Synthesis, antifungal activity and QSAR of some novel carboxylic acid amides

Article abstract of DOI:10.3390/molecules20034071

A series of novel aromatic carboxylic acid amides were synthesized and tested for their activities against six phytopathogenic fungi by an in vitro mycelia growth inhibition assay. Most of them displayed moderate to good activity. Among them N-(2-(1H-indazol-1-yl)phenyl)-2-(trifluoromethyl)benzamide (3c) exhibited the highest antifungal activity against Pythium aphanidermatum (EC₅₀ = 16.75 μg/mL) and Rhizoctonia solani (EC₅₀ = 19.19 μg/mL), compared to the reference compound boscalid with EC₅₀ values of 10.68 and 14.47 μg/mL, respectively. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were employed to develop a three-dimensional quantitative structure-activity relationship model for the activity of the compounds. In the molecular docking, a fluorine atom and the carbonyl oxygen atom of 3c formed hydrogen bonds toward the hydroxyl hydrogens of TYR58 and TRP173.

Full text of DOI:10.3390/molecules20034071

Molecules 2015, 20, 4071-4087; doi:10.3390/molecules20034071
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Antifungal Activity and QSAR of Some Novel
Carboxylic Acid Amides
Shijie Du, Huizhe Lu *, Dongyan Yang, Hong Li, Xilin Gu, Chuan Wan, Changqing Jia,
Mian Wang, Xiuyun Li and Zhaohai Qin *
Department of Applied Chemistry, College of Science, China Agricultural University,
Beijing 100193, China; E-Mails: dsj5216@163.com (S.D.); yangdy@cau.edu.cn (D.Y.);
lihong129106@163.com (H.L.); david9351@126.com (X.G.); wanchuan2011@163.com (C.W.);
cauchangqing@gmail.com (C.J.); woookooo@cau.edu.cn (M.W.); lixiuyun0115@163.com (X.L.)
*
Authors to whom correspondence should be addressed; E-Mails: luhz@cau.edu.cn (H.L.);
qinzhaohai@263.net (Z.Q.); Tel.: +86-10-62732507 (H.L.); Tel./Fax: +86-10-62732958 (Z.Q.).
Academic Editor: Derek J. McPhee
Received: 31 December 2014 / Accepted: 2 March 2015 / Published: 4 March 2015
Abstract: A series of novel aromatic carboxylic acid amides were synthesized and tested for
their activities against six phytopathogenic fungi by an in vitro mycelia growth inhibition
assay. Most of them displayed moderate to good activity. Among them N-(2-(1H-indazol-1-
yl)phenyl)-2-(trifluoromethyl)benzamide (3c) exhibited the highest antifungal activity
against Pythium aphanidermatum (EC50 = 16.75 µg/mL) and Rhizoctonia solani
(
EC50 = 19.19 µg/mL), compared to the reference compound boscalid with EC50 values of
0.68 and 14.47 µg/mL, respectively. Comparative molecular field analysis (CoMFA) and
1
comparative molecular similarity indices analysis (CoMSIA) were employed to develop a
three-dimensional quantitative structure-activity relationship model for the activity of the
compounds. In the molecular docking, a fluorine atom and the carbonyl oxygen atom of 3c
formed hydrogen bonds toward the hydroxyl hydrogens of TYR58 and TRP173.
Keywords: amide fungicides; boscalid; QSAR; SDHIs; molecular docking

Molecules 2015, 20
. Introduction
4072
1
Amide fungicides, an old type of fungicide, still play an important role in the agricultural chemistry
field nowadays [1]. Some excellent commercial fungicides including boscalid (BASF, 2003), tiadinil
Nihon Nohyaku Co., Ltd., 2004), fluxapyroxad (BASF, 2012) and benzovindiflupyr (Syngenta, 2012)
(
belong to this class. Recently, many aspects of structural transformation and structure-activity
relationship studies have been reported. Wen [2] and coworkers changed the biphenyl in the structure of
boscalid to a diphenyl ether, and thus obtained target compounds with some extent of antifungal
activities. Liu et al. synthesized a series of N-substituted pyridine amide fungicides exhibiting good
activity to Magnaporthe oryzae and Blumeria graminis f. sp. Tritici [3]. Ye et al. changed the chlorine
atom of boscalid to a methylthio or aromatic sulfur, and obtained some compounds with good activity
to Rhizoctonia solani and Sclerotinia sclerotiorum [4].
Amide fungicides target the complex II (succinate dehydrogenase, SDH) in the mitochondrial
respiratory chain [5]. As SDH inhibitors (SDHIs), they can inhibit the growth and even cause the death
of pathogens by disrupting the mitochondrial tricarboxylic acid cycle [6] and then interfering with their
respiration [7]. After analyzing the structure of the known SDHIs encompassing furametpyr, boscalid,
fluxapyroxad, bixafen, penthiopyrad, penflufen, sedaxane, isopyrazam, benzovindiflupyr, we found that
the aromatic amines share an auxiliary group with a five to nine atoms bridge and ortho-substituents. The
alignment of the three-dimensional structures of carboxin, flutolanil, boscalid and penflufen (Figure 1)
demonstrates that the common chemical features discussed above superimpose well, suggesting an
identical binding mode at SDH. By summarizing the chemical structure of all commercial aromatic
amides fungicides, Dehne found that they share common chemical features, which are essential for
fungicidal activity, and they hence bind to their target in the same manner [8].
Figure 1. Structural alignment of the four kinds of SDHI fungicides (adapted from [8]).
In most previous studies, the ortho of the amide group on the benzene ring is attached to a C atom
(
Figure 2a), which is an important part in some commercial fungicides. Based on bioisosterism [9,10]
and computational docking experiments [8], we introduced a N atom to replace the C atom and thus

Molecules 2015, 20
4073
designed and synthesized the target compounds (Figure 2b). Bioassays showed that some target
molecules exhibited good antifungal activity and might be useful as potential lead compounds.
O
R1
O
N
O
Ar
NH
NH
NH Cl
N
C
R2
R2
Cl
boscalid
(a)
(b)
Figure 2. Design strategy of the target compounds. (a) Most general molecular structure.
b) The designed compound.
(
2
. Results and Discussion
2.1. Synthesis of Compounds
The synthetic route to the target compounds is shown in Scheme 1. The chlorine atom in
-chloro-2-nitrobenzene was replaced by an amino group via an aromatic nucleophilic substitution
1
reaction, giving the compound I. Then the nitro group in compound I was reduced with hydrazine hydrate
to provide the key intermediates II, which were subsequently acylated to produce the target amides III.
O
O
NO2 H R
NO2
R2
NH
NH
R
1
2
2
NH
2
-NH
2
H
2
O
R
1
Cl
K
2
CO
3
Pd-C
CH OH
Cl
R
2
R
2
HMTA
CuI
3
DMF
I
II
III
Cl
R1=
R2=
N
Cl
N
CF3
1a,1b,1c,1e,1f,1h,1j
a,2b,2d,2e,2g,2h,2j
N
Cl
O
1
.
2.
3.
O
4.
5.
2
O
N
3a,3b,3c,3d,3e,3f
N
N
N
N
N
N
N
N
N
N
i.
4a,4b,4e,4f,4g,4h,4j
5
b,5f,5h
a.
b.
c.
d.
e.
f.
g.
h.
j.
Scheme 1. Synthetic pathway for the target compounds.
2
.2. In Vitro Antifungal Activity
Table 1 summarizes the bioassay results. Overall, the target molecules showed different levels of
antifungal activity. The inhibitory activity of the target molecules to P. aphanidermatum and R. solani
were higher than against the other four fungi. Compounds 1c, 1i, 3c exhibited wide spectrum antifungal
activity and compound 1c displayed excellent activities against Colletotrichum orbiculare, Rhizoctonia
solani, Pythium aphanidermatum and Botrytis cinerea, with 60.43%, 75.19%, 74.14% and 79.27%
inhibition rate, respectively. Moreover, some compounds showed highly specific pathogen activity,
whereby compounds 1h and 3e exhibited activities against Rhizoctonia solani with 67.75% and 69.08%
inhibition rates, respectively.

Molecules 2015, 20
Table 1. In vitro antifungal activity of the target compounds against different
4074
−1
phyto-pathogenic fungi (at 50 μg·mL ).
a
Inhibition rate (%)
Compd.
W. A. b
11.11
39.84
60.43
24.66
56.10
44.99
51.22
8.94
B. B.
30.53
48.09
27.10
61.83
55.98
57.25
54.71
6.11
R. S.
17.56
59.92
75.19
55.73
47.71
67.75
74.24
43.51
51.53
21.95
39.69
50.95
58.78
55.34
42.56
49.24
75.57
39.89
69.08
43.51
36.45
50.57
50.95
33.88
21.04
31.15
24.59
43.99
60.11
55.46
78.73
P. I.
P. A.
21.17
37.62
74.14
51.89
60.58
48.48
42.89
32.22
15.27
24.58
49.41
46.65
42.27
43.39
42.89
39.79
84.17
34.20
38.18
49.10
10.61
44.13
57.79
18.49
39.73
39.04
38.58
40.87
40.87
42.47
81.10
B. C.
12.50
26.22
79.27
43.29
31.30
44.51
51.83
12.23
14.23
14.23
*
1
a
33.15
43.67
18.89
35.83
42.50
41.33
45.07
17.72
41.33
30.11
27.07
30.81
34.32
38.99
31.04
38.29
36.42
34.32
33.61
36.65
17.72
40.63
30.11
24.05
31.30
32.32
29.52
40.20
32.32
31.30
56.50
1
b
1
1
c
e
1
f
1
h
1
i
2a
2b
2d
46.61
19.24
23.31
15.99
50.95
42.55
32.93
48.78
47.97
35.23
43.63
57.32
11.38
37.13
27.10
50.38
*
25.70
16.79
11.20
*
2
e
2
j
12.50
21.34
14.02
11.59
46.34
49.59
36.38
23.78
37.20
*
2
g
40.71
19.59
31.30
38.93
56.49
24.94
28.24
23.66
18.32
32.57
32.44
28.04
37.09
44.75
47.54
54.97
52.65
47.42
58.58
2h
3a
3b
3
c
3
d
3
e
3
f
4
a
4
b
18.90
*
4
e
4
4
j
f
0.00
63.33
43.75
20.00
40.94
49.58
48.75
88.60
4
g
13.74
*
4
5
h
b
41.60
35.11
37.40
71.54
5
f
5
h
Boscalid
a
The tests were repeated three times, and the average inhibition rate is given. b W.A. = Colletotrichum
orbiculare; B. B. = Botryosphaeria berengeriana; R. S. = Rhizoctonia solani; P. I. = Phytophthora infestans
Mont.) De Bary; P. A. = Pythium aphanidermatum; B. C. = Botrytis cinerea; * No antifungal activity
(
In order to study the structure-activity relationship of the target compounds further, we choose
Rhizoctonia solani and Pythium aphanidermatum for precise virulence measurements. As we can see
from Table 2, all of the synthetic compounds exhibited considerable antifungal effects toward the two
fungi. The compounds 1c and 3c exhibited the best fungicidal activity against the two fungi, with
−1
−1
−1
−1
corresponding EC50 values of 19.95 μg·mL , 17.99 μg·mL and 16.75 μg·mL , 19.19 μg·mL ,

Molecules 2015, 20
4075
respectively. The antifungal activity of both compounds was close to that of the control compound
boscalid. They also had broad antifungal spectrum, so they have potential value as secondary lead
compounds for further research.
Table 2. Measured and predicted antifungal activities of target compounds against Pythium
aphanidermatum and Rhizoctonia solani.
Pythium aphanidermatum
Rhizoctonia solani
EC50
Predicted pEC50
CoMFA CoMSIA (μg·mL )
EC50
Predicted pEC50
CoMFA CoMSIA
No.
pEC50
pEC50
−
1
−1
(
μg·mL )
114.20
74.90
19.95
82.78
91.47
94.23
93.29
87.12
166.37
117.20
52.20
56.23
71.40
79.90
82.29
80.49
16.75
77.04
95.50
69.35
169.82
84.10
35.31
79.43
154.88
141.25
151.35
31.29
66.10
41.68
10.68
1
a
3.94
4.13
4.70
4.08
4.04
4.03
4.03
4.06
3.78
3.93
4.28
4.25
4.15
4.10
4.08
4.09
4.78
4.11
4.02
4.16
3.77
4.08
4.45
4.10
3.81
3.85
3.82
4.50
4.18
4.38
4.97
4.002
3.971
4.687
4.170
3.983
4.019
4.155
3.973
3.953
4.013
4.194
4.240
4.019
3.997
4.093
4.121
4.871
4.051
4.048
4.111
3.895
3.895
4.364
4.231
3.892
3.971
3.869
4.388
4.201
4.315
4.960
3.964
3.967
4.677
4.042
3.953
3.993
4.217
3.972
3.992
3.953
4.219
4.240
4.030
4.016
4.061
4.174
4.804
4.033
4.059
4.140
3.834
3.931
4.467
4.227
3.937
3.965
3.910
4.433
4.098
4.405
4.938
141.25
74.99
17.99
77.98
89.95
78.16
82.79
108.52
89.63
163.22
103.51
50.12
76.91
84.92
110.41
87.9
3.85
4.12
4.74
4.11
4.05
4.11
4.08
3.96
4.05
3.79
3.99
4.30
4.11
4.07
3.96
4.06
4.72
4.04
3.99
3.99
3.92
4.07
4.11
4.27
3.77
3.99
4.10
3.99
4.14
4.02
4.84
3.880
4.037
4.729
4.080
3.990
4.035
4.085
3.856
4.039
3.830
3.960
4.301
4.083
4.050
3.923
3.990
4.170
4.006
3.998
3.954
3.927
4.001
4.047
4.316
3.939
4.018
4.012
3.957
4.142
4.022
4.896
3.810
4.023
4.683
4.065
4.012
4.032
4.075
3.820
4.029
3.839
3.979
4.323
4.039
4.037
3.897
4.002
4.704
4.051
4.016
3.991
3.861
4.026
4.028
4.350
3.992
4.034
4.013
4.054
4.194
3.969
4.860
1
b
1
1
1
c
e
f
1
h
1
2
i
a
2
2
b
d
2
2
2
e
j
g
2
h
3
a
3
b
3
c
19.19
90.57
101.39
102.09
121.37
85.9
3
d
3
3
4
e
f
a
4
b
4
4
4
e
j
f
78.51
53.7
170.56
101.39
79.62
103.04
71.94
95.72
14.47
4
g
4
5
h
b
5
f
5
h
Boscalid
From the high bioactivity of compounds 1c and 3c which contain an indazolyl group, we speculated
that this group might have an essential influence on their biological activity. In recent years, indazole
compounds have received much attention in the field of bioactive chemicals [11], especially in medicine,
where they are known for anti-aggregatory and vasorelaxant activity, anticancer effects, and
antimicrobial and antiparasitic properties [12]. Our studies now show that the indazolyl moiety also has
potential use in agrochemicals.

Molecules 2015, 20
.3. Quantitative Structure-Activity Relationship (QSAR) Analyses
During biological screening, CoMFA and CoMSIA models of the new compounds were constructed
4076
2
using Sybyl 7.3 to find some clues about the structure-activity relationships. Compound 3c was the
template molecule used for constructing models for P. aphanidermatum activity. Some important
2
parameters of the models were as follows: (A) the CoMFA model: q = 0.543 at 3 components,
2
2
2
r = 0.814, F = 73.392; (B) the CoMSIA model: q = 0.502 at 3 components, r = 0.893, F = 75.480. For
2
R. solani, some parameters of the models are as follows: (A) the CoMFA model: q = 0.61 at 3
2
2
2
components, r = 0.936, F = 73.446; (B) the CoMSIA model: q = 0.63 at 3 components, r = 0.888,
F = 93.894. The three-dimensional contour maps of the CoMFA and CoMSIA are shown in Figure 3.
Figure 3. (a–d): Fitting of 3c to P. aphanidermatum. (a) CoMFA steric field. (b) CoMFA
electrostatic field. (c) CoMSIA electrostatic field. (d) CoMSIA H acceptor field.
(
(
e–h): fitting of 3c to R. solani. (e) CoMFA steric field. (f) CoMFA electrostatic field.
g) CoMSIA electrostatic field. (h) CoMSIA H acceptor field. In the electrostatic field, the
positively charged favored regions are shown in blue, and negatively charge favored regions
are shown in red. In the sterically favored and disfavored region are shown in green and
yellow respectively. In the hydrogen bond acceptor field, the favored regions are shown in
magenta, and negatively charge favored regions are shown in red.
In the three-dimensional contour maps of the CoMFA of compounds 3c to P. aphanidermatum and
R. solani. Some areas were covered with yellow blocks, which indicates that a large substituent in this
position will likely reduce the biological activity of the compound. Green regions substantially covered
the indazole and the benzene (Figure 3a,e), meaning that the introduction of greater steric hindrance
groups at these positions was beneficial to improving the activity. In the CoMSIA and CoMFA
electrostatic field, carbonyl and trifluoromethyl groups on the benzene ring were red blocks

Molecules 2015, 20
4077
(
Figure 3b,c,f,g), indicating that the introduction of negatively charged groups to the active position was
beneficial for the antifungal activity. In the hydrogen acceptor field (Figure 3d,h), the results showed
that more hydrogen bonds should improve the biological activity. As a hydrogen acceptor, the fluorine
in the trifluoromethyl and the nitrogen atom in the pyrazole are helpful for forming hydrogen bonds,
so taking together all this information, it can explain why 3c showed better antifungal activity than the
other compounds.
2.4. Molecular Docking
In an effort to elucidate the possible antifungal mechanism of these compounds, molecular docking
of compound 3c to the binding site of SDH (pdb code: 2FBW [13,14]) pdb was performed. Boscalid was
docked as a reference at the same time. The three-dimensional schematic diagrams clearly explained the
possible optimal combination between the ligands and receptor protein (Figure 4).
Figure 4. Surflex-Docking of boscalid to complex II. (a) Connolly surface of complex II
with boscalid shown as a stick model. (b) Interaction of boscalid and amino acid residues
near the ligands (3D diagram). (c) Interaction of boscalid and amino acid residues near the
ligands (2D diagram). (d) Connolly surface of complex II with compounds 3c shown as a
stick model. (e) Interaction of compounds 3c and amino acid residues near the ligands (3D
diagram). (f) Interaction of compounds 3c and amino acid residues near the ligands (2D
diagram). The orange dotted line circles show the amino acids that participated in hydrogen
bonding. The magenta dotted semicircle show the amino acids that participated in the van
der Waals interactions. The hydrogen bond interactions are shown as green dotted lines.

Molecules 2015, 20
4078
Boscalid and compound 3c adopted similar conformations and locations in the active site. The
docking total scores were 5.25 and 6.87, respectively. They were both well bound to the receptor protein
with their amino hydrogen toward the carboxyl oxygen of TRP173, and the carboxyl oxygen forming
hydrogen bonds toward the hydroxyl hydrogen of TYR58. At the same time, an additional hydrogen
bond was formed between the fluorine atom of compound 3c and the hydroxyl hydrogen of TYR58.
Two-dimensional diagrams are displayed as Figure 4c,f in which all of the amino acid residues TRP32,
MET36, SER39, ILE40, AGR43, PRO169, HIS216, ILE218 interacted with the ligand, including weak
interactions such as van der Waals interactions and polar interactions. Thus, a stable complex
between compound 3c and SDH was formed based on these interactions. The docking results were
consistent with previous studies comparing boscalid and other SDHIs (such as 3-nitropropionic acid,
2-thenoyltrifluoroacetone, carboxin) [15].
Resistant fungal genotype analysis verified that most of those key residues involved in forming the
binding cavity were related to resistance formation [16].
3. Experimental Section
3.1. General Information
For all reactions, the solvents and chemical reagents were of analytical or synthetic grade obtained
from Sinopharm Chemical Reagent Beijing Co., Ltd. (Beijing, China), and were used without
purification. Column chromatography purification was performed using silica gel. Melting points were
determined using a B-III microscope (Beijing Technical Instrument Co., Beijing China), and the
thermometer was uncorrected. NMR spectra were obtained using an Avance DPX300 spectrometer
(
Bruker, Billerica, MA, USA) with tetramethylsilane (TMS) as the internal standard. High resolution
mass spectrometry data were obtained with an Accurate-Mass-Q-TOF MS 6520 system equipped with
an electrospray ionization (ESI) source (Agilent, Santa Clara, CA, USA).
3.2. Synthesis of Compounds
3
.2.1. Synthesis of Compounds I: 1-(2-Nitrophenyl) Piperidine
In a 100 mL flask, 1-chloro-2-nitrobenzene (3.94 g, 0.025 mol), piperidine (2.55 g, 0.030 mol—we
take R = piperidine as the example, the same method was used to synthesize and purify the rest of the
1
reactants), potassium carbonate (6.9 g, 0.05 mol), HMTA (0.15 g), and CuI (0.2 g) were dissolved in
DMF (50 mL), and the mixture was heated to 130 °C and refluxed for 24 h [17]. A same volume of water
was then slowly added to the solution which was extracted with ethyl acetate (100 mL × 3). The
combined organic layer was dried over anhydrous magnesium sulfate and evaporated under reduced
pressure to give a crude product that was purified by column chromatography (EtOAc-PE = 10:1) to
1
give the title compound as an orange powder, yield 75%; mp 76–77 °C; H-NMR (CDCl
3
) δ 7.74 (dd,
J = 8.1, 1.6 Hz, 1H), 7.43 (ddd, J = 8.4, 7.3, 1.7 Hz, 1H), 7.11 (dd, J = 8.3, 1.2 Hz, 1H), 6.95 (ddd,
J = 8.2, 7.3, 1.3 Hz, 1H), 3.09–2.95 (m, 4H), 1.84–1.66 (m, 4H), 1.64–1.52 (m, 2H).

Molecules 2015, 20
.2.2. Synthesis of Compounds II: 2-(Piperidin-1-yl)aniline
In an 100 mL three-necked flask equipped with a dropping funnel, 1-(2-nitrophenyl)piperidine (4.0 g)
4079
3
and ethanol (50 mL) were mixed and heated to reflux. Palladized charcoal (0.1 g, 5%, previously
moistened with alcohol) was added. Next 80% hydrazine hydrate (15 mL) was added from a dropping
funnel during 30 min. The reaction was continued for 8 h and then cooled [18,19]. The solid was filtered
off and the filtrate was concentrated to give a crude product that was recrystallized from ethyl acetate and
petroleum ether solution (15:1) to afford the title compound as a white powder, yield 95%; mp 48–50 °C;
1H-NMR (CDCl
) δ 7.02–6.95 (m, 1H), 6.95–6.86 (m, 1H), 6.82–6.63 (m, 2H), 3.97 (s, 2H), 2.83 (s,
3
4
H), 1.77–1.63 (m, 4H), 1.57 (d, J = 5.0 Hz, 2H).
3
.2.3. Synthesis of Compounds III: 2-Chloro-N-(2-(piperidin-1-yl)phenyl)nicotinamide (1b)
A solution of 2-(piperidin-1-yl)aniline (0.2 g, 0.025 mol), dichloromethane (25 mL) and triethylamine
(
0.5 mL) was added to a 50 mL flask and cooled to 0 °C in an ice bath. After ten minutes,
-chloronicotinoyl chloride (0.22 g) was added [20,21]. The mixture was stirred for 30 min, and then
2
concentrated under reduced pressure to give a crude product. The pure 2-chloro-N-(2-(piperidin-1-yl)
phenyl) nicotinamide (1b) was obtained by column chromatography (EtOAc:PE = 8:1) purification.
1
yield 88%; yellow powder; mp 76–77 °C; H-NMR (CDCl
3
) δ 9.77 (s, 1H, NH), 8.53 (dd, J = 4.8,
2
7
2
1
.0 Hz, 2H, pyridyl-H), 8.22 (dd, J = 7.7, 2.0 Hz, 1H, pyridyl-H), 7.42 (dd, J = 7.7, 4.8 Hz, 1H, Ar-H),
.25–7.06 (m, 3H, Ar-H), 2.93 (s, 2H, CH ), 2.71 (s, 2H, CH ), 1.88–1.64 (m, 2H, CH ), 1.44–1.22 (m,
) δ 162.10, 150.81, 146.72, 142.92, 139.71,
2
2
2
1
3
H, CH
2
), 0.97 (d, J = 6.4 Hz, 2H, CH
2
); C-NMR (CDCl
3
33.20, 131.83, 125.24, 124.31, 122.62, 120.93, 119.21, 53.90, 26.44, 23.62; HRMS (ESI), m/z calcd
+
for C17
H19ClN
3
O (M+H) 316.1212, found 316.1211.
The following compounds were similarly prepared:
1
2
-Chloro-N-(2-morpholinophenyl)nicotinamide (1a). Yield 78%; white powder; mp 81–83 °C; H-NMR
CDCl ) δ 9.81 (s, 1H, NH), 8.60 (dd, J = 8.0, 1.5 Hz, 1H, pyridyl-H), 8.54 (dd, J = 4.7, 2.0 Hz, 1H,
pyridyl-H), 8.27 (dd, J = 7.7, 2.0 Hz, 1H, pyridyl-H), 7.44 (dd, J = 7.7, 4.7 Hz, 1H), 7.36–7.22 (m, 2H,
(
3
1
3
Ar-H), 7.17 (td, J = 7.6, 1.6 Hz, 1H, Ar-H), 4.01–3.72 (m, 4H, CH
CDCl ) δ 163.71, 150.50, 146.42, 142.74, 140.12, 133.31, 131.24, 125.14, 122.34, 122.43, 120.81,
19.62, 62.92, 55.43; HRMS (ESI), m/z calcd for C16
2
), 3.16–2.68 (m, 4H, CH
2
). C-NMR
(
3
+
1
H16ClN
3
O (M+H) 318.1004, found 318.1000.
2
N-(2-(1H-Indazol-1-yl)phenyl)-2-chloronicotinamide (1c). Yield 85%; yellow powder; mp 110–112 °C;
1H-NMR (CDCl
) δ 9.90 (s, 1H, NH), 8.63 (d, J = 8.2 Hz, 1H, pyridyl-H), 8.45 (dd, J = 4.8, 2.0 Hz, 1H,
3
pyridyl-H), 8.24 (d, J = 0.9 Hz, 1H, pyridyl-H), 8.02 (dd, J = 7.6, 1.9 Hz, 1H, indazole-H), 7.81 (dt,
J = 8.1, 1.0 Hz, 1H, Ar-H), 7.60 (dd, J = 3.0, 1.2 Hz, 1H, Ar-H), 7.57 (dd, J = 2.3, 1.3 Hz, 1H, Ar-H),
1
3
7
1
1
.55–7.42 (m, 2H, Ar-H), 7.39–7.19 (m, 3H, Ar-H); C-NMR (CDCl ) δ 162.70, 150.85, 147.00, 139.76,
3
39.17, 135.98, 132.10, 131.40, 128.97, 128.26, 127.68, 124.82, 124.60, 124.16, 123.38, 122.34, 121.89,
+
21.14, 110.27; HRMS (ESI), m/z calcd for C19
H
13ClN
4
O (M+H) 349.0851, found 349.0851.
2
-Chloro-N-(2-(pyrrolidin-1-yl)phenyl)nicotinamide (1e). Yield 86%; white powder; mp 136–138 °C;
) δ 9.66 (s, 1H, NH), 8.52 (dd, J = 4.7, 2.0 Hz, 1H, pyridyl-H), 8.47 (dd, J = 7.8,
1H-NMR (CDCl
3

Molecules 2015, 20
4080
1
7
2
1
3
.9 Hz, 1H, pyridyl-H), 8.30 (dd, J = 7.7, 2.0 Hz, 1H, pyridyl-H), 7.42 (dd, J = 7.7, 4.7 Hz, 1H, Ar-H),
.23 (d, J = 2.2 Hz, 1H, Ar-H), 7.16 (td, J = 7.1, 2.0 Hz, 2H, Ar-H), 3.05 (t, J = 6.6 Hz, 4H, CH ),
) δ 161.90, 150.82, 146.62, 140.45, 140.11, 133.13, 131.43,
2
1
3
.09–1.78 (m, 4H, CH
2
); C-NMR (CDCl
3
24.63, 124.37, 122.68, 120.17, 120.10, 52.93, 24.20; HRMS (ESI), m/z calcd for C16
H
16ClN
O (M+H)+
3
02.1052, found 302.1055.
2
-Chloro-N-(2-(4-methylpiperidin-1-yl)phenyl)nicotinamide (1f). Yield 77%; white powder; mp
1
120–122 °C; H-NMR (CDCl
3
) δ 9.76 (s, 1H, NH), 8.56 (dd, J = 8.0, 1.6 Hz, 1H, pyridyl-H), 8.53 (dd,
J = 4.8, 2.0 Hz, 1H, pyridyl-H), 8.21 (dd, J = 7.7, 2.0 Hz, 1H, pyridyl-H), 7.42 (dd, J = 7.7, 4.8 Hz, 1H,
Ar-H), 7.18 (dddd, J = 15.1, 9.2, 6.7, 2.8 Hz, 3H, Ar-H), 2.91–2.73 (m, 4H, CH ), 1.76–1.64 (m, 4H,
) δ 162.06, 150.83, 146.63,
2
1
3
CH
2
), 1.45 (m, J = 6.5, 1H, CH), 0.86 (d, 2H, J = 6.5,CH3); C-NMR (CDCl
3
1
42.57, 139.81, 133.29, 131.74, 125.20, 124.23, 122.61, 120.92, 119.19, 53.30, 34.73, 30.18, 21.56;
+
HRMS (ESI), m/z calcd for C18
H20ClN
3
O (M+H) 330.1363, found 330.1368.
2
-Chloro-N-(2-(3-methylpiperidin-1-yl)phenyl)nicotinamide (1h). Yield 76%; white powder; mp
1
9
2–94 °C; H-NMR (CDCl ) δ 9.71 (s, 1H, NH), 8.56 (dd, J = 7.9, 1.4 Hz, 1H, pyridyl-H), 8.52 (dd,
3
J = 4.8, 2.0 Hz, 1H, pyridyl-H), 8.20 (dd, J = 7.7, 2.0 Hz, 1H, pyridyl-H), 7.41 (dd, J = 7.7, 4.8 Hz, 1H,
Ar-H), 7.26–7.08 (m, 3H, Ar-H), 3.01–2.82 (m, 2H, CH ), 2.63 (td, J = 11.4, 2.9 Hz, 1H, CH),
), 1.03 (dd, J = 11.8, 2.5 Hz, 1H, CH), 0.88 (d,
) δ 162.13, 150.79, 146.62, 142.54, 139.58, 133.24, 131.86,
2
2
.42–2.22 (m, 1H, CH), 1.89–1.61 (m, 4H, CH
2
13
J = 6.5 Hz, 3H, CH
3
); C-NMR (CDCl
3
1
25.18, 124.24, 122.60, 120.93, 119.13, 61.05, 53.29, 32.19, 31.64, 25.85, 19.06; HRMS (ESI), m/z
+
calcd for C18
H
20ClN O (M+H) 330.1362, found 330.1368.
3
2
9
2
7
4
1
-Chloro-N-(2-(3-methylpiperidin-1-yl)phenyl)nicotinamide (1i). Yield 83%; white powder; mp
1
0–92 °C; H-NMR (CDCl
.0 Hz, 1H, pyridyl-H), 8.20 (dd, J = 7.7, 2.0 Hz, 1H, pyridyl-H), 7.42 (dd, J = 7.7, 4.8 Hz, 1H, Ar-H),
.25–7.08 (m, 3H, Ar-H), 2.90 (d, J = 7.2 Hz, 2H, CH ), 2.24 (t, J = 11.0 Hz, 2H, CH), 1.89–1.72 (m,
); C-NMR (CDCl ) δ 162.12, 150.79, 146.56, 142.28, 139.62,
3
) δ 9.71 (s, 1H, NH), 8.61–8.54 (m, 1H, pyridyl-H), 8.52 (dd, J = 4.8,
2
1
3
H, CH
2
), 0.88 (d, J = 6.4 Hz, 6H, CH
3
3
33.25, 131.87, 125.20, 124.23, 122.61, 120.93, 120.82, 119.14, 60.62, 41.47, 31.75, 19.04; HRMS
+
(
ESI), m/z calcd for C19
H22ClN
3
O (M+H) 344.1527, found 344.1524.
1
6
-Chloro-N-(2-morpholinophenyl)nicotinamide (2a). Yield 75%; white powder; mp 133–135 °C; H-NMR
) δ 9.60 (s, 1H, NH), 9.09 (ddd, J = 9.5, 2.4, 0.7 Hz, 1H, pyridyl-H), 8.92 (dd, J = 2.5, 0.7 Hz,
H, pyridyl-H), 8.68–8.44 (m, 1H, pyridyl-H), 8.39–8.17 (m, 2H, Ar-H), 7.54 (ddd, J = 8.3, 6.4, 0.7 Hz,
(CDCl
3
1
1
1
3
H, Ar-H), 7.22–7.11 (m, 1H, Ar-H), 4.07–3.58 (m, 4H, CH
2
), 3.10–2.69 (m, 4H, CH ). C-NMR
2
(
CDCl
3
) δ162.51, 154.26, 147.47, 140.62, 137.81, 132.48, 129.62, 124.61, 124.43, 124.30, 120.21,
+
1
19.03, 66.91, 53.24; HRMS (ESI), m/z calcd for C16
H16ClN
3
O
2
(M+H) 318.1004, found 318.1000.
6
-Chloro-N-(2-(piperidin-1-yl)phenyl)nicotinamide (2b). Yield 76%; white powder; mp 126–128 °C;
) δ 9.70 (s, 1H, NH), 8.92 (dd, J = 2.5, 0.6 Hz, 1H, pyridyl-H), 8.50 (dd, J = 7.9,
.5 Hz, 1H, pyridyl-H), 8.28 (dd, J = 8.3, 2.5 Hz, 1H, pyridyl-H), 7.51 (dd, J = 8.3, 0.7 Hz, 1H, Ar-H),
.24–7.05 (m, 3H, Ar-H), 2.92–2.71 (m, 4H, CH ), 1.84–1.69 (m, 4H, CH ), 1.61 (d, J = 16.7 Hz, 2H,
) δ 162.17, 151.01, 147.62, 142.52, 141.21, 134.12, 132.20, 126.24, 124.81,
1H-NMR (CDCl
3
1
7
2
2
1
3
CH
2
). C-NMR (CDCl
3

Molecules 2015, 20
4081
O (M+H) 316.1211,
+
1
22.42, 121.05, 120.11, 55.87, 27.64, 24.66; HRMS (ESI), m/z calcd for C17
H18ClN
3
found 316.1205.
6
1
1
-Chloro-N-(2-(2,6-dimethylmorpholino)phenyl)nicotinamide (2d). Yield 77%; yellow powder; mp
1
47–148 °C; H-NMR (CDCl
3
) δ 9.65 (s, 1H, NH), 8.88 (d, J = 2.5 Hz, 1H, pyridyl-H), 8.58–8.46 (m,
H, pyridyl-H), 8.25 (dd, J = 8.3, 2.5 Hz, 1H, pyridyl-H), 7.51 (d, J = 8.3 Hz, 1H, Ar-H), 7.17 (ddd,
), 2.82 (d, J = 10.8 Hz, 2H,
). C-NMR (CDCl ) δ162.45, 152.37, 146.31,
J = 15.3, 9.6, 4.6 Hz, 3H, Ar-H), 3.84 (dtd, J = 12.6, 6.3, 4.2 Hz, 2H, CH
2
1
3
CH
2
), 2.69–2.45 (m, 2H, CH), 1.23 (d, J = 6.3 Hz, 6H, CH
3
3
1
40.60, 137.75, 133.52, 131.61, 125.18, 124.43, 124.14, 121.41, 120.13, 69.14, 56.57, 17.56; HRMS
+
(
ESI), m/z calcd for C18
H20ClN
3
O
2
(M+H) 346.1317, found 346.1312.
6
-Chloro-N-(2-(pyrrolidin-1-yl)phenyl)nicotinamide (2e). Yield 81%; white powder; mp 102–104 °C;
) δ 9.28 (s, 1H, NH), 8.85 (d, J = 2.1 Hz, 1H, pyridyl-H), 8.36 (d, J = 6.9 Hz, 1H,
pyridyl-H), 8.22 (dd, J = 8.3, 2.4 Hz, 1H, pyridyl-H), 7.48 (d, J = 8.3 Hz, 1H, Ar-H), 7.33–7.01 (m, 3H,
1H-NMR (CDCl
3
1
3
Ar-H), 3.05 (d, J = 5.7 Hz, 4H, CH
2
), 2.20–1.84 (m, 4H, CH
2
); C-NMR (CDCl ) δ 161.56, 154.06,
3
1
47.43, 140.32, 137.81, 132.39, 129.60, 124.60, 124.33, 124.20, 120.11, 119.63, 52.53, 24.24; HRMS
+
(
ESI), m/z calcd for C16
H
16ClN O (M+H) 302.1051, found 302.1055.
3
6
-Chloro-N-(2-(2-methylpiperidin-1-yl)phenyl)nicotinamide (2g) Yield 82%; white powder; mp
1
1
00–102 °C; H-NMR (CDCl
3
) δ 10.22 (s, 1H, NH), 8.92 (dd, J = 2.5, 0.7 Hz, 1H, pyridyl-H), 8.55 (dd,
J = 8.0, 1.4 Hz, 1H, pyridyl-H), 8.28 (dd, J = 8.3, 2.5 Hz, 1H, pyridyl-H), 7.51 (dd, J = 8.3, 0.7 Hz, 1H,
Ar-H), 7.26 (dq, J = 4.3, 1.6 Hz, 2H, Ar-H), 7.13 (td, J = 7.6, 1.6 Hz, 1H, Ar-H), 3.04–2.81 (m, 2H,
CH
2
), 2.69 (td, J = 11.6, 2.6 Hz, 1H, CH), 1.98–1.74 (m, 2H, CH
2
), 1.71–1.32 (m, 4H, CH
2
), 0.81 (d, J
1
3
=
6.2 Hz, 3H, CH
3
); C-NMR (CDCl
3
) δ 161.11, 154.05, 147.26, 140.40, 137.96, 134.96, 129.73, 125.94,
1
24.41, 123.86, 122.59, 118.36, 56.29, 55.30, 35.37, 27.23, 24.18, 19.97; HRMS (ESI), m/z calcd for
+
C
18
H20ClN
3
O (M+H) 330.1363, found 330.1368.
6
-Chloro-N-(2-(3-methylpiperidin-1-yl)phenyl)nicotinamide (2h) Yield 85%; white powder; mp
1
9
0–91 °C; H-NMR (CDCl
3
) δ 9.71 (s, 1H, NH), 8.90 (dd, J = 2.5, 0.6 Hz, 1H, pyridyl-H), 8.69–8.42
(
m, 1H, pyridyl-H), 8.27 (dd, J = 8.3, 2.5 Hz, 1H, pyridyl-H), 7.51 (dd, J = 8.3, 0.7 Hz, 1H, Ar-H),
7
.25–7.06 (m, 3H, Ar-H), 3.01–2.87 (m, 2H, CH
.92–1.81 (m, 2H, CH ), 1.80–1.57 (m, 2H, CH ), 1.09 (d, J = 9.8 Hz, 1H, CH), 0.93 (d, J = 6.5 Hz, 3H,
); C-NMR (CDCl ) δ 161.31, 154.09, 147.25, 142.27, 137.96, 132.96, 129.68, 125.31, 124.41,
2
), 2.77–2.57 (m, 1H, CH
2
), 2.49–2.29 (m, 1H, CH ),
2
1
2
2
1
3
CH
3
3
1
24.10, 120.70, 118.82, 60.75, 53.21, 32.33, 32.19, 26.40, 19.10; HRMS (ESI), m/z calcd for
+
C
18
H20ClN
3
O (M+H) 330.1363, found 330.1368.
N-(2-(1H-Pyrrol-1-yl)phenyl)-6-chloronicotinamide (2j). Yield 81%; yellow powder; mp 150–152 °C;
1H-NMR (CDCl
) δ 8.60–8.50 (m, 2H, pyridyl-H), 7.95 (dd, J = 8.3, 2.6 Hz, 1H, pyridyl-H), 7.63 (s, 1H,
NH), 7.53–7.35 (m, 3H, Ar-H), 7.31–7.20 (m, H, Ar-H), 6.84 (t, J = 2.1 Hz, 2H, pyrrole-H), 6.47 (t, J =
3
1
3
2
1
.1 Hz, 2H, pyrrole-H). C-NMR (CDCl ) δ 161.22, 154.12, 147.40, 141.32, 138.12, 132.34, 129.42,
3
24.57, 124.23, 124.05, 120.19, 120.86, 119.41, 111.27; HRMS (ESI), m/z calcd for C16
H
12ClN O
3
+
(
M+H) 298.0742, found 298.0742.

Molecules 2015, 20
4082
N-(2-Morpholinophenyl)-2-(trifluoromethyl)benzamide (3a). Yield 86%; yellow powder; mp
1
1
12–114 °C; H-NMR (300 MHz, CDCl
3
) δ 8.93 (s, 1H, NH), 8.54 (d, J = 8.1 Hz, 1H, Ar-H), 7.78 (d,
J = 7.5 Hz, 1H, Ar-H), 7.65 (dd, J = 4.8, 3.1 Hz, 3H, Ar-H), 7.26–7.21 (m, 2H, Ar-H), 7.19–7.11 (m,
1
3
1
1
5
H, Ar-H), 3.88–3.60 (m, 4H, CH
2
), 2.96–2.76 (m, 4H, CH
2
); C-NMR (CDCl ) δ 165.17, 140.87,
3
35.92, 133.36, 132.00, 129.87, 128.14, 126.61, 126.37, 126.31, 125.81, 124.26, 120.81, 119.54, 67.10,
+
2.47; HRMS (ESI), m/z calcd for C18
H
17
F
3
N
2
O
2
(M+H) 351.1310, found 351.1315.
N-(2-(Piperidin-1-yl)phenyl)-2-(trifluoromethyl)benzamide (3b). Yield 77%; brown powder; mp
1
8
1
6
1
3
0–82 °C; H-NMR (CDCl
3
) δ 8.92 (s, 1H, NH), 8.50 (dd, J = 6.8, 2.8 Hz, 1H, Ar-H), 7.77 (d, J = 7.7 Hz,
), 1.78–1.37 (m,
) δ 165.18, 142.68, 133.19, 131.91, 129.74, 127.89, 126.48, 126.41, 126.35,
H, Ar-H), 7.71–7.53 (m, 3H, Ar-H), 7.24–7.06 (m, 3H, Ar-H), 2.98–2.61 (m, 4H, CH
2
1
3
H, CH
2
); C-NMR (CDCl
3
24.91, 124.04, 120.45, 119.23, 53.56, 26.43, 23.60; HRMS (ESI), m/z calcd for C19
H
19
F
3
N
O (M+H)+
2
49.1518, found 349.1522.
N-(2-(1H-Indazol-1-yl)phenyl)-2-(trifluoromethyl)benzamide (3c). Yield 76%; yellow powder; mp
1
1
39–141 °C; H-NMR (CDCl
3
) δ 9.55 (s, 1H, NH), 8.68-8.50 (m, 1H, Ar-H), 8.15 (d, J = 0.8 Hz, 1H,
lndazole-H), 7.83–7.76 (m, 1H, Ar-H), 7.74–7.67 (m, 1H, Ar-H), 7.63 (dd, J = 8.5, 0.9 Hz, 1H, Ar-H),
.60–7.51 (m, 3H, Ar-H), 7.51–7.41 (m, 3H, Ar-H), 7.36–7.28 (m, 1H, Ar-H), 7.27–7.21 (m, 1H, Ar-H);
13C-NMR (CDCl
) δ 165.39, 139.66, 135.65, 132.12, 131.77, 129.89, 128.95, 128.15, 127.71, 127.69,
27.63, 126.46, 126.40, 124.66, 124.22, 124.11, 123.54, 121.90, 121.08, 110.47; HRMS (ESI), m/z calcd
7
3
1
+
for C21
H
14
F
3
N O (M+H) 382.115, found 382.1162.
3
N-(2-(2,6-Dimethylmorpholino)phenyl)-2-(trifluoromethyl)benzamide (3d). Yield 86%; yellow powder;
1
mp 100–102 °C; H-NMR (CDCl
3
) δ 8.86 (s, 1H, NH), 8.59–8.42 (m, 1H, Ar-H), 7.77 (d, J = 7.4 Hz,
), 2.77 (d,
); C-NMR (CDCl ) δ
1
H, Ar-H), 7.72–7.57 (m, 3H, Ar-H), 7.25–7.03 (m, 3H, Ar-H), 3.73–3.54 (m, 2H, CH
2
1
3
J = 10.8 Hz, 2H, CH
2
), 2.60–2.36 (m, 2H, CH
2
), 1.17 (d, J = 6.3 Hz, 6H, CH
3
3
1
65.18, 140.62, 133.29, 131.98, 129.84, 128.07, 127.00, 126.36, 126.30, 125.68, 124.27, 120.77, 119.60,
+
7
2.04, 57.97, 18.54; HRMS (ESI), m/z calcd for C20
H
21
F
3
N O
2 2
(M+H) 379.1621, found 379.1628.
N-(2-(Pyrrolidin-1-yl)phenyl)-2-(trifluoromethyl)benzamide (3e). Yield 88%; white powder; mp
1
1
30–131 °C; H-NMR (MHz, CDCl
3
) δ 8.61 (s, 1H, NH), 8.41 (dd, J = 7.6, 1.8 Hz, 1H, Ar-H), 7.77 (d,
J = 7.5 Hz, 1H, Ar-H), 7.63 (dd, J = 12.8, 4.1 Hz, 3H, Ar-H), 7.14 (ddd, J = 14.2, 6.8, 2.0 Hz, 3H,
1
3
Ar-H), 3.01 (t, J = 6.6 Hz, 4H, CH
2
), 1.98–1.77 (m, 4H, CH
2
); C-NMR (CDCl
3
) δ 165.36, 140.42,
1
32.78, 131.90, 129.79, 129.69, 128.13, 126.36, 126.29, 124.55, 124.18, 120.58, 119.66, 52.59, 24.15;
+
HRMS (ESI), m/z calcd for C18
H
17
F
3
2
N O (M+H) 335.1361, found 335.1366.
N-(2-(4-Methylpiperidin-1-yl)phenyl)-2-(trifluoromethyl)benzamide (3f). Yield 74%; white powder; mp
1
7
7–79 °C; H-NMR (CDCl
3
) δ 8.88 (s, 1H, NH), 8.63-8.39 (m, 1H, Ar-H), 7.78 (d, J = 7.5 Hz, 1H,
), 2.69 (dd,
), 1.54–1.41 (m, 1H, CH), 1.28–1.04 (m, 2H,
) δ 165.19, 142.43, 133.18, 131.91, 129.75, 127.90,
Ar-H), 7.72–7.55 (m, 3H, Ar-H), 7.24–7.04 (m, 3H, Ar-H), 2.94 (d, J = 11.8 Hz, 2H, CH
J = 11.8, 2.3 Hz, 2H, CH ), 1.71 (d, J = 10.8 Hz, 2H, CH
CH ), 0.92 (d, J = 6.5 Hz, 3H, CH
2
2
2
1
3
2
3
); C-NMR (CDCl
3
1
26.89, 126.43, 126.36, 126.30, 124.91, 124.05, 120.43, 119.30, 52.94, 34.78, 30.13, 21.49; HRMS
+
(
ESI), m/z calcd for C20
H
21
F
3
N
2
O (M+H) 363.1675, found 363.1675.

Molecules 2015, 20
4083
2
-Chloro-N-(2-morpholinophenyl)isonicotinamide (4a). Yield 76%; yellow powder; mp 134–136 °C;
1H-NMR (CDCl
) δ 9.62 (s, 1H, NH), 8.61 (dd, J = 5.1, 0.6 Hz, 1H, pyridyl-H), 8.51 (d, J = 8.0 Hz, 1H,
3
pyridyl-H), 7.81 (d, J = 0.8 Hz, 1H, pyridyl-H), 7.71–7.60 (m, 1H, Ar-H), 7.32–7.00 (m, 3H, Ar-H),
1
3
4
1
.07–3.78 (m, 4H, CH
2
), 3.13–2.45 (m, 4H, CH
2
). C-NMR (CDCl ) δ161.51, 152.23, 150.35, 146.14,
3
41.24, 132.27, 125.18, 124.34, 121.56, 120.54, 120.24, 119.07, 63.32, 56.40; HRMS (ESI), m/z calcd
+
for C16
H16ClN
3
O
2
(M+H) 318.1004, found 318.1005.
2
-Chloro-N-(2-(piperidin-1-yl)phenyl)isonicotinamide (4b). Yield 84%; white powder; mp 90–92 °C;
1H-NMR (CDCl
) δ 9.71 (s, 1H, NH), 8.60 (d, J = 5.1 Hz, 1H, pyridyl-H), 8.55–8.45 (m, 1H,
pyridyl-H), 7.83 (d, J = 0.8 Hz, 1H, pyridyl-H), 7.66 (dd, J = 5.1, 1.5 Hz, 1H, Ar-H), 7.25–7.09 (m, 3H,
Ar-H), 2.93–2.80 (m, 4H, CH ), 1.78 (dd, J = 10.7, 5.4 Hz, 4H, CH ), 1.63 (d, J = 18.5 Hz, 2H, CH );
3
2
2
2
13C-NMR (CDCl
) δ 160.80, 152.53, 150.40, 145.08, 142.64, 132.57, 125.26, 124.43, 121.86, 120.67,
3
+
1
19.04, 118.89, 53.64, 26.94, 23.65; HRMS (ESI), m/z calcd for C17
H18ClN
3
O (M+H) 316.1211,
found 316.1211.
2
1
1
-Chloro-N-(2-(pyrrolidin-1-yl)phenyl)isonicotinamide (4e). Yield 80%; yellow powder; mp
1
3
08–110 °C; H-NMR (CDCl ) δ 9.28 (s, 1H, NH), 8.58 (d, J = 5.1 Hz, 1H, pyridyl-H), 8.36 (d, J = 7.3 Hz,
H, pyridyl-H), 7.77 (s, 1H, pyridyl-H), 7.62 (d, J = 5.1 Hz, 1H, Ar-H), 7.31–7.11 (m, 3H, Ar-H), 3.07
1
3
(
t, J = 6.1 Hz, 4H, CH
2
), 2.20–1.84 (m, 4H, CH
2
). C-NMR (CDCl ) δ 160.45, 151.83, 150.35, 145.45,
3
1
42.26, 132.37, 125.26, 124.85, 121.57, 121.16, 119.04, 118.89, 53.35, 23.83; HRMS (ESI), m/z calcd
+
for C16
H16ClN
3
O (M+H) 302.1055, found 302.1054.
2
-Chloro-N-(2-(4-methylpiperidin-1-yl)phenyl)isonicotinamide (4f). Yield 85%; white powder; mp
1
8
0–82 °C; H-NMR (CDCl ) δ 9.72 (s, 1H, NH), 8.60 (dd, J = 5.1, 0.7 Hz, 1H, pyridyl-H), 8.48 (dd,
3
J = 7.9, 1.5 Hz, 1H, pyridyl-H), 7.82 (dd, J = 1.5, 0.7 Hz, 1H, pyridyl-H), 7.68 (dd, J = 5.1, 1.5 Hz, 1H,
Ar-H), 7.26–7.09 (m, 3H, Ar-H), 2.97 (d, J = 12.0 Hz, 2H, CH ), 2.76 (td, J = 11.7, 2.3 Hz, 2H, CH ),
), 1.71–1.53 (m, 1H, CH), 1.46-1.20 (m, 2H, CH ), 1.05 (d, J = 6.5 Hz,
) δ 160.41, 152.37, 150.25, 145.42, 140.46, 134.74, 125.72, 124.24, 122.59,
2
2
1
3
1
3
.87 (d, J = 13.1 Hz, 2H, CH
2
2
1
3
H, CH
3
). C-NMR (CDCl
3
21.63, 119.44, 118.72, 56.52, 35.71, 33.20, 20.14; HRMS (ESI), m/z calcd for C18
H
20ClN
O (M+H)+
3
30.1368, found 330.1368.
2
-Chloro-N-(2-(2-methylpiperidin-1-yl)phenyl)isonicotinamide (4g). Yield 78%; yellow powder; mp
1
8
2–84 °C; H-NMR (CDCl
3
) δ 10.25 (s, 1H, NH), 8.60 (dd, J = 5.1, 0.7 Hz, 1H, pyridyl-H), 8.54 (dd,
J = 7.9, 1.3 Hz, 1H, pyridyl-H), 7.83 (dd, J = 1.5, 0.7 Hz, 1H, pyridyl-H), 7.66 (dd, J = 5.1, 1.5 Hz, 1H,
Ar-H), 7.31–7.20 (m, 2H, Ar-H), 7.14 (td, J = 7.6, 1.6 Hz, 1H, Ar-H), 3.03–2.82 (m, 2H, CH ), 2.71 (td,
J = 11.5, 2.5 Hz, 1H, CH), 1.98–1.87 (m, 2H, CH ), 1.65–1.35 (m, 4H, CH ), 0.82 (d, J = 6.2 Hz, 3H,
) δ 160.56, 152.53, 150.39, 145.14, 140.50, 134.65, 125.96, 124.19, 122.66,
2
2
2
1
3
CH
3
); C-NMR (CDCl
3
1
21.89, 119.00, 118.40,56.27, 55.22, 35.43, 27.27, 24.20, 19.96; HRMS (ESI), m/z calcd for
+
C
18
H20ClN
3
O (M+H) 330.1367, found 330.1368.
2
8
1
-Chloro-N-(2-(3-methylpiperidin-1-yl)phenyl)isonicotinamide (4h). Yield 83%; white powder; mp
1
3
1–83 °C; H-NMR (CDCl ) δ 9.70 (s, 1H, NH), 8.59 (d, J = 5.1 Hz, 1H, pyridyl-H), 8.49 (d, J = 8.2 Hz,
H, pyridyl-H), 7.82 (s, 1H, pyridyl-H), 7.65 (dd, J = 5.1, 1.4 Hz, 1H, Ar-H), 7.24–7.05 (m, 3H, Ar-H),

Molecules 2015, 20
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2
.93 (d, J = 11.4 Hz, 2H, CH
m, 2H, CH ), 1.69 (d, J = 12.7 Hz, 2H, CH
13C-NMR (CDCl
) δ 160.86, 152.50, 150.39, 145.11, 142.35, 132.61, 125.30, 124.45, 121.84, 120.71,
19.05, 118.95, 60.66, 53.24, 32.28, 32.15, 26.32, 19.05; HRMS (ESI), m/z calcd for C18 20ClN O
2
), 2.67 (td, J = 11.4, 2.2 Hz, 1H, CH
2
), 2.54–2.31 (m, 1H, CH
2
), 1.95–1.83
(
2
2
), 1.20–1.04 (m, 1H, CH), 0.96 (d, J = 6.6 Hz, 3H, CH );
3
3
1
H
3
+
(
M+H) 330.1367, found 330.1368.
N-(2-(1H-Pyrrol-1-yl)phenyl)-2-chloroisonicotinamide (4j). Yield 82%; yellow powder; mp
1
1
20–122 °C; H-NMR (CDCl
3
) δ 8.51 (dd, J = 12.6, 6.2 Hz, 2H, pyridyl-H), 7.66 (s, 1H, NH), 7.53 (t,
J = 2.7 Hz, 1H, pyridyl-H), 7.50–7.37 (m, 2H, Ar-H), 7.35–7.20 (m, 2H, Ar-H), 6.85 (t, J = 2.1 Hz, 2H,
1
3
pyrrole-H), 6.50 (t, J = 2.1 Hz, 2H, pyrrole-H). C-NMR (CDCl
3
) δ 162.11, 152.43, 151.42, 146.55,
1
42.24, 132.14, 126.20, 124.53, 121.19, 120.16, 119.04, 118.89, 118.42, 110.63; HRMS (ESI), m/z calcd
+
for C16
H
12ClN O (M+H) 298.0742, found 298.0742.
3
1
2
-Methyl-N-(2-(piperidin-1-yl)phenyl)furan-3-carboxamide (5b). Yield 80%; yellow paste; H-NMR
CDCl ) δ 9.12 (s, 1H, NH), 8.48 (d, J = 8.1 Hz, 1H, Ar-H), 7.33 (d, J = 2.1 Hz, 1H, furan-H), 7.22–7.11
m, 2H, Ar-H), 7.06 (dd, J = 7.6, 1.5 Hz, 1H, Ar-H), 6.59 (d, J = 1.9 Hz, 1H, furan-H), 2.95–2.76 (m,
(
3
(
1
3
4
1
H, CH
2
), 2.68 (m, 2H, CH
2
), 1.86–1.70 (m, 4H, CH
2
), 1.63 (s, 2H, CH
2
); C-NMR (CDCl ) δ 161.37,
3
57.44, 140.24, 133.49, 125.07, 123.17, 120.33, 116.19, 107.92, 53.57, 26.86, 23.71, 13.41; HRMS
+
(
ESI), m/z calcd for C17
H
20
N
2
O
2
(M+H) 285.1596, found 285.1598.
2
-Methyl-N-(2-(4-methylpiperidin-1-yl)phenyl)furan-3-carboxamide (5f). Yield 84%; yellow paste;
) δ 9.06 (s, 1H, NH), 8.48 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.33 (d, J = 2.0 Hz, 1H,
furan-H), 7.23–7.10 (m, 2H, Ar-H), 7.04 (td, J = 7.6, 1.6 Hz, 1H, Ar-H), 6.56 (d, J = 2.0 Hz, 1H,
furan-H), 2.97 (d, J = 11.9 Hz, 2H, CH ), 2.72 (dt, J = 6.7, 3.4 Hz, 2H, CH ), 1.82 (d, J = 14.3 Hz, 2H,
CH ), 1.65–1.50 (m, 1H, CH), 1.35 (ddd, J = 15.3, 11.9, 3.9 Hz, 2H, CH ), 1.03 (d, J = 6.5 Hz, 3H, CH );
13C-NMR (CDCl
) δ 161.34, 157.37, 141.88, 140.25, 133.53, 125.01, 123.12, 120.31, 118.74, 116.23,
07.86, 77.21, 76.79, 76.36, 52.91, 35.31, 30.23, 21.71, 13.42; HRMS (ESI), m/z calcd for C18
1H-NMR (CDCl
3
2
2
2
2
3
3
1
H
22
N
2
O
2
+
(
M+H) 299.1752, found 299.1754.
2
-Methyl-N-(2-(3-methylpiperidin-1-yl)phenyl)furan-3-carboxamide (5h). Yield 76%; yellow paste;
) δ 9.10 (s, 1H, NH), 8.53–8.40 (m, 1H, Ar-H), 7.32 (d, J = 2.0 Hz, 1H, furan-H),
.22–7.11 (m, 2H, Ar-H), 7.06 (dd, J = 7.6, 1.6 Hz, 1H, Ar-H), 6.56 (d, J = 2.0 Hz, 1H, furan-H), 2.93
d, J = 11.2 Hz, 2H, CH ), 2.66–2.56 (m, 1H, CH ), 2.43–2.28 (m, 1H, CH ), 1.83 (ddd, J = 9.8, 7.4, 2.5
Hz, 4H, CH ), 1.17–1.03 (m, 1H, CH), 0.94 (d, J = 6.5 Hz, 3H, CH
57.44, 141.89, 140.22, 133.60, 125.07, 123.11, 120.40, 118.69, 116.21, 107.86, 77.21, 76.78, 76.36,
1H-NMR (CDCl
3
7
(
2
2
2
1
3
2
3
); C-NMR (CDCl
3
) δ 161.34,
(M+H)+
1
6
2
0.64, 53.15, 32.34, 32.29, 26.41, 19.13, 13.40; HRMS (ESI), m/z calcd for C18
H
22
N
2
O
2
99.1753, found 299.1754.
3
.3. Bioassays
The six kinds of test fungi were provided by the Laboratory of Institute of Plant Protection, Chinese
Academy of Agricultural Sciences (Beijing, China). After retrieval from the storage tube, the strains
were incubated on potato dextrose agar (PDA) at 25 °C for several days to get new mycelia for the

Molecules 2015, 20
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antifungal tests. The fungicidal activity of the target compounds was tested in vitro against the six plant
pathogenic fungi using the mycelia growth inhibition method [22]. The tested compounds were dissolved
−1
in DMSO to prepare a 10 mg·mL stock solution before mixing with PDA. The media containing
−1
compounds at a concentration of 50 μg·mL were then poured into sterilized Petri dishes for initial
screening. After two days at 25 °C, the colony diameter of each strain was measured. Percentage
inhibition rate was calculated as (1 − a/b) × 100%, where a represents the colony diameter in the Petri
dishes with tested compounds and b is the mean colony diameter in control Petri dishes. Each test was
−1
−1
repeated three times. The 10 mg·mL solution was diluted to 200, 100, 50, 25, 12.5, 6.25 μg·mL and
the above experiments were repeated, the inhibition rates were calculated separately. The EC50 values
were calculated using SPSS Statistics v17.0.
3
.4. QSAR Analyses
D QSAR analyses were performed to predict the favorable and unfavorable moieties for improved
3
bioactivity using the comparative molecular field analysis (CoMFA) and the comparative molecular
similarity indices analysis (CoMSIA) models in the SYBYL 7.3 software [23]. CoMFA models were
generated using the Sybyl 7.3 package on a Linux system. In total, 21 compounds obtained from synthesis
were used to create a data set in which the bioactivity of all compounds was determined (Table 1) against
Pythium aphanidermatum and Rhizoctonia solani. The pEC50 values were used for constructing the
models. Three-dimensional molecular structures were built using the SKETCH module in Sybyl 7.3,
while structural energy minimization was performed with the Tripos force field until a gradient
convergence of 0.05 kcal/(mol A) was achieved. Gasteiger−Hückel charges were calculated and used to
construct the CoMFA models [24].
3.5. Molecular Docking
Docking was performed with Surflex-Dock (SYBYL 7.3). The molecular structures were
energetically minimized using Tripos with 1000 iterations and a minimum gradient of 0.005. The
Gasteiger−Hückel charges of ligands were assigned. All bound water and ligands were eliminated from
the protein, and the polar hydrogen atoms and the Kollman-united charges were added to the proteins.
4
. Conclusions
Based on bioisosterism and the computational docking experiments of commercial amide fungicides,
we designed and synthesized a series of novel aromatic amides by exchanging the biphenyl group of
boscalid for a nitrogen-containing heterocyclic ring. The bioassays showed that all of the target
molecules exhibited considerable in vitro antibacterial activity against the six kinds of fungi. Compounds
1
c and 3c with an indazolyl group exhibited higher inhibition activity than others, indicating the potential
value of this moiety in agrochemical applications. A molecular docking study showed that the total
docking score of compound 3c was higher than that of boscalid and the binding of the indazolyl aniline
moiety to the receptor was tighter than that of a phenylaniline in the SDH ligand binding pocket. The
indazolyl group was thus very favorable for the binding of compound 3c with SDH; although compound
3
c exhibited slightly lower activity than boscalid, indicating that improving some physical and chemical

Molecules 2015, 20
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properties of compound 3c, such as penetration, might improve its bioactivity. Further synthesis and
structural optimization studies are ongoing in our laboratory.
Acknowledgments
This work was supported by the National Technology R&D Program in the 12th Five year plan of
China (grant No. 2011BAE06B02-21).
Author Contributions
Z.Q. designed research; S.D., X.G., C.W., C.J. and M.W. performed the synthesis research; H.L. and
X.L. performed the bioassay research and analyzed the data; S.D., H.Z.L. and D.Y. wrote the paper. All
authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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©
2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/4.0/).
(