Design, Synthesis, and Anti-HIV-1 Evaluation of a Novel Series of 1,2,3,4-Tetrahydropyrimidine-5-Carboxylic Acid Derivatives

Article abstract of DOI:10.1002/cbdv.201700502

A series of tetrahydropyrimidine derivatives (2a – 2l) were designed, synthesized, and screened for anti-HIV-1 properties based on the structures of HIV-1 gp41 binding site inhibitors, NB-2 and NB-64. A computational study was performed to predict the pharmacodynamics, pharmacokinetics, and drug-likeness features of the studied molecules. Docking studies revealed that the carboxylic acid group in the molecules forms salt bridges with either Lys574 or Arg579. Physiochemical properties (e.g., molecular weight, number of hydrogen bond donors, number of hydrogen bond acceptors, and number of rotatable bonds) of the synthesized compounds confirmed and exhibited that these compounds were within the range set by Lipinski's rule of five. Compounds 2e and 2k with 4-chlorophenyl substituent and 4-methylphenyl group at C(4) position of the tetrahydropyrimidine ring was the most potent one among the tested compounds. This suggests that these compounds may serve as leads for development of novel small-molecule HIV-1 inhibitors.

Full text of DOI:10.1002/cbdv.201700502

Article Type: Full Paper
Design, Synthesis and Anti-HIV-1 Evaluation of a Novel Series of
1,2,3,4-Tetrahydropyrimidine-5-carboxylic acid Derivatives
Saghi Sepehri,a,b Sepehr Soleymani,c Rezvan Zabihollahi,c Mohammad R. Aghasadeghi,c Mehdi Sadat,c
Lotfollah Saghaie,a Hamid R. Memarian,d Afshin Fassihia,*
aDepartment of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan
University of Medical Sciences, 81746-73461, Isfahan, Iran
E-mail: fassihi@pharm.mui.ac.ir (A. Fassihi)
bDepartment of Medicinal Chemistry, School of Pharmacy, Ardabil University of Medical Sciences,
Ardabil, 5618953141, Iran
cDepartment of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
dDepartment of Chemistry, Faculty of Sciences, University of Isfahan, 81746-73441 Isfahan, Iran
A series of tetrahydropyrimidine derivatives (2a-l) were designed, synthesized and screened for anti-
HIV-1 properties based on the structures of HIV-1 gp41 binding site inhibitors, NB-2 and NB-64. A
computational study was performed to predict the pharmacodynamics, pharmacokinetics and drug-
likeness features of the studied molecules. Docking studies revealed that the carboxylic acid group in
the molecules forms salt bridges with either Lys574 or Arg579. Physiochemical properties (e.g.
molecular weight, number of hydrogen bond donors, number of hydrogen bond acceptors and
number of rotatable bonds) of the synthesized compounds confirmed and exhibited that these
compounds were within the range set by Lipinski’s rule of five. Compounds 2e and 2k with 4-
chlorophenyl substituent and 4-methylphenyl group at C4 position of the tetrahydropyrimidine ring
was the most potent one among the tested compounds. This suggests that these compounds may
serve as leads for development of novel small-molecule HIV-1 inhibitors.
Keywords: Synthesis •Anti-HIV agents • Tetrahydropyrimidine derivatives • Gp41
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Introduction
One of the possible targets in the treatment of HIV-1 infection lies in the early steps of viral life cycle,
including the process of entry and fusion of viral membrane into the host cell membrane which is
mediated by two glycoproteins, gp120 and gp41.[1]
The transmembrane subunit of HIV-1 envelope (Env) glycoprotein, gp41, plays an important
role in viral fusion. HIV entry is a complex process and involves three separate steps: viral
attachment, co-receptor binding and finally fusion.[2] This process begins with the binding of gp120
protein to the CD4 and chemokine co-receptors CXCR4 or CCR5. Consequently, these interactions
induce a conformational change in gp41 from a pre-fusogenic form to a fusogenic six-helix bundle (6-
HB) structure; thus, allowing the introduction of the viral genome into the host cell.[3-5] The gp41
ectodomain contains a transmembrane zone responsible for attachment to the host cell membrane,
two heptad repeat regions which are N-terminal heptad repeats (NHR) and C-terminal heptad
repeats (CHR). NHRs form a central trimeric coiled-coil while three helices of CHRs are packed in an
anti-parallel fashion into the highly preserved hydrophobic grooves of the central trimeric coiled-coil
to form a trimer of helical hairpins that makes up the six-helix bundle.[6]
Compounds with inhibitory activity on HIV-1 mediated membrane fusion may bind to the gp41
NHR and/or CHR regions and block the 6-HB formation.[7] Lately, several small molecule gp41
inhibitors have been developed as non-peptide small-molecule candidates. In this regard, various
[8-19]
sets of chemical scaffolds have been proposed as gp41 inhibitors
amongst, two pyrrol
derivatives, NB-2 and NB-64 (Figure 1), were introduced as novel HIV-1 fusion inhibitors by high-
throughput screening method in 2004. These compounds bind to the gp41 protein via ionic and
hydrophobic interactions. These two pyrrole derivatives block the formation of the 6HB at low
micromolar concentration.[10,20]
Some main structural similarities can be recognized between NB-2 and NB-64 as known gp-41
inhibitors and Biginelli-type tetrahydropyrimidines for anti-HIV-1 activity (Figure 1).
HO
HO
O
O
Cl
R
HO
O
HO
H3C
NH
N
N
H3C
CH3
N
H
O
NB-64
NB-2
(2a-l)
Figure 1. Carboxylic acid group is in red, hydrophobic moieties are shown inorange and blue.
Hydrophilic groupsare represented in green
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Most of the gp41 inhibitors such as NB-2 and NB-64 have a -COOH group in their scaffold to
bind electrostatically to positively charged residues (Lys574 or Arg579) situated in the groove
pocket. It is reported that the elimination of the -COOH group will decrease the anti-HIV-1 potency
of the compound.[16] We have previously reported the moderate anti-HIV-1 activity of some
Biginelli-type tetrahydropyrimidines (THPM, 6-methyl-2-oxo-4-aryl-1,2,3,4-tetrahydropyrimidine-5-
carboxylate esters).[20] Tetrahydropyrimidines substituted with a carboxylic acid derivative at the 5
position have the proper functionality which is supposed to significantly influence the binding
orientation, because it is predicted to form a salt bridge with Lys574 or Arg579 on the surface of the
binding site (compounds 2a-l, Figure 1, shown in red squares). Aromatic rings, as hydrophobic
moieties, have shown good interaction with the hydrophobic pocket of the gp41 active site and have
improved the potency of inhibitors.[15] Substituted phenyl rings on the C-4 position of THPM ring
could fulfill the interactions with the hydrophobic pocket located in the gp41 active site (compounds
2a-l, Figure 1, yellow circles). Some other hydrophobic groups in the THPM structure are able to
interact with hydrophobic amino acids situated in the active site similar to the hydrophobic groups in
NB compounds (compounds 2a-l, Figure 1, blue squares). An important structural aspect of NB-2 and
NB-64 which is involved in the interaction with hydrophobic binding surface of the gp41 is the
intersecting two-ring system consisted of pyrrol and substituted phenyl rings interconnected by a
single bond. The two-ring molecular system of NB compounds has an analogue in
tetrahydropyrimidine compounds: the substituted phenyl ring bisecting the tetrahydropyrimidine
moiety. The possibility of hydrogen bonding increases the compound’s potency.[15] Hydrogens
attached to N-1 and N-3 of the THPM ring are assumed to take part in such hydrogen bonding with
the hydrophilic amino acids (compounds 2a-l, Figure 1, green circles).
In this paper, in continuation of our previous report on the anti-HIV-1 activity of the Biginelli-
type tetrahydropyrimidines, we describe the design, synthesis and HIV-1 inhibitory activity of a novel
series of tetrahydropyrimidine analogues, 6-methyl-2-oxo-4-aryl-1,2,3,4-tetrahydropyrimidine-5-
carboxylic acids. Intermolecular interactions of the studied compounds with the putative viral
receptor, gp41, are also investigated in this research using molecular docking simulations.
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Results and Discussion
Chemistry
The synthesis of 6-methyl-2-oxo-4-aryl-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid derivatives
(2a-l) was performed as outlined in Scheme 1. Biginelli reaction between the corresponding
aldehydes, benzyl 3-oxobutanoate and urea provided 6-methyl-2-oxo-4-aryl-1,2,3,4-
tetrahydropyrimidine-5-carboxylate esters (1a-l). This reaction was performed in refluxing absolute
ethanol in the presence of the catalytic amounts of Co(HSO4)2. Benzyl 3-oxobutanoate was prepared
according to the modified method of Clemens via condensation of 2,2,6-trimethyl-1,3-dioxine-4-one
with benzyl alcohol in refluxing xylene.[21] Catalytic transfer hydrogenation of 1a-l in the presence of
catalytic amounts of Pd/C (10% w/w) and ammonium formate in dry methanol yielded 2a-l (Scheme
1
1). Chemical structures were confirmed by H-NMR, FT-IR, mass spectroscopy, and elemental
analysis. FT-IR spectra of the final compounds exhibited characteristic absorption peaks for the
ureide N-H bonds at 3102-3260 cm-1 and 3222-3396 cm-1. A broad peak of carboxylic acid OH group
and a signal of carboxylic acid C-O stretch band were observed at around 2500-3500 cm-1 and 1230-
1290 cm-1, respectively. Presence of carboxylic acid C=O group was confirmed by a strong absorption
1
at 1686-1709 cm-1. H-NMR spectra revealed a broad singlet at 11.60-12.24 ppm demonstrating the
existence of carboxylic acid group in the final compounds. The presence of the THPM ring was
confirmed by two peaks in 8.85-9.75 ppm and 7.37-8.08 ppm belonging to ureide N-H bonds and a
1
peak at 4.01-5.21 ppm attributable to the C-4 proton of the THPM ring. Other H-NMR spectral
signals were in accordance with the proposed structures.
Scheme 1. Synthesis of compounds (2a-l). Reagents and conditions: (i) xylene, reflux, 3.5 h, (ii) EtOH,
reflux, 24-48 h, (iii) MeOH, Pd/C (10%), HCO2NH4, room temperature, 24-48 h, (iv) NaOH 0.5 M,
vigorously stirred then acidified by HCl 2 M to pH 4-5
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Chemical structures of the prepared compounds are summarized in Table 1.
Table 1. General structure and structural details of the prepared compounds
Compound
Ar
R
Mol. formula
Mol. weight
1a
1b
1c
1d
1e
1f
Phenyl
4-Dimethylaminophenyl
4-Nitrophenyl
2-Furyl
C5H6CH2
C5H6CH2
C5H6CH2
C5H6CH2
C5H6CH2
C5H6CH2
C5H6CH2
C5H6CH2
C5H6CH2
C5H6CH2
C5H6CH2
C5H6CH2
C19H18N2O3
C21H23N3O3
C19H17N3O5
C17H16N2O4
C19H17ClN2O3
C19H17FN2O3
C19H17ClN2O3
C21H20N2O3
C19H17N3O5
C17H16N2O3S
C20H20N2O3
C19H18N2O4
322.36
365.43
367.36
312.32
356.81
340.35
356.81
348.40
367.36
328.39
336.39
338.36
4-Chlorophenyl
4-Florophenyl
2-Chlorophenyl
Styryl
1g
1h
1i
3-Nitrophenyl
2-Thienyl
1j
1k
1l
4-Methylphenyl
2-Hydroxyphenyl
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2a
2b
2c
2d
2e
2f
Phenyl
4-Dimethylaminophenyl
4-Aminophenyl
2-Furyl
H
H
H
H
H
H
H
H
H
H
H
H
C12H12N2O3
C14H17N3O3
C12H13N3O3
C10H10N2O4
C12H11ClN2O3
C12H11FN2O3
C12H11ClN2O3
C14H14N2O3
C12H13N3O3
C10H10N2O3S
C13H14N2O3
C12H12N2O4
232.24
275.31
247.25
222.20
266.68
250.23
266.68
258.28
247.25
238.27
246.27
248.24
4-Chlorophenyl
4-Florophenyl
2-Chlorophenyl
Styryl
2g
2h
2i
3-Aminophenyl
2-Thienyl
2j
2k
2l
4-Methylphenyl
2-Hydroxyphenyl
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Biological assay
In this study, a series novel 1,2,3,4-THPM derivatives were synthesized and assessed for anti-HIV-1
activity in terms of inhibitory activity against HIV-1 replication in HEK cell cultures. All the
synthesized molecules were also tested for their cytotoxicity on MT-2 cell line by XTT assay. The 5-
carboxylate benzyl ester analogues (1a-l) were also subjected to both these assays.
The main focus of this study was to assess the HIV-1 growth inhibitory activity of 1,2,3,4-
dihydropyrimidine-based compounds with the aim of finding important structural prerequisites for
this property. The evaluated compounds did not exhibit significant cytotoxicity at concentration of
100 μM. Moderate HIV-1 replication inhibitory activities at 100 μM were recorded for the
synthesized analogues. These results are summarized in Table 2.
Compounds 1e, 1i, 2e and 2k were found to be the most potent anti-HIV-1 agents among the
studied compounds, 1a-l and 2a-l. They had moderate HIV-1 replication inhibitory activity in range
42.6-52.3% at 100 μM.The other derivatives were less active. Moreover, almost all compounds
displayed high cell viability. Compound 2k showed the lowest cell viability among others (59.41% at
100 µM). It can be concluded from the data in Table 2 that the intermediate compounds 1a-l were
mostly weaker anti-HIV-1 agents and the inhibiting activity of 2a-l series will be discussed as follows.
Molecules possessing phenyl and styryl groups at C4 position of THPM ring (2a and 2h) showed
lower activity compared to the other compounds bearing different substituents at this position. The
inhibition values for 2e and 2k as the most potent compounds in 2a-l series revealed that 4-
chlorophenyl substituent at C4 position of 1,2,3,4-tetrahydropyrimidine (2e) was more favorable for
HIV-1 inhibitory activity than 4-methylphenyl group (2k). Replacing 4-chlorophenyl ring with 4-
flurophenyl decreased the activity. It seems that the higher lipophilicity character has constructive
impacts on both hydrophobic interactions with receptor and virus accessibility, i.e., host cell
membrane permeability of the compounds. The lipophilicity feature of these compounds
determined as CLogP by MolinspirationWebME Editor1.16. is supportive for this idea: CLogP for 2e is
2.06 and for 2f is 1.54.
Compound 2g with 2-chlorophenyl substitution at C4 position of THPM exhibited lower anti-
HIV-1 compared with compound 2e, which has a 4-chlorophenyl derivative. An explanation for this
difference is provided in the computational studies section.
Displacement of amino substitution from meta to para position diminished the activity. This will
be explained in terms of ligand-gp41 interaction in the next section.
Replacement of 2-furyl moiety at C4 position of 2d with 2-thienyl in 2j decreased the activity.
Compound 2g with 2-chlorophenyl at C4 position displayed better anti-HIV-1 activity compared
to compound 2l with
2-hydroxyphenyl moiety. This can be attributed to the better cell
membrane permeation of 2g cross the infected host cell to exert its anti-HIV-1 activity. This is in
accordance with the differences between CLogP values of these compounds.
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Table 2. Anti-HIV-1 activity of the prepared compounds
100 µM
(%) inhibition of P24 expression
23.7± 1.2
Compound
(%) cell viability
1a
1b
1c
1d
1e
1f
93.7± 1.9
90.9± 1.7
100± 5.3
100± 5.3
100± 1.6
100± 1.8
97.14± 4.3
0± 4.4
5.7± 2.8
36.6± 3.7
22.5± 2.5
47.5± 2.5
4.7± 1.8
18.7± 3.7
0± 1.2
1g
1h
1i
52.2± 1.2
0± 2.5
69.0± 9.1
0± 5.1
1j
1k
1l
0±1.7
0±2.0
0±2.5
0±4.1
2a
2b
2c
2d
2e
2f
1.84± 1.2
35.1± 4.0
16.9± 1.6
41.2± 3.7
49.5± 2.5
28.8± 3.7
40.2± 1.2
4.5± 4.2
41.2±9.8
28.8± 3.7
100± 4.36
97.8± 1.4
100± 3.2
100± 5.3
98.9± 0.6
100± 5.06
76.7± 3.5
65.9± 10.1
87.4± 2.1
70.7± 0.51
2g
2h
2i
2j
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2k
2l
42.6± 2.4
36.2± 3.7
100
59.4± 1.2
75.1± 5.1
100
BMS-806
In silico studies
In silico calculation of physicochemical parameters and toxicity studies
As a part of our investigation, a computational study was performed for estimating drug-likeness
and bioavailability of compounds. A prerequisite for any bioactive molecule to be developed as a
therapeutic agent is good oral bioavailability.[22] Significant predictors of good oral bioavailability are
good intestinal absorption, molecular flexibility (measured by the number of rotatable bonds (n-
ROTB) and low polar surface area or total hydrogen bond count (sum of donors and acceptors).[22,23]
Lipinski predicted the ''drug-likeness'' of potential molecular targets in formulating his “Rule of
Five”.[24] This rule states that most molecules with good membrane permeability should have
partition coefficient values (logP) in the octanol-water system ≤ 5, molecular weight (MW) ≤ 500,
number of hydrogen bond acceptors ≤ 10 and number of hydrogen bond donors ≤ 5. Drug-likeness
can be assumed as a subtle balance between the molecular properties of a compound that directly
influence its pharmacokinetics i.e. its absorption, distribution, metabolism, and excretion (ADME) in
human body.[25] Numerical values for CLogP (calculated LogP), LogS (the logarithm of aqueous
solubility in terms of mol/L), topological polar surface area (TPSA, a physicochemical property
describing the polarity of molecules), number of H-bond donors, number of H-bond acceptors, and
number of rotatable bonds are given in Tables 3 and 4. TPSA is a key aspect in transport across
membranes. It has been known as a good parameter for prediction of passive drug transport
through membrane. It is validated by the good correlation of TPSA with published absorption data.
These data are acquired from different kinds of drug absorbance, including human intestinal
absorption, blood brain barrier crossing, and Caco-2 monolayer penetration.[26] Passively-absorbed
molecules with a TPSA of greater than 140 Å2 tend to be poor at permeating cell membranes; thus,
they show low oral bioavailability.[26,27] Zhao et al. reported equation (1) that can predict the amount
of oral drug absorption.[28]
%ABS = 109 - (0.345 × TPSA)
Eq. (1)
All of the designed compounds in the present study possessed TPSA < 124 Å2 and exhibited ABS
> 66% (Table 4). Thus, it seems that they could have a good capacity for penetrating cell
membranes.
Our analysis of data depicted in Table 3 reveals that all of the synthesized derivatives had low
MW, favorable CLogP, and favorable hydrogen bond-donating and acceptable capabilities. This in
turn fits with the Lipinski’s rule of five indicating that these compounds may have good absorption
or permeability properties through the biological membranes. A low CLogP in the range of 0-3 is
necessary to decrease the toxicity and increase the bioavailability for optimal oral absorption. All of
the studied compounds had CLogP values within this range (Table 3).
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Solubility is another general physicochemical factor in drug discovery. An indication of the solubility
of compounds is LogS value. The aqueous solubility of a compound remarkably influences its
distribution and absorption characteristics. More than 80% of the drugs on the market have a logS
value greater than -4 mol/L.[29] As shown in Table 4, compounds 1c, 1e, 1g, 1h and 1i had lower
calculated solubility, less than -4 mol/L, whereas the rest of the compounds had higher calculated
solubility, -1.90 to -3.98 mol/L.
Table 3. Calculated pharmacokinetic parameters for the prepared compounds
Compound
CLogP a
n-ROTB b
HBA c
HBD d
Volumee
1a
1b
1c
1d
1e
1f
3.59
3.69
3.55
2.85
4.27
3.75
4.22
4.35
3.52
3.49
4.04
3.53
1.38
1.48
0.46
0.64
2.06
1.54
2.01
5
6
6
5
5
5
5
6
6
5
5
5
2
3
2
2
2
2
2
5
6
8
6
5
5
5
5
8
5
5
6
5
6
6
6
5
5
5
2
2
2
2
2
2
2
2
2
2
2
3
3
3
5
3
3
3
3
293.97
339.88
317.31
275.54
307.51
298.90
307.51
321.39
317.31
284.69
310.54
301.99
204.80
250.70
216.09
186.37
218.33
209.73
218.33
1g
1h
1i
1j
1k
1l
2a
2b
2c
2d
2e
2f
2g
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2h
2i
2.13
0.43
1.28
1.83
1.32
3
2
2
2
2
5
6
5
5
6
3
5
3
3
4
232.21
216.09
195.51
221.36
212.81
2j
2k
2l
aCLogP, logarithm of compound partition coefficient between n-octanol and water;
bn-ROTB, number of rotatable bonds; cHBA, number of hydrogen bond acceptors;
dHBD, number of hydrogen bond donors, eÅ3.
Lipinski's violations for all compounds were equal to 0.
Table 4. Prediction of the studied anti-HIV-1 compounds as drugs
Compound
LogSa
TPSAb
%ABSc
Drug-likeness
Drug-scored
1a
1b
1c
1d
1e
1f
-3.67
-3.71
-4.31
-3.35
-4.41
-3.98
-4.41
-4.49
-4.31
67.43
70.67
113.25
80.57
67.43
67.43
67.43
67.43
113.25
85.73
84.61
69.93
81.20
85.73
85.73
85.73
85.73
69.93
-8.44
-9.22
-8.52
-8.71
-7.13
-8.83
-8.19
-9.78
-8.73
0.4
0.24
0.13
0.43
0.35
0.39
0.35
0.35
0.22
1g
1h
1i
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1j
1k
1l
-3.68
-4.01
-3.37
-2.22
-2.26
-2.30
-1.90
-2.95
-2.53
-2.95
-3.04
-2.86
-2.23
-2.56
-1.92
95.67
67.43
87.66
78.43
81.67
124.25
91.57
78.43
78.43
78.43
78.43
124.25
78.43
78.43
98.66
75.99
85.73
78.75
81.94
80.82
66.13
77.41
81.94
81.94
81.94
81.94
66.13
81.94
81.94
74.96
-7.07
-9.64
-8.62
3.41
2.50
3.55
3.16
4.70
2.98
3.57
2.03
-0.44
4.70
2.10
3.07
0.41
0.38
0.42
0.93
0.54
0.93
0.94
0.90
0.91
0.89
0.85
0.38
0.94
0.88
0.93
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
aLogarithm of aqueous solubility measured in M; b Topological polar surface area in
cPercentage of Human oral absorption in GI; d Drug score combines drug likeness,
Å2;
CLogP, logS,
molecular weight and toxicity risks;
None were predicted mutagen exept for 1c.
None were predicted tumorigen exept for 1b and 2b.
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None were predicted irritant.
None were predicted to have reproductive effects except for 1c, 1i and 2i.
Hydrogen-bonding ability has also been known as an important factor for describing drug
permeability.[24,25] All the synthesized compounds possessed a satisfactory number of proton donor
(≤ 5) and proton acceptor (≤ 8) groups to construct efficient interactions with the hydrophilic amino
acids of the active site (Table 3).
Decreased molecular flexibility measured by the number of rotatable bonds (optimally below 8)
is another important predictor of good oral bioavailability.[22,23] According to analysis of data
depicted in Table 3, all of the synthesized derivatives had a low number of rotatable bonds (<5) as
they obtain a high to moderate degree of structural rigidity.
Toxicity risk (mutagenicity, tumorogenicity, irritation, and reproductive effects) are signals
alarming that the designed compounds may be harmful concerning the risk category specified. Most
of the compounds did not show any potential of toxicity in toxicity risk assessment (Table4).
In Osiris calculations the drug-likeness prediction is a fragment-based approach. A positive
drug-likeness value states that a molecule contains mainly fragments which are often present in
commercial drugs. Osiris study displayed that compounds 2e and 2j had high positive drug-likeness
values, and the fragments of these compounds had a contribution for drug-like activities. The drug-
score prediction process in Osiris explorer depends on the combination of drug-likeness, CLogP,
LogS, MW, and toxicity risks in one neat amount. This factor may be used to judge about the
compound's overall potential as a qualified drug.[29] The studied compounds revealed moderate to
good drug score introducing them as potentially safe lead compounds. Drug-likeness and drug-score
values of the designed compounds are presented in Table 4.
Molecular docking studies
Molecular docking simulations and analysis of the binding modes of the designed inhibitors within
gp41 binding site were performed to rationalize the anti-HIV-1 activity results. AutoDock is a
qualified docking program that has facilitated the process of drug design (The AutoDock Web site.
http://autodock.scripps.edu).[30]
At the first step of docking, it was focused on the exploration of the binding mode of both NB-2
and NB-64 as potent gp41 inhibitors within the entire protein via a blind-docking procedure to
validate the applied docking protocol. To get the detailed procedure, readers are referred to our
previous report.[20] Then 1,2,3,4-THPM derivatives were docked into the binding site of gp41. All
these compounds have a chiral center at the C-4 position of the tetrahydropyrimidine ring. Thus,
both R and S enantiomers were considered in the molecular docking simulation studies. Docking
results consisting of electrostatic, hydrophobic, hydrogen bonds, and predicted docking affinities of
molecules are summarized in Tables 5-10.
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Table 5. Estimated hydrogen bonds for the bestdocked conformations (R-enantiomer)
Participant
amino acid
Gln567
Gln567
Lys574
Involved atom
of the ligand
(THPM) N-3-H
(THPM) N-3-H
(Nitro) O
Involved atom
of the amino acid
C=O
Optimized hydrogen
bond distances (Å)
Compound
1a
1b
1c
2.81
2.75
1.63
2.74
1.99
2.35
2.39
1.88
2.14
2.68
2.28
2.79
2.58
1.78
2.03
C=O
+
NH3
Gln567
Lys574
(THPM) N-1-H
(Furyl) O
C=O
1d
+
NH3
1e
1f
1g
1h
1i
Gln567
Gln567
Gln567
Gln567
Lys574
(THPM) N-3-H
(THPM) N-3-H
(THPM) N-3-H
(THPM) N-1-H
(Nitro) O
C=O
C=O
C=O
C=O
+
NH3
1j
Gln567
Gln567
Gln567
Gln567
Lys574
(THPM) N-3-H
(THPM) N-3-H
(Phenol) OH
(THPM) N-1-H
(Acid) OH
C=O
C=O
C=O
NH2
C=O
1k
1l
2a
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Gln567
Lys574
Lys574
Gln575
Gln567
Lys574
Gln567
Lys574
Gln567
Lys574
Gln567
Lys574
Gln567
Lys574
Lys574
Gln567
Gln567
Lys574
2.11
1.95
2.87
2.78
1.57
1.67
2.03
1.57
2.59
2.37
1.51
1.54
1.93
1.74
2.97
2.31
1.98
1.82
(THPM) N-1-H
(Acid) OH
NH2
C=O
C=O
NH2
NH2
C=O
NH2
C=O
NH2
C=O
NH2
C=O
NH2
C=O
2b
2c
2d
2e
2f
(Acid) OH
(Carbonyl) O
(THPM) N-1-H
(Acid) OH
(THPM) N-1-H
(Acid) OH
(THPM) N-1-H
(Acid) OH
(THPM) N-1-H
(Acid) OH
2g
2h
2i
(THPM) N-1-H
(Acid) OH
+
(Acid) OH
NH3
NH2
NH2
C=O
(THPM) N-1-H
(THPM) N-1-H
(Acid) OH
2j
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Gln567
Lys574
Gln567
Lys574
Gln575
Lys574
Trp571
2.12
2.02
2.12
1.79
1.84
1.98
2.98
(THPM) N-1-H
(Acid) OH
NH2
C=O
NH2
C=O
C=O
2k
2l
(Phenol) OH
(Acid) OH
(THPM) N-1-H
(Carbonyl) O
(Carbonyl) O
+
NH3
BMS-806
NH2
Table 6. Estimated hydrogen bonds for the bestdocked conformations (S-enantiomer)
Participant
amino acid
-
Involved atom
of the ligand
-
Involved atom
Optimized hydrogen
bond distances (Å)
Compound
of the amino acid
1a
1b
1c
-
-
Gln567
Lys574
Gln567
Lys574
Gln567
Gln567
2.91
1.63
2.14
1.99
2.35
2.39
(THPM) N-3-H
(Nitro) O
C=O
+
NH3
(THPM) N-1-H
(Furyl) O
C=O
1d
+
NH3
1e
1f
(THPM) N-3-H
(THPM) N-3-H
C=O
C=O
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1g
1h
1i
Gln567
Gln567
Lys574
Gln567
Gln567
Gln567
Gln567
Gln567
Lys574
Gln567
Lys574
Gln575
Lys574
Gln567
Lys574
Gln567
Lys574
Gln567
1.88
2.25
1.87
2.34
2.94
2.78
2.83
1.85
1.96
2.00
1.78
2.32
1.95
2.22
1.76
2.32
1.75
2.13
(THPM) N-3-H
(THPM) N-1-H
(Nitro) O
C=O
C=O
+
NH3
C=O
C=O
C=O
C=O
NH2
C=O
NH2
C=O
NH2
C=O
NH2
C=O
NH2
C=O
NH2
1j
(THPM) N-3-H
(THPM) N-3-H
(THPM) N-3-H
(Phenol) OH
(THPM) N-1-H
(Acid) OH
1k
1l
2a
2b
2c
2d
(THPM) N-1-H
(Acid) OH
(Carbonyl) O
(Acid) OH
(THPM) N-1-H
(Acid) OH
(THPM) N-1-H
(Acid) OH
2e
2f
(THPM) N-1-H
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Lys574
Gln567
Lys574
Gln567
Lys574
Lys574
Gln567
Gln567
Lys574
Gln567
Lys574
Gln567
Lys574
Gln575
Lys574
Trp571
1.89
1.21
1.61
2.06
1.95
1.89
2.03
2.57
2.05
1.83
1.55
2.43
1.43
2.21
1.98
2.98
(Acid) OH
(THPM) N-1-H
(Acid) OH
C=O
NH2
C=O
NH2
C=O
2g
2h
2i
(THPM) N-1-H
(Acid) OH
+
(Acid) OH
NH3
NH2
NH2
C=O
NH2
C=O
NH2
C=O
C=O
(THPM) N-1-H
(THPM) N-1-H
(Acid) OH
2j
(THPM) N-1-H
(Acid) OH
2k
(Phenol) OH
(Acid) OH
2l
(THPM) N-1-H
(Carbonyl) O
(Carbonyl) O
+
NH3
NH2
BMS-806
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Table 7. Estimated electrostatic, hydrophobic, π-π stacking and cation-π interactions for the best
docked conformations (R-enantiomer)
π-π Stacking
interaction
Compound Electrostatic interaction
Hydrophobic interaction
-
Lys574, Gln567, Trp571, Trp631,
Val570, Gln575
1a
Trp571
-
Lys574, Gln567, Trp571, Gln575,
Val570, Leu568, Trp631
1b
Trp571
Lys574
Lys574, Gln567, Trp571, Leu568,
Trp631
1c
Trp631
-
Lys574, Gln567, Trp571, Leu568,
Trp631, Val570
1d
Trp571
-
Lys574, Gln567, Trp571, Leu568,
Trp631, Val570, Gln575
1e
-
-
Lys574, Gln567, Trp571, Leu568,
Trp631, Val570, Gln575
1f
-
-
Gln567, Trp571, Leu568, Trp631,
Val570, Glu634, Leu568, Glu630
1g
-
-
Lys574, Gln567, Trp571, Leu568,
Trp631, Gln575
1h
Trp571
Lys574
Lys574, Gln567, Trp571, Glu630,
Leu568, Trp631, Gln575, Glu634,
Val570
1i
Trp571
-
Lys574, Gln567, Trp571, Glu630,
Leu568, Trp631, Val570
1j
-
-
Lys574, Gln567, Trp571, Val570,
Leu568, Trp631, Gln575
1k
Trp631
-
Lys574, Gln567, Trp571, Glu630,
Leu568, Trp631, Val570
1l
-
Lys574
Lys574, Gln567, Trp571, Gln575,
Val570
2a
-
-
-
2b
2c
Lys574
Lys574
Lys574, Gln567, Trp571, Leu568
Lys574, Gln567, Trp571, Gln575,
Val570
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Lys574
Lys574
Lys574
Lys574
Lys574
Lys574
Lys574
Lys574
Lys574
Lys574, Gln567, Trp571, Gln575,
Val570
2d
-
Lys574, Gln567, Trp571, Gln575,
Val570
2e
-
Lys574, Gln567, Trp571, Gln575,
Val570
2f
-
Lys574, Gln567, Trp571, Gln575,
Val570
2g
-
Lys574, Gln567, Trp571, Gln575,
Val570
2h
Trp571
Lys574, Gln567, Trp571, Gln575,
Val570
2i
-
-
-
-
-
Lys574, Gln567, Trp571, Gln575,
Val570
2j
2k
Lys574, Gln567, Trp571, Gln575,
Val570
Lys574, Gln567, Trp571, Gln575,
Val570
2l
Lys574, Gln567, Trp571, Leu568,
Glu634, Trp631
BMS-806
-
Only compound 1l showed cation- π interactions with Lys574.
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Table 8. Estimated electrostatic, hydrophobic, π-π stacking and cation-π interactions for the best
docked conformations (S-enantiomer)
π-π Stacking
interaction
Compound Electrostatic interaction
Hydrophobic interaction
-
Glu634, Gln567, Trp571, Leu568,
Trp631
1a
Trp571
-
Lys574, Gln567, Trp571, Gln575,
Val570, Leu568, Trp631
1b
-
Lys574
Lys574, Gln567, Trp571, Leu568,
Trp631, Val570, Glu630, Glu634
1c
Trp631
-
Lys574, Gln567, Trp571, Leu568,
Trp631, Val570
1d
Trp571
-
Lys574, Gln567, Trp571, Glu634,
Leu568, Trp631, Val570
1e
-
-
Lys574, Gln567, Trp571, Leu568,
Trp631, Val570
1f
Trp631
-
Lys574, Gln567, Trp571, Glu630,
Leu568, Trp631, Val570
1g
-
-
Lys574, Gln567, Trp571, Val570,
Leu568, Trp631, Gln575
1h
-
Lys574
Lys574, Gln567, Trp571, Glu630,
Leu568, Trp631
1i
-
-
Lys574, Gln567, Trp571, Gln575,
Leu568, Trp631, Val570
1j
Trp571
-
Lys574, Gln567, Trp571, Leu568,
Trp631
1k
-
-
-
-
-
-
Lys574, Gln567, Trp571, Leu568,
Trp631
1l
Lys574
Lys574, Gln567, Trp571, Gln575,
Val570
2a
Lys574
Lys574, Gln567, Trp571, Gln575,
Val570
2b
Lys574
Lys574, Gln567, Trp571, Gln575,
Val570
2c
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Lys574
Lys574
Lys574
Lys574
Lys574
Lys574
Lys574
Lys574
Lys574
Lys574, Gln567, Trp571, Gln575,
Val570
2d
-
Lys574, Gln567, Trp571, Gln575,
Val570
2e
-
Lys574, Gln567, Trp571, Gln575,
Val570
2f
-
Lys574, Gln567, Trp571, Gln575,
Val570
2g
-
Lys574, Gln567, Trp571, Gln575,
Val570
2h
Trp571
Lys574, Gln567, Trp571, Gln575,
Val570
2i
-
-
-
-
-
Lys574, Gln567, Trp571, Gln575,
Val570
2j
2k
Lys574, Gln567, Trp571, Gln575,
Val570
Lys574, Gln567, Trp571, Gln575,
Val570
2l
Lys574, Gln567, Trp571, Leu568,
Glu634, Trp631
BMS-806
-
Only compounds 1f, 1k and 1l showed cation- π interactions with Lys574.
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Table 9. Docking energy results for the best docked conformations (S-enantiomer) docked into the
fusion HIV-1 virus target
(gp41, PDB ID: 1AIK)
ΔGbinding
Intermol energy
(kcal/mol)
Electrostatic energy
(kcal/mol)
Vdw_hb_desolv energy
(kal/mol)
Compound
(kcal/mol)
1a
1b
1c
1d
1e
1f
-5.94
-6.31
-6.71
-5.75
-6.25
-5.85
-5.89
-6.33
-6.97
-5.76
-6.17
-6.48
-5.31
-5.45
-6.70
-7.32
-8.35
-6.69
-7.32
-7.03
-7.05
-7.46
-8.39
-6.94
-7.16
-7.62
-6.11
-6.02
-0.58
-0.10
-2.15
-0.11
-0.39
-0.30
-0.41
-0.19
-1.91
-0.14
-0.22
-0.48
-0.12
-1.63
-6.12
-7.21
-6.20
-6.57
-6.93
-6.74
-6.64
-7.27
-6.48
-6.80
-6.95
-7.14
5.87
1g
1h
1i
1j
1k
1l
2a
2b
-4.40
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2c
-5.15
-4.48
-5.50
-5.31
-5.14
-5.38
-5.25
-4.85
-5.68
-5.23
-6.42
-5.73
-5.39
-6.03
-5.74
-5.91
-6.31
-6.16
-5.44
-5.90
-5.81
-7.61
-1.71
-1.29
-1.52
-1.62
-1.72
-1.56
-1.20
-1.17
-1.61
-1.34
-0.19
-4.02
-4.11
-4.51
-4.12
-4.19
-4.75
-4.97
-4.27
-4.29
-4.47
-7.42
2d
2e
2f
2g
2h
2i
2j
2k
2l
BMS-806
Table 10. Docking energy results for the best docked conformations (R-enantiomer) docked into the
fusion HIV-1 virus target
(gp41, PDB ID: 1AIK)
ΔGbinding
Intermol energy
(kcal/mol)
Electrostatic energy
(kcal/mol)
Vdw_hb_desolv energy
(kal/mol)
Compound
1a
(kcal/mol)
-5.79
-7.02
-0.06
-6.97
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1b
1c
1d
1e
1f
-6.23
-6.92
-5.48
-6.02
-5.64
-5.72
-6.55
-7.05
-5.52
-5.91
-6.87
-4.87
-5.19
-4.58
-4.36
-5.33
-4.84
-4.55
-7.23
-8.24
-6.63
-7.37
-7.05
-7.03
-7.53
-8.69
-6.98
-7.23
-7.38
-5.58
-5.65
-5.61
-5.45
-5.60
-5.26
-5.49
-0.04
-2.13
-0.24
-0.06
-0.10
-0.48
-0.05
-1.89
-0.39
-0.03
-0.30
-0.04
-1.29
-1.38
-1.68
-1.14
-1.54
-1.15
-7.19
-6.11
-6.39
-7.31
-6.95
-6.54
-7.48
-6.79
-6.58
-7.20
-7.08
-4.73
-4.36
-4.23
-3.77
-4.46
-3.73
-4.33
1g
1h
1i
1j
1k
1l
2a
2b
2c
2d
2e
2f
2g
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2h
-5.01
-4.79
-4.43
-5.39
-4.64
-6.42
-6.33
-6.01
-5.69
-5.49
-5.68
-7.61
-1.39
-1.55
-1.70
-1.53
-1.78
-0.19
-4.95
-4.46
-3.99
-3.96
-3.91
-7.42
2i
2j
2k
2l
BMS-806
No significant differences were observed between the ΔGbinding of the R and S enantiomers of
the intermediate compounds, 1a-l. The interaction modes of the two enantiomers were almost the
same with some minor exeptions. The greatest differences were as follows:
S enantiomer of
1a unlike the R enantiomer did not participate in hydrogen bonds with the gp41 receptor. R
enantiomers of 1b, 1f and 1j, and S enantiomer of 1i and 1k exhibited no π-π interactions with the
receptor while the other enantiomers of these compounds were involved in such interactions. No
cation-π interactions were observed for the R enantiomer of 1f and 1k unlike the S enantiomers of
the compounds. The rest of the differences were some minor ones in type of the gp41 residues
involved in the hydrophobic interactions. For the final compounds, 2a-l, not only the ΔGbinding but
also the interaction modes of the R and S enantiomers with the gp41 binding site were almost
similar.
Analysis of the results of molecular docking studies revealed them to be almost confirming of
the biological activity results. The most potent compound (2e) had an inhibition percentage equal to
49.50 in 100 µM. Docking studies demonstrated that 2e participates in key interactions with gp41
binding site residues: an electrostatic contact with Lys574 and an H-bond with Gln567. Detailed
analysis of hydrophobic binding patterns showed that an interaction exists between methyl group of
1,2,3,4-THPM ring and Val570. Moreover, 4-chlorophenyl substituent of the C-4 position is
responsible for further hydrophobic interactions with Trp571, Gln575 and Lys574 in the hydrophobic
pocket. Replacement of the methyl substituent in compound 2e with Cl produced compound 2k
which was the most active final compound after 2e. This compound contributes in H-bond and
electrostatic contact with Gln567 and Lys574, respectively. Furthermore, investigating hydrophobic
binding patterns showed that these interactions occurred between 2k and Trp571, Gln575 and
Val570 residues. Interactions of 2e and 2k with the binding site amino acids are shown in Figure 2.
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Figure 2. Docked conformations of 2e S-enantiomer (A) and 2k S-enantiomer (B) in the gp41
hydrophobic cavity showing possible interactions with the neighboring hydrophobic residues and
Lys574.
As it was mentioned earlier, molecules possessing phenyl and styryl groups at C-4 position of
THPM ring (2a and 2h) showed lower activity (Table 2). The lower anti HIV-1 activity of 2h might be
attributed to the improper geometric orientation of styryl group in the binding site of gp41. This can
be seen in Figure 3 by superimposing 2h and 2e (the most active compound of the series) in the
gp41 binding site.
Figure 3. Superimposition of 2e S-enantiomer (blue sticks) and 2h S-enantiomer (violet sticks) from
docking results in gp41 binding site
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4-Chlorophenyl aromatic group at the C-4 position of 1,2,3,4-THPM ring in 2e provided a more
potent compound compared to 2-chlorophenyl substituent in 2g. The docking results were
supportive of this observation (Figure 4). In fact, the binding mode of compound 2g showed that the
intermolecular interactions of the ortho-Cl with the neighboring residues were fewer compared with
meta-Cl. This can be attributed to a steric hindrance leading to a change in the orientation of 2-
chlorophenyl moiety of 2g and subsequent weak interactions.
Figure 4. The superimposition of 2e S-enantiomer (blue) with 2g S-enantiomer (violet) in gp41
groove
Replacement of 4-chlorophenyl at C4 position of 1,2,3,4-THPM ring in 2e with 4-flurophenyl in
2f decreased the activity. According to the CLogP data, 2e is more lipophilic than 2f. Furthermore,
the molecular volume of 2e is more than 2f (218.33 Å3 and 209.73 Å3, respectively), thus compound
2e would occupy a larger space in the hydrophobic pocket and show better anti-HIV-1 activity.
Replacing 2-chlorophenyl (2g) with 2-hydroxyphenyl ring (2l) at the C-4 position of 1,2,3,4-
THPM decreased the activity. Based on the previous researches, analogues with increased
hydrophilic character were equal or slightly improved in potency, but addition of polar groups had a
negative effect.[14] Thus, 2-chlorophenyl group provided additional hydrophobic contacts to residues
at binding site. Moreover, docking results revealed that compound 2l formed H-bonds with Gln567,
Gln575 and Lys574, while compound 2g participated in hydrogen bonding to Gln567 and Lys574
(Figure 5).
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Figure 5. H-bonds and hydrophobic interactions of 2g S-enantiomer (A) and 2l S-enantiomer (B) in
gp41 binding site
Compound 2b with 4-dimethyaminophenyl showed higher activity than 2c with 4-amine phenyl
group. It seemed that due to existence of a bulk group at C4 position of 2b, this compound has a
bigger volume than 2c (250.70 Å3 for 2b and 216.09 Å3 for 2c); thus 2b would occupy more space in
the hydrophobic pocket thus shows better anti-HIV-1 activity.
Compound 2c with an amino group at para position of phenyl ring of C4 showed weaker activity
than 2i having amino group at meta position. According to the docking results, amine group at the
meta position in 2i forms H-bond with Gln567 residue but amine group at para position in 2c does
not form any H-bonds with this residue.
Compound 1i had the most negative ΔGbinding value compared with other compounds. Thus, this
molecule binds firmly to the receptor. It is obvious from its molecular structure that electrostatic
interactions with the receptor are responsible for this. A nitro group shares electrostatic interaction
with the positively charged residue Lys574. Moreover, this compound shows the highest activity
compared with other compounds (52.25% inhibition at 100 µM) in series 1a-l. It is accepted that the
presence of a meta carboxylate group on N-substituted pyrrol ring of gp41 inhibitors favors the
receptor interaction.[10] The meta nitro group on the phenyl ring located at the C4 position in 1i takes
part in the same electrostatic interaction with gp41 binding site. The para nitro analogue (1c) has a
higher ΔGbinding value and a lower electrostatic contribution than 1i. This may account for the
differences between the anti-HIV potency of these compounds.
Ligand-residue binding energies and docked poses indicated that compound 2h might
participate in favorable π-π stacking contacts with Trp571. Nearly parallel orientation of aromatic
rings (Trp571 indol ring and ligand phenyl ring) might be responsible for the detected π-π stacking
interaction (Figure 6).
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Figure 6. Nearly parallel orientation of 2h S-enantiomer phenyl and Trp571 indol rings and possible
π-π stacking interaction
All of the 2a-l compounds participated in electrostatic interactions with Lys574 via their C-5-
COOH group and most of them took part in hydrophobic interactions with Val570 via their C-6
methyl groups.
BMS-806 and related compounds are low-molecular-weight inhibitors of HIV-1 entry and
attachment (the first steps of HIV-1 infection) that were identified by a viral infection-based screen.
BMS-806 was shown to be specific for HIV-1, with no activity against HIV-2 or simian
immunodeficiency virus.[31] This compound as the control HIV-1 glycoprotein inhibitor was used in
this the work. Compound BMS-806 was docked into the gp41 binding site and showed interactions
almost similar to NB-2 and the synthesized derivatives. This compound formed H-bonds with Lys574
and Trp571. It was not able to interact electrostatically with the receptor since it lacks the proper
functionalities (Figure 7).
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