Design, synthesis, antifungal activity and 3D-QSAR study of novel pyrazole carboxamide and niacinamide derivatives containing benzimidazole moiety

Article abstract of DOI:10.1039/c8nj05150j

A series of novel pyrazole carboxamide and niacinamide derivatives containing a benzimidazole moiety were designed and synthesized as antifungal candidate agents. All target compounds were characterized by FTIR, ¹H NMR, ¹³C NMR, HRMS and elemental analysis techniques. The structure of compound T1 was further confirmed by single crystal X-ray diffraction analysis. The antifungal activities of the target compounds were evaluated in vitro against four phytopathogenic fungi (namely Botrytis cinerea, Rhizoctonia solani, Fusarium graminearum and Alternaria solani) by the mycelium growth inhibition method. The bioassay results indicated that some of the compounds exhibited good antifungal activity against B. cinerea at 100 μg ML^?1 compared to other three fungi. In order to better explore the structure-activity relationship (SAR), the EC₅₀ values of target compounds against B. cinerea were measured and assessed. Subsequently, a 3D quantitative structure-activity relationship (3D-QSAR) study was carried out using the comparative molecular field analysis (CoMFA) technique based on the inhibitory activities of tested compounds against B. cinerea. Molecular modelling results revealed fine predictive ability with cross-validated q² and non-cross-validated r² values of 0.578 and 0.850, respectively.

Full text of DOI:10.1039/c8nj05150j

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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
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Design, synthesis, antifungal activity and 3D-QSAR study of novel
pyrazole carboxamide and niacinamide derivatives containing
benzimidazole moiety
Received 00th January 20xx,
Accepted 00th January 20xx
Wei-Jie Si,abc Xiao-bin Wang,ab Min Chen,ab Meng-Qi Wang,a Ai-Min Lua and Chun-Long Yang*abc
DOI: 10.1039/x0xx00000x
www.rsc.org/
A series of novel pyrazole carboxamide and niacinamide derivatives containing a benzimidazole moiety were designed and
1
synthesized as the antifungal candidate agents. All the target compounds were characterized by FTIR, H NMR, 13C NMR ,
HRMS spectra and elemental analysis. The structure of compound T1 was further confirmed by single crystal X-ray
diffraction analysis. The antifungal activities of the target compounds were evaluated in vitro against four phytopathogenic
fungi (namely Botrytis cinerea, Rhizoctonia solani, Fusarium graminearum and Alternaria solani) by the mycelium growth
inhibition method. The bioassay results indicated that some of the compounds exhibited good antifungal activity against B.
cinerea at 100 ug/mL compared to other three fungi. In order to better explore the structure-activity relationship (SAR),
the EC50 values of target compounds against B. cinerea were further tested and assessed. Subsequently, a 3D quantitative
structure–activity relationship (3D-QSAR) study was carried out using the comparative molecular field analysis (CoMFA)
technique based on the inhibitory activities against B. cinerea. The analysis results from the molecular model revealed fine
predictive ability with cross-validated q2 and non-cross-validated r2 values of 0.578 and 0.850, respectively.
The benzimidazole derivatives have been attracting the
attention of organic chemists and pharmacologists for new
drug discovery owing to their broad spectrum biological
Introduction
Plant diseases caused by fungi are increasingly recognized as
a worldwide threat to crop safety and food security, and has
been exacerbated by the increasing frequency of agricultural
activities and global trade.1,2 The use of synthetic pesticides
has restrained the occurrence and development of plant
fungal diseases, improved the yield and quality of crops,3 and
reduced agricultural economic losses by fighting against
highly destructive plant pathogens, such as Botrytis cinerea,4
Rhizoctonia solani,5 Fusarium graminearum,6 and Alternaria
solani.7 For example, Botrytis cinerea, as an important
pathogen of nursery plants, vegetables, ornamental, field,
and orchard crops, infects its host plants in all climate areas
of the world, infects mainly upper plant parts at pre and post-
harvest stages and causes serious damage.8,9 However, the
emergence of drug-resistance, environmental hazards and
other problems objectively indicated the urgent need for
novel fungicidal candidates.10,11
activity and extensive applications,12 such as, anticancer,13
anti-inflammatory,14 antiviral,15 anti-tubercular,16
antibacterial,17 and antifungal activity.18 In addition, several
commercial fungicides containing the benzimidazole scaffold
have been applied in the plant disease control, including
carbendazim, thiabendazole, and benomyl (Fig. 1).
Meanwhile, pyrazole and pyridine, as the biologically active
substructures, are usually found in the structures of
fungicides. For example, furametpyr ( Sumitomo Chemical Co.,
Ltd., 1997) contains
a 5-chloro-1,3-dimethyl-4-pyrazole
a. Department of Chemistry, College of Sciences, Nanjing Agricultural University,
Nanjing 210095, China.
b. Jiangsu Key Laboratory of Pesticide Science, Nanjing Agricultural University,
Nanjing 210095, China.
c. Key Laboratory of Monitoring and Management of Crop Diseases and Pest
Insects, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095,
China.
d. E-mail: ycl@njau.edu.cn. Tel: +86-25-84395207.
†Electronic Supplementary Information (ESI) available: The copies of 1H NMR,
13C NMR, and HRMS spectrograms for all the title compounds
can be found in the ESI. See DOI: 10.1039/x0xx00000x
Fig. 1 Commercial fungicides containing benzimidazole, pyrazole or
pyridine moiety.
This journal is © The Royal Society of Chemistry 20xx
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carboxamide derivatives, such as the compounds VAiewanArdticBle,Oanllsinoe
3
4
5
6
7
8
9
F
DOI: 10.1039/C8NJ05150J
possessed fungistatic activity (Fig. 2).19,20
O
O
O
In the present work, the strategy is to design a novel type of
carboxamide compounds with new vitality in antifungal
activity via the combination of benzimidazole scaffold with a
pyrazole or pyridine moiety through a bridge of formyl aniline
group (Fig. 3). In order to better explore the relationship
between the effects on the bioactivity caused by structural
changes, the pyrazole-4-carboxamide, pyrazole-5-carboxamide
and niacinamide derivatives with different substituent groups
were synthesized respectively. The antifungal activities of the
synthesized compounds were evaluated in vitro against four
important phytopathogenic fungi, namely B. cinerea, R. solani,
F. graminearum and A. solani. The 3D-QSAR based on the
inhibitory activities of target compounds against B. cinerea
was also discussed in detail.
NH
NH
F2HC
F2HC
F2HC
NH
F
N
N
N
N
N
N
F
F
Cl
Bixafen
Cl
Fluxapyroxad
Sedaxane
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O
Cl
O
H3COOC
N
O
N
H
N
H
N
N
N
OCH2CH3
A
B
Fig. 2 Representative pyrazole carboxamide compounds with antifungal
activities.
R4
R2
O
O
O
R3
N
H
N
H
N
H
N
N
N
N
N
N
NH
Cl
N
NH
R1
N
NH
R1
Results and discussion
Chemistry
R1
The synthetic approaches for the two different series of
R1=H, Cl, Me; R2=Me, CF3; R3=Me, CH2CHMe2; R4=H, Cl, SH.
pyrazole-4-carbonyl chlorides
5 and pyrazole-5-carbonyl
T7-T12
T13-T20
T1-T6
chlorides 12 are outlined in Scheme 1. The intermediates
pyrazole-4-carbonyl chlorides 5a and 5b were prepared using
an optimized four-step procedure.21,22 The raw material
methylhydrazine was firstly condensed with ethyl acetoacetate
1a or ethyl 4,4,4-trifluoroacetoacetate 1b to form 5-hydroxyl-
1H-pyrazoles 2a and 2b, which were treated respectively with
POCl3 and DMF (Vilsmeier-Haack chloroformylation) to give
pyrazole-4-formaldehydes 3a and 3b. The compound 3 was
then treated with potassium permanganate to provide
pyrazole-4-carboxylic acids 4a and 4b. The intermediates 5
were finally obtained by the reaction of the acids 4 with
thionyl chloride.
The intermediates pyrazole-5-carbonyl chlorides 12a and
12b were prepared using an optimized five-step
procedure.23,24 The raw material diethyl oxalate 6 was firstly
reacted with acetone 7a or 4-methyl-2-pentanone 7b in the
presence of sodium ethoxide to produce ethyl 2,4-
dioxovalerate 8a and ethyl 6-methyl-2,4-dioxoheptanoate 8b,
which were further reacted respectively with hydrazine
Fig. 3 General structural formulas of target compounds T1-T20.
moiety (Fig 1); penthiopyrad ( Mitsui Chemicals Co., Ltd., 1996)
contains a 1-methyl-3-trifluoromethyl-4-pyrazole moiety (Fig
1); boscalid ( BASF, 2003) contains a 2-chloropyridine moiety
(Fig. 1), and these substructures are both common
pharmacophores in drug development.
The carboxamide fungicides, as a class of well-known
fungicides, still play an important role in the agricultural field
nowadays, and they have been recently developed with novel
structures and superior performance. Sedaxane (Syngenta),
fluxapyroxad (BASF), and bixafen (Bayer) were the newly
registered fungicides in 2017 and all belonged to the amide
compounds (Fig. 2). It is worth noting that most of the
commercially available pyrazole amide fungicides are pyrazole-
4-carboxamide derivatives. In other words, they possess a
carboxamide functional group in 4-position of pyrazole ring. In
addition, some studies have shown that pyrazole-5-
CHO
COOH
Cl
COCl
Cl
R2
R2
O
O
R2
R2
i
iv
iii
ii
5a R2=Me,
5b R2=CF3.
OH
Cl
R2
O
N
N
N
N
N
N
N
N
1
2
3
4
5
O
O
O
O
R3
O
O
R3
COCl
O
R3
COOH
viii
ix
v
vi
vii
O
O
O
R3
O
+
R3
N
R3
N
N
N
N
N
N
O
N
H
O
8
9
10
12
12a R3=Me, 12b R3=CH2CHMe2.
6
7
11
Scheme 1 Synthetic route to pyrazole intermediates 5 and 12. Reagents and conditions: (i) methyl hydrazine, EtOH, 65 °C for 3 h, then reflux for 5 h;
(ii) POCl3, DMF, rt for 1 h, 55 °C for 2 h, then 100 °C for 5 h; (iii) KMnO4, H2O, 80 °C, 4 h; (iv) SOCl2, reflux, 6 h; (v) EtONa, anhydrous EtOH, -5 °C, 3 h;
(vi) hydrazine hydrate, EtOH, 0 °C, 1 h; (vii) Me2SO4, DMF, 60 °C, 3 h; (viii) NaOH, THF, H2O, 70 °C, 2 h, (iX) SOCl2, reflux, 6 h.
This journal is © The Royal Society of Chemistry 20xx
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hydrate to give ethyl pyrazole-5-carboxylates 9. Dimethyl
ARTICLE
1
2
3
4
View Article Online
DOI: 10.1039/C8NJ05150J
sulfate was used for the methylation reaction with 9 to give
5
6
7
8
R1
NH2
ethyl 1-methyl-1H-pyrazole-5-carboxylates 10, which were
then hydrolyzed to give the pyrazole-5-carboxylic acids 11. The
intermediates 12 were finally obtained by the reaction of the
acids 11 with thionyl chloride.
CHO
NO2
i
SO3Na
NH2
NO2 OH
ii
9
13
14
In above two synthetic routes, different raw materials and
reaction conditions were used to synthesize the pyrazole
intermediates pyrazole-4-carbonyl chlorides and pyrazole-5-
carbonyl chlorides. The most key steps were the cyclization
reactions of different pyrazole rings. When methylhydrazine was
used for the cyclization of compound 2, it was necessary to
maintain the relatively high temperature (reflux) and long reaction
time (5 h). When hydrazine hydrate was used for the cyclization
of compound 9, it is necessary to maintain the relatively low
temperature (−5 °C) and short reaction time (3 h).
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11
12
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R1
R1
N
N
iii
N
N
H
H
H2N
O2N
R1 = H, Cl, CH3
15
16
Scheme
2
Synthetic route to benzimidazole intermediates 16.
Reagents and conditions: (i) NaHSO3, EtOH, H2O, rt, 15 min; (ii) DMF,
reflux, 3 h; (iii) SnCl2·2H2O, EtOH, reflux, 0.5-1 h.
Another kind of key intermediates 2-(benzimidazol-2-
yl)phenylamines 16 were obtained by using an optimized
three-step procedure.25,26 As displayed in Scheme 2, the raw
material 2-nitrobenzaldehyde 13 was firstly reacted with
R2
O
R2
COCl
N
H
N
N
i
N
N
N
NH
R1
Cl
5
sodium
bisulfite
to
prepare
sodium
hydroxy(2-
T1-T6
nitrophenyl)methanesulfonate 14. This salt was reacted
respectively with three substituted o-phenylenediamines to
give the cyclized products 15. After the reduction reaction
using stannous chloride, the intermediates 16 were obtained
in the yields of 40% above.
The target compounds T1−T20 were finally synthesized by
the amidation reaction. The synthetic route was shown in
Scheme 3. Two amidation methods were used to synthesize
the target compounds. When the pyrazole carboxamide
derivatives T1−T12 were synthesized, the pyrazole carbonyl
chlorides 5 and 12 were reacted respectively with the
benzimidazole intermediates in the presence of Et3N. When
the niacinamide derivatives T13−T20 were synthesized, the
substituted nicotinic acids were directly used to react with
different benzimidazole intermediates in the presence of TBTU
(O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
R1=H, Cl, Me;
R2=Me, CF3.
O
NH2
R3
COCl
R3
N
H
N
NH
R1
N
N
N
N
N
NH
R1
12
T7-T12
i
R1=H, Cl, Me;
R3=Me, CH2CHMe2.
16
R4
O
COOH
R4
N
H
N
N
N
NH
R1
ii
T13-T20
R1=H, Cl, Me;
R4=H, Cl, SH.
tetrafluoroborate) and Et3N.
Crystal structure of compound T1
Scheme 3 Synthetic route to target compounds T1-T20. Reagents and
conditions: (i) Et3N, CH2Cl2, rt, overnight; (ii) TBTU, Et3N, CH2Cl2, rt,
overnight.
The structure of compound T1 was further studied using the
single crystal X-ray analysis. The corresponding crystal
structure was shown by Fig. 4. The crystal data for T1:
monoclinic, a = 9.2973(4) Å, b = 16.7097(7) Å, c = 11.0503(5) Å,
α = 90 °, β = 94.760(4) °, γ = 90 °, V = 1710.80(13) Å3, Z = 4,
space group P21/c (no.14), μ = 0.242 mm-1, Dcalc =1.420 g/cm3,
7766 reflections measured (4.4≤2θ≤52.8), 4000 unique (Rint
=
0.048) were used in all calculations. The final R1 was 0.0428
(I>2σ(I)) and wR(F2) was 0.1021. The crystallographic data have
been deposited with the Cambridge Crystallographic Data
Centre. The deposition number was CCDC1865306.
Antifungal activity
The antifungal activities of target compounds T1−T20 against
four fungi were assayed with hymexazol as the positive control
at 100 ug/mL. As can be seen from table 1 that the target
Fig 4. Crystal structure diagram of compound T1.
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compounds showed better inhibitory activity against B. cinerea
View Article Online
DOI: 10.1039/C8NJ05150J
Table 1 Inhibitory rates of target compounds T1-T20 against four phytopathogenic fungi at 100 ug/mL
R1
R2
R3
R4
B. cinerea
R. solani
F. graminearum A. solani
6
7
8
9
Compound
T1
T2
T3
T4
T5
T6
T7
H
H
Me
CF3
Me
CF3
Me
CF3
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
H
Cl
SH
H
Cl
SH
H
Cl
−
65.53±0.40
83.11±0.21
72.37±1.30
79.68±0.31
85.62±0.34
85.39±0.21
43.38±0.42
45.66±0.49
44.98±0.17
52.28±1.08
52.28±1.08
41.55±0.61
52.05±1.29
88.13±0.56
44.75±0.42
32.42±0.61
55.02±2.93
35.39±0.70
31.74±0.91
74.66±0.50
100.00
64.34±0.31
64.84±0.22
62.34±0.17
69.08±0.33
64.34±0.54
62.84±0.31
38.65±0.86
30.17±1.41
39.90±0.65
36.66±0.21
34.91±1.09
45.38±0.50
40.15±0.02
69.82±0.17
28.67±0.88
33.91±1.87
44.89±0.98
22.69±0.67
26.43±0.60
67.33±0.48
72.82±0.17
39.21±0.34
52.37±0.17
51.58±0.42
52.63±0.05
45.52±0.34
47.37±0.21
21.05±0.00
18.42±0.21
22.36±0.17
25.79±0.06
17.36±0.42
25.00±0.22
21.05±0.04
41.58±0.26
19.47±0.37
15.52±0.22
24.21±0.37
29.21±0.17
14.73±0.06
43.15±0.07
68.16±0.17
29.71±0.37
34.57±0.31
52.57±0.21
58.28±0.49
66.00±0.17
68.28±0.67
21.42±0.40
29.14±1.38
24.57±0.08
34.00±0.22
62.57±0.31
30.85±0.21
22.85±0.07
40.00±0.21
30.28±0.21
24.57±0.03
34.28±0.21
25.14±0.21
28.00±0.63
58.85±0.04
55.43±0.37
Cl
Cl
Me
Me
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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Me
CH2CHMe2
Me
CH2CHMe2
Me
CH2CHMe2
−
−
−
−
−
−
−
−
−
T8
T9
H
Cl
Cl
Me
Me
H
T10
T11
T12
T13
T14
T15
T16
T17
T18
T19
T20
Hymexazol
H
H
Cl
Cl
Cl
Me
Me
−
−
−
than the other three fungi. Most of the compounds presented
moderate to good antifungal activity against B. cinerea in vitro. cinerea with EC50 values of 18.24 and 10.16 ug/mL,
In particular, among the compounds tested, T2, T5, T6 and T14 respectively. When substituted pyrazoles were introduced, the
exhibited excellent activities with the inhibition rates as high target compounds with pyrazole-4-carboxamide moiety
as 83.11%, 85.62%, 85.39% and 88.13%. Additionally, some showed higher activities than the target compounds with
compounds also exhibited obvious inhibitory activity against R. pyrazole-5-carboxamide moiety. For example, the activities of
solani and A. solani. The inhibition rates of compounds T1−T6, T1 and T2 were better than those of T7 and T8, the activities of
T14, and T20 exceeded 60% against R. solani. And the T3 and T4 were better than those of T9 and T10, and the
inhibition rates of compounds T4−T6, T11 and T20 ranged activities of T5 and T6 were better than those of T11 and T12.
from 58.28% to 68.28% against A. solani, which were better In the target compounds containing pyrazole-4-carboxamide
than 55.43% of the control hymexazol.
moiety, the compounds with CF3 group at R2 position were
Based on the results of the preliminary screening, the EC50 more active than the compounds with CH3 group at R2
values of the title compounds against B. cinerea were position. That is to say, the compounds T2, T4 and T6 showed
evaluated and summarized in Table 2. The compounds T5 and better activity than T1, T3 and T5, respectively. When
T6 exhibited encouraging antifungal activities against B. substituted pyridines were introduced, the activities of targets
with 2-chlorine-niacinamide were superior than other
niacinamide products. For example, the activity of compound
T14 was superior to that of T13 and T15, the activity of
compound T17 was superior to that of T16 and T18, and the
Table 2 EC50 values of target compounds T1-T20 against B. cinerea
Compound
T1
T2
T3
T4
T5
T6
T7
T8
Regression equation
y = 1.5740x+2.2330
y = 1.8298x+2.1726
y = 1.2895x+2.9740
y = 1.5838x+2.5535
y = 1.3517x+3.2953
y = 0.9867x+4.0063
y = 1.5565x+1.9229
y = 1.7151x+1.4811
y = 0.9127x+2.9113
y = 1.0533x+2.5913
y = 1.6850x+1.5565
y = 1.4714x+2.0347
y = 1.6975x+1.7669
y = 1.6931x+2.5975
y = 1.6027x+1.5433
y = 1.3403x+2.2538
y = 1.2197x+2.5777
y = 1.6049x+1.6830
y = 1.1698x+2.4282
y = 1.4928x+2.6659
y = 0.6466x+4.2544
EC50 (ug/ml)
57.27±0.40
35.09±0.32
37.25±0.29
35.04±0.34
18.24±0.36
10.16±0.26
94.82±0.42
112.63±0.29
194.25±0.18
193.61±0.16
110.57±0.48
103.57±0.14
80.28±0.39
26.24±0.37
143.48±0.27
111.92±0.38
96.81±0.33
116.64±0.42
157.92±0.34
36.60±0.24
14.22±0.05
r
0.9812
0.9878
0.9795
0.9821
0.9716
0.9726
0.9787
0.9914
0.9881
0.9930
0.9761
0.9972
0.9847
0.9808
0.9912
0.9760
0.9779
0.9802
0.9754
0.9897
0.9920
T9
T10
T11
T12
T13
T14
T15
T16
T17
T18
T19
T20
Hymexazol
Fig. 5 Alignment of all title compounds to common template molecule T6.
J. Name., 2013, 00, 1-3 | 4
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activity of T20 was superior to that of T19. Furthermore, the
Cl-substituted
View Article Online
DOI: 10.1039/C8NJ05150J
Table 3 Experimental and predicted pEC50 results against B. cinerea
model were 0.578 with 3 ONC, 0.850, 0.166, and 22.661,
respectively. The values of q2 and r2 in the CoMFA model were
better than the standard reference values of 0.5 (q2) and 0.8
(r2), which manifested the good predictive ability of the
obtained 3D-QSAR model.27,28 Meanwhile, as shown in Table 4,
the contributions of steric field and electrostatic field to the
CoMFA model were 0.516 and 0.484 respectively, which
suggested that the bioactivity was mainly determined by the
steric interactions. Using the obtained CoMFA model as a test
tool, the predicted pEC50 values of title compounds agsinst B.
cinerea in the training and test sets were predicted and
presented in Table 3. As presented in Fig. 6, the correlation
between the predicted and experimental pEC50 values in the
CoMFA model also demonstrated the good predictive ability of
the obtained CoMFA model.
Compound
T1
T2*
T3
T4
T5
T6
T7
T8
T9*
T10
T11
T12*
T13
T14*
T15
T16
T17
T18
T19
T20
Experimentala
3.804
4.077
4.030
4.112
4.318
4.629
3.543
3.520
3.274
3.323
3.494
3.573
3.592
4.123
3.382
3.493
3.596
3.513
3.318
3.995
Predictedb
3.996
4.306
3.950
4.257
4.198
4.512
3.435
3.433
3.392
3.386
3.624
3.626
3.434
3.610
3.544
3.389
3.565
3.501
3.633
3.809
Residualc
-0.192
-0.229
0.080
-0.146
0.120
0.118
0.108
0.088
-0.118
-0.063
-0.130
-0.054
0.159
0.513
-0.161
0.104
0.031
0.012
-0.314
0.187
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CoMFA contour map analysis
In the CoMFA contour maps of the steric (Fig. 7A) and
electrostatic (Fig. 7B) fields, the title compound T6, which
possesses the best antifungal activity against B. cinerea, are
shown inside to better understand the structural features of
synthesized compounds. As shown in Fig. 7A, there are two
larger yellow contours closely around the pyrazole scaffold,
which indicates that the bulky substituted groups at these
regions are unfavorable for their biological activity. This can
explain that the compounds T8, T10 and T12 bearing an
a Experimental pEC50
b Predicted pEC50 by CoMFA;
;
c Relative error of experimentally predicted pEC50
;
* Samples of test set.
Table 4 Statistical parameters for CoMFA model
Statistical parameter
CoMFA Validation criteria
q2 a
0.578
3
> 0.5
ONC b
r2 c
0.850
0.166
22.661
> 0.8
SEEd
F e
Fraction of field contributions f
Steric
0.516
0.484
Electrostatic
a Cross-validated correlation;
b Optimum number of components;
c Noncross-validated correlation;
d Standard error of estimate;
e F-test value;
f Field contributions: steric and electrostatic.
compounds were superior to the H and SH-substituted
compounds. In other words, the compounds fell into order by
activity as T14>T13>T15, and T17>T16>T18.
Fig. 6 Correlation between predicted and experimental pEC50 values in
CoMFA model.
Performance of CoMFA model
A total of sixteen title compounds were randomly chosen as
the training set, and the remaining four compounds (labeled
with asterisks) were used as the test set. The 3D structures of
twenty title molecules were aligned to the common template
molecule of T6 with the best antifungal activity against B.
cinerea and are showed on Fig. 5. Based on the experimental
pEC50 values of the training set in Table 3, the CoMFA model
was developed for the 3D-QSAR study, and their calculated
statistical parameters are shown in Table 4. The results in
Table 4 indicated that the cross-validated coefficient q2, non-
cross-validated correlation coefficient r2, SEE and F of the
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Fig. 7 CoMFA contour maps of steric and electrostatic fields. (A)
Sterically favored areas for bioactivity are in green, and sterically
disfavored areas for bioactivity are in yellow; (B) Electron-withdrawing
favored areas for bioactivity are in red, and electron-withdrawing
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DOI: 10.1039/C8NJ05150J
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isobutyl at the 3-position of pyrazole-5-yl scaffold had Technologies 6540 UHD Accurate-Mass Q-TOF LC/MS using ESI.
notgood fungicidal activity. Meanwhile, the Fig. 7A shows two All the reagents were of pure analytical grade and used
green contours around the 1-and 3-positions of pyrazole directly without further treatment unless otherwise noted.
scaffold, which suggests that introducing suitable bulky
Synthesis of pyrazole-4-carbonyl chlorides 5
substitutes into these regions are favorable for their antifungal
activity. It was confirmed that the compounds T1−T6 bearing a The solution of 40% methylhydrazine (0.06 mol) was heated to
methyl at 1-position and a methyl or trifluoromethyl at 3- 65 °C, and the raw material ethyl acetoacetate 1a or ethyl
position of pyrazole-4-yl scaffold exhibited higher antifungal 4,4,4-trifluoroacetoacetate 1b (0.05 mol) was added dropwise.
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activity.
The reaction mixture was maintained at 65 °C for 3 h, and then
As shown in Fig. 7B, a large blue contour near the 1-position it was heated to reflux for another 5 h. The solvent was
of pyrazole scaffold indicates that electron-donating evaporated under reduced pressure to afford the yellow solids
substitutes at this region are helpful for the increase of 1,3-dimethyl-5-hydroxyl-1H-pyrazole 2a (75.4%) and 1-methyl-
antifungal activity, such as the compounds T1−T6. A large red 3-trifluoromethyl-5-hydroxyl-1H-pyrazole 2b (80.3%).
contour between the carbonyl group and the 3-position of
To a violently stirred cold solution (−5°C) of DMF (0.1 mol)
pyrazole ring indicates the carboxamide group played a pivotal was added dropwise phosphorus oxychloride (0.15 mol). The
role in maintaining their antifungal activity. Meanwhile, the resulting mixture was stirred at room temperature for 1 h. To
large red contour also demonstrates that introducing a the above mixture was added the compound 2 (0.05 mol) in
electron-withdrawing group at the 3-position of pyridine ring portions, it was then heated to 55 °C for 2 h and stirred at
would increase their bioactivity. For example, the compounds 100 °C for another 5 h. After cooling to room temperature, the
T14 and T20 containing chlorine atoms at the R4 position of mixture was poured into ice-water (200 mL), and the resulting
pyridine ring showed good antifungal activity against B. precipitate was collected by filtration. The filtrate was
cinerea. In addition, a small blue contour near the 6-position of continuously extracted with ethyl acetate (3 × 50 mL), and the
benzimidazole scaffold indicates that the electron-donating organic layer was dried over anhydrous magnesium sulfate.
substitutes are favorable for their biological activity, which The solvent was removed under reduced pressure to give a
agrees with the fact that the title compounds T5 and T6 brown solid. The solid obtained above in two portions was
contributed the highest antifungal activity.
collected together as the products 5-chloro-1,3-dimethyl-1H-
pyrazole-4-formaldehyde 3a (76.3%) and 5-chloro-1-methyl-3-
trifluoromethyl-1H-pyrazole-4-formaldehyde 3b (79.6%).
The compound 3 (0.025 mol), acetone (5 mL), and water (25
mL) were added in a 250 mL three-neck round-bottom flask.
The solution was stirred and slowly warmed up to 60 °C. Then,
a solution of potassium permanganate (0.0275 mol) and water
(50 mL) was added dropwise, and heated to 80 °C for 4 h. The
solution was cooled and filtered, concentrated hydrochloric acid
was slowly added until the pH<2. A large amount of white solid
was precipitated, filtered, washed with water, and dried to
give the white solids 5-chloro-1,3-dimethyl-1H-pyrazole-4-
carboxylic acid 4a (70.2%) and 5-chloro-1-methyl-3-
trifluoromethyl-1H-pyrazole-4-carboxylic acid 4b (76.1%).
The pyrazole-4-carboxylic acids 4 were reacted respectively
with thionyl chloride under reflux for 6 h. The excessive thionyl
chloride was removed under reduced pressure to obtain 5-
chloro-1,3-dimethyl-1H-pyrazole-4-carbonyl chloride 5a and 5-
chloro-1-methyl-3-trifluoromethyl-1H-pyrazole-4-carbonyl
chloride 5b.
Conclusion
In summary, the pyrazole carboxamide and niacinamide
derivatives containing a benzimidazole moiety were prepared
and evaluated for their antifungal activities against four
pathogenic fungi. The structures of these compounds were
well identified by spectral data and single crystal X-ray
diffraction. The bioassay results showed that some of the
compounds possessed good antifungal activity against B.
cinerea in vitro. In particular, the compounds T5 and T6
exhibited high antifungal activities with EC50 values of 18.24
and 10.16 ug/mL, respectively. The established CoMFA model
based on the anti-B. cinerea activities of the target compounds
showed fine predictive ability. The present work demonstrated
that pyrazole carboxamide and niacinamide derivatives
containing a benzimidazole moiety can effectively control the
fungus B. cinerea. Further structural optimization of the target
compounds and other related experiments are ongoing in our
laboratory.
Synthesis of pyrazole-5-carbonyl chlorides 12
The mixed solution of diethyl oxalate 6 (0.105mol) and
acetone 7a (or 4-methyl-2-pentanone 7b) (0.1 mol) was added
slowly to the cooled (−10°C) solution of sodium ethoxide
(0.128mol) and ethonal (100 mL). The obtained mixture was
stirred at −5°C for 3 h and then poured into ice water, adjusted
pH to 4 with 10% dilute hydrochloric acid, and extracted with
ethyl acetate. The organic layer was washed twice with water
and dried over anhydrous Na2SO4, filtered and concentrated
under reduced pressure to give the yellow liquids ethyl 2,4-
Experimental
The melting points of the products were determined using a
SMP50 Automatic Melting Point Apparatus and were not
corrected. The H NMR and 13C NMR spectra were captured
with a Bruker Avance III 400 NMR spectrometer at room
temperature with TMS as the internal standard. The Tin-layer
chromatography (TLC) was performed on silica gel GF254. The
high resolution mass spectrometer was recorded on an Agilent
1
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dioxovalerate 8a
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(
85.8%) and ethyl 6-methyl-2,4- Synthesis of target compounds T1−T20
View Article Online
dioxoheptanoate 8b (80.4%).
The pyrazole carbonyl chloride
5
oDrOI1: 120.10(339/Cm8mNJo0l5)150inJ
The compound 8 (0.05 mol) was dissolved in ethanol (50 mL)
and cooled to −10°C. 80% Hydrated hydrazine (0.0625 mol)
was added dropwise to the above solution, and kept stirring
below 0°C for 1 h. The solvent was evaporated under reduced
pressure. Water (100 mL) was added to the residue, and the
mixture was extracted with ethyl acetate (3 × 20 mL). The
organic layer was washed twice with water, dried over
anhydrous Na2SO4, filtered, and concentrated under pressure
to give the pale yellow solids ethyl 3-methyl-1H-pyrazole-5-
carboxylate 9a (88.4%) and ethyl 3-isobutyl-1H-pyrazole-5-
carboxylate 9b (73.8%).
The compound 9 (0.04mol) and dimethyl sulfate (0.048 mol)
were dissolved in DMF, and the solution was stirred at 60°C for
3 h. The progress of reaction was monitored by TLC. After
complete disappearance of the starting material, the reaction
solution was poured into water (100 mL) and extracted with
ethyl acetate (3 × 25 mL). The organic phase was washed twice
with water, dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure to give ethyl 1,3-
dimethyl-1H-pyrazole-5-carboxylate 10a (85.1%) and ethyl 1-
methyl-3-isobutyl-1H-pyrazole-5-carboxylate 10b (77.3%).
20% Sodium hydroxide solution (0.12 mmol) was added to a
solution of compound 10 (0.1 mmol) in THF, stirred at 70 °C for
2 h. The solution was concentrated in vacuo to remove most of
the THF. The pH was adjusted to 3−4 with 10% dilute
hydrochloric acid. The mixture was filtered, and the filter cake
was washed with water to give 1,3-dimethyl-1H-pyrazole-5-
carboxylic acid 11a (68.4%) and 1-methyl-3-isobutyl-1H-
pyrazole-5-carboxylic acid 11b (65.9%). Finally, the same
synthesis method as the intermediates 5 was used to prepare
1,3-dimethyl-1H-pyrazole-5-carbonyl chloride 12a and 1-
methyl-3-isobutyl-1H-pyrazole-5-carbonyl chloride 12b.
dichloromethane (25 mL) was slowly added to a solution of
corresponding benzimidazole-2-phenylamine 16 (3 mmol) and
triethylamine (6 mmol) in dichloromethane at a controlled
temperature of 0−5 °C. The reaction mixture was stirred at
room temperature overnight and then was washed with water,
dried, and evaporated under reduced pressure to obtain a
solid. The solid was purified by column chromatography to give
desired product the pyrazole carboxamide derivative
containing a benzimidazole moiety. The yields of obtained
target compounds T1−T12 ranged from 41.3% to 70.6%.
The nicotinic acid (3 mmol) and TBTU (3.6 mmol) were
added to dichloromethane (30mL). Triethylamine (6 mmol)
was then added dropwise, and the mixture was stirred for 30
min. After the corresponding benzimidazolee-2-phenylamine
16 (3 mmol) was added to the mixture, stirring was continued
overnight at room temperature. The reaction mixture was
then filtered. The resulting solid was washed with water and
dried to give a target product the niacinamide derivative
containing a benzimidazole moiety. The yields of obtained
target compounds T13−T20 ranged from 42.0% to 80.5%.
N-(2-(1H-Benzimidazol-2-yl)phenyl)-5-chloro-1,3-dimethyl-
1H-pyrazole-4-carboxamide (T1): White solid, yield 70.6%, mp
240-242 °C; IR (KBr) (νmax/cm–1): 3288, 2863, 1646, 1625, 1587,
9
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a
1
1542, 1429, 1366, 1328, 1146, 968, 902, 554. H NMR (400
MHz, DMSO-d6) δ: 13.32 (s, 1H), 13.22 (s, 1H), 8.78 (d, J = 8.4
Hz, 1H), 8.25–8.11 (m, 1H), 7.64 (dd, J = 15.7, 7.2 Hz, 2H), 7.54
(t, J = 7.8 Hz, 1H), 7.32 (t, J = 7.6 Hz, 3H), 3.87 (s, 3H), 2.48 (s,
3H). 13C NMR (101 MHz, DMSO-d6) δ: 160.6, 151.0, 148.2,
142.4, 138.6, 134.0, 131.0, 127.8, 127.5, 123.8, 123.5, 122.7,
121.2, 118.8, 116.3, 113.6, 111.9, 36.6, 14.2. HRMS (ESI) Calcd
for C19H17ClN5O [M+H]+: 366.11161, found: 366.11169. Anal.
Calcd for C, 62.38; H, 4.41; N, 19.14. Found: C, 62.65; H, 4.80; N,
18.80%.
Synthesis of 2-(benzimidazol-2-yl)phenylamines 16
N-(2-(1H-Benzimidazol-2-yl)phenyl)-5-chloro-1-methyl-3-
2-Nitrobenzaldehyde 13 (0.05 mol) and sodium bisulfite (0.05
mol) were dissolved in ethanol (30 mL) and water (30 mL),
respectively, and then mixed and stirred for 15 min at room
temperature. The reaction mixture was filtered to obtain
sodium hydroxy(2-nitrophenyl)methanesulfonate 14. The
resulting salt (0.02 mol) and o-phenylenediamine (0.02 mol) in
DMF (60 mL) were refluxed for 3 h in an oil bath. After the
reaction mixture was poured onto crushed ice, 2-(2-
nitrophenyl)-1H-benzimidazole 15 was separated by filtration
and recrystallized from a methanol/water mixture (V/V = 1:1).
The mixture of compound 15 (0.05 mol) and SnCl2·2H2O (0.25
mol) in ethanol (50 mL) was refluxed for 0.5−1 h. The reaction
mixture was cooled to room temperature. And 10% NaOH
solution was then added in small portions in an ice-water bath
until the mixture became strongly alkaline (pH>11). The
resulting mixture was extracted with ethyl acetate (4 × 100
mL). The combined extracts were washed with brine, dried
over anhydrous Na2SO4, filtered and evaporated in vacuo to
give 2-(1H-benzimidazol-2-yl)phenylamine 16a (45.4%), 2-(6-
chloro-1H-benzimidazol-2-yl)phenylamine 16b (48.7%), and 2-
(6-methyl-1H-benzimidazol-2-yl)phenylamine 16c (40.2%).
(trifluoromethyl)-1H-pyrazole-4-carboxamide
(T2):
Pale
yellow solid, yield 58.4%, mp 254-256 °C; IR (KBr) (νmax/cm–1):
3247, 2934, 1661, 1610, 1587, 1533, 1485, 1450, 1432, 1372,
1325,1274, 1188, 1149, 1024, 959, 905, 751. 1H NMR (400
MHz, DMSO-d6) δ: 13.92 (s, 1H), 13.30 (s, 1H), 8.80 (d, J = 8.3
Hz, 1H), 8.30–8.14 (m, 1H), 7.66 (s, 2H), 7.61–7.53 (m, 1H),
7.42–7.34 (m, 1H), 7.30 (s, 2H), 4.07 (s, 3H). 13C NMR (101 MHz,
DMSO-d6) δ: 157.9, 150.8, 139.2 (q, J= 38.3 Hz) 138.1, 131.2,
129.0, 127.8, 124.2, 122.1, 120.9, 119.5, 116.3, 115.2, 38.0.
HRMS (ESI) Calcd for C19H14ClF3N5O [M+H]+: 420.08335, found:
420.08344. Anal. Calcd for C, 54.36; H, 3.12; N, 16.68. Found: C,
54.17; H, 3.49; N, 16.27%.
5-Chloro-N-(2-(6-chloro-1H-benzimidazol-2-yl)phenyl)-1,3-
dimethyl-1H-pyrazole-4-carboxamide (T3): White solid, yield
59.6%, mp 231-233 °C; IR (KBr) (νmax/cm–1): 3452, 3205, 1673,
1631, 1590, 1527, 1471, 1432, 1378, 1310, 1274, 1060, 932,
905, 807, 733. 1H NMR (400 MHz, DMSO-d6) δ: 13.38 (s, 1H),
13.04 (s, 1H), 8.75 (d, J = 8.3 Hz, 1H), 8.20-8.04 (m, 1H), 7.77-
7.59 (m, 2H), 7.55 (t, J = 7.8 Hz, 1H), 7.32 (t, J = 7.6 Hz, 2H),
3.87 (s, 3H), 2.45 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ:
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160.6, 152.3, 148.4, 138.6, 131.3, 128.0, 127.4, 123.6, 121.3, J = 5.6, 2.7 Hz, 1H), 7.63 (dd, J = 5.6, 2.6 Hz, 1H), 7.54 (t, J = 7.6
View Article Online
13
116.0, 113.4, 36.6, 14.2. HRMS (ESI) Calcd for C19H16Cl2N5O Hz, 1H), 7.34 (m, 3H), 7.07 (s, 1H), 4.09 (sD,O3IH: 1)0, .210.3393/C(s8,N3JH05).150CJ
[M+H]+: 400.07264, found: 400.07245. Anal. Calcd for C, 57.01; NMR (101 MHz, DMSO-d6) δ: 158.2, 151.2, 146.3, 142.2, 138.5,
H, 3.78; N, 17.50. Found: C, 57.39; H, 4.22; N, 17.28%.
5-Chloro-N-(2-(6-chloro-1H-benzimidazol-2-yl)phenyl)-1-
methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (T4): [M+H]+: 332.15059, found: 332.15085. Anal. Calcd for C, 68.87;
White solid, yield 41.5%, mp 225-227 °C; IR (KBr) (νmax/cm–1): H, 5.17; N, 21.13. Found: C, 68.85; H, 5.48; N, 20.73%.
136.6, 133.8, 131.1, 127.6, 124.0, 123.5, 122.9, 120.1, 118.4,
115.8, 112.0, 107.1, 39.1, 13.5. HRMS (ESI) Calcd for C19H18N5O
3407, 3226, 1649, 1619, 1539, 1485, 1450, 1399, 1325, 1191,
N-(2-(1H-Benzimidazol-2-yl)phenyl)-3-isobutyl-1-methyl-
10
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12
13
14
15
16
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1155, 1057, 1033, 751. 1H NMR (400 MHz, DMSO-d6) δ: 13.62 1H-pyrazole-5-carboxamide (T8): White solid, yield 51.4%, mp
(s, 1H), 13.44 (s, 1H), 8.78 (d, J = 7.8 Hz, 1H), 8.19 (dd, J = 7.9, 213-215 °C; IR (KBr) (νmax/cm–1): 3410, 3110, 1634, 1613, 1599,
1.0 Hz, 1H), 7.71 (s, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.62–7.55 (m, 1551, 1438, 1405, 1316, 1277, 762, 736, 620. 1H NMR (400
1H), 7.37 (dd, J = 11.2, 4.1 Hz, 1H), 7.31 (dd, J = 8.5, 1.3 Hz, 1H), MHz, DMSO-d6) δ: 13.88 (s, 1H), 13.45 (s, 1H), 8.82 (d, J = 7.8
4.06 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 157.9, 152.1, Hz, 1H), 8.22 (t, J = 13.6 Hz, 1H), 7.71–7.61 (m, 2H), 7.54 (dd, J
139.2 (q, J= 38.3Hz), 138.1, 131.5, 129.1, 127.9, 124.2, 122.1, = 11.5, 4.2 Hz, 1H), 7.32 (dd, J = 8.8, 6.5 Hz, 3H), 7.07 (s, 1H),
121.0, 119.5, 116.0, 115.0, 38.1. HRMS (ESI) Calcd for 4.11 (s, 3H), 2.56 (d, J = 7.0 Hz, 2H), 1.99 (m, 1H), 1.01 (s, 3H),
C19H13Cl2F3N5O [M+H]+: 454.04438, found: 454.04425. Anal. 0.99 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 158.3, 151.3,
Calcd for C, 50.24; H, 2.66; N, 15.42. Found: C, 50.65; H, 3.08; N, 150.0, 142.2, 138.5, 136.5, 133.9, 131.1, 127.9, 123.9, 123.6,
15.26%.
5-Chloro-1,3-dimethyl-N-(2-(6-methyl-1H-benzimidazol-2-
122.8, 120.2, 118.1, 115.9, 112.2, 106.8, 39.2, 37.1, 28.9, 22.8.
HRMS (ESI) Calcd for C22H24N5O [M+H]+: 374.19754,
yl)phenyl)-1H-pyrazole-4-carboxamide (T5): White solid, yield found:.374.19775. Anal. Calcd for C, 70.76; H, 6.21; N, 18.75.
53.0%, mp 220-222 °C; IR (KBr) (νmax/cm–1): 3280, 2860, 1643, Found: C, 70.71; H, 6.09; N, 18.95%.
1613, 1590, 1551, 1524, 1485, 1429, 1363, 1310, 1265, 962.
N-(2-(6-Chloro-1H-benzimidazol-2-yl)phenyl)-1,3-dimethyl-
902, 762, 632, 599. 1H NMR (400 MHz, DMSO-d6) δ: 13.32 (s, 1H-pyrazole-5-carboxamide (T9): White solid, yield 41.3%, mp
1H), 13.07 (d, J = 16.9 Hz, 1H), 8.81–8.71 (m, 1H), 8.13 (d, J = 254-256 °C; IR (KBr) (νmax/cm–1): 3410, 2949, 1634, 1616, 1592,
7.9 Hz, 1H), 7.49 (dd, J = 16.1, 8.6 Hz, 2H), 7.40 (d, J = 19.1 Hz, 1544, 1446, 1422, 1321, 1288, 937, 777, 634. 1H NMR (400
1H), 7.30 (t, J = 7.6 Hz, 1H), 7.10 (dd, J = 19.3, 8.1 Hz, 1H), 3.86 MHz, DMSO-d6) δ: 13.57 (d, J = 34.8 Hz, 1H), 13.44 (s, 1H), 8.77
(s, 3H), 2.46 (t, J = 7.2 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) δ: (d, J = 8.3 Hz, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.79 – 7.47 (m, 3H),
160.6, 150.9, 150.5, 148.1 (d, J = 10.7 Hz), 142.8, 140.62, 138.5, 7.32 (dd, J = 16.1, 8.4 Hz, 2H), 6.98 (s, 1H), 4.07 (s, 3H), 2.31 (s,
134.3, 133.3, 132.0, 131.8, 130.7, 127.6 (d, J = 12.8 Hz), 125.3, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 158.0, 146.1, 138.5,
124.3, 123.5, 121.2, 118.4 (d, J = 7.4 Hz), 116.4, 113.6, 111.5 (d, 136.4, 131.2, 127.6, 123.3, 120.1, 115.2, 107.0, 39.1, 13.5.
J = 14.2 Hz), 36.6, 21.7 (d, J = 17.4 Hz), 14.2. HRMS (ESI) Calcd HRMS (ESI) Calcd for C19H17ClN5O [M+H]+: 366.11161, found:
for C20H19ClN5O [M+H]+: 380.12726, found: 380.12711. Anal. 366.11182. Anal. Calcd for C, 62.38; H, 4.41; N, 19.14. Found: C,
Calcd for C, 63.24; H, 4.78; N, 18.44. Found: C, 63.68; H, 5.12; N, 62.78; H, 4.84; N, 18.80%.
18.19%.
5-Chloro-1-methyl-N-(2-(6-methyl-1H-benzimidazol-2-
yl)phenyl)-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide
N-(2-(6-Chloro-1H-benzimidazol-2-yl)phenyl)-3-isobutyl-1-
methyl-1H-pyrazole-5-carboxamide (T10): White solid, yield
52.4%, mp 194-195 °C; IR (KBr) (νmax/cm–1): 3327, 2946, 1652,
(T6): White solid, yield 65.1%, mp 160-162 °C; IR (KBr) 1631, 1616, 1587, 1548, 1447, 1423, 1325, 1304, 1063, 932,
(νmax/cm–1): 3416, 3235, 1652, 1613, 1587, 1536, 1488, 1432, 765. 1H NMR (400 MHz, DMSO-d6) δ: 13.58 (s, 1H), 13.40 (s,
1402, 1316, 1271, 1185, 1149, 1024, 899, 774. 1H NMR (400 1H), 8.76 (d, J = 8.3 Hz, 1H), 8.17 – 8.05 (m, 1H), 7.65–7.57 (m,
MHz, DMSO-d6) δ: 13.90 (s, 1H), 13.11 (d, J = 17.2 Hz, 1H), 2H), 7.58–7.47 (m, 1H), 7.40–7.18 (m, 2H), 6.95 (s, 1H), 4.08 (s,
8.81–8.73 (m, 1H), 8.17 (dd, J = 7.9, 1.1 Hz, 1H), 7.61 – 7.31 (m, 3H), 2.53 (d, J = 3.8 Hz, 2H), 1.97 (td, J = 13.3, 6.6 Hz, 1H), 1.01
4H), 7.11 (dd, J = 22.5, 8.2 Hz, 1H), 4.06 (d, J = 5.9 Hz, 3H), 2.45 (s, 3H), 0.99 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 158.2,
(d, J = 11.7 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 157.9, 152.6, 149.9, 138.5, 136.4, 131.3, 127.7, 127.5, 123.4, 120.2,
150.7, 150.3, 142.6, 140.4,139.1 (dd, J = 38.1 Hz,5.4 Hz) 138.0 115.5, 106.6, 39.1, 37.1, 28.8, 22.8. HRMS (ESI) Calcd for
(d, J = 2.2 Hz), 134.1, 133.5, 131.9 (d, J = 5.1 Hz), 131.0 (d, J = C22H23ClN5O [M+H]+: 408.15856, found: 408.15866. Anal. Calcd
6.1 Hz), 129.1 (d, J = 9.0 Hz), 127.6, 125.4, 124.2 (d, J = 13.9 Hz), for C, 64.78; H, 5.44; N, 17.17. Found: C, 64.98; H, 5.82; N,
122.1, 120.8 (d, J = 5.9 Hz), 119.5, 118.7 (d, J = 3.2 Hz), 116.4, 16.83%.
115.2, 111.5 (d, J = 11.8 Hz), 38.0 (d, J = 4.8 Hz), 21.8, 21.6.
1,3-Dimethyl-N-(2-(6-methyl-1H-benzimidazol-2-yl)phenyl)-
HRMS (ESI) Calcd for C20H16ClF3N5O [M+H]+: 434.09900, found: 1H-pyrazole-5-carboxamide (T11): White solid, yield 66.1%,
434.09889. Anal. Calcd for C, 55.37; H, 3.49; N, 16.14. Found: C, mp 256-258 °C; IR (KBr) (νmax/cm–1): 3309, 2914, 1685, 1640,
55.86; H, 3.79; N, 15.95%.
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1616, 1593, 1542, 1485, 1435, 1319, 1280, 1021, 751. H NMR
N-(2-(1H-Benzimidazol-2-yl)phenyl)-1,3-dimethyl-1H-
(400 MHz, DMSO-d6) δ: 13.90 (s, 1H), 13.13 (d, J = 13.6 Hz, 1H),
pyrazole-5-carboxamide (T7): White solid, yield 53.5%, mp 8.79 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 7.9 Hz, 1H), 7.64–7.37 (m,
245-247 °C; IR (KBr) (νmax/cm–1): 3199, 2961, 1679, 1631, 1613, 3H), 7.30 (t, J = 7.5 Hz, 1H), 7.15 (t, J = 8.2 Hz, 1H), 7.05 (s, 1H),
1599, 1554, 1435, 1363, 1313, 1277, 1247, 1057, 1018, 962, 4.09 (s, 3H), 2.48 (s, 3H), 2.32 (d, J = 3.8 Hz, 3H). 13C NMR (101
771, 730. 1H NMR (400 MHz, DMSO-d6) δ: 13.90 (s, 1H), 13.29 MHz, DMSO-d6) δ: 158.2, 151.0, 150.7, 146.2, 142.6, 140.4,
(s, 1H), 8.80 (d, J = 8.3 Hz, 1H), 8.18 (d, J = 7.4 Hz, 1H), 7.75 (dd, 138.4 (d, J = 4.8 Hz), 136.6, 134.1, 133.4, 131.9 (d, J = 5.4 Hz),
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130.9 (d, J = 5.4 Hz), 127.5, 125.4, 124.4, 123.4, 120.1, 118.0 (d, 7.88 (d, J = 5.4 Hz, 1H), 7.62–7.45 (m, 3H), 7.31 (dd, J = 14.9,
View Article Online
J = 12.4 Hz), 115.9, 111.6 (d, J = 15.5 Hz), 107.1, 21.8 (d, J = 7.4 Hz, 1H), 7.23 (d, J = 8.7 Hz, 2H), 6.94D(tO,I:J1=0.160.369H/Cz8,N1JH05).15103CJ
10.0 Hz), 13.5. HRMS (ESI) Calcd for C20H20N5O [M+H]+: NMR (101 MHz, DMSO-d6) δ: 174.8, 165.0, 150.7, 140.7, 139.0,
346.16624, found: 346.16632. Anal. Calcd for C, 69.55; H, 5.54; 138.8, 138.2, 130.9, 128.5, 123.9, 121.5, 118.0, 113.1. HRMS
N, 20.28. Found: C, 69.24; H, 5.56; N, 20.03%.
3-Isobutyl-1-methyl-N-(2-(6-methyl-1H-benzimidazol-2-
(ESI) Calcd for C19H15N4OS [M+H]+: 347.09611, found:
347.09616. Anal. Calcd for C, 65.88; H, 4.07; N, 16.17. Found: C,
yl)phenyl)-1H-pyrazole-5-carboxamide (T12): White solid, 65.39; H, 3.89; N, 15.92%.
yield 51.7%, mp 180-182 °C; IR (KBr) (νmax/cm–1): 3324, 2952,
N-(2-(6-Chloro-1H-benzimidazol-2-yl)phenyl)nicotinamide
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1640, 1619, 1589, 1550, 1491, 1431, 1407, 1306, 1268, 1169, (T16): White solid, yield 65.3%, mp 300-302 °C; IR (KBr)
1062, 1015, 967, 595. 1H NMR (400 MHz, DMSO-d6) δ: 13.91 (d, (νmax/cm–1): 3324, 2896, 1661, 1634, 1504, 1548, 1480, 1441,
J = 34.3 Hz, 1H), 13.15 (d, J = 17.1 Hz, 1H), 8.80 (dd, J = 8.2, 3.5 1390, 1325, 1262, 1060, 965, 768, 727, 599. H NMR (400 MHz,
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Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 7.59–7.48 (m, 2H), 7.43 (d, J = DMSO-d6) δ: 13.88 (s, 1H), 13.46 (s, 1H), 9.34 (d, J = 1.7 Hz, 1H),
23.1 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.16 (t, J = 8.0 Hz, 1H), 8.87 (dd, J = 4.6, 3.6 Hz, 2H), 8.51 (d, J = 8.0 Hz, 1H), 8.15 (d, J =
7.06 (d, J = 10.7 Hz, 1H), 4.10 (s, 3H), 2.58–2.53 (m, 2H), 2.47 (d, 7.9 Hz, 1H), 7.75 (dt, J = 14.5, 7.3 Hz, 1H), 7.66 (s, 2H), 7.57 (t, J
J = 9.7 Hz, 3H), 2.08–1.92 (m, 1H), 1.01 (dd, J = 10.9, 6.6 Hz, = 7.8 Hz, 1H), 7.32 (t, J = 7.2 Hz, 2H). 13C NMR (101 MHz,
6H). 13C NMR (101 MHz, DMSO-d6) δ: 158.3 (d, J = 4.2 Hz), DMSO-d6) δ: 163.6, 152.8, 152.5, 148.9, 138.6, 135.3, 131.5,
151.1, 150.7, 149.9 (d, J = 6.0 Hz), 142.5, 140.4, 138.4 (d, J = 130.6, 127.7, 124.4, 123.7, 120.4, 115.6. HRMS (ESI) Calcd for
8.6 Hz), 136.5 (d, J = 7.7 Hz), 134.1, 133.5, 131.8 (d, J = 11.4 Hz), C19H14ClN4O [M+H]+: 349.08507, found: 349.08527. Anal. Calcd
130.9 (d, J = 5.9 Hz), 127.5, 125.3, 124.4, 123.5, 120.1, 117.9, for C, 65.43; H, 3.76; N, 16.06. Found: C, 65.75; H, 4.01; N,
117.7, 116.0 (d, J = 3.3 Hz), 111.6 (d, J = 15.1 Hz), 106.7 (d, J = 16.22%.
9.7 Hz), 37.1, 28.9 (d, J = 5.4 Hz), 22.8 (d, J = 2.7 Hz), 21.7 (d, J =
2-Chloro-N-(2-(6-chloro-1H-benzimidazol-2-
16.3 Hz). HRMS (ESI) Calcd for C23H26N5O [M+H]+: 388.21319, yl)phenyl)niacinamide (T17): White solid, yield 49.1%, mp
found: 388.21304. Anal. Calcd for C, 71.29; H, 6.50; N, 18.07. 228-230 °C; IR (KBr) (νmax/cm–1): 3419, 2993, 1699, 1640, 1616,
Found: C, 71.29; H, 6.53; N, 17.85%.
1538 , 1479, 1431, 1348, 1309, 1146, 1056, 747. 1H NMR (400
N-(2-(1H-Benzimidazol-2-yl)phenyl)niacinamide
(T13): MHz, DMSO-d6) δ: 13.53 (d, J = 25.1 H, 1H), 13.43 (s, s, 1H),
White solid, yield 69.6%, mp 235-237 °C; IR (KBr) (νmax/cm–1): 8.78 (d, J = 8.3 Hz, 1H), 8.65 (d, J = 4.6 Hz, 1H), 8.31 (d, J = 7.6
3410, 3288, 1634, 1619, 1599, 1545, 1485, 1435, 1402, 1319, Hz, 1H), 8.17 (d, J = 7.9 Hz, 1H), 7.71 (td, J = 8.3, 5.0 Hz, 1H),
1197, 965. 1H NMR (400 MHz, DMSO-d6) δ: 14.23 (s, 1H), 7.61 (t, J = 8.0 Hz, 2H), 7.50 – 7.35 (m, 2H), 7.28 (dd, J = 29.8,
13.34 (s, 1H), 9.41 (d, J = 1.6 Hz, 1H), 8.96–8.85 (m, 2H), 8.58 (d, 8.5 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ: 163.8, 151.7,
J = 8.0 Hz, 1H), 8.22 (d, J = 7.9 Hz, 1H), 7.77 (dd, J = 7.5, 5.1 Hz, 147.2, 138.9, 138.2, 132.8, 131.6, 128.0, 124.3, 123.9, 120.7,
2H), 7.64 (d, J = 7.0 Hz, 1H), 7.58 (t, J = 7.9 Hz, 1H), 7.39–7.29 118.0, 116.2. HRMS (ESI) Calcd for C19H13Cl2N4O [M+H]+:
(m, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 163.8, 152.9, 151.3, 383.04609, found: 383.04617. Anal. Calcd for C, 59.55; H, 3.16;
149.1, 142.1, 138.6, 135.3, 133.8, 131.2, 130.8, 127.6, 124.3, N, 14.62. Found: C, 59.75; H, 3.50; N, 14.39%.
124.0, 123.8, 122.9, 120.4, 118.5, 116.1, 112.0. HRMS (ESI)
N-(2-(6-Chloro-1H-benzimidazol-2-yl)phenyl)-2-
Calcd for C19H15N4O [M+H]+: 315.12404, found: 315.12411. mercaptoniacinamide (T18): Yellow solid, yield 43.8%, mp
Anal. Calcd for C, 72.60; H, 4.49; N, 17.82. Found: C, 72.29; H, 215-217 °C; IR (KBr) (νmax/cm–1): 3416, 3309, 2905, 1637, 1587,
4.62; N, 17.48%.
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1539, 1485, 1426, 1396, 1331, 1247, 1155, 1057, 742. H NMR
N-(2-(1H-Benzimidazol-2-yl)phenyl)-2-chloroniacinamide
(400 MHz, DMSO-d6) δ: 13.93 (s, 1H), 13.26 (s, 1H), 13.18 (s,
(T14): White solid, yield 80.5%, mp 203-205 °C; IR (KBr) 1H), 8.67 (d, J = 8.2 Hz, 1H), 8.11 – 7.99 (m, 2H), 7.90 (d, J = 5.3
(νmax/cm–1): 3229, 2991, 1700, 1628, 1599, 1548, 1485, 1435, Hz, 1H), 7.56 (d, J = 9.4 Hz, 3H), 7.32 (dd, J = 15.5, 8.0 Hz, 1H),
1399, 1319, 1277, 1244, 1137, 1072, 745. 1H NMR (400 MHz, 7.26 (s, 1H), 6.96 (t, J = 6.7 Hz, 1H). 13C NMR (101 MHz, DMSO-
DMSO-d6) δ: 13.87 (s, 1H), 13.28 (s, 1H), 8.82 (d, J = 8.3 Hz, 1H), d6) δ: 174.6, 164.8, 141.0, 139.7, 138.1, 138.0, 131.2, 128.9,
8.70–8.63 (m, 1H), 8.38–8.30 (m, 1H), 8.21 (d, J = 7.8 Hz, 1H), 124.1, 121.9, 118.3, 113.2. HRMS (ESI) Calcd for C19H14ClN4OS
7.71 (dd, J = 7.5, 4.9 Hz, 1H), 7.66–7.56 (m, 2H), 7.46–7.35 (m, [M+H]+: 381.05714, found: 381.05685. Anal. Calcd for C, 59.92;
2H), 7.30 (t, J = 7.5 Hz, 1H), 7.23 (t, J = 7.5 Hz, 1H). 13C NMR H, 3.44; N, 14.71. Found: C, 59.97; H, 3.92; N, 14.28%.
(101 MHz, DMSO-d6) δ: 163.8, 151.7, 151.0, 147.2, 142.1,
N-(2-(6-Methyl-1H-benzimidazol-2-yl)phenyl)niacinamide
139.0, 138.2, 133.8, 133.0, 131.3, 127.8, 124.3, 124.0, 123.8, (T19): White solid, yield 72.4%, mp 259-260 °C; IR (KBr)
122.8, 120.6, 118.7, 116.5, 112.0. HRMS (ESI) Calcd for (νmax/cm–1): 3324, 2854, 1661, 1634, 1616, 1593, 1551, 1485,
C19H14ClN4O [M+H]+: 349.08507, found: 349.08511. Anal. Calcd 1441, 1396, 1322, 1024, 765. 1H NMR (400 MHz, DMSO-d6) δ:
for C, 65.43; H, 3.76; N, 16.06. Found: C, 65.82; H, 4.14; N, 14.24 (s, 1H), 13.17 (d, J = 16.8 Hz, 1H), 9.46–9.35 (m, 1H),
15.77%.
N-(2-(1H-Benzimidazol-2-yl)phenyl)-2-
8.98–8.80 (m, 2H), 8.56 (d, J = 8.0 Hz, 1H), 8.19 (d, J = 7.9 Hz,
1H), 7.76 (dt, J = 8.0, 5.1 Hz, 1H), 7.65–7.38 (m, 3H), 7.33 (t, J =
mercaptoniacinamide (T15): Yellow solid, yield 42.0%, mp 7.3 Hz, 1H), 7.15 (t, J = 9.3 Hz, 1H), 2.48 (s, 3H). 13C NMR (101
207-209 °C; IR (KBr) (νmax/cm–1): 3416, 2955, 1661, 1575, 1527, MHz, DMSO-d6) δ: 163.7, 152.8, 151.1, 150.7, 149.1 (d, J= 8.8
1432, 1322, 1298, 1241, 1137, 1075, 757. 1H NMR (400 MHz, Hz), 142.5, 140.3, 138.5 (d, J= 4.4 Hz), 135.4 (d, J= 7.3 Hz),
DMSO-d6) δ: 13.89 (s, 1H), 13.33 (s, 1H), 13.12 (s, 1H), 8.70 (d, 134.0, 133.5, 131.9, 131.8, 130.9 (d, J= 6.9 Hz), 130.8, 127.4,
J = 8.1 Hz, 1H), 8.08 (d, J = 7.6 Hz, 1H), 7.99 (d, J = 6.8 Hz, 1H), 125.5, 124.4, 124.3 (d, J= 3.9 Hz), 123.7, 120.3, 118.1 (d, J= 8.7
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Hz), 116.2, 111.6, 111.5, 21.8, 21.7. HRMS (ESI) Calcd for these fungi were calculated as I (%)=[(C−T)/ (C−0.5)]×100,
View Article Online
C20H17N4O [M+H]+: 329.13969, found: 329.13992. Anal. Calcd where C represents the diameter of coloDnOyIo: 1n0.u10n3t9r/eCa8tNeJd05P1D50AJ,
for C, 73.15; H, 4.91; N, 17.06. Found: C, 72.84; H, 4.92; N, T represents the diameter of colony on treated PDA, and I
16.89%.
2-Chloro-N-(2-(6-methyl-1H-benzimidazol-2-
represents the inhibition rate. Furthermore, the fungicidal
activities of these compounds against B. cinerea were further
yl)phenyl)niacinamide (T20): White solid, yield 70.3%, mp assessed. As per the preliminary test record, these compounds
150-152 °C; IR (KBr) (νmax/cm–1): 3249, 2919, 1699, 1635, 1613, were dissolved in DMSO and diluted with the medium to
1600, 1549, 1484, 1431, 1405, 1388, 1314, 1269, 1148, 1064, obtain different final concentration grades. The colony
799, 753. 1H NMR (400 MHz, DMSO-d6) δ: 13.90 (s, 1H), 13.14 diameters were measured, and the inhibition percentages
(d, J = 17.8 Hz, 1H), 8.82 (d, J = 8.1 Hz, 1H), 8.66 (d, J = 3.0 Hz, relative to the blank control were calculated. The EC50 values
1H), 8.33 (dd, J = 7.6, 1.7 Hz, 1H), 8.18 (d, J = 7.9 Hz, 1H), 7.71 were finally obtained via linear-regression analysis.
(dd, J = 12.1, 6.1 Hz, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.50–7.34 (m, 3D-QSAR analysis
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2H), 7.32–7.18 (m, 1H), 7.08 (dd, J = 30.7, 8.1 Hz, 1H), 2.42 (d, J
The molecular modeling and CoMFA analysis were performed
using SYBYL 2.0 software. The 3D structures of all molecules
were built using the Sketch Molecule function in SYBYL. The
initial structural optimization was conducted using the
Gasteiger−Huckel charge, Tripos force field, and Powell
conjugate gradient algorithm with a convergence criterion of
0.005 kcal/mol Å.32
= 19.7 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 163.8, 151.7,
147.2, 138.9, 138.1, 133.0, 131.1, 127.6, 124.2, 123.9, 120.5,
118.3, 116.5, 111.6, 21.7 (d, J= 21.2Hz). HRMS (ESI) Calcd for
C20H16ClN4O [M+H]+: 363.10072, found: 363.10059. Anal. Calcd
for C, 66.21; H, 4.17; N, 15.44. Found: C, 66.62; H, 4.42; N,
15.29%.
Crystallographic study
The 3D-QSAR modeling was derived using the partial least
The single crystal of T1 was obtained from the mixed liquor of square (PLS) analysis. The interaction energies between a
chloroform and ethyl acetate (V/V = 2:3). The single crystal X- probe atom and all compounds were computed at surrounding
ray diffraction data were collected on an Agilent SuperNova points by using a volume dependent lattice with a 2.0 grid
(Dual, Cu at zero, AtlasS2) diffractometer at 100.00(10) K using spacing (default in SYBYL).33 The CoMFA descriptors were used
Mo Kα radiation (λ = 0.71073 Å) by the ω scan mode. The as independent variables, and the experimental pEC50 values
program CrysAlisPro was used for integration of the diffraction were presented as the dependent variables. The performance
profiles.29 The structure was solved by direct methods using of the models was evaluated by leave-one-out cross-validation,
the SHELXS program of the SHELXTL package and refined by and the optimal number of components (ONC) was
full-matrix least-squares methods with SHELXL.30 All non- determined with the highest cross-validated q2.34,35 The non-
hydrogen atoms of the compound T1 were refined with cross-validated correlation coefficient r2, standard error of
anisotropic thermal parameters. All hydrogen atoms were estimate (SEE), and F were subsequently calculated according
observed and placed at their calculated positions with a fixed to the definitions in the SYBYL 2.0 package. The contour maps
value of their isotropic displacement parameters.
In vitro antifungal bioassay
and standard deviations of CoMFA were generated by using
PLS coefficients.
The antifungal activities were evaluated against four
pathogenic fungi (B. cinerea, R. solani and F. graminearum, A.
solani) in vitro with a mycelia growth test on potato dextrose
agar medium (PDA).2,31 The mother liquor (22.5 mg/mL) was
prepared by dissolving the compound (18 mg) in DMSO (0.8
mL). 0.2 ML of mother liquor was mixed with sterile molten
PDA (45 mL) to obtain the PDA with the test compound at final
concentration of 100 ug/mL. The portions of PDA with
different compounds were poured into three Petri dishes (90
mm in diameter) on average (15 mL/dish), on which 5-mm
mycelial disks of the four fungi were placed at the center. The
disks were obtained from a pure PDA culture plate by
punching at the edge of the actively growing mycelia colony.
Each treatment condition was produced in three replicates.
The commercial fungicide hymexazol was served as the
positive control.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by the National Nature Science
Foundation of China (No. 31772209) and the Fundamental
Research Funds for the Central Universities of China (No.
KYTZ201604 and KJSY201607).
Notes and references
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Design, synthesis, antifungal activity and 3D-QSAR study of novel pyrazole carboxamide and
niacinamide derivatives containing benzimidazole moiety
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Wei-Jie Si, Xiao-bin Wang, Min Chen, Meng-Qi Wang, Ai-Min Lu and Chun-Long Yang
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The synthesized pyrazole carboxamide and niacinamide derivatives containing a benzimidazole moiety showed effectively inhibition
against the fungus B. cinerea. The 3D-QSAR model was built and revealed fine predictive ability.