Molecular design and the optimum synthetic route of the compounds with multi-pyrazole and its derivatives and the potential application in antibacterial agents

Accepted Manuscript

Molecular design and the optimum synthetic route of the compounds with multi-

pyrazole and its derivatives and the potential application in antibacterial agents

Ji-Xiao Wang, Zi-Ran Zhu, Feng-Ying Bai, Xin-Yu Wang, Xiao-Xi Zhang,

Yong-Heng Xing

PII:

S0277-5387(15)00336-8

DOI:

http://dx.doi.org/10.1016/j.poly.2015.06.020

POLY 11367

Reference:

Polyhedron

To appear in:

Received Date:

Accepted Date:

22 April 2015

12 June 2015

Please cite this article as: J-X. Wang, Z-R. Zhu, F-Y. Bai, X-Y. Wang, X-X. Zhang, Y-H. Xing, Molecular design

and the optimum synthetic route of the compounds with multi-pyrazole and its derivatives and the potential

application in antibacterial agents, Polyhedron (2015), doi: http://dx.doi.org/10.1016/j.poly.2015.06.020

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers

we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and

review of the resulting proof before it is published in its final form. Please note that during the production process

errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Molecular design and the optimum synthetic route of the

compounds with multi-pyrazole and its derivatives and the

potential application in antibacterial agents

Ji-Xiao Wang^a, Zi-Ran Zhu^b, Feng-Ying Bai^c, Xin-Yu Wang^a,

Xiao-Xi Zhang^a, Yong-Heng Xing^a∗

^aCollege of Chemistry and Chemical Engineering, Liaoning Normal University, Huanghe Road

850^#, Dalian City, 116029, P.R. China.

^bDepartment of Biochemistry, Dalian Medical University, Dalian, 116044, P.R. China

^cCollege of Life Science, Liaoning Normal University, Dalian 116029, P.R. China.

^∗Co-corresponding authors.

E-mail: xingyongheng2000@163.com (Y. H. Xing)

1

Abstract

Molecular design and efficient synthetic procedures have been

developed for pyrazole and its derivatives with different linkers. In

particular, twelve compounds with –I or –NO₂substituted groups on the

pyrazole ring were synthesized for the first time. These compounds are

characterized by element analysis, IR, HNMR, M.P. and X-ray diffraction.

In addition, some compounds and corresponding complexes were also

assayed in vitro for their ability to inhibit the growth of representative

Gram-positive bacteria, Gram-negative bacteria and the fungus. It was

worthwhile to note that some compounds could be used as potential

antibacterial agents.

Keywords: Pyrazole and its derivatives; N-heterocyclic ligands;

Antibacterial activity; Crystal structure

2

1 Introduction

Pyrazole and its derivatives as a crucial family member of N-

heterocyclic ligands are used as pesticides and medicinal preparations

because of their high biological activities[1-4]. In the mean time, it is

also found widespread applications in the fields of supramolecular

chemistry, crystal engineering, materials sciences, sensors, biochemistry,

catalysis etc.[5-12]. In the recent years, with the further study of

multidentate ligands, there is a growing interest in molecular design of

multi-pyrazole compounds and its derivatives due to larger π-conjugated

system, good planarity and stable structure[13-17]. During the past two

decades, some complexes with multi-pyrazolyl compound as ligands have

been

reported,

for

instance,

[Zn(L₂)(SCN)₂]

(L₂=

1,4-bis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzene),

Co(bpz*eaT)(SCN)₂

(bpz*eaT:

2,4-dimethyl-1H-pyrazol-1-yl)-6-diethylamino-1,3,5-triazine),

[Ag₂(Lp)_1.5(NO₃)](NO₃) [Cu₂(µ-Lp)(H₂O)₆](SiF₆)₂⋅(H₂O)₄Ag(µ-Lp)NO₃

(Lp=

p−[CH(pz)₂]₂C₆H₄),

Hg(TpzT)(SCN)₂·H₂O

(TPzT:

2,4,6-tri(pyrazole-1-yl)-1,3,5-triazine), etc.[18-22].

Most of the

complexes were reported about their characteristics, including spectrum,

structures, thermal properties, luminescent properties and synthesis.

However, there are few investigations about biological activity for the

complexes with multi-pyrazole and its derivatives as ligands. Obviously,

3

it became a hot issue that efficiently synthesizing of multi-pyrazole and its

derivatives compounds for exploring the application of the complexes in

the field of biochemistry.

Here, we have synthesized firstly twelve multi - pyrazolyl compounds

with -I or -NO₂substituted groups and explored a series of optimization

synthetic procedures for preparing multi - pyrazolyl compounds by the

reaction of pyrazole and its derivatives with different linkers in two kinds

of solvents: dimethylsulfoxide (DMSO)-potassium hydroxide (KOH) and

tetrahydrofuran (THF). Compared with traditional procedures[23-30],

we have achieved the goals of maximizing reaction efficiency and

minimizing chemical wastes. We have developed four types synthetic

system, including multi - pyrazolyl compounds (scheme 1): alkane

–pyrazole derivatives, benzene – dipyrazole derivatives and benzene –

tetrapyrazole derivatives in the superbasic DMSO-KOH medium; triazine

– pyrazole derivatives in the THF. In addition, in long time for exploring

synthetic procedures, we found that steric hindrance, substituted groups

and the condition of the reaction play a vital role in the targets of

separating high yield and purity compounds. At the same time, some

new compounds and the complexes reported by our group are assayed in

vitro for their biological activity and some of them exhibit excellent ability

of antibacterial activity.

4

Scheme 1: Four kinds of pyrazole and its derivatives: (a) alkane –pyrazole derivatives; (b) benzene

– dipyrazole derivatives; (c) benzene – tetrapyrazole derivatives; (d) triazine – pyrazole derivatives

2. Results and discussion

2.1 Synthesis

We began to explore the optimum synthetic route of alkane – pyrazole

derivatives by controlling the reaction of pyrazole, 1,1,2,2 –

tetrabromoethane (TBE) and potassium hydroxide (KOH) in 4:1:4 molar

ratios and prolonging deprotonation reaction time in the mixed system of

pyrazole and KOH in dimethylsulfoxide (DMSO) at 80 °C. That is the

5

solution of TBE in DMSO was added dropwise into mixed solution and

stirred continuously for 5 h. Through a series of follow-up treatments,

the target product 1a was obtained in 71.4 %, which was much higher

yield and low side reactions with TBE than that reported in literature [24].

To obtain high yield and reduce reaction time of compound 1c, we

attempted to choose 4-iodo-1H-pyrazole as nucleophile to replace starting

material “1a” in the literature[23] to synthesize “1c”, and the synthetic

condition was similar to that of compound 1a, the result found that we

have obtained successfully target product 1c and achieved expected yield.

Scheme 2. The synthetic route of the compounds with alkane – pyrazole derivatives 1a-1c

In addition, we have also studied the optimum synthetic method of

benzene – dipyrazole derivatives and corresponding complexes[31].

That is the mixture of pyrazole (or its derivatives) and KOH in DMSO

was vigorous stirred for making pyrazole complete deprotonation and then

6

reacted with the solution of 1,4-bis(bromomethyl)benzene in DMSO.

After a series of following steps, we obtained compounds 2a and 2b. It

was considered that 1H-pyrazole occurs readily iodination (nitration)

substituted reaction on the C4 atom and coordination modes of the

compounds would be varied via introduction of different substituents onto

the pyrazole rings, we choose pyrazole derivatives with iodination or

nitration group as nucleophile and synthesized successfully four new

compounds 2c - 2f with -I and NO₂groups by the similar synthetic method

of the compounds 2a - 2b.

Scheme 3 The synthetic route of the compounds with benzene – tetrapyrazole derivatives 2a-2f

The facile methods of synthesizing benzene – tetrapyrazole derivatives

were shown in Scheme 4. The optimization route ideology was similar

to that of alkane – pyrazole derivatives: that is controlling primary

materials molar ratio and prolonging completely deprotonation reaction

time. We obtained higher yields and the products, which were readily

isolated by dilution with water and filtration. The additional advantages

7

was avoiding the use of toxic phosgene in the traditional route, shortening

the reaction time of pyrazole with 1,4-bis(dibromomethyl)benzene and the

procedure was more benign from the ecological viewpoint. In addition,

we also chosen pyrazole derivatives with -I and NO₂groups as

nucleophile to synthesize successfully a series of new compounds 3c – 3f.

Through a lot of experimental results, we found that substituted groups

and steric hindrance from different substituted groups on the pyrazole ring

play a vital role in synthesizing benzene– dipyrazole/tetrapyrazole

derivatives. So, synthesizing of the corresponding compounds with CH₃

group on the pyrazole ring would be more difficult than the other

compounds and need more time. In addition, it needed more time to

synthesize benzene – tetrapyrazole derivatives compounds due to the

increasing number of pyrazole and its derivatives.

Scheme 4. The synthetic route of the compounds with benzene – tetrapyrazole derivatives 3a-3f

In order to further investigate synthetic method and explore new

compounds, we have also studied the synthetic procedures of

1,3,5-triazine – pyrazole derivatives. The triazine – pyrazole derivatives

8

were prepared by using a mild, convenient, timesaving and general

method with high yield of the product (Scheme 5). We adjusted adding

sequence of raw materials and modified reaction condition to obtain 4a:

that is 3, 5-dimethylpyrazol was added gradually into the stirring

completely the THF solution with cyanuric chloride at room temperature,

then, triethylamine was added dropwise to a mixture solution of

3,5-dimethylpyrazole and cyanuric chloride in the THF at room

temperature. After stirring for 1 h at room temperature, the solution was

refluxed at 80 °C for 5 h and then filtered to get desired compound. We

attempted to change 3,5-dimethylpyrazole into pyrazole derivatives for

obtaining new compounds and synthesized successfully 4b – 4e for the

first time with the same reaction condition. In the mean time, we used

the similar optimum reaction condition to obtain 4f – 4g. For completely

deprotonation of the pyrazole, the pyrazole in the anhydrous THF was

added dropwise into completely mixed solution of NaH in the anhydrous

THF at room temperature for 1 h. Subsequently, the anhydrous THF

with cyanuric chloride was added dropwise into the refluxing mixture and

the mixture refluxed at 70 °C for 5 h, at last, we obtained successfully a

series of novel compounds 4h – 4k with the similar reaction route. In the

exploration of synthetic routes, both the adding order of raw materials and

different reaction temperature during the synthesizing process were

essential factors.

9

Scheme 5. The synthetic route of the compounds with triazine – pyrazole derivatives 4a-4k

2.2 Structural description

The complexes with multi - pyrazole and its derivatives were

reported in many literatures, but few compounds with – I and -NO₂

substituted groups were studied. Here, we reported mainly some

compounds with the multi - pyrazole of the - I and -NO₂substituted

groups. Crystallographic data and the structure refinement were given in

Table 1. The selected bond lengths and bond angles were listed in Table

S1.

C₁₄H₁₀N₁₂O₈·2DMSO (1b). Compound 1b is crystallized in the

triclinic system with P-1 space group. X-ray single crystal analysis

indicates that the asymmetric unit of the compound 1b consists of an half

of the compound 1b molecule and one free DMSO molecule (Fig. 1a).

10

Four 4-nitro-pyrazolyl moieties are linked by the “-CH-CH-“ moiety to

form a N-heterocyclic compound which has trans-symmetrical molecular

configuration. There are three kinds of hydrogen bondings among the

carbon atoms from pyrazole or CH-CH moiety and the oxygen atom from

the lattice DMSO molecule to form a trifurcated hydrogen bond (Fig. 1b):

C2-H2···O5^#1, C4-H4···O5^#1and C5-H5···O5^#1[C2-H2···O5^#1, 3.123(7)

Å, 131.1°; C4-H4···O5^#1, 3.076(6), 139.7°; C5-H5···O5^#1, 3.188(7),

130.5°, #1: 2-x, 1-y, 1-z.].

a

b

Fig.1 (a) molecular structure of compound 1b; (b) a view of hydrogen bonds in 1b. (a

part of hydrogen atoms are omitted for clarity) (#1: 2-x, 1-y, 1-z).

C₁₄H₁₀N₈I₄·2DMF (1c). Single crystal X-ray diffraction analysis

reveals that compound 1c crystallizes in the monoclinic, space group

P2₁/n. Similarly, the asymmetric unit of compound 1c contains half a 1c

molecule and one lattice DMF molecule (Fig. 2a). Four 4-iodo-pyrazole

moieties are linked by the “-CH-CH-“ moiety to form a N-heterocyclic

compound which has trans-symmetrical molecular configuration. In

11

addition, in the molecular packing of the compound 1c, there are hydrogen

bondings to occur, as illustrated in Fig. 2b: hydrogen bonding between the

carbon from the pyrazole and the oxygen from the lattice DMF molecule,

C5-H5A···O5^#2(C5-H5A···O5^#2: 3.214(7) Å, 167.0°, #2: 1-x, 1-y, 2-z.).

a

b

Fig.2 (a) molecular structure of compound 1c; (b) a view of hydrogen bonds in 1c. (a

part of hydrogen atoms are omitted for clarity) (#2: 1-x, 1-y, 2-z).

C₁₄H₁₀N₈I₄·2DMF (1c-1). We obtained compound 1c-1 which is

different from compound 1c in crystal system and space group,

temperature re-crystallized for the two compounds is distinct. 1c-1 is

crystallized in the triclinic system with P-1 space group. The asymmetric

unit of the compound 1c-1 is made up of an half of the 1c-1 molecule and

one lattice DMF molecule (Fig.3). The -CH-CH- moiety links four

4-iodo-pyrazole moieties to form a N-heterocyclic compound with

trans-symmetrical molecular configuration.

12

Fig.3 molecular structure of compound 1c-1.(a part of hydrogen atoms are omitted for

clarity)

C₁₄H₁₂N₄I₂(2c). Single crystal X-ray diffraction analysis reveals that

compound 2c crystallizes in the monoclinic, space group P2₁/n. The

asymmetric unit of compound 2c contains an half of 2c molecule (Fig. 4a).

Two 4-iodo-pyrazole moieties are linked by the para-xylene moiety to

form a N-heterocyclic compound with the structural feature of chair

conformation. The dihedral angle between benzene ring and pyrazole

ring is 76.51(34)°.

C₁₈H₂₀N₄I₂(2d). Compound 2d is crystallized in the monoclinic

system with P2₁/c space group. X-ray single crystal analysis indicates

that the asymmetric unit of the compound 2d consists an half of 2d

molecule (Fig. 4b).

The para-xylene moiety links two

4-iodo-3,5-dimethyl-pyrazole moieties to form a N-heterocyclic

compound which has the structural of tensional chair conformation. The

13

dihedral angle between benzene ring and pyrazole ring is 84.56(0.41)°.

a

b

Fig.4 (a) molecular structure of compound 2c; (b) molecular structure of compound 2d

(a part of hydrogen atoms are omitted for clarity)

C₁₄H₁₂N₆O₄(2e) Single crystal X-ray diffraction analysis reveals that

compound 2e crystallizes in the monoclinic system with space group P2₁/c.

Structural analysis shows that there is an half of the 2e molecule in the

asymmetric unit (Fig. 5a). Two 4-nitro-pyrazole moieties are linked by

para-xylene moiety to form a N-heterocyclic compound which has the

structural of chair conformation. The dihedral angle between benzene

ring and pyrazole ring is 74.69(16)°. In addition, the adjacent molecules

are further linked by intermolecular weak hydrogen bonding interactions

between carbon atom from the pyrazole ring and oxygen atom of the nitro

group (C7–H7···O2^#3, 3.220(4) Ǻ, 162.6°, #3: 1-x, 1-y, -z) to afford a

zigzag chain along ac plane ( Fig. 5b).

14

a

b

Fig.5 (a) molecular structure of compound 2e (b) a view of hydrogen bonds in 2e. (a

part of hydrogen atoms are omitted for clarity) (#3: 1-x, 1-y, -z).

C₂₀H₁₄N₈I₄(3c) Single crystal X-ray diffraction analysis reveals that

compound 3c crystallizes in the triclinic, space group P1.

The

asymmetric unit of compound 3c contains an half of 3c molecule (Fig.

6(a)). Four 4-iodo-pyrazole moieties are linked by a para-xylene moiety

to form the N-heterocyclic compound with the trans-symmetrical structure.

The dihedral angles between benzene ring and pyrazole ring are

72.07(54)° and 75.12(53)°, the dihedral angle between pyrazole ring with

pyrazole ring is 68.93(32)°.

C₁₃H₁₄N₈I_2.(4b) Compound 4b is crystallizing in monoclinic, space

group P2₁/c. The asymmetric unit of compound 4b consists an half of

the 4b molecule (Fig. 6(b)).

The triazine moity links two

4-iodo-pyrazole moieties to form a N-heterocyclic compound. The

dihedral angles between triazine ring and pyrazole ring are 3.51(28)° and

15

10.81(29)°.

a

b

Fig. 6 (a) molecular structure of compound 3c (b) molecular structure of compound 4b

(a part of hydrogen atoms are omitted for clarity)

By comparison of these structural characterization, it is found that the

pyrazole ring and linkers are not in the same plane: (i) for the alkane –

pyrazole derivatives and benzene – pyrazole derivatives, the pyrazole rings

are linked by the linkers easily to form the N-heterocyclic compound of

multi-coordination sites, which has trans-symmetrical structure or the

characteristic of chair conformation; (ii) the small dihedral angle between

triazine ring and pyrazole ring is formed easily in triazine – pyrazole

derivatives. By comparison of corresponding bond lengths on the

pyrazole ring (Table 2), it is found that the bond lengths of C-N and C-C

are in the close range in the corresponding compounds, especially, the

distances of C_pz– I and C_pz- N_nitroare close very much.

16

Table1 Crystallographic data for compounds

compounds

1b

1c

1c-1

2c

2d

2e

3c

4b

formula

M (g mol⁻¹)

Crystal system

Space group

a/Å

C₁₈H₂₂N₁₂O₁₀S₂

630.60

C₂₀H₂₄I₄N₁₀O₂

944.09

Monoclinic

P2₁/c

C₂₀H₂₄I₄N₁₀O₂

944.09

C₁₄H₁₂I₂N₄

490.08

Triclinic

P-1

C₁₈H₂₀I₂N₄

546.18

Monoclinic

P2₁/c

C₁₄H₁₂N₆O₄

328.30

Monoclinic

P2₁/c

C₂₀H₁₄I₄N₈

873.99

Triclinic

P1

C₁₃H₁₄I₂N₈

536.12

Monoclinic

P2₁/c

Triclinic

P-1

Triclinic

P1

8.6812(17)

8.9094(11)

5.6438(8)

4.5306(10)

7.5347(11)

11.151(2)

5.4245(8)

8.644(2)

b/Å

c/Å

9.5183(19)

10.382(2)

89.222(2)

67.749(2)

64.594(2)

705.7(2)

1

11.0749(13)

16.5832(16)

90

115.053(5)

90

9.6032(14)

14.003(2)

76.079(2)

83.613(2)

83.802(2)

729.48(18)

1

8.2058(18)

11.009(2)

73.492(2)

82.107(3)

75.042(3)

378.22(14)

1

14.945(2)

8.7180(12)

90

95.782(2)

90

4.5954(9)

14.707(3)

90

106.20(3)

90

11.6688(18)

12.4309(19)

99.592(2)

91.753(2)

98.072(2)

767.0(2)

1

16.631(4)

12.285(3)

90

91.006(3)

90

α (°)

β (°)

γ (°)

V (Å³)

Z

1482.3(3)

2

976.7(2)

2

723.7(2)

2

1765.9(7)

4

D_calc(g cm⁻³)

1.484

2.115

2.149

2.152

1.857

1.507

1.892

2.017

crystal size (mm)

0.31×0.23×0.19

0.64x0.55x0.49 0.55x0.25x0.09 0.67x0.19x0.17 0.53x0.21x0.15 0.46x0.31x0.26 0.45x0.37x0.22

0.46x0.14x0.07

F(000)

µ(Mo-Kα)/mm^-1

326

0.261

884

4.240

442

4.308

230

4.153

524

3.227

340

0.115

402

4.083

1016

3.573

2.15 to 27.03

2.28 to 24.00

2.19 to 26.00

2.66 to 25.00

2.72 to 25.00

3.81 to 27.45

2.22 to 25.00

2.06 to 26.65

θ (°)

Reflections collected

4155

6651

3786

1875

4795

6409

3705

9666

Independent reflections

2995(1937)

2329(2075)

2784(2303)

1299(1224)

1706(1527)

1641(961)

2654(2272)

3637(2631)

(I>2σ(I))

Parameters

∆(ρ) (e Å^–3)

192

2.796, -0.682

167

0.510, -1.063

175

0.978, -1.169

91

111

0.425, -1.879

109

0.156, -0.221

152

1.832, -1.229

210

0.464, -0.720

0.844, -1.384

17

Goodness of fit

1.070

1.083

1.084

1.266

1.210

1.053

1.084

1.041

R^a

0.0997(0.1366)^b

0.2837(0.3225)^b

0.0311(0.0359)^b0.0382(0.0470)^b0.0342(0.0359)^b0.0412(0.0449)^b0.0582(0.1053)^b0.0521(0.0593)^b0.0351(0.0572)^b

0.0707(0.0731)^b0.0926(0.0978)^b0.1060(0.1071)^b0.1043(0.1070)^b0.1198(0.1371)^b0.1891(0.1970)^b0.0734(0.0828)^b

a

wR₂

*^aR = Σ Fo − Fc / Σ  Fo , wR₂= {Σ[w(F_o−F_c²)²] / Σ[w(F_o) c^1/2; [F_o> 4σ(F_o)].

2

2 2

^bBased on all data

Table 2. Comparison of related bond lengths (Å) for compounds

compounds

C_pz-C_pz

C_pz=C_pz

C_pz-N_pz

C_pz=N_pz

N_pz-N_pz

C_pz-I

C_pz-N_nitro

1.403(8), 1.386(7)

1.391(8), 1.393(7)

1.395(7), 1.400(7)

1.383(8)

1.370(8), 1.366(7)

1.359(7), 1.363(6)

1.369(7), 1.359(7)

1.379(8)

1.340(6), 1.342(6)

1.344(6), 1.344(6)

1.348(6), 1.355(6)

1.343(8)

1.317(7), 1.327(7)

1.322(7), 1.318(6)

1.320(7), 1.318(7)

1.328(8)

1.366(6), 1.363(6)

1.359(5), 1.363(5)

1.365(5), 1.358(6)

1.360(7)

1.432(7),1.437(6)

1b

1c

2.080(5), 2.067(5)

2.068(5), 2.071(5)

2.066(6)

1c-1

2c

1.403(7)

1.369(7)

1.349(6)

1.337(7)

1.362(5)

2.073(5)

2d

2e

1.384(3)

1.373(3)

1.321(3)

1.365(3)

1.417(3)

1.383(13),1.381(12)

1.404(6), 1.394(7)

1.374(12),1.373(14)

1.342(6), 1.344(6)

1.350(10),1.358(10)

1.370(5), 1.357(6)

1.321(12),1.358(13)

1.314(6), 1.328(6)

1.359(9), 1.329(10)

1.363(5), 1.376(5)

2.087(8),2.058(9)

2.077(5), 2.068(5)

3c

4b

18

2.3 Antibacterial activity of the compounds and complexes 1-5

Antibacterial activity of the compounds and the complexes[31] were

assayed in vitro for their ability to inhibit the growth of representative

Gram-positive bacteria (Staphyloccocus aureus and Bacillus subtilis),

Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria

and the fungus Candida albicans. The susceptibilities of certain strains

of bacteria and a fungus to the compounds and their complexes were

evaluated by measuring the area of the bacteria-inhibiting ring: S = π (R²

– r²) (R: radius of the bacteria-inhibiting ring; r: the radius of one piece of

the filter paper). The results are given in Table 3 and Table S2 for the

compounds and complexes.

In our work, we observed that compound 2b and complexes 1 - 5 have

varying degrees of antibacterial activities for all the bacteria and fungus.

Most of them were found to be more active against Gram-positive

bacteria than Gram-negative bacteria. Compounds 3a and 3e exhibited

only the inhibiting effect to the Gram-positive bacteria and compound 3b

displayed better inhibiting effect to the Bacillus subtilis. In the

experiments, it was worthwhile to note that (i) all of the compounds and

complexes with antibacterial activities demonstrated the inhibiting effect

in a dose-dependent manner; (ii) all of them showed the inhibiting effect

to the Staphyloccocus aureus, their inhibitory effect was ordered as: 5 >

3e > 2 > 4 > 1 > 3 > 2b > 3a > 3b.

Based on the data in the Table 3, it was found that 3- and 5- positions

of the pyrazole ring were substituted by electron-donating -CH₃group,

which would make the electron cloud density of pyrazole ring increasing,

resulting in compounds exhibiting antibacterial activity in the

benzene–dipyrazole derivatives.

In which -NO₂group was a

withdrawing electron group and -I group was the cation with large ion

19

radius. Both reduced the electron density of the pyrazole ring. The

benzene-tetrapyrazole derivatives showed only antibacterial activity

against Gram-positive bacteria and their inhibitory effect was ordered as:

3e > 3a > 3b (the substituted groups on the pyrazole ring for 3e was NO₂,

for 3b was -CH₃and no substituted group for 3a). According to the

structure of the compound, it was found that the antibacterial activity

enhanced with the electron cloud density decreasing in the benzene -

tetrapyrazole derivatives. The larger steric hindrance could result in low

antibacterial activity of the compounds (3c, 3d and 3f). While the

complexes exhibited common antibacterial activity against all the four

bacteria and fungus. It was interesting to find that metal ions can

improve inhibitory ability of the compound or made the compound to

exhibit better antibacterial activity. At the same time, we also found that

the kind of the metal ion would affect the result for the antibacterial

activity. Complexes 2 and 3 were of similar structures with different

central metal ions, but they exhibited respective excellence of the

antibacterial activity against Gram-positive bacteria, Gram-negative

bacteria and the fungus. The analysis result of antibacterial activity for

some triazine – pyrazole derivatives found they have not any antibacterial

activity (Table S2).

Table 3. Antibacterial activities for some the compounds and related complexes 1-5

Dose

(µg)

480

240

120

60

480

240

120

60

Area of the zone ( mm²)

compound

P. aeruginosa

S. aureus

0.00

160.37

79.51

0.00

B. subtilis

E. coil

0.00

19.63

12.78

0.00

C. albicans

0.00

79.51

40.84

33.45

15.58

0.00

19.63

0.00

71.72

33.45

26.67

0.00

2a

2b

480

240

120

60

0.00

2c

0.00

480

2d

20

240

120

60

0.00

480

240

120

60

480

240

120

60

0.00

2e

2f

480

240

120

60

480

240

120

60

480

240

120

60

480

240

120

60

480

240

120

60

480

240

120

60

480

240

120

60

480

240

120

60

131.85

104.89

87.96

55.70

71.72

12.78

0.00

108.57

63.62

48.41

0.00

867.80

517.70

222.14

141.37

0.00

222.14

123.05

71.72

40.84

0.00

141.37

104.89

66.93

40.84

48.41

26.67

0.00

123.05

87.96

63.62

26.67

79.51

48.41

12.78

0.00

131.85

104.89

87.96

0.00

87.96

40.84

26.67

0.00

201.06

180.67

79.51

63.62

19.63

0.00

160.37

123.05

55.70

25.63

79.51

48.41

0.00

3a

3b

3c

3d

3e

3f

1

0.00

431.81

255.38

180.67

79.51

0.00

244.35

122.87

71.56

26.53

473.97

244.57

141.37

87.96

201.06

123.05

87.96

26.67

290.19

201.06

141.37

79.51

563.00

391.22

364.52

290.19

190.47

104.89

71.72

19.63

267.04

185.55

87.96

19.63

201.06

141.37

87.96

0.00

201.06

104.89

87.96

40.84

487.79

445.13

314.75

302.38

2

0.00

480

240

120

60

480

240

120

60

233.26

160.37

19.63

0.00

87.96

40.84

211.83

141.37

104.89

123.05

211.83

104.89

26.67

0.00

79.51

40.84

26.67

326.57

222.14

180.67

79.51

3

4

240

120

60

5

30

21

3. Conclusions

We have developed a practical and efficient route for synthesizing

compounds with multi - pyrazole and its derivatives and successfully

synthesized twelve novel pyrazole compounds with -I or -NO₂substituted

group. In the mean time, antibacterial activities of the some compounds

and complexes are assayed. It is found that some of them could be used

as potential antibacterial agents.

4. Experimental section

4.1 Materials and General procedure

All chemicals purchased were of reagent grade or better and were used

without further purification. Biological reagent: Staphyloccocus aureus,

Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Candida

albicans were obtained from Dalian Medical University, Department of

Biochemistry. The tryptone and yeast extract were purchased from

OXOID.

The gelatine was purchased from BIOSHARP.

The

complexes 1 – 5 were synthesized by the method of the literature[31].

The infrared spectra were recorded on a JASCO FT/IR-480 PLUS Fourier

Transform spectrometer with pressed KBr pellets in the range 200–4000

cm^-1. Melting points were determined by X-5 precision melting point

1

apparatus. The H NMR and ¹³C NMR spectra were recorded on

BrukerAV-500 apparatus (CDCl₃or DMSO-d₆as solvent, TMS internal

standard). Elemental analyses for C, H, and N were carried out on a

Perkin Elmer 240C automatic analyzer.

4.2 X-ray single crystal structural determinations

Suitable single crystals of compounds were mounted on glass fibers for

X-ray measurement, respectively. Reflection data were collected at

room temperature on a Bruker AXS SMART APEX II CCD

diffractermeter with graphite-monochromatized Mo-Ka radiation (λ=

0.71073 Å) and a ω scan mode. All the measured independent

22

reflections (I>2σ(I)) were used in the structural analyses, and

semi-empirical absorption corrections were applied using SADABS

program[32]. The structures were solved by the direct method using

SHELXL-97 program[33].

4.3 Antibacterial activity methods

The bacteria-inhibiting ring method was applied to measure the

anti-bacterial effects of benzene - pyrazole derivatives, a part of triazine -

pyrazole derivatives and complexes 1 - 5 on the growths of the bacterium

strains of Staphyloccocus aureus, Bacillus subtilis, Escherichia coli,

Pseudomonas aeruginosa and Candida albicans.

The five types of

strains were made into bacterial culture plates by the four section line

method and the plates were incubated at 37 °C for 17 h. The single

colony was shook cultivation (37 °C, 160 r/min) in 5 mL of the LB liquid

medium for 17 h. The bacterial suspensions were measured the

absorbance at 600nm and diluted to OD₆₀₀= 0.1 (8.5 × 10⁹CFU/L).

Then, 100 µL of the suspensions were coated evenly on the LB solid plate

surface and put one piece of filter paper (diameter = 6mm) with autoclave

and drying on the center of the plate. 10 µL of DMSO was added into

the filter paper as negative control and four pieces of the filter paper were

pasted evenly around it in the periphery. The four pieces of the filter

paper were added respectively 10µL of different concentrations samples

from high to low in clockwise. The plate was incubated at 37 °C in the

incubator for 17 h, and then measured the diameter of the

bacteria-inhibiting ring (accurate to 1 mm).

4.4 Preparation of alkane – pyrazole derivatives 1a - 1c

4.4.1 1,1,2,2-tetra(1H-pyrazol-1-yl)ethane (1a). A mixture of 13.88 g

(0.2 mol) of pyrazole, 13.66 g (0.2 mol) of finely powdered potassium

hydroxide, and 50 ml of DMSO was stirred at 80 °C. After 1 h, a

23

solution of 5.83 mL (0.05 mol) of TBE in 50 mL of DMSO was added

dropwise to the resulting suspension, the mixture continue to stir for 5 h.

Then, the reaction mixture was allowed to cool then diluted with 1000

mL of ice water. The precipitate was filtered off, washed with water and

dried under reduced pressure. Obtaining as a white solid in 71.4 %

(10.50 g), m. p.: 271–273 °C. IR (KBr, ν/cm^-1): 3136, 3117, 2994,

1521, 1310, 1052. ¹HNMR (500 MHz, CDCl₃, δ/ppm): 6.13 (t, 4H,

4-H-pz), 7.47 (d, 4H, 3-H-pz), 7.65 (d, 4H, 5-H-pz), 7.83 (s, 2H). Calc.

for C₁₄H₁₄N₈: C, C, 57.13; H,4.79; N, 38.07. Found: 57.09; H, 4.67; N,

38.26 %.

4.4.2 1,1,2,2-tetrakis(4-nitro-1H-pyrazol-1-yl)ethane (1b). A mixture

of 0.29 g (1.00 mmol) of compound 1a, 1.3 ml of 68% nitric acid (20.0

mmol of HNO₃), and 6 ml of 98% sulfuric acid was kept for 24 h at room

temperature. The mixture was then diluted with an ice–water mixture

(150 mL). The precipitate was filtered off, washed with water and dried

under reduced pressure. Compound 1b was dissolved in 5 mL DMSO at

room temperature and the crystals for X-ray measurement were obtained

after 3 days. Obtaining as a white solid in 81 % (0.33 g), m. p.: ＞

280 °C. IR (KBr, ν/cm^-1): 3141, 3109, 3012, 1514, 1313, 1003, 1542,

1329. ¹HNMR (500 MHz, DMSO-d₆, δ/ppm): 9.22(s, 4H, 5-H-pz),

8.77(s, 2H, CH), 8.44(s, 4H, 3-H-pz). ¹³CNMR (125 MHz, DMSO-d₆,

δ/ ppm): 137.55, 135.99, 131.85, 71.69. Calc. for C₁₄H₁₀N₁₂O₈: C, 35.45;

H,2.13; N, 35.44. Found: C, 35.37; H, 2.07; N, 35.61 %.

4.4.3 1,1,2,2-tetrakis(4-iodo-1H-pyrazol-1-yl)ethane (1c). A mixture of

3.88 g (0.02 mol) of 4-iodo-1H-pyrazole, 1.37 g (0.02 mol) of finely

powdered potassium hydroxide, and 15 mL of DMSO was stirred at

80 °C. After 30 min, a solution of 0.58 mL (0.005 mol) of TBE in 10

mL of DMSO was added dropwise to the resulting suspension, the

24

mixture was stirred for 2 h. The reaction mixture was allowed to cool

then diluted with 1000 mL of ice water. The precipitate was filtered off,

washed with water and dried under reduced pressure. We obtained

single crystals 1c and 1c-1 for X-ray measurement with different

recrystallization condition. Compound 1c was dissolved in 10 mL DMF

at room temperature to obtain single crystals 1c for X-ray measurement

after 5 days. Compound 1c was dissolved in 10 mL DMF at 50 °C for

2h and then the solution was stored at room temperature to obtain single

crystals 1c-1 for X-ray measurement after 5 days. Obtaining as a white

solid in 86.7 % (3.46 g), m. p.: ＞280 °C. IR (KBr, ν/cm^-1): 3125, 3112,

2987, 1510, 1310, 1107, 611. ¹HNMR (500 MHz, DMSO-d₆, δ/ppm):

8.28(s, 2H, CH), 8.27(s, 4H, 5-H-pz), 7.57(s, 4H, 3-H-pz). ¹³CNMR

(125 MHz, DMSO-d₆, δ/ppm): 145.39, 135.95, 71.62, 59.05. Calc. for

C₁₄H₁₀N₈I₄: C, 21.07; H,1.26; N, 14.04 Found: C, 21.16; H, 1.15; N,

14.31.%.

4.5 General experimental procedures for preparation of benzene –

dipyrazole derivatives 2a-2f

4.5.1 1,4-bis((1H-pyrazol-1-yl)methyl)benzene (2a). A mixture of 1.02

g (15 mmol) of pyrazole, 1.02 g (15 mmol) of finely powdered potassium

hydroxide, and 10 ml of DMSO was stirred at 80 °C. After 1 h, a

solution of 1.98 g (7.5 mmol) of 1,4-bis(bromomethyl)benzene in 10 mL

of DMSO was added dropwise to the resulting suspension, the mixture

was stirred for 5 h. The reaction mixture was allowed to cool then

diluted with 200 mL of water. The precipitate was filtered off, washed

with the mixture of ethanol and water, dried under reduced pressure.

Obtaining as a white powder in 68.9 % (1.23 g), m. p.: 111.5-112.1°C.

IR (KBr, ν/cm^-1): 3116, 3090, 2934, 2857, 1611 1516, 1451, 1516 1285,

1051, 808,. ¹HNMR (500 MHz, CDCl₃, δ/ppm): 7.53(d, 2H, H⁵-pz,

J=1.7Hz), 7.36(d, 2H, H³-pz, J=2.2Hz), 7.17(s, 4H, ph), 6.27(t, 2H, H4-pz,

25

J=2.1Hz), 5.30(s, 4H, CH₂). ¹³CNMR (125 MHz, CDCl₃, δ/ppm):

139.62, 136.55, 129.24, 128.04, 106.02, 55.52. Calc. for C₁₄H₁₄N₄:

C,70.57; H,5.92; N, 23.51. Found: C, 70.49; H, 5.88; N, 23.63%.

4.5.2 1,4-bis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzene (2b).

A

mixture of 1.47 g (15 mmol) of 3,5-dimethylpyrazole, 1.02 g (15 mmol)

of potassium hydroxide, and 10 ml of DMSO was stirred at 80 °C for 1 h.

A solution of 1.98 g (7.5 mmol) of 1,4-bis(dibromomethyl)benzene in 10

ml of DMSO was added dropwise, the mixture was stirred for 8 h at

80 °C, diluted with 200 ml of water, and the precipitate was filtered

off ,washed with the mixture of ethanol and water, dried under reduced

pressure. Obtaining a white powder in 61.2 % (1.35 g), m. p.:

101-102°C. IR (KBr, ν/cm^-1): 3123, 3030, 2934, 2860, 1610 1548, 1461,

1511 1308, 1026,797. ¹HNMR (500 MHz, CDCl₃, δ/ppm): 7.00(s, 4H,

ph), 5.82(s, 2H, H⁴-pz), 5.16(s, 4H, CH₂), 2.22(s, 6H, 5-CH₃-pz), 2.12(s,

6H, 3-CH₃-pz). ¹³CNMR (125 MHz, CDCl₃, δ/ppm): 147.58, 139.11,

136.68, 126.92, 105.54, 52.23, 13.48, 11.07. Calc. for C₁₈H₂₂N₄: C,

73.44; H, 7.53; N, 19.03. Found: C, 73.57; H,7.32; N, 19.11 %.

4.5.3 1,4-bis((4-iodo-1H-pyrazol-1-yl)methyl)benzene (2c). Compound

2c could be obtained by the similar synthetic procedures as compound 2a,

but the starting material 4-iodo-1H-pyrazol (2.19 g, 15 mmol) instead of

pyrazole. The single crystals 2c for X-ray measurement were obtained

from methanol at room temperature after 3 days. Obtaining a white

solid in the yield of 76.5 % (2.81 g), m. p.: 157-158°C. IR (KBr, ν/cm^-1):

3121, 2986, 2946, 1594, 1515, 1438, 1505, 1279, 1108, 801. ¹HNMR

(500 MHz, CDCl₃, δ/ppm): 7.53(s, 2H, H⁵-pz), 7.40(s, 2H, H³-pz), 7.20(s,

4H, ph), 5.28(s, 4H, CH₂). ¹³CNMR (125 MHz, CDCl₃, δ/ppm): 144.76,

136.10, 133.67, 128.37, 56.62, 56.01. Calc. for C₁₄H₁₂N₄I₂: C, 34.31; H,

2.47; N, 11.43. Found: C, 34.23; H, 2.55; N, 11.38 %.

26

4.5.4 1,4-bis((4-iodo-3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzene (2d).

Compound 2d could be obtained by the similar synthetic procedures as

compound 2b, but the starting material 4-iodo-3,5-dimethyl-1H-pyrazole

(3.33 g, 15 mmol) instead of 3,5-dimethylpyrazole. The single crystals

2d for X-ray measurement were obtained from methanol at room

temperature after 3 days. Obtaining a white solid with yield of 88.4 %

(3.63 g), m. p.: 188-191°C. IR (KBr, ν/cm^-1): 3015, 2986, 2946, 1638,

1529, 1473, 1516, 1311, 1060, 799. ¹HNMR (500 MHz, CDCl₃, δ/ppm):

7.02(s, 4H, ph), 5.23(s, 4H, CH₂), 2.23(s, 6H, 5-CH₃-pz), 2.16(s, 6H,

3-CH₃-pz). ¹³CNMR (125 MHz, CDCl₃, δ/ppm): 149.55, 140.68, 136.29,

127.16, 63.27, 53.59, 14.04, 12.04. Calc. for C₁₈H₂₀N₄I₂: C, 39.58; H,

3.69; N, 10.26. Found: C, 39.46; H, 3.55; N, 10.31 %.

4.5.5 1,4-bis((4-nitro-1H-pyrazol-1-yl)methyl)benzene (2e). Compound

2e could be obtained by the similar synthetic procedures as compound 2a,

but the starting material 4-nitro-1H-pyrazole (1.70 g, 15 mmol) instead of

pyrazole. The single crystals 2e for X-ray measurement were obtained

from methanol at room temperature after 3 days. Obtaining a yellow

solid in 65.4 % (1.61 g), m. p.: 269.9-271°C. IR (KBr, ν/cm^-1): 3139,

3119, 2923, 2852, 1605, 1530, 1435, 1498, 1306, 1004, 824. ¹HNMR

(500 MHz, DMSO-d₆, δ/ppm): 9.02(s, 2H, H⁵-pz), 8.25(s, 2H, H³-pz),

7.34(s, 4H, ph), 5.39(s, 4H, CH₂). ¹³CNMR (125 MHz, DMSO-d₆,

δ/ppm): 135.91, 135.68, 135.04, 130.58, 128.36, 55.37. Calc. for

C₁₄H₁₂N₆O₄: C, 51.22; H, 3.68; N, 25.60. Found: C, 51.18; H, 3.59; N,

25.68 %.

4.5.6 1,4-bis((4-nitro-3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzene (2f).

Compound 2f could be obtained by the similar synthetic procedures as

compound 2b, but the starting material 4-nitro-3,5-dimethyl-1H-pyrazol

(2.12 g, 15 mmol) instead of 3,5-dimethylpyrazole. Obtaining a yellow

solid in 61.8 % (1.78 g), m. p.: 188-191°C. IR (KBr, ν/cm^-1):

27

3041(=CH); 2991, 2851(CH₃); 1630, 1561, 1443(ph); 1488, 1308,

999(pz); 813(=CH). ¹HNMR (500 MHz, DMSO-d₆, δ/ppm): 7.13(s, 4H,

ph), 5.24(s, 4H, CH₂), 2.55(s, 6H, 5-CH₃-pz), 2.52(s, 6H, 3-CH₃-pz).

¹³CNMR (125 MHz, DMSO-d₆, δ/ppm): 146.38, 140.29, 135.26, 127.67,

53.20, 14.14, 11.69. Calc. for C₁₈H₂₀N₆O₄: C, 56.24; H, 5.24; N, 21.86.

Found: C, 56.18; H, 5.17; N, 21.94 %.

4.6 General experimental procedures for preparation of benzene –

tetrapyrazole derivatives 3a-3f

4.6.1 1,4-bis(di(1H-pyrazol-1-yl)methyl)benzene (3a). A mixture of

1.02 g (15 mmol) of pyrazole, 1.02 g (15 mmol) of finely powdered

potassium hydroxide, and 10 ml of DMSO was stirred at 80 °C. After 1

h, a solution of 1.98 g (3.75 mmol) of 1,4-bis(dibromomethyl)benzene in

10 mL of DMSO was added dropwise to the resulting suspension, the

mixture was stirred for 8 h. The reaction mixture was allowed to cool

then diluted with 200 mL of water. The precipitate was filtered off,

washed with the mixture of ethanol and water, dried under reduced

pressure. Obtaining a yellow solid in 78 % (1.08 g), m. p.: 168-169°C.

IR (KBr, ν/cm^-1): 3132, 3017, 2963, 2939, 1622, 1513, 1431, 1513, 1360,

1043, 797. ¹HNMR (500 MHz, DMSO-d₆, δ/ppm): 7.72(s, 2H, CH),

7.62(d, 4H, H⁵-pz, J=1.6Hz), 7.54(d, 4H, H³-pz, J=2.4Hz), 7.02(s, 4H, ph),

6.33(t, 4H, H⁴-pz, J₁=2.15, J₂=2.05Hz). ¹³CNMR (125 MHz, CDCl₃,

δ/ppm): 140.90, 137.52, 129.78, 127.50, 106.79, 77.36. Calc. for

C₂₀H₁₈N₈: C, 64.85; H, 4.90; N, 30.25. Found: C, 64.77; H, 5.05; N,

30.18 %.

4.6.2 1,4-bis(bis(3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzene (3b).

Compound 3b could be obtained by the similar synthetic procedures as

compound 3a, but the starting material 3,5-dimethylpyrazole (1.47 g 15

mmol) instead of pyrazole. Obtaining a yellow solid in 86.7 % (1.16 g),

m. p.: 102-103 °C. IR (KBr, ν/cm^-1): 3132, 3024, 2946, 2922, 1619,

28

1557, 1464, 1514, 1301, 1033, 837. ¹HNMR (500 MHz, CDCl₃, δ/ppm):

7.60(s, 2H, CH), 6.89(s, 4H, ph), 5.83(s, 4H, H⁴-pz), 2.19(s, 24H, CH₃).

¹³CNMR (125 MHz, CDCl₃, δ/ppm): 148.80, 141.02, 137.12, 127.14,

106.91, 73.64, 13.70, 11.84. Calc. for C₂₈H₃₄N₈: C, 69.68; H, 7.10; N,

23.22. Found: C, 69.61; H, 7.23; N, 23.16 %.

4.6.3

1,4-bis(bis(4-iodo-1H-pyrazol-1-yl)methyl)benzene

(3c).

Compound 3c could be obtained by the similar synthetic procedures as

compound 3a, but the starting material 4-iodo-1H-pyrazol (2.19 g, 15

mmol) instead of pyrazole.

The single crystals 3c for X-ray

measurement were obtained from ethyl acetate at room temperature after

3 days. Obtaining a white solid in 87.0 % (2.85 g), m. p.: 154-155 °C.

IR (KBr, ν/cm^-1): 3125, 3090, 2960, 1594, 1514, 1432, 1318, 1022, 795.

¹HNMR (500 MHz, DMSO-d₆, δ/ppm): 8.10(s, 2H, CH), 8.04(s, 4H,

H⁵-pz), 7.71(s, 4H, H³-pz), 7.18(s, 4H, ph). ¹³CNMR (125 MHz,

DMSO-d₆, δ/ppm): 145.19, 136.99, 134.64, 127.50, 75.77, 59.04. Calc.

for C₂₀H₁₄N₈I₄: C, 27.48; H, 1.61; N, 12.82. Found: C, 27.61; H, 1.48; N,

12.71 %.

4.6.4

1,4-bis(bis(4-iodo-3,5-dimethy-1H-pyrazol-1-yl)methyl)benzene

(3d). Compound 3d could be obtained by the similar synthetic

procedures as compound 3a, but the starting material

4-iodo-3,5-dimethyl-1H-pyrazole (3.33 g, 15 mmol) instead of pyrazole.

Obtaining a white solid in 85% (3.13 g), m. p.: 186-189 °C. IR (KBr,

ν/cm^-1): 3101, 2954, 2922, 1617, 1538, 1471, 1515, 1300, 1060, 781.

¹HNMR (500 MHz, CDCl₃, δ/ppm): 7.67(s, 2H, CH), 6.92(s, 4H, ph),

2.24(s, 12H, 5-CH₃-pz), 2.20(s, 12H, 3-CH₃-pz). ¹³CNMR (125 MHz,

CDCl₃, δ/ppm): 150.66, 142.38, 136.30, 127.28, 75.14, 66.00, 14.29,

12.98. Calc. for C₂₈H₃₀N₈I₄: C, 34.10; H, 3.07; N, 11.36. Found: C,

34.16; H, 2.89; N, 11.27 %.

4.6.5

1,4-bis(bis(4-nitro-1H-pyrazol-1-yl)methyl)benzene

(3e).

29

Article Doi

Molecular design and the optimum synthetic route of the compounds with multi-pyrazole and its derivatives and the potential application in antibacterial agents

DOI: 10.1016/j.poly.2015.06.020

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.poly.2015.06.020

Full text of DOI:10.1016/j.poly.2015.06.020

Products guided by the article

R&D Labs maybe for 1073267-94-3

Relevant to this article

Hot Product