Design, synthesis, molecular docking, biological evaluations and QSAR studies of novel dichloroacetate analogues as anticancer agent

Article abstract of DOI:10.1016/j.molstruc.2020.128689

Dichloroacetate (DCA) as a mitochondria-targeting small molecule, through inhibition of pyruvate dehydrogenase kinases (PDK_1-4), promotes mitochondria-regulated apoptosis and hence, inhibits tumour growth and reduces its proliferation. In this study, a series of novel N-aryl-2,2-dichloroacetamide and aryl-2,2-dichloroacetate derivatives were designed and synthesized. Their cytotoxic activities against various human cancer cell lines including A549, HCA-7, MCF-7, MDA-MB-231, KB and SKOV3 were evaluated. These compounds showed satisfactory potencies with much higher anticancer activity than the parent compound DCA, against the studied cancer cell lines. Molecular docking studies were also done to find their binding site and types of their interactions with PDKs isoenzymes. Among the synthesized compounds, 2,2-dichloro-N-(9,10-dioxo-9,10-dihydroanthracen-1-yl)acetamide (f1) can also induce A549 cells apoptosis. Therefore, compound f1 might have a potential value for further study in drug development. QSAR studies of this class of compounds were also explored using a collection of chemometrics methods.

Full text of DOI:10.1016/j.molstruc.2020.128689

Journal Pre-proof
Design, synthesis, molecular docking, biological evaluations and QSAR studies of
novel dichloroacetate analogues as anticancer agent
Masood Fereidoonnezhad, S. Mohammad Hossein Tabaei, Amirhossein Sakhteman,
Hassan Seradj, Zeinab Faghih, Zahra Faghih, Ayyub Mojaddami, Batool Sadeghian,
Zahra Rezaei
PII:
S0022-2860(20)31014-0
DOI:
https://doi.org/10.1016/j.molstruc.2020.128689
MOLSTR 128689
Reference:
To appear in: Journal of Molecular Structure
Received Date: 22 April 2020
Revised Date: 9 June 2020
Accepted Date: 10 June 2020
Please cite this article as: M. Fereidoonnezhad, S.M.H. Tabaei, A. Sakhteman, H. Seradj, Z. Faghih,
Z. Faghih, A. Mojaddami, B. Sadeghian, Z. Rezaei, Design, synthesis, molecular docking, biological
evaluations and QSAR studies of novel dichloroacetate analogues as anticancer agent, Journal of
Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.128689.
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Design, Synthesis, Molecular Docking, Biological Evaluations and QSAR
studies of Novel Dichloroacetate Analogues as Anticancer Agent
a,b
b
b
Masood Fereidoonnezhad , S. Mohammad Hossein Tabaei , Amirhossein Sakhteman ,
b
b
c
a,b
b
Hassan Seradj , Zeinab Faghih , Zahra Faghih Ayyub Mojaddami , Batool Sadeghian ,
,
b
Zahra Rezaei *
a
Department of Medicinal Chemistry, School of Pharmacy, Ahvaz Jundishapur University of
Medical Sciences, Ahvaz, Iran.
b
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Centre, School of
Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.
c
Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical
Sciences, Shiraz, Iran.
Masood Fereidoonnezhad: PhD student, Writing- Original draft preparation
S. Mohammad Hossein Tabaei: Conceptualization, Supervison, methodology
Amirhossein Sakhteman: Software, molecular modeling supervision
Hassan Seradj: Visualization, Spectroscopic Data curation
Zeinab Faghih: Assistant in synthesis of compounds
Zahra Faghih: Supervison, biological assay
Ayyub Mojaddami: Assistant for molecular modeling study
Batool Sadeghian: Reviewing and Editing
Zahra Rezaei: Conceptualization, Supervison, Project administration

Design, Synthesis, Molecular Docking, Biological Evaluations and QSAR studies of Novel
Dichloroacetate Analogues as Anticancer Agents
Masood Fereidoonnezhad, S. Mohammad Hossein Tabaei, Amirhossein Sakhteman, Hassan
Seradj, Zeinab Faghih, Zahra Faghih, Ayyub Mojaddami, Batool Sadeghian, Zahra Rezaei*
A series of novel N-aryl-
2
and
,2-dichloroacetamide
aryl-2,2-
dichloroacetate
derivatives
designed
were
and
synthesized.
cytotoxic
Their
activities
against various human
cancer cell lines
including A549, HCA-7,
MCF-7, MDA-MB-231,
KB and SKOV3 were evaluated.Apoptosis, Molecular docking and QSAR studies of them were
also explored.

Design, Synthesis, Molecular Docking, Biological Evaluations and QSAR
studies of Novel Dichloroacetate Analogues as Anticancer Agent
a,b
b
Masood Fereidoonnezhad , S. Mohammad Hossein Tabaei , Amirhossein
b
b
b
c
a,b
Sakhteman , Hassan Seradj , Zeinab Faghih , Zahra Faghih Ayyub Mojaddami ,
,
b
b
Batool Sadeghian , Zahra Rezaei *
a
Department of Medicinal Chemistry, School of Pharmacy, Ahvaz Jundishapur University of
Medical Sciences, Ahvaz, Iran.
b
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Centre, School
of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.
c
Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical
Sciences, Shiraz, Iran.
*
Corresponding author. Tel.: +98 713 2425305; Fax: +98 7132424128; Email: rezaeiza@sums.ac.ir

Abstract
Dichloroacetate (DCA) as a mitochondria-targeting small molecule, through inhibition of
pyruvate dehydrogenase kinases (PDK ), promotes mitochondria-regulated apoptosis and
1
-4
hence, inhibits tumour growth and reduces its proliferation. In this study, a series of novel N-
aryl-2,2-dichloroacetamide and aryl-2,2-dichloroacetate derivatives were designed and
synthesized. Their cytotoxic activities against various human cancer cell lines including
A549, HCA-7, MCF-7, MDA-MB-231, KB and SKOV3 were evaluated. These compounds
showed satisfactory potencies with much higher anticancer activity than the parent compound
DCA, against the studied cancer cell lines. Molecular docking studies were also done to find
their binding site and types of their interactions with PDKs isoenzymes. Among the
synthesized compounds, 2,2-dichloro-N-(9,10-dioxo-9,10-dihydroanthracen-1-yl)acetamide
(f1) can also induce A549 cells apoptosis. Therefore, compound f1 might have a potential
value for further study in drug development. QSAR studies of this class of compounds were
also explored using a collection of chemometrics methods.
Keywords: Dichloroacetate (DCA), N-aryl-2,2-dichloroacetamide, Aryl-2,2-dichloroacetate,
Cytotoxic activity, Docking, apoptosis.

1
. Introduction
Dichloroacetate (DCA) as a metabolic modulator for the treatment of lactic acidosis has
recently been proposed as a novel and relatively non-toxic anticancer agent that can reverse
the glycolytic phenotype in cancer cells t through the inhibition of pyruvate dehydrogenase
kinases (PDKs) [1-4]. The orphan drug DCA which combating cancer by metabolic targeting,
selectively promotes mitochondria-regulated apoptosis and inhibits tumour growth in
preclinical models by shifting the glucose metabolism in cancer cells from anaerobic to
aerobic glycolysis [4, 5]. It does not have severe side effects and drug resistance and had no
effect on the proliferation and induction of apoptosis in non-malignant cells [6]. Moreover, it
has been reported that DCA has a Synergistic effect on other active chemotherapeutic agents
as well as radiotherapy, and results in the reduction of their adverse effects. [7-9].
However, in comparison to the other commercially available anticancer drugs, DCA
therapeutic dosage is very high and also it has not ideal cytotoxic activity against different
cancer cell lines which was evaluated [10, 11]. Also Trapella et al. synthesized several DCA
derivatives to increase bioavailability and cellular uptake of DCA [12]. Hence, it’s
indispensable to modify the DCA structure to enhance its cytotoxic activity as well as
bioavailibity.
Recently, yang et al. synthesized a series of N-phenyl dichloroacetamide derivatives which
showed satisfactory anticancer activity with ideal IC50 against human gastric carcinoma
(
BGC-823), human oral epidermoid carcinoma (KB), human nonsmall cell lung cancer
A549), and human liver carcinoma (BEL-7402) cell lines [13]. These compounds induce
(
cancer cell apoptosis and have a low toxicity in mice. A series of N-arylphenyl-2,2-
dichloroacetamide analogues was also synthesized by Li, Tianwen, et al. [14] which showed
potent cytotoxic activity against not only A549 cells but also H460 (large cell lung cancer
cell line) and KB-3-1 (Human epidermoid carcinoma cell line) cells.
As a continuing effort to find out and screen more active DCA analogues, in the present
study, a series of novel N-aryl-2,2-dichloroacetamide and aryl-2,2-dichloroacetate derivatives
were synthesized and their cytotoxic activities against various human cancer cell lines
including A549 (human lung adenocarcinoma epithelial cell line), HCA-7 (human colon
cancer cell line), MCF-7 (breast adenocarcinoma), MDA-MB-231 (breast adenocarcinoma),
KB (human oral epidermoid carcinoma cell line) and SKOV3 (ovarian cancer cell line) were
evaluated. One of the best cytotoxic compound i.e. f1, was also used to induce A549 cells
apoptosis. Molecular docking studies of them were also done to find their binding site to four

PDKs isoenzymes. Hence, the important amino acids inside the active site cavity that are in
charge of essential interactions with PDKs were determined. Finally, the structure-activity of
these compounds were investigated using a series of well-known chemometrics methods like
multiple linear regression (MLR), factor analysis-based multiple linear regression (FA-
MLR), principal component regression (PCR) and partial least squared included in variable
selection genetic algorithm (GA-PLS).
2
. Experimental section
2
.1. Drug design
More than 100 N-Aryl-2,2-dichloroacetamide and Aryl-2,2-dichloroacetate derivatives were
designed based on Scheme 1. The two dimensional structures of the ligands were drawn
using ACD chemsketch software. The ligands were subjected to minimization procedures by
means of an in house TCL script [15-17] using Hyperchem (Version 8, Hypercube Inc.,
Gainesville, FL, USA). Each ligand was optimized with different minimization methods such
+
as molecular mechanics method (MM ) and then quantum based semiemprical method
(AM1) using Hyperchem package. For doing docking, the output structures were thereafter
converted to PDBQT using MGLtools 1.5.6 [18].
Scheme 1. Chemical structure of N-Aryl-2,2-dichloroacetamide and Aryl-2,2-dichloroacetate
derivatives used in molecular docking study.
2
.2. Drug-likeness analysis
In the drug design field of medicinal chemistry, drug-likeness rules can be quite effective and
efficient. As shown in table 1, using DruLiTo [19], an open source virtual screening tool,

drug-likeness descriptors such as logP, AlogP, H-Bond Acceptor (HBA), H-Bond Donor
HBD), Molecular Weight (MW), Total Polar Surface Area (TPSA), Atom Molar
(
Refractivity (AMR), number of Rotatable Bond (nRB), and nHB (number of Hydrogen
Bond) for all of the ligands were calculated. Those compounds which successfully passed the
drug-likeness filter especially Lipinski's rule of five were subjected to molecular docking
studies.
2
.3. Chemistry
All chemicals were obtained from Sigma Aldrich or Merck chemical companies. The
Progress of the reactions was followed by TLC using silica gel polygrams SIL G/UV 254
plates or by GC using a Bruker gas chromatograph 450-GC, equipped with a flame ionization
detector (FID) and a 3-meter length capillary column CP-SIL 5CB and nitrogen as the carrier
1
gas. IR spectra were run on a Bruker’s VERTEX 70 Series FT-IR Spectrometers. The H
1
3
NMR and C NMR spectra were recorded on a Bruker Avance DPX 300 FT-NMR
1
13
spectrometer ( H NMR: TMS at 0.00 ppm, CDCl at 7.26 ppm, DMSO-d at 2.50 ppm; C
3
6
NMR: CDCl at 77.23 ppm, DMSO-d at 39.51 ppm). All yields refer to isolated products.
3
6
Evaporation of solvents was performed at reduced pressure, with a Buchi rotary evaporator.
The general melting points were recorded on an electrothermal digital melting point
apparatus. Mass spectra were recorded on a Finnigan MAT 8430 mass spectrometer
operating at an electron energy of 70 eV.
2.4. General procedure for N-Aryl-2,2-dichloroacetamide derivatives (compounds f1-f27)
synthesis
N-Aryl-2,2-dichloroacetamide analogues were synthesized from the respective amine
derivatives, and dichloroacetyl chloride in a base free condition. In a flask, amine derivatives
(10 mmol, 1.0 equiv) were dissolved in toluene. In the case of the insoluble amines in
toluene, acetonitrile or mixture of acetonitrile/DMF was applied as the solvent. Then
dichloroacetyl chloride (13 mmol, 1.3 equiv) was added to the flask. More basic amine
derivatives were reacted in room temperature and less basic of them needed reflux
temperature. It should be noted that in case of N,N’-(aryl)bis(2,2-dichloroacetamide
derivatives, more than 2 equiv of dichloroacetyl chloride were used. The progress of the
reaction was monitored by TLC (n-hexane/ethyl acetate). After 0.5-7 h, the crude products

were washed with sodium bicarbonate and then the solvent and excess dichloroacetyl
chloride was allowed to evaporate under vacuum. The residual powder was N-Aryl-2,2-
1
13
dichloroacetamide derivatives, with a 75-98% yield. The melting point, IR, H-NMR, C-
NMR and mass of all the compounds were obtained.
As we reported previously [17], this reaction can also be done in another synthetic method.
This synthetic method ignored the use of toluene as a toxic solvent and carried out in a
solvent free conditions without using of high reflux temperature and the final product was
just free base not hydrochloride salt.
2
.5. General procedure for Aryl-2,2-dichloroacetate derivatives (compounds f28-f38)
synthesis
Aryl-2,2-dichloroacetate analogues were synthesized from the respective phenol derivatives,
and dichloroacetyl chloride in the presence of a base. In a flask, phenol derivatives (10 mmol,
1
.0 equiv) were dissolved in toluene and 10 mmol base (potassium carbonate or
triethylamine) was added to the mixture. In the case of the insoluble phenols in toluene,
acetonitrile or mixture of acetonitrile/DMF was applied as the solvent. Then dichloroacetyl
chloride (13 mmol, 1.3 equiv) was added to the flask and stirred at reflux temperature. The
progress of the reaction was monitored by TLC (n-hexane/ethyl acetate). After 1-8 h,
evaporation of the solvent and excess dichloroacetyl chloride under vacuum followed by
chromatography on a short column of silica gel using n-hexane/ethyl acetate (5/1) as eluent
gave the products. The obtained liquid was Aryl-2,2-dichloroacetate derivatives, with a 62-
1
13
8
2% yield. The IR, H-NMR, C-NMR and mass of all the compounds were obtained.
2
.4.1. 2,2-dichloro-N-(9,10-dioxo-9,10-dihydroanthracen-1-yl)acetamide (f1)
°
-1
Orange-yellow solid, yield: 92%, mp: 167-170 C, IR (KBr) ν (cm ): 3443.7 (N-H stretch,
amide), 3112 (C-H, aromatic), 2998.9 (C-H, aliphatic), 1698.9, 1673.7 (C=O, ketones),
1
1
642.4 (C=O, amide), 1581.2, 1523 (C=C, alkene), 1280.4 (C-N stretch, aromatic); H NMR
(300 MHz, Chloroform-d) δ = 8.47 (s, 1H), 8.41 – 8.29 (m, 3H), 8.26 (dd, J=4.5, 2.3, 2H),
1
3
7
.88 – 7.76 (m, 2H), 6.11 (s, 1H). C NMR (75 MHz, Chloroform-d) δ 186.9, 182.3, 164.9,
1
38.8, 134.4, 133.9, 133.8, 133.7, 133.5, 133.2, 126.7, 126.6, 124.4, 122.3, 119.2, 67.0; MS
+
m/z (%): 333 (10) [M ], 250 (100), 223.1 (10), 195 (5), 166.1 (10), 151 (15), 141 (14), 105
(4), 76 (4).

2
.4.2. 2,2-dichloro-N-(9,10-dioxo-9,10-dihydroanthracen-2-yl)acetamide (f2)
°
-1
Tan solid, yield: 95%, mp: 179-182 , IR (KBr) ν (cm ): 3338.6 (N-H stretch, amide), 3067.9
(C-H, aromatic), 2994.9 (C-H, aliphatic), 1726.53 (C=O, ketones), 1668.8 (C=O, amide),
1
1
585.9, 1536.5 (C=C, alkene), 1232.5 (C-N stretch, aromatic); H NMR (300 MHz,
Chloroform-d) δ = 13.35 (s, 1H), 9.06 (dd, J=8.5, 1.0, 1H), 8.36 (m, 1H), 8.33 – 8.25 (m,
1
3
1
H), 8.17 (dd, J=7.7, 1.0, 1H), 7.89 – 7.79 (m, 3H), 6.13 (s, 1H). C NMR (75 MHz,
Chloroform-d) δ 182.1, 180.0, 165.2, 141.3, 133.8, 133.1, 132.7, 128.3, 127.6, 127.2, 127.0,
+
1
21.2, 118.2, 67.9; MS m/z (%): 333 (70) [M ], 250.1 (100), 222.1 (32), 207 (63), 194.1 (30),
64 (13), 151 (53), 139 (41), 75.9 (17), 63 (8).
1
2
.4.3. N-(acridin-9-yl)-2,2-dichloroacetamide (f3)
°
-1
Yellow solid, yield: 90%, mp: 369-371 C, IR (KBr) ν (cm ): 3259.6 (N–H stretch, amide),
3
1
142 (N, pyridine), 3086.5 (C-H, aromatic), 2966.7 (C-H, aliphatic), 1662.6 (C=O, amide),
1
591.6, 1542.8 (C=C, alkene), 1269.2 (C-N stretch, aromatic), H NMR (300 MHz, DMSO-
d6) δ = 10.20 (s, 1H), 8.77 (d, J=8.6, 2H), 8.17 – 7.81 (m, 4H), 7.53 (ddd, J=8.2, 5.9, 1.9,
1
3
2
H), 6.04 (s, 1H). C NMR (75 MHz, DMSO-d ) δ 166.1, 157.6, 139.1, 135.8, 124.7, 124.2,
6
+
1
18.7, 111.3, 70.7; MS m/z (%): 304.1 (65) [M ], 194.1 (100), 166 (15), 140 (10), 129 (3), 97
(5), 76 (4), 66.1 (5).
2
.4.4. 2-(4-(2,2-dichloroacetamido)phenyl)acetic acid (f4)
°
-1
Cream solid, Yield: 90%, mp: 103-105 C, IR (KBr) ν (cm ): 3303.4, (N–H stretch, amide),
3
206 (O–H stretch, carboxylic acid), 3082.7(C-H, aromatic), 2990.1 (C-H, aliphatic), 1705.4
(C=O, carboxylic acid), 1681.6 (C=O, amide), 1551.8 (C=C, alkene), 1296.5 (C–N stretch,
1
aromatic); H NMR (300 MHz, DMSO-d ) δ = 10.64 (s, 1H), 7.94 (s, 1H), 7.54 (d, J=8.2,
6
1
3
2
1
H), 7.25 (d, J=8.2, 2H), 6.66 (s, 1H), 3.61 (s, 2H). C NMR (75 MHz, DMSO-d ) δ 174.0,
6
+
65.2, 135.9, 130.4, 129.8, 121.7, 67.9, 40.2; MS m/z (%): 261 (60) [M ], 216 (65), 178
(100), 150 (23), 132 (10), 107 (35), 89 (10), 77 (30), 51.1 (8).
2
.4.5. (4-(2,2-dichloroacetamido)benzoyl)glycine (f5)
°
-1
Brown solid, yield: 88%, mp: 157-160 C, IR (KBr) ν (cm ): 3276.6 (N–H stretch, amide),
3
186.1 (O–H stretch, carboxylic acid), 3104.9, 3002.2 (C-H, aromatic), 2983.8 (C-H,
aliphatic), 1787.3 (C=O, carboxylic acid), 1698.5, 1601.2 (C=O, amide), 1537.8 (C=C,
1
aromatic), 1330.5 (C–N stretch, aromatic); H NMR (300 MHz, DMSO-d ) δ = 11.67 (s, 1H),
6
1
0.08 (s, 1H), 8.93 (s, 1H), 7.87 (app.d, J=7.6, 2H), 7.26 (d, J=7.6, 2H), 7.05 (s, 1H), 3.84 (s,

1
3
2
4
H); C NMR (75 MHz, DMSO-d ) δ 172.3, 166.9, 165.2, 137.7, 128.2, 127.5, 122.5, 67.9,
6
+
1.7; MS m/z (%): 305.1 (24) [M ], 277 (5), 236 (58), 201 (20), 172.1 (10), 131 (18), 76.9
(100), 51.1 (70).
2
.4.6. 4-(2,2-dichloroacetamido)benzamide (f6)
°
-1
White solid, yield: 91%, mp: 47-50 C, IR (KBr) ν (cm ): 3438.0, 3226.5 (N–H stretch,
amide), 3010.8 (C-H, aromatic), 2973.4 (C-H, aliphatic), 1720.2, 1651.5 (C=O, amide),
1
1
539.8, 1508.7 (C=C, alkene), 1251.3 (C–N stretch, aromatic); H NMR (300 MHz,
Chloroform-d) δ = 9.99 (s, 1H), 8.95 (s, 2H), 7.78 (d, J=8.6, 2H), 7.59 (d, J=8.6, 2H), 5.93 (s,
1
3
1
H). C NMR (75 MHz, Chloroform-d) δ 167.6, 165.2, 137.8, 129.5, 127.3, 123.7, 67.9; MS
+
m/z (%): 246 (50) [M ], 229.9 (45), 163 (90), 145 (100), 135 (15), 117 (32), 107 (25), 90
(24), 82.9 (30), 76 (21), 63.1 (15), 48 (20).
2
.4.7. 2,2-dichloro-N-(thiazol-2-yl)acetamide (f7)
°
-1
Beige solid, yield: 95%, mp: 120-122 C, IR (KBr) ν (cm ): 3297.4 (N–H stretch, amide),
3
062.86 (C-H, aromatic), 2993.9 (C-H, aliphatic), 1706.3 (C=O, amide), 1673.7 (C=O,
1
amide), 1575.5 (-C=N, thiazole), 1058.6 (N=C-S-C=N), 682 (C-S-C); H NMR (300 MHz,
1
3
Chloroform-d) δ = 14.38 (s, 1H), 7.68 (d, J=3.9, 1H), 7.21 (d, J=3.9, 1H), 6.42 (s, 1H). C
+
NMR (75 MHz, CDCl ) δ 169.8, 164.7, 133.2, 112.1, 68.3; MS m/z (%): 209.9 (35) [M ],
3
1
26.9 (27), 111 (15), 100 (100), 83 (15), 76 (10), 55.1 (16), 45.1 (12).
2
.4.8. 2,2-dichloro-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)acetamide
(
f8)
Ivory solid, yield: 95%, mp: 175-178 C, IR (KBr) ν (cm ): 3164.7 (N–H stretch, amide),
082.6 (C-H, aromatic), 2982.6 (C-H, aliphatic), 1712.7 (C=O, pyrazole), 1610.7 (C=O,
°
-1
3
amide), 1544.5 (C=C, alkene), 1317.8 (C–N stretch, aromatic), 1201.5 (C–N stretch,
1
aliphatic); H NMR (300 MHz, Chloroform-d) δ = 10.73 (s, 1H), 7.68 – 7.49 (m, 3H), 7.38
1
3
(
m, 2H), 6.55 (s, 1H), 3.47 (s, 3H), 2.43 (s, 3H). C NMR (75 MHz, Chloroform-d) δ 163.4,
1
62.0, 142.5, 135.3, 129.3, 126.9, 123.4, 122.3, 67.7, 36.9, 10.7; MS m/z (%): 313.1 (25)
+
[
M ], 230.1 (100), 202.1 (5), 83 (25), 56 (51).
2
.4.9. 2
,2-dichloro-N-(pyridine-3-yl)acetamide (f9)
°
-1
Brown solid, yield: 97%, mp: 175-180 C, IR (KBr) ν (cm ): 3244.3 (Pyridine amine), 3216
(N–H stretch, amide), 3086.9 (CH-aromatic), 2946.3 (C-H, aliphatic), 1655.5 (C=O, amide),

1
1
563.9 (C=C, alkene); H NMR (300 MHz, DMSO-d ) δ = 12.62 (s, 1H), 9.24 – 9.17 (s, 1H),
6
1
3
8
.68 (dd, J=18.6, 6.9, 2H), 8.05 (dd, J=8.4, 5.6, 1H), 6.97 (s, 1H). C NMR (75 MHz,
+
DMSO-d ) δ 165.2, 144.7, 141.8, 134.4, 130.6, 124.6, 67.9; MS m/z (%): 204.1 (50) [M ],
6
1
34.1 (5), 121.2 (100), 105.1 (4), 94.1 (12), 83 (10), 78.1 (62), 66.1 (9), 51.2 (8).
2
.4.10. 2,2-dichloro-N-(pyridine-2-yl)acetamide (f10)
°
-1
White solid, yield: 98%, mp: 149-151 C, IR (KBr) ν (cm ): 3244.3 (Pyridine amine), 3216
(N–H stretch, amide), 3086.9 (CH-aromatic), 2946.3 (C-H, aliphatic), 1655.5 (C=O, amide),
1
1
8
1
563.9 (C=C, alkene); H NMR (300 MHz, DMSO-d ) δ = 11.24 (s, 1H), 8.45 – 8.32 (m, 1H),
6
1
3
.12 – 7.77 (m, 2H), 7.32 (m, 1H), 6.67 (s, 1H). C NMR (75 MHz, DMSO-d ) δ 164.9,
6
52.9, 147.9, 138.3, 118.0, 114.3, 67.9; MS m/z (%): 203.9 (25), 169 (50), 121 (100), 105
(5), 94 (25), 83 (7), 78 (100), 67 (10), 51 (10).
2
.4.11. 2,2-dichloro-N-(5-methylisoxazol-3-yl)acetamide (f11)
°
-1
White-Peach solid, Yield: 96%, mp: 139-141 C, IR (KBr) ν (cm ): 3219.8 (N–H stretch,
amide), 3093.2 (C-H, aromatic), 2977.8 (C-H, aliphatic), 1632.8 (C=O, amide), 1568.3,
1
1
483.36 (C=C, aromatic), 1318.5 (C-N, aromatic), 1029.6 (C-O, stretch); H NMR (300
1
3
MHz, DMSO-d ) δ = 11.74 (s, 1H), 6.63 (s, 1H), 6.59 (s, 1H), 2.41 (s, 3H); C NMR (75
6
+
MHz, DMSO-d ) δ 165.5, 162.6, 159.8, 98.1, 68.3, 12.1; MS m/z (%): 207.8 [M ], (100),
6
1
92.9 (61), 173 (52), 164.9 (38), 155 (54), 145 (43), 124.8 (100), 109 (85), 97.9 (100), 84
(54), 76 (34), 70 (100), 55.1 (60).
2
.4.12. 2,2-dichloro-1-morpholinoethan-1-one (f12)
°
-1
White solid, yield: 92%, mp: 69-71 C, IR (KBr) ν (cm ): 3063.5 (C-H, aromatic), 2983.6 (C-
1
H, aliphatic), 1634.5 (C=O, amide), 1031.3 (C-O, stretch); H NMR (300 MHz, Chloroform-
1
3
d) δ = 6.93 (s, 1H), 3.61 (m, 4H), 3.56 (m, 4H); C NMR (75 MHz, Chloroform-d) δ 169.4,
+
6
6.7, 66.5, 45.0; MS m/z (%): 196.9 (10) [M ], 182 (5), 162 (8), 148.9 (4), 131.9 (10), 114
(100), 86 (20), 76 (8), 70 (35), 63 (5), 56.1 (24), 48 (8).
2
.4.13. 2
,4-dichloro-6-(2,2-dichloroacetamido)benzoic acid (f13)
°
-1
Mild brown solid, Yield: 85%, mp: 174-176 C, IR (KBr) ν (cm ): 3311.3 (N–H stretch,
amide), 3079.6 (O–H stretch, carboxylic acid), 2987.7 (C-H, aliphatic), 1695.7 (C=O,
carboxylic acid), 1680.5 (C=O, amide), 1564.6, 1524.6 (C=C, alkene), 1294.9 (C–N stretch,
1
aromatic); H NMR (300 MHz, Chloroform-d) δ = 10.75 (s, 1H), 8.46 (d, J=1.9, 1H), 7.37 (d,

1
3
J=1.9, 1H), 6.07 (s, 1H); C NMR (75 MHz, DMSO-d6) δ 169.4, 165.3, 143.9, 135.2, 134.6,
+
1
27.0, 118.1, 117.4, 67.0; MS m/z (%): 316.9 (8) [M ], 293.1 (10), 278 (13), 213.9 (55), 205
(
45), 186.9 (100), 159.9 (63), 124 (24), 108.9 (7), 100 (20), 83.9 (48), 76 (18), 63 (15), 48
33).
(
2
.4.14. 2- (2
,2-dichloroacetamido)-4,5-difluorobenzoic acid (f14)
1
Yield: 91%, gelly, H NMR (300 MHz, DMSO-d ) δ = 13.13 (s, 1H), 12.18 (s, 1H), 8.40 (dd,
6
1
3
J=13.2, 7.5, 1H), 7.98 – 7.80 (m, 1H), 6.82 (s, 1H). C NMR (75 MHz, Chloroform-d) δ =
69.5 (d, J=2.8), 165.4 , 154.4 (d, J=20.0), 151.0 (d, J=20.0), 149.4 (d, J=20.0), 146.0 (d,
1
J=19.5), 138.6 (dd, J=8.0, 2.8), 120.2 (dd, J=20.0, 8.0), 115.1 (dd, J=8.0, 3.4), 108.6 (dd,
+
J=20.0, 8.0); MS m/z (%): 283 (10) [M ], 200 (15), 182 (100), 116 (5), 155 (7), 126 (20), 101
(10), 83 (5).
2
.4.15. 2
,2-dichloro-N-(2-cyanophenyl)acetamide (f15)
°
-1
White solid, yield: 93%, mp: 184-187 C, IR (KBr) ν (cm ): 3252.2 (N–H stretch, amide),
3
1
066.7 (C-H, aromatic), 2956.4 (C-H, aliphatic), 2228.6 (C≡N stretch), 1689.8 (C=O, amide),
1
608, 1582.1, 1540.7 (C=C, alkene), 1286.4 (C-N stretch, aromatic); H NMR (300 MHz,
DMSO-d ) δ = 11.25 (s, 1H), 7.92 – 7.68 (m, 1H), 7.60 – 7.11 (m, 1H), 6.91 (s, 1H), 6.83 (d,
6
1
3
J=8.3, 1H), 6.67 – 6.55 (m, 1H). C NMR (75 MHz, DMSO-d ) δ 165.3, 141.0, 132.8, 131.1,
6
+
1
23.3, 122.3, 117.1, 100.2, 67.0; MS m/z (%): 228 (20) [M ], 145 (100), 129 (13), 117 (20),
02 (11), 90 (14), 76 (10), 63 (6).
1
2
.4.16. 2,2-dichloro-N-(4-chloro-2-methylphenyl)acetamide (f16)
°
-1
White solid, Yield: 98%, mp: 152-154 C, IR (KBr) ν (cm ): 3254.4 (N–H stretch, amide),
3
054.4 (C-H, aromatic), 2923.6 (C-H, aliphatic), 1678.7 (C=O, amide), 1581.9, 1543.9 (C=C,
1
alkene), 1245.2 (C-N stretch, aromatic); H NMR (300 MHz, Chloroform-d) δ = 8.06 (s, 1H),
1
3
7
.92 (d, J=1.5, 1H), 7.21 – 7.05 (m, 2H), 6.07 (s, 1H), 2.29 (s, 3H). C NMR (75 MHz
Chloroform-d) δ 165.3, 136.6, 134.2, 130.4, 129.3, 126.9, 125.1, 67.0, 17.9; MS m/z (%):
+
2
51 (25) [M ], 180 (7), 168 (100), 140 (30), 125 (28), 104 (7), 89 (10), 77.1 (22), 51.1 (6).
2
.4.17. 2,2-dichloro-N-(2,4-dimethoxyphenyl)acetamide (f17)
°
-1
Black solid, yield: 92%, mp: 110-112 C, IR (KBr) ν (cm ): 3255.5 (N–H stretch, amide),
081.5 (C-H, aromatic), 2940.3 (C-H, aliphatic), 1680.6 (C=O, amide), 1554.3, 1507.4 (C=C,
3

1
alkene), 1279.7 (C-N stretch, aromatic); H NMR (300 MHz, DMSO-d ) δ = 9.67 (s, 1H),
6
7
3
6
.78 (dd, J=8.8, 4.0, 1H), 6.81 (s, 1H), 6.60 (s, 1H), 6.48 (dd, J=8.8, 2.6, 1H), 3.84 (s, 3H),
1
3
.75 (s, 3H). C NMR (75 MHz, DMSO-d ) δ 165.3, 161.6, 152.1, 125.1, 122.7, 108.3, 99.1,
6
+
7.0, 55.6, 54.6; MS m/z (%): 263.2 (100) [M ], 248.1 (80), 152.1 (60), 138.1 (56), 125.1
(20), 110.1 (35), 95.1 (15), 79.1 (10).
2
.4.18. 2,2-dichloro-N-(3,4-dimethoxyphenyl)acetamide (f18)
°
-1
Black solid, Yield: 90%, mp: 102-105 C, IR (KBr) ν (cm ): 3256.3 (N–H stretch, amide),
3
092.2 (C-H, aromatic), 2970.1 (C-H, aliphatic), 1681.3 (C=O, amide), 1551.2, 1511.3 (C=C,
1
alkene), 1312.4 (C-N stretch, aromatic); H NMR (300 MHz, Chloroform-d) δ = 8.23 (s, 1H),
7
3
6
1
.27 (d, J=2.3, 1H), 6.97 (dd, J=8.6, 2.4, 1H), 6.84 (s, 1H), 6.09 (s, 1H), 3.87 (s, 3H), 3.87 (s,
1
3
H). C NMR (75 MHz, Chloroform-d) δ 165.2, 147.3, 141.9, 135.9, 117.6, 115.3, 107.4,
+
7.9, 56.7, 56.0; MS m/z (%): 263.1 (100) [M ], 228.1 (8), 180.1 (15), 165 (20), 152.1 (100),
38 (17), 122.1 (20), 109.1 (14), 93.1 (15), 79.1 (13), 52.1 (6).
2
.4.19. 2
,2-dichloro-N-(3,4,5-trimethoxyphenyl)acetamide (f19)
°
-1
Sunset yellow solid, Yield: 90%, mp: 119-121 C, IR (KBr) ν (cm ): 3253.7 (N–H stretch,
amide), 3069.1 (C-H, aromatic), 2940 (C-H, aliphatic), 1680.2 (C=O, amide), 1545.5, 1509.3
1
(
C=C, alkene), 1331.1 (C-N stretch, aromatic); H NMR (300 MHz, Chloroform-d) δ = 8.65
1
3
(
s, 1H), 6.88 (s, 2H), 6.19 (s, 1H), 3.81 (s, 3H), 3.79 (s, 6H). C NMR (75 MHz,
Chloroform-d) δ 165.2, 155.3, 136.9, 135.8, 102.3, 67.9, 60.7, 56.1; MS m/z (%): 293.2 (70)
+
[
M ], 278.2 (100), 182.1 (20), 140.1 (15), 68.1 (8).
2
.4.20. 2,2-dichloro-N-(5-chloro-2-methoxyphenyl)acetamide (f20)
°
-1
White solid, Yield: 94%, mp: 112-115 C, IR (KBr) ν (cm ): 3328.1, 3281.4 (N–H stretch,
amide), 3057.79 (C-H, aromatic), 2976.68 (C-H, aliphatic), 1682.14 (C=O, amide), 1597.4,
1
1
486.8 (C=C, alkene), 1330.4 (C-N, aromatic), 1021.8 (C-O, stretch) ; H NMR (300 MHz,
DMSO-d ) δ = 10.12 (s, 1H), 7.73 (d, J=1.9, 1H), 7.19 (dd, J=7.5, 2.0, 1H), 7.01 (s, 1H), 6.84
6
1
3
(
d, J=7.5, 1H), 3.86 (s, 3H). C NMR (75 MHz, Chloroform-d) δ 165.3, 145.9, 128.2,
+
1
28.1, 123.9, 120.9, 112.7, 67.0, 56.3; MS m/z (%): 267.1 (60) [M ], 184.1 (100), 169.1 (55),
56.1 (25), 142.1 (30), 114.1 (13), 99.1 (15), 93.2 (20), 78.1 (16), 63.2 (10), 51.2 (10).
1
2
.4.21. 2,2-dichloro-N-(4-hydroxyphenyl)acetamide (f21)

°
-1
White solid, yield: 95%, mp: 122-125 C, IR (KBr) ν (cm ): 3452.5 (O–H stretch, phenol),
241.4 (N–H stretch, amide), 3069.5 (C-H, aromatic), 2998.9 (C-H, aliphatic), 1673.5 (C=O,
3
amide), 1556.9, 1509.3 (C=C, alkene), 1348 (C-N stretch, aromatic), 1222.8 (C-O stretch);
1
H NMR (300 MHz, DMSO-d ) δ = 10.54 (s, 1H), 9.57 (s, 1H), 7.13 (d, J=8.1, 2H), 6.99 (d,
6
1
3
J=8.2, 2H), 6.57 (s, 1H). C NMR (75 MHz, DMSO-d ) δ 165.2, 153.5, 133.1, 123.1, 115.6,
6
+
6
7.9; MS m/z (%): 219 (35) [M ], 136 (30), 109 (100), 80 (53), 65 (8), 53.1 (16).
2
.4.22. 2,2-dichloro-N-(3-hydroxyphenyl)acetamide (f22)
°
-1
White solid, Yield: 92%, mp: 137-140 C, IR (KBr) ν (cm ): 3361.3 (O–H stretch, phenol),
3
252.8 (N–H stretch, amide), 3089.5 (C-H, aromatic), 2921.4 (C-H, aliphatic), 1684.3 (C=O,
1
amide), 1586.3, 1519.5 (C=C, alkene), 1286 (C-N stretch, aromatic), 1214.3 (C-O stretch); H
NMR (300 MHz, DMSO-d ) δ = 10.62 (s, 1H), 9.66 (s, 1H), 7.29 – 7.16 (m, 2H), 7.07 (d,
6
1
3
J=8.2, 1H), 6.65 (s, 1H), 6.64 – 6.61 (m, 1H); C NMR (75 MHz, DMSO-d ) δ 165.2, 158.5,
6
+
1
39.1, 129.7, 116.2, 110.4, 106.6, 67.9; MS m/z (%): 219.1 (55) [M ], 148 (14), 136.2 (100),
08.1 (36), 93.1 (35), 81.1 (15), 65.1 (15).
1
2
.4.23. 2,2-dichloro-N-(2-hydroxyphenyl)acetamide (f23)
°
-1
White solid, Yield: 88%, mp: 117-119 C, IR (KBr) ν (cm ): 3337.3 (O–H stretch, phenol),
3
253.5 (N–H stretch, amide), 3043.5 (C-H, aromatic), 2873.6 (C-H, aliphatic), 1681.5 (C=O,
amide), 1552.9, 1457.4 (C=C, alkene), 1342.3 (C-N stretch, aromatic), 1231.5 (C-O stretch);
1
H NMR (300 MHz, DMSO-d ) δ = 10.42 (s, 1H), 10.05 (s, 1H), 8.36 (dd, J=7.5, 2.0, 1H),
6
7
.15 (td, J=7.5, 2.0, 1H), 7.04 (s, 1H), 7.04 (dd, J=14.9, 2.0, 1H), 6.76 (dd, J=7.5, 2.0, 1H);
1
3
C NMR (75 MHz, DMSO-d ) δ 165.3, 148.6, 127.6, 125.3, 121.9, 121.6, 115.9, 67.0; MS
6
+
m/z (%): 219.1 (58) [M ], 183.1 (5), 148.1 (20), 136.2 (100), 120.1 (5), 108.1 (37), 81.1 (14),
6
5.1 (15), 53.2 (5).
2
.4.24. N,N’-(2-nitro-1,4-phenylene)bis(2,2-dichloroacetamide) (f24)
°
-1
Brown solid, yield: 92%, mp: 175-177 C, IR (KBr) ν (cm ): 3472.1, 3296.4 (N–H stretch,
amide), 3086.7 (C-H, aromatic), 2969.1 (C-H, aliphatic), 1703.1, 1685.3 (C=O, amide),
1
1
1
1
6
596.2 (C=C, alkene), 1518.4, 1347.7 (C-NO , aromatic); H NMR (300 MHz, DMSO-d ) δ =
2
6
1.38 (s, 1H), 11.05 (s, 1H), 8.55 – 8.41 (m, 2H), 8.11 (d, J=1.6, 1H), 6.81 (s, 1H), 6.72 (s,
1
3
H). C NMR (75 MHz, DMSO-d ) δ 165.3, 165.2, 138.1, 137.1, 134.8, 123.0, 123.0, 116.9,
6
+
7.9, 67.0; MS m/z (%): 374.9 (100) [M ], 328.9 (70), 310 (5), 290 (30), 262 (22), 246 (100),

2
11 (10), 181 (30), 166 (6), 152 (100), 136 (45), 119 (35), 105 (55), 82.9 (95), 63 (16), 52
(21).
2
.4.25. N,N’-(naphthalene-1,5-diyl)bis(2,2-dichloroacetamide) (f25)
°
-1
White solid, Yield: 91%, mp: 340-343 C, IR (KBr) ν (cm ): 3249.4, 3225.5 (N–H stretch,
amide), 3042.4 (C-H, aromatic), 2971.4 (C-H, aliphatic), 1676.6 (C=O, amide), 1557.1, 1504
1
(
C=C, alkene), 1347.1, 1316.9 (C-N stretch, aromatic); H NMR (300 MHz, DMSO-d ) δ =
6
1
3
1
0.77 (s, 1H), 7.96 (d, J=8.1, 1H), 7.68 (dq, J=15.7, 7.6, 7.1, 2H), 6.89 (s, 1H). C NMR (75
MHz, DMSO-d ) δ 164.9, 138.4, 125.3, 125.2, 122.9, 118.9, 67.0; MS m/z (%): 380 (100)
6
+
[
M ], 343 (7), 307 (5), 295 (25), 263 (43), 247.9 (35), 223 (21), 197.1 (30), 184 (22), 152
40), 140 (32), 114.1 (18), 102 (39), 83 (26), 63 (10).
(
2
.4.26. N,N’-(methylenebis(4,1-phenylene))bis(2,2-dichloroacetamide) (f26)
°
-1
White solid, yield: 91%, mp: 177-180 C, IR (KBr) ν (cm ): 3245.5 (N–H stretch, amide),
3
054.2 (C-H, aromatic), 2895.4 (C-H, aliphatic), 1677.0 (C=O, amide), 1598.2, 1543.8 (C=C,
1
alkene), 1341.5 (C-N stretch, aromatic); H NMR (300 MHz, DMSO-d ) δ = 10.66 (s, 1H),
6
1
3
7
.52 (d, J=8.3, 2H), 7.21 (d, J=8.3, 2H), 6.60 (s, 1H), 3.88 (s, 1H). C NMR (75 MHz,
+
DMSO-d ) δ 165.2, 137.0, 134.1, 129.2, 121.8, 67.9, 42.0; MS m/z (%): 420 (56) [M ], 385
6
(
15), 347.1 (34), 335 (5), 311 (5), 263 (5), 216.1 (15), 195.2 (10), 180 (15), 165.1 (11), 152
15), 132 (100), 107 (21), 82.9 (13), 63 (4).
(
2
.4.27. N,N’-(oxybis(4,1-phenylene))bis(2,2-dichloroacetamide) (f27)
°
-1
Pastel pink solid, Yield: 92%, mp: 218-220 C, IR (KBr) ν (cm ): 3263.8 (N–H stretch,
amide), 3003.8 (C-H, aromatic), 2846.9 (C-H, aliphatic), 1675.9 (C=O, amide), 1554.5,
1
1
502.1 (C=C, alkene), 1339.2 (C-N stretch, aromatic); H NMR (300 MHz, DMSO-d ) δ =
6
1
3
1
1.09 (s, 1H), 7.65 (d, J=9.0, 2H), 6.86 (d, J=8.8, 2H), 6.79 (s, 1H). C NMR (75 MHz,
+
DMSO-d ) δ 165.2, 155.1, 134.9, 121.1, 119.8, 67.9MS m/z (%): 421.9 (23) [M ], 387 (10),
6
3
45 (30), 310 (5), 200 (100), 171.1 (23), 156 (5), 108 (32), 93 (8), 80 (16), 65.1 (11).
2
.5.1. 2,2-dichloro-N-cyclohexylacetamide (f28)
°
-1
White solid, Yield: 90%, mp: 88-90 C, IR (KBr) ν (cm ): 3211.1 (N–H stretch, amide),
2
953.3 (C-H, aliphatic), 1679.9 (C=O, amide), 1566.8 (C=C, alkene), 1211.5 (C-N stretch,
1
aliphatic); H NMR (300 MHz, Chloroform-d) δ = 8.66 (s, 1H), 6.18 (s, 1H), 3.83 (s, 1H),

1
3
2
.28 – 2.11 (m, 4H), 1.94 – 1.55 (m, 2H), 1.60 – 1.33 (m, 4H). C NMR (75 MHz,
+
Chloroform-d) δ 166.1, 67.1, 49.3, 33.0, 26.0, 25.1; MS m/z (%): 209.1 (5) [M ], 174.1 (15),
1
66 (35), 128 (55), 83.2 (100), 76.1 (5), 67.2 (20), 55.2 (35), 48.2 (4).
2
.5.2. Phenyl 2,2-dichloroacetate (f29)
-1
Yield: 82%, yellow liquid, IR (KBr) ν (cm ): 3039.1 (C-H, aromatic), 2943.3 (C-H,
aliphatic), 1759.5 (C=O, ester), 1511.9, 1450 (C=C, alkene), 1149.6 (C-O stretch, aromatic);
1
H NMR (300 MHz, Chloroform-d) δ = 7.48 – 7.40 (m, 1H), 7.37 (m, 2H), 7.19 – 7.11 (m,
1
3
2
6
5
H), 6.52 (s, 1H); C NMR (75 MHz, Chloroform-d) δ 163.2, 150.7, 129.4, 124.5, 121.3,
+
4.6; MS m/z (%): 204.1 (16) [M ], 159 (11), 139 (10), 112 (70), 96 (25), 77 (8), 83 (45),
7.1 (13).
2
.5.3. 4-(tert-butyl)phenyl 2,2-dichloroacetate (f30)
-1
Yield: 84%, yellow liquid, IR (KBr) ν (cm ): 3029.1 (C-H, aromatic), 2965.2 (C-H,
aliphatic), 1768.7 (C=O, ester), 1506.9, 1473 (C=C, alkene), 1168.8 (C-O stretch, aromatic);
1
H NMR (300 MHz, Chloroform-d) δ = 7.49 – 7.32 (m, 2H), 7.15 – 6.99 (m, 2H), 6.15 (s,
1
3
1
6
9
H), 1.30 (s, 9H). C NMR (75 MHz, Chloroform-d) δ 162.7, 150.9, 147.6, 126.5, 121.0,
+
4.6, 34.2, 31.1; MS m/z (%): 260.1 (15) [M ], 245.1 (90), 135.2 (100), 121.1 (4), 107.1 (14),
1.1 (20), 65.1 (5), 48.1 (4).
2
.5.4. 2-methoxyphenyl 2,2-dichloroacetate (f31)
-1
Yield: 80%, red wine liquid, IR (KBr) ν (cm ): 3089.3 (C-H, aromatic), 2924.1 (C-H,
aliphatic), 1739.6 (C=O, ester), 1585, 1541.4 (C=C, alkene), 1249 (C-O stretch, aromatic),
1
1
7
1
146.5 (C-O stretch, aliphatic); H NMR (300 MHz, Chloroform-d) δ = 7.26 – 7.06 (m, 3H),
1
3
.02 – 6.86 (m, 1H), 6.70 (s, 1H), 3.90 (s, 3H); C NMR (75 MHz, Chloroform-d) δ 162.7,
+
51.5, 142.7, 124.9, 122.5, 121.9, 114.8, 63.9, 55.9; MS m/z (%): 234 (25) [M ], 171 (5), 124
(100), 107 (24), 95 (24), 83.0 (30), 77 (12), 63 (7), 52.1 (6).
2
.5.5. 3-methoxyphenyl 2,2-dichloroacetate (f32)
-1
Yield: 84%, black liquid, IR (KBr) ν (cm ): 3071.1 (C-H, aromatic), 2967.2 (C-H, aliphatic),
1
746.7 (C=O, ester), 1598, 1501.2 (C=C, alkene), 1257 (C-O stretch, aromatic), 1108.2 (C-O
1
stretch, aliphatic); H NMR (300 MHz, Chloroform-d) δ = 7.31 (t, J=8.2, 1H), 6.84 (ddd,
J=8.3, 2.4, 1.0, 1H), 6.77 (ddd, J=8.1, 2.2, 0.8, 1H), 6.72 (d, J=2.3, 1H), 6.15 (s, 1H), 3.80 (s,
1
3
3
H); C NMR (75 MHz, Chloroform-d) δ 162.8, 160.2, 153.0, 130.1, 116.6, 111.4, 106.5,

+
6
4.1, 55.4; MS m/z (%): 234 (32) [M ], 171 (5), 124 (85), 107 (34), 95 (26), 83.0 (35), 63
(11).
2
.5.6. 2-chlorophenyl 2,2-dichloroacetate (f33)
-1
Yield: 71%, yellow liquid, IR (KBr) ν (cm ): 3058.2 (C-H, aromatic), 2962.4 (C-H,
aliphatic), 1763.7 (C=O, ester), 1584.4, 1476.5 (C=C, alkene), 1208.4 (C-O stretch,
1
aromatic); H NMR (300 MHz, Chloroform-d) δ = 7.42 (dd, J=7.0, 2.4, 1H), 7.28 – 7.09 (m,
1
3
2
1
1
H), 7.05 (dd, J=7.0, 2.4, 1H), 6.82 (s, 1H). C NMR (75 MHz, Chloroform-d) δ 162.7,
+
49.1, 130.6, 128.2, 127.3, 125.5, 122.8, 63.9; MS m/z (%): 239.4 (17) [M ], 162.1 (88),
33.1 (25), 126.1 (7), 98.1 (20), 83.1 (32), 73.1 (12), 63.2 (30), 48.2 (15).
2
.5.7. 2,6-dichlorophenyl 2,2-dichloroacetate (f34)
-1
Yield: 65%, yellow liquid, IR (KBr) ν (cm ): 3095.2 (C-H, aromatic), 2968.5 (C-H,
1
aliphatic), 1767.9 (C=O, ester), 1582.3, 1473 (C=C, alkene), 1247 (C-O stretch, aromatic); H
1
3
NMR (300 MHz, Chloroform-d) δ = 7.03 (d, J=7.5, 2H), 6.82 (t, J=7.5, 1H), 6.20 (s, 1H); C
NMR (75 MHz, Chloroform-d) δ 163.1, 146.1, 130.6, 129.6, 127.0, 63.9; MS m/z (%): 271.9
+
(
32) [M ], 242.1 (15), 149 (10), 122.1 (100), 91.1 (35), 77.1 (37), 65 (12), 51 (10).
2
.5.8. 2,4-dichlorophenyl 2,2-dichloroacetate (f35)
-1
Yield: 65%, yellow liquid, IR (KBr) ν (cm ): 3026.4 (C-H, aromatic), 2958.6 (C-H,
aliphatic), 1701.5 (C=O, ester), 1595.4, 1476.1 (C=C, alkene), 1265.2 (C-O stretch,
1
aromatic); H NMR (300 MHz, Chloroform-d) δ = 7.45 (d, J=2.4, 1H), 7.28 (dd, J=5.6, 2.7,
1
3
1
1
1
H), 6.97 (d, J=8.7, 1H), 6.24 (s, 1H); C NMR (75 MHz, Chloroform-d) δ 162.7, 148.7,
+
30.8, 129.5, 128.4, 128.0, 122.0, 63.9; MS m/z (%): 274.0 (17) [M ], 245.1 (11), 209 (5),
62.1 (95), 145 (13), 133.1 (23), 126.1 (7), 109.1 (5), 98.1 (20), 83.1 (32), 73.1 (12), 63.2
(30), 48.2 (13).
2
.5.9. 4-nitrophenyl 2,2-dichloroacetate (f36)
-1
Yield: 83%, yellow liquid, IR (KBr) ν (cm ): 3085.1 (C-H, aromatic), 2962.7 (C-H,
aliphatic), 1787.0 (C=O, ester), 1592, 1523.4 (C=C, alkene), 1495.4, 1336.5 (C-NO2,
1
aromatic), 1227.2 (C-O stretch, aromatic); H NMR (300 MHz, Chloroform-d) δ = 8.11 –
1
3
7
1
.96 (m, 2H), 7.05 – 6.90 (m, 2H), 6.25 (s, 1H); C NMR (75 MHz, Chloroform-d) δ 162.7,
+
53.3, 146.2, 125.2, 122.0, 64.6; MS m/z (%): 249 (14) [M ], 186 (10), 166 (9), 139 (67), 123
(100), 109 (38), 101.1 (5), 93 (10), 82.9 (65), 76 (20), 63 (33), 50.1 (10).

2
.5.10. 4-fluorophenyl 2,2-dichloroacetate (f37)
-1
Yield: 81%, yellow liquid, IR (KBr) ν (cm ): 3067.1 (C-H, aromatic), 2993.4 (C-H,
aliphatic), 1766.5 (C=O, ester), 1501.9, 1474.2 (C=C, alkene), 1225.2 (C-O stretch,
1
13
aromatic); H NMR (300 MHz, Chloroform-d) δ = 7.40 – 7.19 (m, 4H), 6.38 (s, 1H); C
NMR (75 MHz, Chloroform-d) δ 162.7, 161.4, 158.0, 150.2 (d, J=2.8), 122.0 (d, J=8.0),
+
1
8
16.2 (d, J=20.0), 64.6; MS m/z (%): 222 (15) [M ], 159 (11), 139 (10), 112.1 (100), 95 (24),
6.1 (10), 83 (50), 75.1 (6), 57.1 (13).
2
.5.11. 4-benzylphenyl 2,2-dichloroacetate (f38)
-1
Yield: 62%, brown liquid, IR (KBr) ν (cm ): 3026 (C-H, aromatic), 2961.2 (C-H, aliphatic),
1
1
751.4 (C=O, ester), 1509.7, 1451.2, 1438 (C=C, alkene), 1165 (C-O stretch, aromatic); H
NMR (300 MHz, Chloroform-d) δ = 7.33 – 7.11 (m, 4H), 7.04 (dd, J=14.2, 8.4, 3H), 6.77 (d,
1
3
J=8.4, 2H), 6.12 (s, 1H), 3.97 (s, 2H); C NMR (75 MHz, Chloroform-d) δ 162.7, 151.6,
+
1
41.6, 134.6, 129.9, 129.1, 128.9, 127.1, 121.1, 64.6, 41.2; MS m/z (%): 294.1 (20) [M ],
84.2 (100), 165.1 (25), 153.1 (15), 128.1 (10), 115.1 (7), 107.1 (15), 91.1 (13), 83 (5), 77.1
1
(10), 63.1 (5), 51.1 (5).
2
2
.6. Molecular docking study
.6.1. Molecular Preparation of the receptors
The 3D crystal structures for four PDK isozymes, PDK1 (2Q8H), PDK2 (2BU8), PDK3
(1Y8O)
and
PDK4
(3D2R)
were
retrieved
from
protein
data
bank
(http://www.rcsb.org/pdb/home/home.do). All water molecules were removed, missing
hydrogens were added and after determining the Kollman united atom charges, non-polar
hydrogens were merged into their corresponding carbons using AutoDock Tools [20].
Finally, desolvation parameters were assigned to each protein atom. Among the three
different search algorithms performed by AutoDock 4.2 the commonly used Lamarckian
Genetic Algorithm (LGA) was applied [21-23]. Subsequently, the enzymes were converted to
PDBQT using MGLTOOLS 1.5.6. The validation of the docking protocol was done
according to the previously published work by our research team [16]
.
2
.6.2. Docking procedure

The docking simulations were carried out by means of an in house batch script
DOCKFACE) [24, 25] for automatic running of AutoDock 4.2 and Vina in parallel mode.
(
An in house application i.e DOCKFACE was designed to facilitate the virtual ligand
screening in stepwise mode including ligand preparation, receptor preparation, grid maps
generation, dpf files preparation and finalization of docking runs [24, 25]. The genetic
algorithm search method was used to find the best pose of each ligand in the active site of the
target enzymes in all Autodock 4.2 experiments [26-28]. The dockings were carried out on
flexible ligands and rigid receptors. For the Lamarckian GA method; 100 No. of GA run,
2
7,000 maximum generations; 2500000 maximum No. of evaluations, 150 population size,
the mutation rate of 0.02; and a crossover rate of 0.8 were used. The grid box parameters
were adjusted based on our previous known docking protocol on PDKs [16]. All visualization
of protein ligand complexes were analyzed using VMD software [29], Autodock tools
program (ADT, Version 1.5.6), PyMOL molecular graphics program (6) and PLIP (fully
automated protein–ligand interaction profiler) [30]. All calculations were run on a core i7
personal computer (CPU at 8 MB) with the Windows 7 operating system.
2
2
.7. Biological assay
.7.1. Cell lines and cell culture
Human lung adenocarcinoma (A549, ATCC ® CCL-185™), colon carcinoma (HCA-7),
breast adenocarcinoma (MCF-7, ATCC® HTB22™ and MDA-MB-231, ATCC® HTB-
2
6™), oral epidermoid carcinoma (KB, ATCC ® CCL-17™) and ovarian cancer (SKOV3,
ATCC® HTB77™) cell lines were obtained from National Cell Bank of Iran (NCBI, Pasteur
Institute, Tehran, Iran). All cell lines were cultured in DMEM culture medium except for
MCF-7 cells which were cultured in RPMI 1640 medium supplemented with 10% fetal
o
bovine serum (FBS) and 1% penicillin-streptomycin and incubated at 37 C in a humidified
CO incubator.
2
Cytotoxic activity of all synthesized compounds was checked by standard 3-(4,5-
dimethylthiazol-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay as described in our
previous publications [31-33]. Briefly, the cells were harvested and plated in 96-well
4
microplates at a density of 1 × 10 cells per well in 100 μl complete culture medium
(containing FBS and antibiotics). After 24h incubation, each cell was treated with different
-4
-7
concentrations of each compound (from 2×10 to 1×10 M) in triplicate manner. Different

concentrations of DCA and doxorubicin were also used as positive controls. Three untreated
wells were considered as negative controls. After 72h, the media were completely removed
and replaced with 100 μl media containing 0.5 mg/ml of MTT solution and incubated for 3-4
hours. Then, media containing MTT were discarded and 150 μl dimethylsulfoxide (DMSO)
was added to each well to dissolve the formazan crystals and were incubated for more 3
hours. After 30 min, the absorbance in individual wells was determined at 570 nm by a Bio-
Rad microplate reader (Model 680). Data were calculated and expressed as the 50%
inhibitory concentrations (IC ). Each experiment was independently repeated three times.
5
0
Data are presented as mean ± SEM.
2
.7.2. Apoptosis assay
3
A549 cells were cultured in 6-well culture plate at concentration of 100 × 10 cells per well
and treated with different concentrations of compound f1 (25 μM, 50 μM, and 100 μM) for
2
4 h and/or 48 h. The cells were trypsinized, collected and resuspended in 1X phosphate
buffered saline (PBS) to be used for apoptosis analysis by Annexin V Apoptosis Detection kit
(eBiosciences, Cat. No. 88-8007) according to the manufacturer’s instruction. Briefly,
6
harvested cells were washed and re-suspend in 1
X
Binding Buffer at 1 × 10 cells per
milliliter and 5 μl of FITC conjugated Annexin V were then added to 100 μl of the cell
suspension. The mixture was incubated 10 min at room temperature in dark. The cells were
washed and re-suspend in 200 μl of 1
X Binding Buffer. Then 5 μl of Propidium Iodide
Staining Solution was added and the cells were acquired by FACSCalibur flow cytometer.
The data were analyzed by FlowJo software.
2
2
.8. QSAR studies
.8.1. Molecular descriptors
To calculate a large number of molecular descriptors, Hyperchem, Gaussian 98 [34] and
Dragon packages [35] were applied. Also, to calculate chemical parameters such as molecular
volume (V), molecular surface area (SA), hydrophobicity (LogP), hydration energy (HE) and
molecular polarizability (MP), Hyerchem software (Version 8, Hypercube Inc., Gainesville,
FL, USA) was used. Similarly, Gaussian 98 software was applied to calculate the most
positive and the negative net atomic charges, highest occupied molecular orbital (HOMO)
and lowest unoccupied molecular orbital (LUMO) energies, the average absolute atomic
charge and molecular dipole moment. According to the equations developed by Thanilaivelan
et al, quantum chemical indices including hardness (η = 0.5 (HOMO+LUMO)); softness

2
1
(
S= ꢁ ), electrophilicity (ω = χ ⁄2η) and electronegativity (ꢂ = −0.5) (HOMO-LUMO) were
ꢀ
calculated [36]. Different topological, geometrical, charge, empirical and constitutional
descriptors for each molecule and also 2D autocorrelations, aromaticity indices, atom-
centered fragments and functional groups were calculated by Dragon.
2
.8.2. Model validation
To validate the regression equation, statistical parameters including correlation coefficient
2
(
R ), root mean square error of cross-validation (RMScv), leave-one-out cross-validation
2
correlation coefficient (Q ), and variance ratio (F) with certain degrees of freedom were
applied. 20% of the molecules were selected as test set (prediction set) molecules to test the
developed model performance. The predictive value of a QSAR model that has not been
taken into account during the process of developing the model should be tested on an external
set of data.
3. Results and discussion
3
.1. Chemistry
The synthesis pathway of N-aryl-2,2-dichloroacetamide and aryl-2,2-dichloroacetate
analogues was illustrated in Scheme 2. At the first step, dichloroacetyl chloride (c) was
prepared using dichloroacetic acid (a) and thionyl chloride (b) in dichloromethane (DCM).
The target analogues were synthesized by the addition of dichloroacetyl chloride (c) to the
appropriate respective amine or phenol derivatives in toluene as the solvent. In the case of the
insoluble amines and phenols, acetonitrile or a mixture of acetonitrile/DMF was applied as
the solvent. After completion of the reaction, the crude products were washed with sodium
bicarbonate and then the solvent and excess dichloroacetyl chloride was allowed to evaporate
under vacuum. The residual powders were N-aryl-2,2-dichloroacetamide derivatives (f1-f27),
with a 75-98% yield. In order to the synthesis of aryl-2,2-dichloroacetate analogues (f28-f38),
after 1-8 h, the solvent and excess dichloroacetyl chloride were evaporated under vacuum and
followed by chromatography on a short column of silica gel using n-hexane/ethyl acetate
(5/1) to give liquid products with a 62-82% yield.

Scheme 2. Synthesis of N-aryl-2,2-dichloroacetamide and aryl 2,2-dichloroacetate derivatives.
Reagents and conditions: i) DCM, r.t.; ii) toluene or acetonitrile or CH CN/DMF, r.t. or reflux, 0.5-
3
7
h; iii) toluene, K CO or NaHCO3 or NEt , reflux, 3-8h.
2 3 3
3
.2. Molecular docking study
In order to determine the affinity and activity of the synthesized compounds and to predict
the binding sites and binding orientation of them to PDKs isoenzymes, molecular docking
was applied. Having completed the docking process, the protein–ligand complex was then
analyzed to investigate the type of interactions. Top ranked binding energies (kcal/mol) in
AutoDock dlg output file were considered as a response in each run. Docking results were
mostly supported by high cluster populations.
The best docking binding energies for the derivatives were acquired. Recently we presented
that the most sensitive isozymes of PDK to DCA and its analogues were PDK2 [16]. The
docking binding energies of all the synthesized compounds on two different PDB structure of
PDK2 were shown in table 1.
Table 1. Molecular descriptor analysis and docking binding energy (kcal/mol) on PDK2 isoenzyme.
1
2
3
4
5
6
Ar
∆E(Kcal/mol)
MW XlogP nDHB nAHB nRB
tPSA
Name

2BTZ 2BU8
O
O
f1
-5.32 -5.56 333
3.55
1
4
2
63.24
f2
f3
-5.41 -5.66 333
-5.17 -5.21 304
3.55
4.55
1
1
4
3
2
4
63.24
41.99
f4
f5
-9.53 -9.79 261
-9.49 -9.77 306
1.92
1.10
2
3
4
6
4
5
66.40
95.50
f6
f7
-4.22 -4.65 246
-5.47 -5.96 210
1.20
1.12
2
1
4
3
3
2
72.19
70.23
f8
f9
-5.32
-5.6
313
2.53
1.30
1
1
5
3
2
2
52.65
41.99
-5.14 -5.27 204
f10
f11
f12
-5.21 -5.35 204
-5.47 -5.67 208
-4.69 -4.62 197
1.80
1.47
0.29
1
1
0
3
4
3
2
2
1
41.99
50.69
29.54

f13
-9.73
-9.9
315
2.55
2
4
3
66.40
f14
f15
f16
-9.15 -9.56 283
-5.63 -5.57 228
-5.03 -5.28 251
2.28
2.25
2.90
2
1
1
4
3
2
3
3
2
66.40
52.89
29.10
f17
f18
-4.49
-4.8
263
1.80
1.80
1
1
4
4
4
4
47.56
47.56
-4.27 -4.41 263
f19
f20
-4.31 -4.47 293
-5.44 -5.36 267
1.36
2.34
1
1
5
3
5
3
56.79
38.33
f21
f22
-4.95
-5.1
219
1.73
1.73
2
2
3
3
2
2
49.33
49.33
-5.53 -5.76 219
-5.39 -5.49 219
f23
1.73
2
3
2
49.33

f24
f25
-9.26 -9.41 373
-5.53 -5.69 378
3.21
3.62
3
2
7
4
4
4
95.50
58.20
f26
f27
f28
-3.18 -2.98 418
-3.22 -3.46 420
-5.11 -5.04 209
4.28
4.48
2.19
2
2
1
4
5
2
6
6
2
58.20
67.43
29.10
f29
f30
f31
-5.16 -5.16 204
-4.09 -4.29 260
-5.79 -6.08 234
3.44
4.54
2.78
0
0
0
2
2
3
3
4
4
26.30
26.30
35.53
f32
f33
f34
-5.13 -5.32 234
-5.43 -5.77 238
-6.05 -6.22 272
2.78
3.32
3.42
0
0
0
3
2
2
4
3
3
35.53
26.30
26.30
f35
f36
-6.07 -6.14 272
-9.19 -9.52 249
3.42
2.99
0
1
2
5
3
3
26.30
63.60

f37
f38
-4.98 -5.08 222
-5.13 -5.05 294
3.18
4.88
0
0
2
2
3
5
26.30
26.30
1
2
3
4
5
6
Docking binding energy
Molecular weight
Number of donor hydrogen bond
Number of acceptor hydrogen bond
Number of rotatable bond
Total polar surface area
The docking binding energies of some compounds such as f1, f3, f5, f8, f11, f13, f17, f18,
f25 and f27 to the other PDK isozymes were also shown in table 2.
Table 2. Docking binding Energy (kcal/mol) on PDK isoenzymes.
1
-4
Name
Docking binding energy (kcal/mol)
2
Q8H
3
D2R
1Y8O
PDK3
2BU8
PDK2
Receptor
PDK4
-5.25
-3.82
-6.05
-5.34
-4.91
-4.27
-5.46
-5.59
-5.85
-4.67
-3.19
PDK1
-6.98
-6.48
-4.84
-6.56
-6.31
-5.83
-5.09
-5.00
-5.70
-5.61
-3.33
f1
f3
f5
-3.45
-3.11
-2.21
-3.07
-2.14
-2.35
-2.49
-3.77
-2.81
-3.41
-2.98
-5.56
-5.21
-5.77
-5.6
-5.67
-6.9
f8
f11
f13
f17
f18
f25
f27
DCA
-4.8
-4.41
-5.69
-3.46
-3.54
The interactions of the synthesized DCA derivatives with four isozymes of PDK were
investigated. As it was depicted in Fig. 1, a hydrogen bond acceptor interaction exists
between the oxygen of amidic carbonyl group of f1 with Arg188 in binding to 2Q8H (PDK1)
receptor. A hydrogen bond acceptor interaction of carbonyl group of quinone ring with
Ser271 was also observed. Meanwhile, a hydrogen bond acceptor interaction exists between
the oxygen of the carbonyl group with Tyr80 and an arene-arene (π-π) interaction between
phenyl ring and His115 in binding to 2BU8 (PDK2) receptor. The most important residues in
binding to 1Y8O (PDK3) target was a hydrogen bond acceptor interaction between the

oxygen of carbonyl group with Tyr149 and an arene-cation interaction between phenyl ring
and Asp152. The most important residues in binding to 3D2R (PDK4) target was a hydrogen
bond acceptor interaction between the oxygen group of quinone carbonyl with Arg166 and
His128. There is also existed a hydrogen bond donor interaction between NH of amide group
with Arg174 and a π-π interaction between phenyl ring and His128.
Fig. 1. Interactions of compound f1 with the residues in the binding site of PDK isoenzymes.
a) PDK1 (2Q8H), b) PDK2 (2BU8), c) PDK3 (1Y8O), d) PDK4 (3D2R).
As it was shown in Fig. 2, The most important residues in binding of f8 to 2Q8H (PDK1)
receptor is a hydrogen bond acceptor interaction between the oxygen of amidic carbonyl
group with Met199, a hydrogen bond acceptor interaction between the carbonyl of the
heterocyclic ring with Arg166 and a weak hydrogen bond interaction between chloro group
and Arg166. The type of interactions of this compound to 2BU8 (PDK2) receptor is a
hydrogen bond acceptor interaction of carbonyl group with Tyr80 and an arene-cation

interaction between phenyl ring and Arg158. The most important residues in binding to
Y8O (PDK3) target was a hydrogen bond acceptor interaction between the carbonyl of
1
heterocyclic ring and oxygen of amidic carbonyl group with Ser239. There is also exists a
hydrogen bond donor interaction between NH of amide group with His243. The most
important residues in binding to 3D2R (PDK4) target was a hydrogen bond acceptor
interaction between the oxygen of amidic carbonyl group with Arg124 and a weak hydrogen
bond interaction between chloro group and Arg166. There is also existed a hydrogen bond
interaction between the hydrogen of the methyl group with His128.
Fig. 2. Interactions of compound f8 with the residues in the binding site of PDK isoenzymes.
a) PDK1 (2Q8H), b) PDK2 (2BU8), c) PDK3 (1Y8O), d) PDK4 (3D2R).
Table 3 shows the participating atom of all the synthesized compounds in binding to PDK2
isoenzyme as the most sensitive isozymes of PDKs.

Table 3. The participating atom in binding to PDK2 isoenzyme.
Name Amino Acid
The participating atom in bonding
Type of Interactions
Ligand
Amino Acid
f1
Tyr80
His115
His115
His115
His115
His115
Arg154
Arg158
Arg112
His115
Tyr80
Phe150
Arg154
Arg112
Arg158
Arg158
Tyr80
His115
His115
His115
Arg154
Ileu111
His115
Arg154
Arg154
Tyr328
His115
His115
His115
Arg154
Arg158
His115
Arg158
His115
Arg154
Arg158
Tyr80
Oxygen of carbonyl
Phenyl ring
OH
H.B
π-π
π-π
π-π
π-π
Imidazole ring
Imidazole ring
Imidazole ring
Imidazole ring
Imidazole ring
NH (side chain)
f2
f3
Phenyl ring
Phenyl ring
Pyridine ring
Phenyl ring
f4
π-π
+
Oxygen of carbonyl
Oxygen of hydroxyl
hydrogen of hydroxyl
Phenyl ring
H.B
H.B
H.B
π-π
2
+
NH (side chain)
2
Oxygen of carbonyl
Imidazole ring
OH
f5
f6
H of Amide
H.B
H.B
H.B
H.B
H.B
Arene-cation
H.B
π-π
Carbonyl of COOH
Amidic carbonyl
H of Amidic nitrogen
Oxygen of DCA carbonyl
Phenyl ring
Oxygen of carbonyl
Pyridine ring
Pyridine ring
Oxazole ring
Oxygen of Morpholine
H of COOH
NH of the main backbone
+
NH (side chain)
Oxygen of carbonyl
NH (side chain)
NH (side chain)
2
+
f7
f8
2
+
2
OH
f9
Imidazole ring
Imidazole ring
Imidazole ring
NH (side chain)
backbone(C=O)
Imidazole ring
NH (side chain)
NH (side chain)
f10
f11
f12
f13
π-π
π-π
H.B
H.B
π-π
+
2
Phenyl ring
O of COOH
Phenyl ring
Amidic carbonyl
Phenyl ring
+
f14
2
H.B
Arene-cation
H.B
π-π
+
2
OH
f15
f16
f17
Imidazole ring
Imidazole ring
Imidazole ring
NH (side chain)
NH (side chain)
Imidazole ring
NH (side chain)
Imidazole ring
NH (side chain)
Phenyl ring
Phenyl ring
π-π
π-π
+
Oxygen of Methoxy
Oxygen of Methoxy
Phenyl ring
Oxygen of Methoxy
Phenyl ring
Oxygen of Methoxy-4
Oxygen of Methoxy-3
Oxygen of Methoxy-5
Phenyl ring
H.B
H.B
π-π
H.B
π-π
H.B
H.B
H.B
π-π
2
+
2
f18
f19
+
2
+
2
+
NH (side chain)
2
OH
f20
f21
His115
His115
Arg158
His115
Imidazole ring
Imidazole ring
NH (side chain)
Imidazole ring
Phenyl ring
4-OH
Phenyl ring
π-π
H.B
π-π
+
2
f22