S
>100
–
N
N
CH3
CH3
Methyl
Ethyl
R
1.5
5.7
–
–
R/S
N
CH3
H3C
38
Methyl
R
8.7
–
N
S
OCH3
N
3
9
0
Methyl
Methyl
S
S
>100
>100
–
–
S
CH3
4
N
41
Methyl
R
1.7
0.025
N
CH3
OCH3
N
N
4
2
3
Ethyl
R/S
>100
>100
–
–
S
S
OCH3
4
Methyl
R
Nevirapine
Trovirdine
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
23
0.8
1.5
0.6
–
0.034
0.007
0.009
0.001
0.0015
0.005
0.002
Delvirdine
HI-240
Efavirenz [27]
Capravirin [28]
TMC-125 [29–31]
–
–
The anti-HIVactivity was measured by determining the inhibition of the HIV-1 strain HTLVIIIB in human PBMCs as previously described in detail [21,22]. The
cell-free RT inhibition assays using recombinant RT (rRT) and the Quan-RT assay kit (Amersham, Arlington Heights, IL) were performed as reported [21,22].
when carboethoxy group was introduced the activity
decreased by 20-fold. Benzothiazolyl, dimethyl pyridyl,
substitutions were also found to be detrimental for these
sets of ethyl substituted compounds.
We next examined the ability of these novel thiourea
compounds to inhibit the replication of the HIV-1 strain
HTLVIIIB in human peripheral blood mononuclear cells
recombinant reverse transcriptase activity. It is also of note
that the racemic ethyl substituted thioureas were very potent
showing nanomolar level activity, implying that these
compounds may be useful as lead compounds for future
studies. In order to evaluate their clinical potential, we
further examined their activity against a multidrug resistant
virus (Table 4).
(
PBMCs). As shown in Table 3, few of the thiourea
As a reference, we have also included the known anti-
viral compounds values for Table 4. Compound 27 was
1400 times more active than nevirapine, and 400 times
more active than delavirdine. Similarly, compound 22 was
250 times more active than nevirapine and 80 times more
active than delavirdine. In order to further evaluate the
potential outcome of these chiral derivatives, a few of these
derivatives inhibited HIV-1 replication at nanomolar con-
centrations. Once again the ‘R’ isomers were more active as
compared to their enantiomers. The thiazolyl compound
(20) was a potent compound with an activity of 0.001 mM in
our assay. The structure activity relationship observed in the
series was almost akin to that observed in the case of

1940
T.K. Venkatachalam et al. / Biochemical Pharmacology 67 (2004) 1933–1946
Table 4
Activity of a-methyl benzylthioureas against multidrug resistance and mutant virus strains of HIV-1
R
S
X
N
N
H
H
Compound
R
Isomer
X
RTMDR (mM)
A17 (mM)
A17V (mM)
N
2
0
1
Ethyl
R/S
1.130
2.0
1.50
S
N
N
Br
Cl
2
Methyl
Methyl
R
R
0.005
0.010
0.010
0.20
2.70
2
2
2
7
10.20
CH3
N
N
Methyl
R
0.001
0.006
0.164
0.196
S
S
CH3
31
Ethyl
R/S
0.321
0.154
Nevirapine
Trovirdine
Delavirdine
HI-240
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1.400
100.0
0.50
50.0
0.20
–
100.0
100.0
100.0
41.0
1.5
–
0.400
–
–
–
–
Efavirenz [27]
Capravirin [28]
TMC-125 [29–31]
–
–
–
–
derivatives were tested against A17, A17 variant virus
strains (Table 4). These compounds show promise indicat-
ing activity in nanomolar to micromolar concentration. As
a whole, a trend emerges that a 5-substitution appears to be
optimal in these thioureas. The stereochemistry also needs
to have a ‘R’ configuration for the best anti-HIV activity.
This is in agreement with our previous hypothesis and
strongly suggests that the stereochemistry of the thiourea
derivatives plays a critical role in determining their activity
towards HIV-1.
the most potent (Table 5). These compounds had nano-
molar IC50 values against recombinant RT in vitro.
In contrast, none of the ‘S’ stereoisomerisms were active
against recombinant RT. Similarly, only the ‘R’ stereoi-
somers of the five a-naphthyl ethyl thiazolyl thiourea
compounds exhibited anti-HIV activity (Table 5). Overall,
the ‘R’ stereoisomers of all 11 compounds inhibited the
recombinant RT in vitro with lower IC50 values than their
enantiomers.
Of the seven compounds whose ‘R’stereoisomers exhib-
ited nanomolar IC50 values against recombinant RT, five
were further evaluated for their ability to inhibit HIV-1
replication in human PBMCs. All five ‘R’ stereoisomers
were active anti-HIVagents and inhibited the replication of
the HIV-1 strains HTLVIIIB (NNRTI-sensitive), A17 (NNI-
resistant, Y181C mutant RT), and A17Var (NNI-resistant,
Y181C ꢀ K103N mutant RT) in human PBMCs at nano-
molar concentrations, whereas their enantiomers were
inactive (Table 6).
In addition we observed that the A17V (Y181C ꢀ
K103N) mutant was quite resistant to these compounds
as seen in Table 6. Furthermore, in comparison with
RTMDR, the Y181C ꢀ K103N mutant displayed high
resistance to all the compounds tested in the present
study. For example, the Y181C ꢀ K103N mutant displays
10 times greater resistance to compound 52, and 50 times
or greater resistance to 44, 46 and 48, respectively.
3
.3. Molecular modeling studies
Molecular modeling studies indicated that the ‘R’
stereoisomer would fit the target NNRTI binding pocket
on HIV-RT much better than its enantiomer (Fig. 1) in the
case of b-methyl phenyl ethyl thiourea derivatives. We
further proposed that a similar trend is anticipated to occur
in the case of a-methyl and a-ethyl benzyl thiourea deriva-
tives. This was further confirmed by extending our exam-
ination towards cyclohexenyl, as well as naphthyl chiral.
This third class of naphthyl substituted chiral PETT
compounds were synthesized using a similar method to
the one described previously (Scheme 2). Among the six
a-naphthyl ethyl pyridyl thiourea compounds, the ‘R’
stereoisomers of compounds with halide (Cl (44), Br
(
46)) and methyl (48) substitutions at the 5-position were

T.K. Venkatachalam et al. / Biochemical Pharmacology 67 (2004) 1933–1946
1941
Scheme 2.
However, our lead compounds 44 and 52 were 3 orders of
magnitude more potent than the standard NNRTI drug
nevirapine against the NNRTI-resistant HIV-1 strains.
Table 6 also shows the activity data observed for com-
pounds against drug resistant laboratory strains of HIV-1 as
well as a lead clinical HIV-1 isolates for AIDS patients.
The structure–activity relationship once again established
that a pyridyl ring with halo substitutions at the 5-position
was essential to retain the anti-HIV activity of these
naphthyl thiourea compounds. This is again consistent
Table 5
Effect of stereochemistry on anti-HIV activity of naphthyl substituted thiourea compounds
X
S
S
N
Y
S
N
Z
N
N
N
N
N
N
N
S
S
H
H
H
H
H
H
Compound
X
Y
Z
Estimated K
i
(mM)
IC50 rRT (mM)
IC50 HTLVIIIB (nM)
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
4 (R)
5 (S)
6 (R)
7 (S)
8 (R)
9 (S)
0 (R)
1 (S)
2 (R)
3 (S)
4 (R)
5 (S)
6 (R)
7 (S)
8 (R)
9 (S)
0 (R)
1 (S)
2 (R)
3 (S)
4 (R)
5 (S)
5-Cl
5-Cl
–
–
0.01
>100
0.01
>100
–
0.58 ꢀ 0.18
>100
1.0 ꢀ 0
–
–
–
5-Br
–
–
0.67 ꢀ 0.20
>100
1.5 ꢀ 0.6
5-Br
5-Me
–
–
–
–
–
0.45 ꢀ 0.06
>100
2.3 ꢀ 0.3
5-Me
–
–
>100
–
–
6-Me
–
–
1.03 ꢀ 0.10
>100
4.0 ꢀ 3.7
6-Me
–
–
–
>100
–
–
4-Me
4-Me
H
–
1.17 ꢀ 0.32
>100
1.0 ꢀ 0
–
–
>100
–
–
–
–
12
–
–
4,6-Dimethyl
4-H
–
–
>100
–
>100
40
–
–
–
4,6-Dimethyl
–
–
>100
–
>100
–
–
–
–
–
–
–
H
H
–
–
–
4-Methylene ethoxy
4-Methylene ethoxy
–
0.89 ꢀ 0.41
>100
54.0 ꢀ 16.0
–
>100
–
–
–
–
–
–
–
–
–
–
–
H
0.44
205.0 ꢀ 95.3
H
>100
–
>100
–
4-Me
4-Me
10.2
>100
–
>100
–
–
–
–
–
–
–
1.21
–
>100
–
>100
–
Efavirenz [27]
Capravirin [28]
–
1.5
5.0
2.0
–
–
TMC-125 [29–31]
–
–
The anti-HIVactivity was measured by determining the inhibition of the HIV-1 strain HTLVIIIB in human PBMCs as previously described in detail. The cell-
free RT inhibition assays using recombinant RT (rRT) and the Quan-RT assay kit (Amersham, Arlington Heights, IL) were performed as reported [21,22].

1942
T.K. Venkatachalam et al. / Biochemical Pharmacology 67 (2004) 1933–1946
Table 6
Activity of lead CNT compounds against drug-resistant HIV-1 laboratory strains and primary HIV-1 isolates from AIDS patients
X
S
S
N
Y
N
N
N
N
N
S
H
H
H
H
Compound
X
Y
IC50 (nM)
a
b
c
d
RTMDR
A17
A17V
Patient HIV-1 isolates (n ¼ 3)
4
4
4
5
4 (R)
6 (R)
8 (R)
2 (R)
5-Cl
5-Br
5-Me
–
–
1 ꢀ 0
1 ꢀ 0
3 ꢀ 0
21 ꢀ 10
155
43 ꢀ 14
105 ꢀ 60
66 ꢀ 22
145 ꢀ 71
>1,000
–
367 ꢀ 141
287 ꢀ 177
543 ꢀ 353
114 ꢀ 50
>10,000
–
5 ꢀ 4
–
1 ꢀ 0
–
4-Me
11 ꢀ 9
11 ꢀ 10
9 ꢀ 5
Nevirapine
AZT
–
–
–
–
55
4 ꢀ 1
The anti-HIV activity was measured by determining the inhibition of the replication of the indicated HIV-1 strains and clinical HIV-1 isolates in human
PBMCs [20,21].
a
V106A, T215Y, L74V.
Y181C.
b
c
Y181C, K103N.
d
The patient HIV-1 isolates were BR/93/020 (L214F), BR/93/019(D67, T215D, K219 Q, L214F) and BR/93/029(L214F) obtained from NIH.
with the earlier results observed for the other chiral deri-
vatives discussed previously.
Our molecular modeling studies again indicated that
compound 52 (the ‘R’ stereoisomer) would fit the target
NNI binding pocket on HIV-RT much better than its
enantiomer (compound 53) (Fig. 2).
Based on our earlier hypothesis on chiral phenyl ethyl
thiourea compounds (Fig. 1) we propose a similar scheme
for naphthyl thiourea compounds. The predictions from
the modeling studies are consistent with the experimental
data obtained in cell-free RT inhibition assays and cellular
HIV inhibition assays. Furthermore, these compounds are
also very active against primary HIV-1 isolates of AIDS
patients (Table 6), again demonstrating the importance
of stereochemistry as well as regiochemistry in their
structure.
The next class of chiral thioureas examined consisted of
two cyclohexyl methyl halopyridyl thiourea compounds,
two a-methyl benzylhalopyridyl thiourea compounds and
one cyclohexyl ethyl thiazolyl thiourea compound. These
were introducedto examinethe effectsofa non-planargroup
on the activity of the thiourea compounds. We followed the
general synthetic procedure as discussed earlier. The iso-
meric purities of a few derivatives were further established
by X-ray crystal structures. The structures of the chiral
bromopyridyl thiourea compounds, N-[1-(1-(1R)-cyclohex-
ylethyl)]-N-[2-(5-bromopyridyl)]thiourea(66) andN-[1-(1-
(
(
1S)-cyclohexylethyl)]-N-[2-(5-bromopyridyl)] thiourea
67), were resolved by X-ray crystallography (Fig. 3).
Both compounds adopt lower energy conformations in
Fig. 2. Compounds 52 and 53 were docked into the NNRTI binding site of
HIV reverse transcriptase. The stick models of 52 (‘R’ isomer, top) and 53
(‘S’ isomer, bottom) were docked into the NNRTI binding site as
lower energy staggered conformations with respect to the naphthyl and
methyl groups on the chiral carbon. Only the ‘R’ isomer 40 provided a
suitable fit into the NNRTI binding pocket. The ‘S’ isomer 41 formed
unfavorable steric interactions with binding pocket residues near the Y181
side chain.
the crystalline state relative to the C14–C7–C8–C13 tor-
sion angle (62.0(5)8) in 66 and the C14–C7–C8–C9 torsion
angle (À62.4(5)8) in 67, with staggering of the cyclohexyl
and methyl groups on the chiral carbon. Our molecular
modeling studies indicated that the ‘R’ stereoisomer

T.K. Venkatachalam et al. / Biochemical Pharmacology 67 (2004) 1933–1946
1943
Fig. 3. X-ray crystal structures of compounds 66 and 67. Thirty percent probability ellipsoids, T ¼ 22 8C. (A) Compound 66: space group: P3
2
2
1
, unit cell:
3
˚
˚
˚
˚
a ¼ 9:2322ð4Þ A, b ¼ 9:2322ð4Þ A, c ¼ 32:9810ð18Þ A, a ¼ 908, b ¼ 908, g ¼ 1208, volume ¼ 2434:5ð2Þ A , Z ¼ 6, y range for data collection ¼ 1:85–
˚
2
8.178 (l ¼ 0:71073 A), total reflections collected ¼ 14,953, independent reflections ¼ 3775 (Rint ¼ 0:030), data/restraints/parameters ¼ 3775/0/173, R1
2
ðI > 2sðIÞÞ ¼ 0:041, wR2 ¼ 0:11, goodness-of-fit on F ¼ 0:986. Absolute structure parameter: 0.019(12). (B) Compound 67: space group: P3
1 1
2 , unit cell:
3
˚
˚
˚
˚
a ¼ 9:2290ð4Þ A, b ¼ 9:2290ð4Þ A, c ¼ 32:979ð2Þ A, a ¼ 908, b ¼ 908, g ¼ 1208, volume ¼ 2432:6ð2Þ A , Z ¼ 6, y range for data collection ¼ 1:85–
˚
2
8.208 (l ¼ 0:71073 A), total reflections collected ¼ 14,952, independent reflections ¼ 3792 (Rint ¼ 0:038), data/restraints/parameters ¼ 3792/0/181, R1
2
ðI > 2sðIÞÞ ¼ 0:041, wR2 ¼ 0:099, goodness-of-fit on F ¼ 0:913. Absolute structure parameter: 0.014(12).
compound 66 would fit the target NNI binding pocket on
HIV-RT much better than its enantiomer 67 (Fig. 4).
Unfavorable steric interactions with the NNI binding
pocket residues near the Y181 side chain would impair
the binding of ‘S’ isomer in a lower energy, ‘‘staggered’’
conformation. This steric hindrance would be relieved
if the ‘S’ isomer adopts an energetically unfavorable
indicated that the methyl group on the chiral carbon of
66 and 68 promotes the strong binding to the NNI binding
pocket via van der Waals contacts with residue V179. Since
˚
this methyl group is 7 A away from Y181 (as measured
from the carbon atom of the methyl group to the Cg position
˚
˚
of the protein residue), 9 A from Y188, 8.5 A from V106,
˚
and 6.5 A from K103, its favorable influence on the binding
‘‘eclipsed’’ conformation. In either case, the estimated
binding energy would be significantly higher for 67, as
reflected by the higher estimated K value (Table 7). Similar
i
of compounds 66 and 68 to RT should not be affected by
frequently encountered mutations involving these residues.
The control compounds with unsubstituted pyridyl rings
were predicted to fit poorly into the NNI binding pocket
(Table 7). The accuracy of the predictions from the model-
ing studies was evaluated in cell free RT inhibition assays.
As presented in Table 7, compounds 66 and 68, with
assumptions could be made in favor of the ‘R’ stereoisomer
in modeling studies of the chiral chloropyridyl thiourea
compounds, N-[1-(1-(1R)-cyclohexylethyl)]-N-[2-(5-chloro-
pyridyl)] thiourea (68) and N-[1-(1-(1S)-cyclohexylethyl)]-
N-[2-(5-chloropyridyl)] thiourea (69). Modeling studies
estimated K values 100-fold lower than those of the ‘S’
i
Fig. 4. Binding of thiourea compounds to NNRTI binding pocket of HIV-RT. Compounds 66 and 67 were docked into the NNRTI binding site of HIV reverse
transcriptase. The stick models of 66 (‘R’ isomer, left) and 67 (‘S’ isomer, right) were docked into the NNRTI binding site as lower energy staggered
conformations with respect to the cyclohexane and methyl groups on the chiral carbon. Only the ‘R’ isomer (66) provided a suitable fit into the NNRTI
binding pocket. The ‘S’ isomer (67) formed unfavorable steric interactions with binding pocket residues near the Y181 sidechain.

1944
T.K. Venkatachalam et al. / Biochemical Pharmacology 67 (2004) 1933–1946
Table 7
Effect of stereochemistry on anti-HIV activity of thiourea compounds
X
X
S
S
S
N
N
N
N
N
N
N
N
N
S
H
H
H
H
H
H
X = Cl [66, 67]
X = Br [68, 69]
X = Cl [21, 22]
X = Br [23, 24]
[72, 73 ]
a
Compound
Isomer
X
Estimated K
i
(mM)
IC50 rRT (mM)
1.2
IC50 HTLVIIIB (mM)
66
67
68
69
70
71
21
23
22
24
72
73
R
S
R
S
S
R
R
S
R
S
R
S
–
–
–
Br
Br
Cl
Cl
H
H
Br
Br
Cl
Cl
–
1.1
>100
1.2
>100
>100
>100
–
0.001
>1
>100
1.4
>100
0.025
>1
>100
>100
>1
>1
1.6
>100
1.2
>100
13.0
0.01
>1
–
–
0.01
ND
–
12.0
>100
–
0.001
>1
–
>100
Efavirenz [27]
–
–
–
–
0.0015
0.005
0.002
Capravirin [28]
TMC-125 [29–31]
–
–
–
–
The anti-HIVactivity was measured by determining the inhibition of the HIV-1 strain HTLVIIIB in human PBMCs as previously described in detail [21,22]. The
cell-free RT inhibition assays using recombinant RT (rRT) and the Quan-RT assay kit (Amersham, Arlington Heights, IL) were performed as reported [21,22].
a
i
K values were estimated based on our previously published procedures [10–12].
stereoisomers, inhibited recombinant RT in vitro with 100-
fold lower IC50 values. We next examined the ability of ‘R’
stereoisomers 66 and 68 to inhibit the replication of the
HIV-1 strain HTLVIIIB in human peripheral blood mono-
nuclear cells (PBMCs). Both compounds inhibited HIV-1
replication with IC values of 0.001 and 0.025 mM, respec-
In fact, compounds 74–81 were inactive (see supplemen-
tary material) demonstrating the critical structural require-
ments for the anti-HIV activity of these chiral thioureas.
Taken together, these results provide evidence that the
stereochemistry of a thiourea compound can profoundly
affect its ability to fit into the NNRTI binding pocket of RT
and therefore its anti-HIVactivity. However, the structure–
activity relationship studies should be interpreted with due
caution since no activity testing was performed on purified
mutant RT proteins.
5
0
tively. In contrast, the IC50 values of the ‘S’ stereoisomers
6
7 and 69 and the control compounds with unsubstituted
pyridyl rings (70 and 71) were >1 mM (Table 7). Similarly
the ‘R’ stereoisomers (but not ‘S’ stereoisomers) of the a-
methyl benzyl halopyridyl compounds 21 and 22 exhibited
potent activity both in cell-free RT inhibition assays and
cellular HIV-1 replication assays (Table 7). The substitution
of the pyridyl ring of 66 and 68 with a thiazolyl ring
3.4. Comparing the anti-HIV activity of the chiral
thiourea derivatives with known anti-HIV agents
(
compound 72) resulted in 10-fold higher K values and
i
0-fold higher IC50 values in cell free RT inhibition assays.
Having established the potent anti-HIV activity of the
chiral thiourea compounds examined in this study, we next
compared them with known anti-HIVagents. For this study
we selected several new NNRTI that are currently under-
going clinical trials. Comparing the activities towards wild
1
The ‘S’ stereoisomer (73) with an estimated K value of
>
i
100 mM did not exhibit any RT inhibitory activity even at
00 mM.
In order to further validate the structure activity relation-
1
RT by Evafirvenz (IC50 ¼ 1:5 nM), Capravarin (IC
¼
50
ship we prepared some of the substituted cyclohexenyl
thiourea compounds and examined their antiviral activity
towards recombinant reverse transcriptase. Introduction of
a methyl group as well as a benzothiazolyl unit in the
structure shown below did not yield active compounds.
5 nM), TMC-125 (IC50 ¼ 2 nM) and our lead compounds,
the methylphenyl substituted compounds 1 (IC50 ¼ 3 nM)
and 3 (IC50 ¼ 1 nM), we find that they have similar
activities (Table 1). However, our lead compounds are
more potent than Evafirvenz against mutant strains.

T.K. Venkatachalam et al. / Biochemical Pharmacology 67 (2004) 1933–1946
1945
Additionally, some of the a-ethylbenzyl substituted thiou-
reas, namely 20, 25 and 26, and a number of the ‘R’ isomer
naphthly substituted thioureas (44, 46, 48, 50, and 52) also
indicted anti-HIV activity with an IC50 ¼ 1 nM against
wild RT. However, unlike the methylphenylthioureas,
the naphthyl derivatives when compared to Evafirvenz
had lower activity against mutant strains. Overall, these
results indicate that further structural modification of
these thiourea compounds may result in potent anti-HIV
agents.
This conclusion parallels our modeling predictions for a
tight fit in the NNRTI binding pocket due to an ‘R’ isomer
methyl substitution of the ethyl linker unit of these thiourea
derivatives.
The toxicity and phamacodynamic features of the chiral
thiourea derivatives will be the subject of future studies in
order to assess their bioavailability before clinical evalua-
tion. It will be important to determine if the in vitro activity
observed for these compounds can be achieved in vivo
without unacceptable toxicity. Based on the results pre-
sented in our earlier publications we believe this goal can
be accomplished [25,26] and further work is in progress
to formulate active thiourea derivatives for preclinical
evaluations.
3
.5. Stability of chiral thiourea compounds
Related to our study on the effectiveness of these chiral
thiourea compounds as anti-HIV agents, we have also
evaluated their stability under varying temperature and
humidity conditions. In brief, the results indicated that
these compounds are quite stable for a period of at least 1
year. Analysis of samples by HPLC also indicated negli-
gible degradation of these compounds. In addition, we
have also evaluated the transport of these types of com-
pounds by examining their pharmacodynamics as well as
pharmacokinetic profiles as reported in our earlier pub-
lications [25,26].
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