Synthesis of Biuret Derivatives as Potential HIV-1 Protease Inhibitors Using (LDHs-g-HMDI-Citric Acid), as a Green Recyclable Catalyst

Article abstract of DOI:10.1002/bkcs.12152

In this study, a novel catalyst based on layered double hydroxides (LDHs) attached by hexamethylene-1,6-diisocyanate (HMDI) and citric acid (LDHs-g-HMDI-Citric acid) is reported and used to increase the yield of biurets synthesis. Biuret derivatives 5a–n were prepared by reaction of several phenyl allophanates (3a–d), which prepared from the reaction of phenyl chloroformate and urea derivatives (2a–d), with variously substituted amines (4a–g) in the presence of LDHs-g-HMDI-Citric acid as a reusable heterogeneous catalyst at reflux condition for 60–180 min. These biurets (5a–n) were evaluated for human immunodeficiency virus type-1 (HIV-1) protease inhibitory activity by HIV-1 p24 antigen ELISA kit and six of them (5n, 5i, 5j, 5 m, 5f, and 5a) showed moderate activity on HIV-1 virus with IC₅₀ values ranging from 55 to 100 μM compared with the azidothymidine as the reference drug (IC₅₀ = 0.11 μM). Results of the in vitro test and docking study were in good correlation.

Full text of DOI:10.1002/bkcs.12152

Article
DOI: 10.1002/bkcs.12152
BULLETIN OF THE
Z. Ghiasifar et al.
KOREAN CHEMICAL SOCIETY
Synthesis of Biuret Derivatives as Potential HIV-1 Protease Inhibitors
Using (LDHs-g-HMDI-Citric Acid), as a Green Recyclable Catalyst
†
§
Zahra Ghiasifar,† Hafezeh Salehabadi,‡ Neda Adibpour,‡, Eskandar Alipour, Farzad Kobarfard,
*
and Mohammad Reza Shoushizadeh¶
†Department of Organic Chemistry, Islamic Azad University Tehran North Branch, Tehran
1651153311, Iran
‡Department of Medicinal Chemistry, School of Pharmacy, Zanjan University of Medical Sciences,
Zanjan 45139-56184, Iran. *E-mail: n.adibpour@gmail.com
§Department of Medicinal Chemistry, School of Pharmacy, Shahid Beheshti University of Medical
Sciences, Tehran 1991953381, Iran
¶Department of Medicinal Chemistry, School of Pharmacy, Ahvaz Jundishapur University of Medical
Sciences, Ahvaz 61357-15794, Iran
Received August 15, 2020, Accepted November 6, 2020
In this study, a novel catalyst based on layered double hydroxides (LDHs) attached by hexamethylene-
1,6-diisocyanate (HMDI) and citric acid (LDHs-g-HMDI-Citric acid) is reported and used to increase the
yield of biurets synthesis. Biuret derivatives 5a–n were prepared by reaction of several phenyl
allophanates (3a–d), which prepared from the reaction of phenyl chloroformate and urea derivatives (2a–
d), with variously substituted amines (4a–g) in the presence of LDHs-g-HMDI-Citric acid as a reusable
heterogeneous catalyst at reflux condition for 60–180 min. These biurets (5a–n) were evaluated for human
immunodeficiency virus type-1 (HIV-1) protease inhibitory activity by HIV-1 p24 antigen ELISA kit and
six of them (5n, 5i, 5j, 5 m, 5f, and 5a) showed moderate activity on HIV-1 virus with IC50 values ranging
from 55 to 100 μM compared with the azidothymidine as the reference drug (IC50 = 0.11 μM). Results of
the in vitro test and docking study were in good correlation.
Keywords: Biurets, Synthesis, Layered double hydroxides catalyst, HIV-1 protease inhibitor, Molecular
docking
Introduction
saquinavir, indinavir, and their analogs, are approved by
the FDA.7 However, in long-term treatment, resistance to
these drugs and several adverse effects such as insulin
resistance, dyslipidemia, cerebrovascular and cardiovascular
diseases, and unacceptable toxicity have been identified for
these inhibitors which limit their clinical use.8-10 Therefore,
finding novel HIV-proteases inhibitors with a novel struc-
ture, improved bioavailability, and low toxicity is compul-
sory to improve the quality of HIV patients.
Today, anti-HIV-1 peptides (AHPs) have an essential
role in the treatment of HIV infection. They mimic many
key enzyme substrates or important epitopes. Their specific
binding to their targets, which results in relatively few off-
target side effects, and also a low potential for systemic
toxicity.11,12 In this regard, biuret derivatives with pseudo
peptide structures could be considered as anti-HIV-1 prote-
ase peptide-like agents. There are several reports on various
pharmacological effects of biuret derivatives, such as inhi-
bition of gastric acid secretion, anti-analgesic, anti-inflam-
matory, anti-nociceptive, anti-leishmaniasis, antitumor, and
hypoglycemic activity.13-16 However, the activity of these
compounds against HIV has not been investigated.
Human immunodeficiency virus type-1 (HIV-1) is a retrovi-
rus capable of suppressing the immune system by
destroying CD4 lymphocytes.1,2 Over time, damages to the
immune system by HIV infection lead to different patho-
logical problems, including severe infections and certain
cancers.3 HIV-1 protease is an enzyme that is crucial for
viral assembly and maturation.4 Viral RNA is translated
into a polypeptide sequence that includes several individual
proteins (reverse transcriptase, transcriptase, protease, and
integrase). The HIV-1 protease (EC 3.4.23.16) is a viral
aspartic protease that can recognize Phe-Pro and Tyr-Pro
sequences and cleaves Gag and Gag-Pol viral polyproteins
into functional enzymes, which provides a proper situation
for HIV-1 survival and assembly.5,6 Therefore, it is possible
to fight HIV-1 infection using the substances that can
inhibit HIV-1 protease activity. Inhibitors of HIV-1 prote-
ase can lead to the production of new noninfectious viruses
with disorganized structures. Therefore, they could be con-
sidered as potential therapeutic agents to treat HIV/AIDS.
To date, numerous HIV-1 protease inhibitors have been
introduced in the literature, and some of them, such as
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In the past few decades, scientists have shown great
interest in developing green chemistry and protecting the
living environment.17 Citric acid is one of the safe and
most environmentally benign agents that is widely used as
a green biocatalyst in one-step synthesis.18-22 It exists espe-
cially in concentrate lemons and limes, which is applied in
the food, beverage, cosmetic, and pharmaceutical indus-
tries.23 Following our previous study on the synthesis of
new biuret compounds,16 an eco-friendly procedure using
modified layered double hydroxides (LDHs), as a green
recyclable heterogeneous catalyst (Scheme 1), was used for
the synthesis of these derivatives in shorter time reaction
and higher yield.
in polymers,34 anti-wear additives in lubricants like motor
oil,35,36 flame retardants,37,38 the formation of interesting
nano-composite materials,39-45 as well as catalysts and pre-
cursors to mixed metal oxide catalysts.46-49 In recent years,
the development of magnetic LDH-based nanoparticles
(MLDHs) with a large surface area as a magnetic
nanocatalyst is in great attention.50,51 There are various
methods for preparing LDHs, such as co-precipitation,52
urea hydrolysis,53 the sol–gel method,54 hydrothermal
synthesis,55 reformation,56 and mechanical milling.57
In this paper, the co-precipitation method was used,
which is the most common and inexpensive “one-pot”
method for the preparation of citric acid grafted
hexamethylene-1,6-diisocyanate-g-supported layered dou-
ble hydroxides (LDHs-g-HMDI-citric acid), as a green
recyclable heterogeneous catalyst for the synthesis of biuret
derivatives (5a–5n). The anti-HIV activity of these com-
pounds was assessed in vitro. A docking study on the
derivatives with the highest inhibitory effect on HIV-1 pro-
tease was also carried out.
LDHs are a class of anionic clay minerals with octahe-
dral layer structure that are represented by the general for-
2+
3+
mula [M1-x
Mx
(OH)2]x+[An-]x/n. mH2O.24,25 In this
formula, M2+ and M3+ indicate any divalent and trivalent
metal cations, respectively, and x is the metal ratio M3
+/(M2+ + M3+).24-26 An− is an interlayer and the exchange-
able anion (organic or inorganic) and m is the amount of
water present in the interlayer area.27,28 Due to the ability
to combine a wide variety of metal cations and anions and
specific structure of LDHs, they have exhibited various
applications.24,25 The applications contain adsorbents, anion
exchangers, medical applications ranging from simple ant-
acids to targeted drug delivery mechanisms,29-33 additives
Experimental
General. All common chemicals for the synthesis step
were purchased from Merck (Darmstadt, Germany).
Reagents used for cell culture were obtained from Gibco
(Waltham, Massachusetts, USA). The single-chain replica-
tion (SCR) HIV-1 was supplied by the Pasteur Institute of
Iran (Tehran, Iran). HIV-1 p24 antigen ELISA kit was pur-
chased from BioMerieux (Marcy l’Etoile, France). Ultra-
pure water was used to prepare all of the aqueous solutions.
Melting points were determined on a MEL-270 Sibata melt-
ing point apparatus and are uncorrected. For Fourier trans-
form infrared spectroscopy (FT-IR) analysis, the FT-IR
spectra were recorded on a Nicolet Magna FT-IR 550 spec-
trometer (Nicolet Instrument Co., WI, USA), using FT-IR
1
grade potassium bromide (KBr). H nuclear magnetic reso-
nance (1HNMR) spectra were recorded on a 400 MHz
Brucker spectrometer using
a
deuterated solvent
(DMSO‑d6, and CDCl3). Chemical shifts (δ) are reported
in parts per million (ppm) relative to tetramethylsilane
(TMS) as internal standard. Mass spectra were obtained
using a Finningan TSQ-70 instrument (Thermo Finnigan
LLC,San Jose, CA). Elemental analyses for C, H, and N
were performed using a Heracus CHN–O rapid elemental
analyzer and the results are within ꢀ0.4% of the theoretical
values. Qualitative analysis of LDHs-g-HMDI-citric acid
was performed by energy-dispersive X-ray spectroscopy
(EDX) on a FESEM-SIGMA instrument (Oberkochen, Ger-
many). Scanning electron microscopy (SEM) micrograph
was acquired on a VEGA 2 TESCAN SEM operated at 15–
20 kV. The curves obtained from thermogravimetric analy-
sis (TGA) were recorded under the air atmosphere using
TGA/DTA PYRIS DIAMOND (Pyris Diamond Perkin-
Elmer, Waltham, MA, USA).
Scheme 1. Preparation of LDHs-g-HMDI-citric acid.
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General Procedure for Synthesis of LDHs-g-HMDI-Cit-
ric Acid Catalyst
(400 MHz, CDCl3) δ: 7.02–7.5 (m, 12H), 9.50 (s, 1H)
ppm. MS (EI) m/z: 533.4 (M+), 402.995, 255, 213, 137,
106, 91. Anal.Calcd for C14H13N3O2: C, 65.87; H, 5.13; N,
16.46. Found C, 65.75; H, 5.18; N, 16.32.
Preparation of Layered Double Hydroxides (LDHs). The
conventional co-precipitation method was used for the syn-
thesis of LDHs in a one-pot reaction.58 Briefly, an aqueous
salt solution was prepared by adding a zinc salt (Zn
(NO3)2.6H2O) and a chromium Salt (Cr(NO3)3.9H2O) with
the Zn/Cr molar ratio of 3:1. Subsequently, the mixture was
added dropwise to NaOH solution (8 M) in a 250 mL
round-bottom flask at 80 ꢁC under vigorous stirring. Then,
the pH of the solution was adjusted to 7.0. The solution
was stirred for 48 h at 80 ꢁC for crystallization. Then, the
product was filtered and washed with deionized water sev-
eral times and dried overnight at 100 ꢁC.
N-Phenyl-N0-(3-phenylpropyl)Imidodicarbonic Diamide
(5b). White solid; Yield 91%; mp 89–91 ꢁC; IR (KBr) νmax
:
3340, 3308, 3043, 2928, 2861, 1701, 1669 cm−1. HNMR
(400 MHz, CDCl3) δ: 1.6–1.9 (m, 2H), 2.5–2.9 (m, 2H),
3.0–3.4 (m, 2H), 6.7–7.4 (m, 10H), 9.5 (br, 1H). MS (EI)
m/z: 297(M+), 193, 117, 93. Anal. Calcd. For
C17H19N3O2: C, 68.67; H, 6.44; N, 14.13. Found: C,
68.47; H, 6.50; N, 14.08.
1
N-Phenyl-N0-[3-(benzo[b]thiazol-2-ylthio)Propyl]
Imidodicarbonic Diamide (5c). Yellow solid; Yield 89%;
mp 105–109 ꢁC; IR (KBr) νmax: 3352, 3317, 3100, 2944,
Anchoring of HMDI onto LDHs Surface (LDHs-g-
HMDI). In order to prepare LDHs-g-HMDI, 1 g of LDHs
was added into 20 mL of anhydrous DMF and stirred at
1703, 1686, 1602 cm−1 1HNMR (400 MHz, CDCl3) δ:
.
2.01–2.04 (m, 2H), 3.2–3.4 (m, 4H), 7.06–7.45 (m, 7H),
7.77 (d, 1H, J = 8.0 Hz), 7.95 (d, 1H, J = 8.0 Hz), 9.38 (s,
1H). MS (EI) m/z: 386 (M+), 293.9, 254.7, 251, 193.6,
180.0, 166.4, 134.4, 93.1. Anal. Calcd. For
C18H18N4O2S2: C, 55.94; H, 4.69; N, 14.50. Found: C,
55.98; H, 4.39, N, 14.65.
room temperature for
2
h. Then, 17 mmol of
hexamethylene-1,6-diisocyanate (HMDI) in 15 mL anhy-
drous DMF was added dropwise into the flask, and the
mixture was stirred for another 3 h. All unreacted HMDI
was removed by filtration and washing with DMF three
times. The product was dried under vacuum overnight.
Preparation of LDHs-g-HMDI-Citric Acid. To a stirred
solution of prepared LDHs-g-HMDI (1 g) in anhydrous
DMF (20 mL) at 70 ꢁC, 15 mL of citric acid solution
(20 mmol) in anhydrous DMF (15 mL) was added
dropwise with continuous stirring for 3 h and then cooled
N,N0-Bis Phenylmethyl Imidodicarbonic Diamide (5d).
White solid; Yield 95%; mp 168–169 ꢁC. Anal.Calcd for
C16H17N3O2: C, 67.83; H, 6.05; N, 14.83. Found C,
67.72; H, 5.07; N, 14.75.
N-(Phenylmethyl)-N0-(2-Phenylethyl)Imidodicarbonic
Diamide (5e). White solid; Yield 94%; mp 155–160 ꢁC.
Anal.Calcd for C17H19N3O2: C, 68.67; H, 6.44; N, 14.13.
Found C, 68.32; H, 6.55; N, 13.97.
ꢁ
to room temperature (25 ꢀ 1 C). The resulting solid prod-
uct was then filtered and washed with anhydrous DMF sev-
eral times and then dried at 70 ꢁC.
N-(Phenylmethyl)-N0-(3-Phenylpropyl)Imidodicarbonic
General Experimental Procedure for the Modified Synthe-
sis of Biuret Derivatives 5a-n Catalyzed by LDHs-g-
HMDI-Citric Acid. As in the previous study, urea deriva-
tives 2a–d were synthesized by the reaction of amine
hydrochlorides 1a–d with potassium isocyanate in water.59
Phenyl allophanates 3a–d were prepared by reaction of
phenyl chloroformate with ureas 2a–d in dichloromethane
in the presence of pyridine at 0 ꢁC.16 A mixture of
allophanates 3a–d (1 mmol) and variously substituted
amines 4a–g (1 mmol) in 10 mL anhydrous dic-
hloromethane in the presence of LDHs-g-HMDI-Citric acid
as catalyst was refluxed for 60–180 min. After completion
of the reaction, which was monitored by thin-layer chroma-
tography, the mixture was allowed to cool at room tempera-
ture and then centrifuged and the supernatant was separated
from the catalyst. After washing with water (three times),
the solvent was evaporated under a vacuum. The products
were purified by preparative thick layer chromatography as
previously reported (Scheme 2) and the structures of the
selected biuret derivatives were admitted on the basis of
their physical and spectral data (FT-IR, 1H NMR, and MS).
Spectral Data for the Biuret Derivatives
Diamide (5f). White solid; Yield 89%; mp 88 ꢁC; IR (KBr)
ν
max: 3345, 3310, 3045, 2940, 2865, 1710, 1660 cm−1
.
1HNMR (400 MHz, CDCl3) δ: 1.80–1.90 (m, 2H), 2.65 (t,
2H, J = 8.0 Hz), 3.2 (t, 2H, J = 8.0 Hz), 4.4 (d, 2H,
J = 8.0 Hz), 7.17–7.35 (m, 10H), 9.45 (s, 1H). MS (EI) m/
z: 311 (M+), 296, 282, 219, 205, 194, 176, 118, 105, 91.
Anal. Calcd. For C18H21N3O2: C, 69.43; H, 6.80; N, 13.49.
Found: C, 69.35; H, 6.85; N, 13.26.
N-(Phenylmethyl)-N0-[2-(Pyridin-2-yl))Ethyl]
Imidodicarbonic Diamide (5g). White solid; Yield 92%;
mp 74 ꢁC; IR (KBr) νmax: 3380, 3266, 3100, 2948, 1698,
1680 cm−1 1HNMR (400 MHz, CDCl3) δ: 2.97 (t, 2H,
.
J = 6.5 Hz), 3.56 (q, 2H, J = 6.0 Hz), 4.35 (d, 2H,
= 5.5 Hz), 7.08–7.10 (m, 1H), 7.13 (d, 1H,
J
J = 7.5 Hz),7.21–7.30 (m, 3H), 7.27–7.3 (m, 2H), 7.55–
7.58 (m,1H), 8.5 (d, 1H, J = 4.0 Hz), 9.46 (s, 1H). MS (EI)
m/z: 298 (M+), 192.1, 149, 121, 106, 93.1. Anal. Calcd.
For C16H18N4O2: C, 64.41; H, 6.08; N, 18.78. Found: C,
64.56; H, 5.95; N, 18.85.
N-(Phenylmethyl)-N0-(2-Methylquinolin-4-yl)
Imidodicarbonic Diamide (5h). White solid; Yield 90%;
mp 161 ꢁC; IR (KBr) νmax: 3415, 3340, 3065, 1702, 1692,
1670, 1600 cm−1. 1HNMR (400 MHz, DMSO-d6) δ: 2.5 (s,
3H), 4.31 (d, 2H, J = 6.3 Hz), 7.09–7.12 (m, 1H), 7.16–
7.21 (m, 4H), 7.31–7.36 (m, 1H), 7.5 (t, 1H, J = 5.72 Hz),
N,N0-Bis Phenyl Imidodicarbonic Diamide (5a). White
solid; Yield 91%; mp 212 ꢁC; IR (KBr) νmax: 3386, 3200,
3130, 3024, 1690, 1605, 1550, 1237 cm−1
.
1HNMR
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Scheme 2. LDHs-g-HMDI-citric acid catalyzed synthesis of biuret derivatives.
7.78 (d, 1H, J = 6.76 Hz), 7.84 (d, 1H, J = 6.72 Hz),7.98
(s, 1H), 9.16 (br, 1H). MS (EI) m/z: 334 (M+), 316, 291,
283, 270, 200, 184, 176, 158, 106, 91. Anal. Calcd. For
C19H18N4O2: C, 68.25; H, 5.43; N, 16.76. Found: C,
68.04; H, 5.49; N, 16.81.
δ: 1.84–1.9 (m, 2H), 2.03–2.08 (m, 2H), 2.68 (t, 2H,
J = 7.6 Hz), 3.20–3.27 (m, 2H), 3.3–3.5 (m, 4H), 7.15–7.2
(m, 3H), 7.25–7.3 (m, 3H),7.38–7.43 (m, 1H),7.73 (d, 1H,
J = 8.0 Hz), 7.9 (d, 1H, J = 8.0 Hz), 9.50 (s, 1H). MS (EI)
m/z: 428 (M+), 350, 336, 308, 294, 268, 250, 224, 194,
181, 166, 136, 108, 91. Anal. Calcd. For
C21H24N4O2S2: C, 58.85; H, 5.64; N, 13.07. Found: C,
58.56; H, 5.53; N, 12.96.
N-(Phenylmethyl)-N0-[3-(Benzo[d]thiazol-2-ylthio)Pro-
pyl]Imidodicarbonic Diamide (5i). Yellow oil; Yield
85%; mp 70–73 ꢁC; IR (KBr) νmax: 3348, 3309, 3080,
2946, 1700, 1688, 1600 cm−1.1HNMR (400 MHz, CDCl3)
δ: 1.9–2.07 (m, 2H), 3.3–3.4 (m, 4H), 4.35 (d, 2H,
j = 6.4 Hz), 7.0–7.5 (m, 7H), 7.65–7.8 (m, 2H), 9.6
(br,1H). MS (EI) m/z: 400 (M+), 292, 283, 250, 203, 194,
166, 105. Anal. Calcd. For C19H20N4O2S2: C, 56.98; H,
5.03; N, 13.99. Found: C, 56.89; H, 5.24; N, 13.78.
N-(3-Phenylpropyl)-N0-[3-(1-Phenyl-1H-tetrazol-5-
ylthio)Propyl]Imidodicarbonic Diamide (5n). White
solid; Yield 88%; mp 90–91 ꢁC; IR (KBr) νmax: 3350,
3310, 3089, 2946, 1700, 1685, 1605 cm−1
.
1HNMR
(400 MHz, CDCl3) δ: 1–1.22(m, 1H), 1.52–2 (m, 4H), 2.5–
2.6 (m, 2H), 3.03–3.35 (m, 5H), 6.9–7.5 (m, 5H), 7.5–7.75
(m, 5H), 9.3 (br, 1H). MS (EI) m/z: 439 (M+), 296, 277,
236, 219, 205, 191, 178, 166, 145, 132, 118, 106, 91. Anal.
Calcd. For C21H25N7O2S: C, 57.38; H, 5.73; N, 22.31.
Found: C, 57.31; H, 5.89; N, 22.25.
N,N0-Bis(2-Phenylethyl)Imidodicarbonic Diamide (5j).
White solid; Yield 96%; mp 115 ꢁC; IR (KBr) νmax: 3359,
3272, 3030, 2944, 1690, 1530, 1230 cm−1
.
1HNMR
(400 MHz, CDCl3) δ: 2.78–2.86 (m,4H), 3.38–3.48 (m,
4H), 7.19–7.39 (m, 12H), 9.4 (s, 1H, NH) MS (EI) m/z:
311 (M+), 219, 205, 190, 162, 147, 119, 105, 91. Anal.
Calcd. For C18H21N3O2: C, 69.43; H, 6.80; N, 13.49.
Found: C, 69.22; H, 6.91; N, 13.54.
Cells and Viruses. Blood samples from three healthy vol-
unteers were collected in heparinized tubes. The volunteers
were seronegative for HIV, HBV, and HCV (approved by
blood transfusion center, Alzahra hospital, Esfahan, Iran).
Peripheral blood mononuclear cells (PBMCs) were isolated
by Lymphodex density centrifugation (1800g, 20 min). The
cells were cultured in a complete medium, containing Ros-
well Park Memorial Institute (RPMI) media supplemented
with 15% fetal bovine serum (FBS), phytohemagglutinin
(PHA), 100 μg/mL penicillin solution, 100 μg/mL strepto-
mycin, and 2 mM L-glutamine. PBMCs were incubated in a
moistened atmosphere with 5% CO2 for 6 h at 37 ꢁC.60
The virus titers were measured using HIV-1 p24 antigen
ELISA kit (BioMerieux, France). The viruses were stored
at −70 ꢁC for later use.
HIV Replication Assay. The anti-HIV-1 activity of biuret
derivatives was studied by the HIV-1 p24 Antigen ELISA
kit according to the manufacturer’s protocol.61 Briefly,
6 × 105 PBMCs were seeded in 24-well plates and infected
with 0.01 mol of SCR HIV-1 subtype A, in 500 μL of cul-
ture medium. Different concentrations of biuret derivatives
(0.1, 1, 10, 50, and 100 μM) were added to each assay sam-
ple and incubated at 37 ꢁC for 12 h. The infected cells were
N-(2-Phenylethyl)-N0-[3-(Benzo[d]thiazol-2-ylthio)Pro-
pyl]Imidodicarbonic Diamide (5k). Yellow solid; Yield
95%; mp 70–73 ꢁC; IR (KBr) νmax: 3348, 3308, 3070,
1
2949, 1701, 1687, 1603 cm−1. HNMR (400 MHz, CDCl3)
δ: 2.03 (t, 2H, J = 6.5 Hz), 2.80 (t, 2H, J = 6.5 Hz), 3.28–
3.37 (m, 4H), 3.4–3.6 (m, 2H), 7.16–7.2 (m, 3H), 7.24–
7.28 (m, 3H), 7.35–7.40 (m, 1H), 7.72 (d, 1H, J = 7.5 Hz),
7.88 (d, 1H, J = 8.0 Hz), 9.6 (br, 1H). MS (EI) m/z: 414
(M+), 309.4, 294.1, 250, 191, 180, 166, 148, 120, 105, 91.
Anal. Calcd. For C20H22N4O2S2: C, 57.95; H, 5.35; N,
13.52. Found: C, 57.91; H, 5.47; N, 13.75.
N,N0-Bis (3-Phenylpropyl)Imidodicarbonic Diamide (5l).
White solid; Yield 93%; mp 85–86 ꢁC. Anal.Calcd.for
C20H25N3O2: C,70.77; H, 7.42; N,12.38. Found: C,
70.47; H, 7.47; N, 12.31.
N-(3-Phenylpropyl)-N0-[3-(Benzo[d]thiazol-2-ylthio)Pro-
pyl]Imidodicarbonic Diamide (5m). Yellow oil; Yield
90%; mp 95–96 ꢁC; IR (KBr) νmax: 3349, 3305, 3075,
1
2949, 1703, 1688, 1605 cm−1. HNMR (400 MHz, CDCl3)
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Synthesis of Biuret Derivatives as Potential HIV-1 Protease Inhibitors
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then washed and overlaid with the medium at different con-
centrations of biuret derivatives. Azidothymidine (AZT)
(0.1, 1, 10, 50, and 100 μM) and DMSO (1%) were used as
positive control and blank. After 3 days of incubation, the
supernatant was collected and transferred to the coated 96-
well plate to quantify the HIV-1 p24 core protein. Optical
density (OD) of viruses was measured at 450 nm by ELISA
reader (Awareness Technology Inc., Florida, USA). The
minimal concentration required to suppress the load of P24
by 50% (IC50) was determined by regression analysis of
the dose–response curve generated from the data.
Inc, Gainesville, FL, USA) was used to add hydrogen
atoms and geometric optimization. The atomic charges
were defined based on Gasteiger-Marsili partial charges.
Each docking job was carried out by 100 runs. All other
parameters and the docking validation were performed
using previously published methods.64 Afterward, the
results obtained from the docking simulations were ranked
based on the lowest binding free energy and were analyzed
to determine the binding modes and orientations of the
selected compounds in the active site of the protein.
Results and Discussion
Docking Studies. Molecular docking studies were per-
formed using AutoDock 4.2 software (The Scripps
Research Institute, La Jolla, CA, USA) to characterize the
binding modes of the selected compounds,62 with HIV-1
protease active site. The crystal structure of HIV-1 protease
(PDB code: 1HPV) was derived from the protein data bank
(www.rcsb.org). The optimized structure of the protein and
ligands were used as the input for the subsequent docking
studies. The AutoDockTools program (ADT; version 1.5.6)
was utilized to prepare input files as well as to view the
results.62 Water molecules were removed from the PDB
file, and polar hydrogens were added to the protein struc-
ture. The Kollman-united charges method was assigned for
all atoms of the enzyme to calculate the partial atomic char-
ges, and grid maps were generated by AutoGrid 4.2 (The
Scripps Research Institute, La Jolla, CA, USA). The chemi-
cal structures of the ligands were generated by Marvin
Sketch version 5.7 (ChemAxon, Budapest, Hungary).63
Characterization of LDHs-g-HMDI-Citric Acid Cata-
lyst. As shown in Scheme 1, the procedure of the prepara-
tion of LDHs-g-HMDI-Citric acid was accomplished in
three steps. LDHs were prepared by co-precipitation of Zn2
+
and Cr3+ salts with the Zn/Cr molar ratio of 3:1. Then,
hexamethylene-1,6-diisocyanate (HMDI) as a linker was
connected to LDHs to obtain functionalized LDHs-g-
HMDI. Finally, the resulting LDHs-g-HMDI was reacted
with citric acid to prepare LDHs-g-HMDI-Citric acid as a
novel acidic catalyst. The structure of LDHs-g-HMDI-Cit-
ric acid catalyst was evaluated precisely using various tech-
niques such as FT-IR, X-ray diffraction (XRD) analysis,
energy dispersive X-ray spectroscopy (EDX), scanning
electron microscopy (SEM), TGA, and derivative thermal
gravimetric (DTG).
FT-IR Spectra. The FT-IR spectra of the catalyst and pre-
HyperChem
software
(version
8.0)(Hypercube,
cursor composites, including LDHs, LDHs-g-HMDI, and
Figure 1. FT-IR spectra of (a) LDHs, (b) LDHs-g-HMDI and (c) LDHs-g-HMDI-citric acid.
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Figure 2. X-ray diffraction pattern of LDHs-g-HMDI-citric acid.
LDHs-g-HMDI-Citric acid, are illustrated in Figure 1. The
characteristic peaks are observed in the region 500–
800 cm−1 declare the vibrating bands belong to the Zn─O
and Cr─O groups in Figure 1(a)–(c). As is illustrated in
Figure 1(b) and (c), the absorption band at 1384 cm−1 is
assigned to the nitrate anions that exist between the layers
of LDHs. The specific peaks at 1651 cm−1 and 1659 cm−1
imply the existence of C═O groups in Figure 1(b) and (c),
respectively. The appearance of an absorption band at
2400 cm−1 in Figure 1(b) is attributed to N═C═O func-
tional group of LDHs-g-HMDI. The fade of this peak in
Figure 1(c) further confirms the successful attachment of
citric acid to the surface of the HMDI-g-LDHs. Also, the
characteristic peaks at 2667 cm−1 and 2941 cm−1 depicted
in Figure 1(b) and (c) can be ascribed to the aliphatic CH2
groups. Also, the O─H bond of acid group (COOH)
absorbs between 2500 and 3300 cm−1, clearly verifies the
successful immobilization of citric acid onto the surface of
LDHs (Figure 1(c)).
Figure 4. SEM micrograph of LDHs-g-HMDI-citric acid.
X-ray Diffraction Studies. The XRD technique was
applied to identify the crystalline structure of synthesized
LDHs-g-HMDI-Citric acid. As can be observed in Figure 2,
the positions and relative diffraction peaks at around 10.04
(1), 19.91 (2), 33.67 (2), 59.81 (2), 70.20 (5), are attributed
to LDHs-g-HMDI-Citric acid. This pattern emphasizes the
effective grafting of citric acid on the LDHs-g-HMDI.
Energy Dispersive X-ray Analysis. The composition ele-
ments of the catalyst (C, N, O, Zn, and Cr) are shown in
the EDX of the typical catalyst sample (Figure 3).
According to Figure 3, the results approve the successful
grafting of citric acid and hexamethylen-1,6-diisocyanate
(HMDI) on the surface of LDHs.
SEM Analysis. The morphology and particle size of the
LDH-g-HMDI-Citric acid were observed by SEM analysis
Figure 3. EDX spectrum of LDHs-g-HMDI-citric acid.
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decomposition of the particles does not occur even at
700 ꢁC. Therefore, the results confirmed a good grafting of
citric acid and HMDI on the surface of LDHs.
The Catalytic Activity of LDHs-g-HMDI-Citric Acid for
the Synthesis of Biuret Derivatives. Initially, to establish
the optimized reaction conditions, the model reactions
between allophanate and phenylethylamine in the presence of
LDHs-g-HMDI-Citric acid was investigated. For this pur-
pose, the effects of several parameters such as different
amounts of catalyst, type of solvent (THF, DCM, Ethanol,
DMF), and temperature on the reaction time as well as yield
of the model reaction were assessed. According to the data
that are presented in Table 1, the best results for the model
synthesis of N,N0-bis(2-phenylethyl)imidodicarbonicdiamide
(5j) were obtained when the reaction was carried out in
DCM and reflux condition using 5 mg of catalyst for 1 h
(entry 7). In addition, increasing the amount of the catalyst to
8 mg showed no progress in the rate and yield of the reaction
(entry 8). It should be noted that the reaction under the same
conditions and absence of catalyst was carried out, and the
product was achieved after a prolonged reaction time with a
lower yield (entry 14). The results proved the vital role of
LDHs-g-HMDI-Citric acid as a suitable catalyst for the syn-
thesis of biurets.
Figure 5. TGA analysis of LDHs-g-HMDI-citric acid.
as shown in Figure 4. As can be observed in Figure 4, the
SEM micrograph of the LDHs-g-HMDI-Citric acid reveals
uniformity and layered structure.
Thermal Gravimetric (TG) and Derivative Thermal
Gravimetric (DTG) Analysis. In Figure 5, the first weight
loss of ca. 5.5% at 170 ꢁC can be attributed to the removal
of residual water attached to the catalyst surface. The sec-
ond weight loss (17%) at about 200–370 ꢁC is likely related
to the removal of the residual organic solvent and
immobilized urethane moiety. Also, the third weight loss of
about 2.5%, which occurred in the thermal range of 370–
500 ꢁC is probably due to the decomposition and removal
of the citric acid moiety from the catalyst. The complete
Furthermore, the reactions of various allophanates and
amines were carried out. As can be seen in Table 2, the
corresponding products (5a–n) were obtained with higher
yields (28–96%) and shorter times (60–180 min) than the
previous study.16
Table 1. Screening the reaction parameters for the production of N,N0-bis(2-phenylethyl)imidodicarbonic diamide.a
Entry
Solvent
Catalyst (mg)
Temperature (ꢁC)
Time (minutes)
Yield (%)b
1
2
3
4
5
6
7
8
THF
3
3
3
3
3
3
5
8
3
3
3
3
r.t
r.t
r.t
r.t
60
Reflux
Reflux
Reflux
60
180
180
180
180
120
90
38
59
25
12
51
87
96
96
45
33
68
59
41
CH2Cl2
EtOH
DMF
THF
CH2Cl2
CH2Cl2
CH2Cl2
EtOH
DMF
THF
EtOH
DMF
60
60
9
120
150
120
120
120
300
10
11
12
13
14
120
Reflux
Reflux
Reflux
Reflux
3
CH2Cl2
No catalyst
Trace
a Conditions: phenyl [(2-phenylethyl)aminocarbonyl]carbamate (1 mmol), phenylethyl amine (1 mmol), solvent (10 mL).
b Isolated pure yield.
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Table 2. Synthesis of biuret derivatives catalyzed by LDHs-g-HMDI-citric acid as catalyst.a
Melting point (ꢁC)
Yield (%)b
Reaction time
(minutes)
Compound
R1
R2
Found
212
89–91
105–109
Reported16 present Reported16
5a
5b
5c
Phenyl
Phenyl
Phenyl
Phenyl
3-Phenylpropyl
3-(Benzo[d]thiazol-2-ylthio)
propyl
60
60
60
208–210
92–93
91
91
89
53
57
105–109
35.6
5d
5e
5f
5 g
5 h
5i
Phenylmethyl
Phenylmethyl
Phenylmethyl
Phenylmethyl
Phenylmethyl
Phenylmethyl
Phenylmethyl
2-Phenylethyl
3-Phenylpropyl
60
60
90
100
120
80
168–169
155–160
88
74
161
70–73
168–169
156–159
90–91
73–74
163
95
94
89
92
90
85
76
73.4
34
58
47.2
23
2-(Pyridin-2-yl)ethyl
2-Methyl-quinoline-4-yl
3-(Benzo[d]thiazol-2-ylthio)
propyl
68–72
5j
5 k
2-Phenylethyl
2-Phenylethyl
2-Phenylethyl
3-(Benzo[d]thiazol-2-ylthio)
propyl
60
100
115
70–73
110–112
70–73
96
95
84
76
5 L
5 m
3-Phenylpropyl
3-Phenylpropyl
3-Phenylpropyl
3-(Benzo[d]thiazol-2-ylthio)
propyl
100
60
85–86
95–96
85–86
93–96
93
90
68.5
47
5n
3-Phenylpropyl
3-(1-Phenyl-1H-tetrazol-
5-ylthio)propyl
100
90–91
89–92
88
28
a Conditions: allophanate (1 mmol), amine (1 mmol), catalyst (5 mg), dichloromethane (10 mL), reflux conditions.
b Isolated pure yield.
A plausible mechanism that can be suggested for the
reaction of various derivatives of allophanates and
amines in the presence of LDHs-g-HMDI-Citric acid is
depicted in Scheme 3. At first, due to the interaction
between the acidic hydrogen of LDHs-g-HMDI-Citric
acid and the oxygen of carbonyl functional group from
Scheme 3. A plausible mechanism for the synthesis of biuret derivatives (5a–n) catalyzed by LDHs-g-HMDI-citric acid.
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Figure 6. Recyclability test of LDHs-g-HMDI-citric acid.
allophanate, the carbonyl site became more active for
nucleophilic addition of amine, and the nucleophilic sub-
stitution was easier. In other words, by lowering the
HOMO (highest occupied molecular orbital) energy level
of amine and approaching its level to the LUMO (lowest
unoccupied molecular orbital) energy level of carbonyl,
the catalyst causes the reaction to take place at a much
higher rate and yield.
Figure 7. Residues involved in the interaction of VX-478 with
HIV-1 protease according to the docking methodology.
At the end of the reactions, the catalyst was isolated by
centrifuge (2000g, 10 min), and the isolated catalyst was
washed with hot ethanol and dried in an oven at 70 ꢁC.
The isolated catalyst particles were reused in subsequent
reactions at least five times without significant loss of cata-
lytic activity (Figure 6). Also, the exposure of the catalyst
to air at room temperature for 2 weeks did not significantly
alter its activity. These results demonstrate the exceptional
stability and reusability of the LDHs-g-HMDI-Citric acid
as a heterogeneous catalyst.
inactive compounds. The above six compounds were
selected for docking studies.
Molecular Docking Studies
Evaluation of the Docking Protocol. To verify the validity
of the docking protocol, initially, the docking of VX-478, a
potent and orally bioavailable inhibitor of the enzyme, over
the HIV-1 protease was investigated. The native ligand (VX-
478) was extracted from the X-ray structure of HIV-1 prote-
ase (PDB: 4XCT) and redocked into the corresponding active
site of protein (according to the mentioned protocol in the
docking studies section). As it is shown in Figure 7, the
obtained results from redocking and the crystallographic
structure are in agreement in terms of the ligand interactions,
position, and orientation in the active pocket of the enzyme.
The root mean square distance (RMSD) of re-docked and co-
crystallized ligand over 4XCT was less than 2.0 Å that con-
firmed the accuracy of docking protocol.65
Biological Evaluation.
The antiviral activities (IC50
values) of the synthesized compounds and azidothymidine
(AZT) as a reference drug are presented in Table 3.
According to the results, compounds 5n, 5i, 5j, 5 m, 5f,
and 5a exhibited antiviral activities (IC50 between 55.09
and 100 μM) against the HIV-1 virus. Other compounds
did not show any significant antiviral activities at the con-
centrations higher than 100 μM and therefore considered as
Binding position and interaction of the active compounds.
Molecular docking is a popular computational method that
is used in the prediction of binding modes of a potential
bioactive compound with its biomolecular target.66 The
synthesized biuret derivatives could be considered as poten-
tial HIV-1 protease inhibitors due to their pseudo peptide
structures. Therefore, the binding energy, position and
interaction of the most active compounds with HIV-1 prote-
ase were investigated by molecular docking studies. The
HIV-1 protease is a homodimeric protease including two
identical 99-residue monomers. Each monomer consists of
the conserved triads (Asp-Thr-Gly) in positions of 25–27
Table 3. IC50 (μM) values of the active compounds.
Compound
IC50 (μM)
5a
5f
5i
5j
5 m
5n
100.0 ꢀ 1.7
94.3 ꢀ 1.3
64.4 ꢀ 0.7
75.7 ꢀ 1.1
85.6 ꢀ 1.2
55.1 ꢀ 0.9
0.11 ꢀ 0.01
Azidothymidine
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Figure 8. 2D plots of the interaction between selected compounds and HIV-1 protease; olive green arrow: H bond interaction, brick red
representation: Hydrophobic site, nitrogen is blue, oxygen is red, carbon is black and sulfur is yellow. (a) 5a, (b) 5f, (c) 5i, (d) 5j, (e) 5 m,
and (f) 5n.
and 250–270, that aspartate amino acids do all of the cata-
lytic activities.67 All the selected compounds were success-
fully docked into the corresponding active site of HIV-1
protease (Figure 8). The binding affinity of the active com-
pounds to HIV1-protease and H- bond interactions with
key residues of active site has been shown in Table 4. Each
compound has one or more atoms with free electron pairs
that able to form hydrogen or electrostatic bond with the
binding pocket of the enzyme. Also, the hydrophobic
groups, such as benzyl moieties, could occupy the binding
pocket by interaction with hydrophobic residues. As shown
in Figure 8, the biuret moiety in the majority of the selected
compounds participates in hydrogen bonding with Asp
25, Gly 27, and Ile 50. The hydrogen bonding of Asp
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Table 4. The docking binding affinity of the selected compounds to HIV1- protease and hydrogen bond interactions.
Binding
Compound
energy
Type of interactions
5a
−6.48
Two hydrogen interactions are possible with
• Amino acid Gly 27B (H-acceptor) with distance about 2.95 Å
• Amino acid Ile 50A (H-donor) with distance about 3.27 Å
Two hydrogen interactions are possible with
• Amino acid Gly 27A (H-acceptor) with distance about 2.87 Å
• Amino acid Asp 25A (H- acceptor) with distance about 2.63 Å
Three hydrogen interactions are possible with
5f
5i
−7.56
−8.40
• Amino acid Gly 27B (H-acceptor) with distance about 2.72 Å
• Amino acid Asp 25A (H- acceptor) with distance about 2.75 Å
• Amino acid Ile 50A (H-donor) with distance about 3.09 Å
Only one hydrogen interaction (H-donor) is possible with amino acid Gly 27B with distance about
2.69 Å
5j
−7.19
−8.55
5 m
Two hydrogen interactions are possible with
• Amino acid Gly 27A (H-acceptor) with distance about 2.76 Å
• Amino acid Asp 25B (H- acceptor) with distance about 2.77 Å
Two hydrogen interactions are possible with
• Amino acid Gly 27B (H-acceptor) with distance about 2.87 Å
• Amino acid Ile 50B (H-donor) with distance about 3.29 Å
Only one hydrogen interaction (H-donor) is possible with amino acid Ile 50B with distance about
3.06 Å
5n
−8.94
−8.89
Azidothymidine
25 with different inhibitors has been previously reported.67
In summary, docking results revealed that all of the selected
compounds could occupy an HIV-1 protease active site.
However, compound 5n showed higher interaction energy.
There is a good correlation between docking analysis and
anti- HIV activity (IC50). Therefore, selected compounds
could serve as lead compounds for developing new poten-
tial HIV-1 protease inhibitor candidates.
2. E. Mugomeri, P. Chatanga, N. Chakane, Afr. J. Tradit. Com-
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The LDHs-g-HMDI-Citric acid was introduced as a new,
efficient, versatile and heterogeneous catalyst for the syn-
thesis of biuret derivatives and fully characterized by FT-
IR, EDX, XRD, SEM, and TGA techniques. The attractive
features of the presented catalyst are high yields of the
products, short reaction times, simple reaction conditions,
efficient recyclability, and reusability of the catalyst. The
HIV-1 inhibitory activity of the synthesized biuret deriva-
tives was investigated by in vitro assay, followed by the
docking study. The results of the in vitro assay were in
good correlation with those of the docking study. In both
studies, compound 5n showed the best activity. It seems
that further exploration of biuret derivatives might lead to
compounds with high HIV-1 protease inhibitory activity.
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