Synthesis and antifungal activity of benzamidine derivatives carrying 1,2,3-triazole moieties

Article abstract of DOI:10.3390/molecules19055674

Eighteen novel benzamidine derivatives containing 1,2,3-triazole moieties were synthesized. The in vitro and in vivo fungicidal acitivities of the title compounds and the arylamidine intermediates against Colletotrichum lagenarium and Botrytis cinerea wer

Full text of DOI:10.3390/molecules19055674

Molecules 2014, 19, 5674-5691; doi:10.3390/molecules19055674
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Antifungal Activity of Benzamidine Derivatives
Carrying 1,2,3-Triazole Moieties
Guangyou Chen, Yiwan Zhou, Chonglin Cai, Jia Lu and Xing Zhang *
Research & Development Center of Biorational Pesticide, Key Laboratory of Plant Protection
Resources and Pest Management of Ministry of Education, Northwest A&F University, Yangling
712100, China
* Author to whom correspondence should be addressed; E-Mail: zhxing1952@126.com;
Tel./Fax: +8-629-870-921-22.
Received: 18 March 2014; in revised form: 20 April 2014 / Accepted: 25 April 2014 /
Published: 2 May 2014
Abstract: Eighteen novel benzamidine derivatives containing 1,2,3-triazole moieties were
synthesized. The in vitro and in vivo fungicidal acitivities of the title compounds and the
arylamidine intermediates against Colletotrichum lagenarium and Botrytis cinerea were
tested. The synthesized benzamidines exhibited weak antifungal activities in vitro against
the tested fungi, but some of the compounds showed excellent activities in vivo to the same
strains. Among the compounds tested, 9b showed 79% efficacy in vivo against
C. lagenarium at a concentration of 200 μg/mL, and the efficacy of compound 16d (90%)
toward the same strain was even superior than that of the commercial fungicide
carbendazim (85%).
Keywords: amidine; 1,2,3-triazole; antifungal activity; synthesis
1. Introduction
The majority of agricultural phytopathogens, especially Botrytis cinerea, the causal agent of the
common disease gray mold on vegetable and fruit crops, have developed resistance to the most
commonly used fungicides all over the world [1]. Application of synthetic fungicides is still the most
efficient tool to control crop diseases. This situation urged us to find new compounds having
agricultural antifungal activities. Amidine derivatives exhibit several significant bioactivities, such as
antitumor [2], trypanocidal [3–5], antiprotozoan [6,7], anti-HIV [8], diuretic, anti-inflammatory,

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analgesic [9], antivirus, fungicidal and bactericidal [10–13] activities. Amidines can also be used in the
synthesis of metallo-organic compounds [14]. Aromatic diamidines has been explored as agents
against a widespread range of microorganisms [15]. In our previous works, aromatic diamidines
were found to have excellent antifungal activities in agriculture, especially for the treatment of gray
mold on tomatoes [16,17]. It was reported that good bioacitivities could be obtained by linking the
arylamidino group with heteorocycle moieties [18–20].
1,2,3-Triazoles are very important heterocycles showing excellent bioactivities in pharmaceutical and
agrochemical applications. Compounds containing 1,2,3-triazoles have been reported for their
antifungal [21–23], antibacterial [24–27], anti-HIV [28], insecticidal [29], herbicidal [30], antiviral [31],
antitumor [32], tuberculosis inhibitory [33], antiprotozoal [34], antimalarial [35], anticancer [36], and
larvicidal [37] activities. The fungicidal activity of the commercial fungicide fluconazole can be improved
by introducing a 1,2,3-triazole moiety into the molecule [38,39]. The presence of the 1,2,3-triazole moiety
in oxazolidinone compounds can increase the protein binding ability and subsequently improve the
antibacterial activities [40]. The compounds formed by linking a 1,2,3-triazole ring and an indole nucleus
together possessed much higher antimicrobial activity compared with the precursors [41]. Another
advantage of 1,2,3-triazoles is that they can be prepared conveniently by Huisgen’s cycloaddition reaction
between azides and terminal alkynes without complicated purification.
In an attempt to find more potent antifungal molecules, a series of new compounds were designed
by linking aromatic amidines and 1,2,3-triazoles. In addition, their in vitro and in vivo biological
activity toward the strains C. lagenarium and B. cinerea were studied in this paper.
2. Results and Discussion
2.1. Chemistry
Target molecules 4a–e were synthesized according to Scheme 1. 4-(Azidomethyl)benzonitrile (2)
was obtained by adding aqueous sodium azide to 4-cyanobenzyl bromide in DMF [42], the reaction
took place quantitatively and the product can be separated by simple extraction. The potentially
explosive benzyl azide 2 should be handled carefully and the precipitation should be avoided by first
adding toluene to the reaction mixture in the end of the reaction. After being dried, 2 can be used as a
solution without evaporation of the solvent. Then compound 2 was reacted with propargyl alcohol
under standard “click chemistry” [43] conditions using a copper sulfate/sodium ascorbate system as the
catalyst. The copper sulfate and sodium ascorbate were dissolved in water before being added to the
reaction mixture to ensure a good dispersion of the resultant cuprous salt. The temperature is crucial in
this step. Little 2 had been converted and the mixture stayed clear when the reaction mixture being
stirred for 48 h at 40 °C. However, plenty of crystals were formed in the flask after being stirred for
6 h at 55 °C when TLC indicated that no 2 remained. The target compound was obtained in high yield
by recrystallization. 4-((4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl) methyl)benzonitrile (3) was
converted into 4 by a Pinner reaction [44]. Anhydrous conditions were important in the synthesis of the
Pinner salt. Compound 3 was dried under 110 °C before use, and absolute ethanol was adopted as both
solvent and reactant. The moisture-sensitive Pinner salt was handled quickly when being filtered and
recrystallized in order to reduce byproducts. The Pinner salt was dissolved in absolute ethanol and kept

Molecules 2014, 19
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under anhydrous conditions before use to avoid the absorption of moisture. Compounds 4a–e were
obtained by the ammonization of the Pinner salt with corresponding amines.
Scheme 1. Synthesis of compounds 4 and 9.
propargyl alcohol, t-BuOH/H2O
CuSO4 ·5H2O, sodium ascorbate
NaN3
N
CN
CN
HO
CN
DMF/H2O
N3
Br
N
N
2
3
1
NH
NH
Amd1=
Amd1=
(4a)
(4b)
C
(4d)
(4e)
Amd1=
C
C
N
H
N
H
i,HCl, abs. C2H5OH
ii Amine
N
NH
NH
N
HO
Amd1
Amd1=
C
N
N
HN
4a-e
NH
N
Amd1=
(4c)
C
Br
Aceton/K2CO3,
reflux, 6hr
N
2, t-BuOH/H2O
CuSO4 ·5H2O, sodium ascorbate
O
O
CN
OH
+
N
N
NO2
NO2
NO2
5
6
7
8
NH
NH
Amd2=
(9a)
C
C
(9d)
(9e)
Amd2=
C
C
N
H
N
H
i,HCl, abs. C2H5OH
ii Amine
N
NH
NH
N
Amd2=
O
Amd2
Amd2=
Amd2=
(9b)
(9c)
N
N
N
H
NO2
NH
N
C
9a-e
Compounds 9a–e were obtained according to the procedure described in Scheme 1. 4-((4-((2-
Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-benzonitrile (8) was obtained by reacting
1-nitro-2-(prop-2-ynyloxy) benzene (7) with 2 under “click chemistry” conditions. Compound 8 was
converted into the corresponding amidines 9 following the same procedure used for 4.
When we applied the method used for the preparation of 4 and 9 in synthesizing 16 and 17, the target
products could not be obtained. Compounds 18 and 19 were obtained in high yields by reacting 10 with
propargyl alcohol or 12, respectively. However, when treated with hydrogen chloride in anhydrous ethanol,
the cyano group on 18 or 19 could not be converted into the corresponding Pinner salt. We surmised that
the unreactiveness of the cyano group on the 3-position of the benzene ring may be attributed to the
electron donating aminomethyl group at the meta position [45], which decreased the electron
density at the position the cyano group is linked to, and made it very difficult for the
HCl-catalyzed Pinner reaction [46] to occur. The synthesis strategy was therefore adjusted as
shown in Scheme 2. The 3-(azidomethyl)benzonitrile was inverted into the corresponding amidines by a
Pinner reaction [42] before the 3-(azidomethyl)-N-benzamidines 15a–e were connected with
1-nitro-4-(prop-2-ynyloxy)benzene (12) or propargyl alcohol using the “click chemistry” method to
obtain the target compounds.
The chemical structures of the target compounds (Table 1) were confirmed by their spectral data
(1H-NMR, 13C-NMR, ESI-MS, and elementary analysis). The 1H-NMR spectra of compounds 4, 9, 16 and
17 showed a single NH signal from the triazole group at 7.95–8.25 ppm [47,48] and the signal of CH (or
CH2) attached to the amidines at 2.97–3.89 ppm [49]. The 13C-NMR spectra of the compounds 4, 9, 15, 16
and 17 showed the signal of C=N in the amidino group at 162–166 ppm [50]. Based on the
abovementioned data, it can be concluded that the structures of the amidines were identified correctly.

Molecules 2014, 19
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Scheme 2. Synthesis of compounds 16 and 17.
Br
Aceton/K2CO3,
O2N
O
O2N
OH
+
reflux, 6hr
12
11
10
NaN3
Amd3
i,HCl, abs. C2H5OH
ii Amine
N3
DMF/H2O
N3
Br
CN
CN
15a-d
14
13
propargyl alcohol, t-BuOH/H2O
CuSO4 H2O, sodium ascorbate
N
Amd3
HO
O2N
N
N
N
16a-d
12, t-BuOH/H2O
N
Amd3
O
CuSO4 H2O, sodium ascorbate
N
N
17a-d
propargyl alcohol, t-BuOH/H2O
CuSO4 H2O, sodium ascorbate
N
i,HCl, abs. C2H5OH
ii Amine
Amd3
HO
10
CN
HO
N
N
N
N
16a-d
18
12, t-BuOH/H2O
N
N
N
i,HCl, abs. C2H5OH
ii Amine
Amd3
CN
O2N
O
10
O2N
O
CuSO4 H2O, sodium ascorbate
N
N
N
17a-d
19
NH
NH
NH
C
C
(15a, 16a, 17a)
(15c, 16c, 17c)
Amd3=
C
C
(15b, 16b, 17b)
HN
N
H
N
H
NH
N
(15d, 16d, 17d)
Table 1. Inhibition activity (%) of the amidines on phytopathogenic fungi a.
Inhibition rate (%)
Compounds
in vitro (200 μg/mL)
in vivo (300 μg/mL)
C. lagenarium B. cinerea C. lagenarium B. cinerea
4a
4b
26
19
17
8
23
14
25
0
49
30
60
0
19
60
23
16
56
24
49
13
35
14
43
68
48
46
4c
4d
4e
10
32
31
61
29
35
12
25
12
10
12
22
30
17
44
14
16
19
24
13
48
57
79
4
9a
9b
9c
9d
17
45
31
61
0
9e
15a
15b
15c
15d
0

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Table 1. Cont.
Inhibition rate (%)
in vitro (200 μg/mL) in vitro (200 μg/mL)
C. lagenarium B. cinerea C. lagenarium B. cinerea
Compounds
16a
16b
25
16
10
26
65
21
32
10
100
15
8
19
56
52
90
52
27
45
53
85
11
32
47
7
16c
5
16d
13
36
19
12
17
100
17a
74
0
17b
17c
45
31
61
17d
Carbendazim
a The data are the average of three duplicated results.
2.2. Antifungal Activity Bioassay
The in vitro and in vivo fungicidal activities of the amidines 4a–e, 9a–e, 16a–d, 17a–d and 15a–d
are listed in Table 1. Some of the compounds showed valuable in vitro inhibitory activity against
C. lagenarium and B. cinerea at a concentration of 200 μg/mL. The test results showed that some of
the title compounds and the intermediates 3-(azidomethyl)-N-benzamidine 15a–d possessed fairly
potent antifungal activities in vivo to both C. lagenarium and B. cinerea at the concentration of
300 μg/mL. The antifungal activity of those compounds was compared with that of the the
commercial fungicide carbendazim at the same concentration. Compounds 9c and 17a exhibited a
significant in vitro inhibition effect against C. lagenarium, but the inhibition activities (control
efficacy of 61% and 65%) were less than those of the fungicide carbendazim (100%). The analysis
above indicated that the variation of the substituent groups on the 1,2,3-triazole ring had a great
effect on the antifungal activity against C. lagenarium. The activities of most compounds in the 9
and 17 series having nitrophenoxyl groups on the 1,2,3-triazole ring were higher than those of the 4
and 16 series with hydroxymethyl ones. For example, the activities of compounds 4c and 4d were
17% and 9%, while the activities of 9c and 9d were increased to 61% and 29%. The cyclization of
azido groups with propargyl alcohol had no obvious effect on the antifungal activities. However,
when propargyl alcohol was replaced with 1-nitro-4-(prop-2-ynyloxy)benzene, the activities were
increased significantly. The substituents on the amidino group and the position of the amidino group
on the phenyl ring also had significant effects on the antifungal activities, but no rules could be
extracted from the activity data. The in vitro activity of the compounds to B. cinerea obeyed the
same rule, however, it was not as obvious as the one drawn from the C. lagenarium data. Some of
the compounds exhibited significant activities in vivo toward C. lagenarium, in which the
compounds 4c, 9a, 9b, 16d and 15b had control efficacy rates of 60%, 57%, 79% and 61%,
respectively. It was noteworthy that compound 16d was found to be even more effective than the
fungicide carbendazim (control efficacy of 85%). For B. cinerea, many compounds showed excellent
in vivo antifungal activities. Compounds 4b, 4e, and 17a and 15b, whose efficacy rates were 60%,
56%, 75%, and 68%, respectively, were found to be much more effective as compared to the

Molecules 2014, 19
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fungicide carbendazim SC (54%) against B. cinerea. The different substituents at the 4-position of
the triazole ring and amidino group had no obvious relationship with the fungicidal activity against
B. cinerea. Most of the compounds exhibited more effective activities in vivo, especially for
B. cinerea, than ones in vitro when compared with the commercial fungicide. The aryl amidine
moieties played a very crucial role in the antifungal activities.
3. Experimental
3.1. General Information
Melting points were measured on an X-4 melting point apparatus (Beijing Tech. Instrument Co.,
Beijing, China), and were not corrected. Mass spectra were obtained on a Thermo-Finnigan
LCQ-Advantage (ESI) instrument (Thermo Electron Corporation, Waltham, MA, USA); 1H-NMR and
13C-NMR spectra were obtained on Bruker 400 MHz or 500 MHz spectrometers (Bruker Corporation,
Billerica, MA, USA) in CDCl3, CDOD3, or (CD3)2SO solution; elemental analysis was performed on
an Elemental Vario-III CHN analyzer (Elementar Analysensysteme GmbH, Hanau, Germany).
Reagents and solvents were all chemically or analytically pure without further purification if not
mentioned. Anhydrous solvents were purified by standard techniques before use. Anhydrous HCl (gas)
was prepared by the addition of concentrated hydrochloric acid dropwise to concentrated sulfuric acid
and dried by the latter. Reaction progress was monitored by TLC on silica gel F254 plates prepared in
the laboratory, and was visualized under UV254 and iodine.
3.2. Synthesis of 4-(Azidomethyl)benzonitrile (2) [42]
4-Cyanobenzyl bromide (1, 45.10 g, 230 mmol) was dissolved in DMF (200 mL) in a flask
equipped with a thermometer, and a solution of NaN3 (19.80 g, 300 mmol) in distilled water (100 mL)
was added to it in batches under ice bath cooling with stirring, keeping the reaction mixture under
15 °C. The reaction mixture was stirred for 2 more h at ambient temperature after the addition of the
sodium azide until TLC indicated no 1 remained. The mixture was transferred to a conical flask and
diluted with toluene (200 mL) and distilled water (600 mL). The mixture was separated in a separating
funnel and the aqueous phase was extracted with toluene (2 × 70 mL). The organic extracts were
combined and washed with brine (70 mL) and water (2 × 70 mL) and dried by MgSO4 overnight. A
solution of 2 in toluene (310 mL) was obtained after filtration. After the solvent was evaporated under
reduced pressure, 2 was obtained as light yellow oil (35.69 g, 98.2%), 1H-NMR (500 MHz, CDCl3) δ:
7.61 (d, J = 8.2 Hz, 2H, benzene-H), 7.41 (d, J = 7.4 Hz, 2H, benzene-H), 4.42 (s, 2H, CH2). 13C-NMR
(125 MHz, CDCl3) δ: 140.97, 132.82, 132.55, 132.55, 129.07, 128.56, 128.56, 128.27, 125.35, 118.55,
111.92, 77.80, 77.55, 77.29, 53.90, 53.90, 21.37.
3.3. Synthesis of 4-((4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl) Benzonitrile (3)
Compound 2 (35.1 g) was dissolved in a mixture of t-BuOH (300 mL) and distilled water (100 mL),
then propargyl alcohol (12.3 g, 230 mmol) was added. The reaction mixture was stirred in a water bath at
60 °C for 48 h after the addition of sodium ascorbate (306 mg, in 3 mL of water) and cupric sulfate
(CuSO4·5H2O) (105 mg, in 2 mL of water). The clear reaction mixture turned light yellow.

Molecules 2014, 19
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Compound 3 (46.65 g, 128 mmol, 81%) was obtained as white needle-like crystals after filtration
and dried under 110 °C for 7 h when most of the solvent was evaporated under vacuum; m.p.
1
136–137 °C. H-NMR (400 MHz, DMSO) δ: 8.09 (s, 1H, triazole-H), 7.86 (d, J = 8.3 Hz, 2H,
benzene-H), 7.46 (d, J = 8.3 Hz, 2H, benzene-H), 5.70 (s, 2H, CH2), 5.22 (t, J = 5.6 Hz, 1H, OH),
13
4.53 (d, J = 5.4 Hz, 2H, CH2). C-NMR (100 MHz, DMSO) δ: 148.97, 142.19, 133.19, 129.10,
123.73, 119.04, 111.34, 55.49, 52.53.
3.4. General Procedure for the Synthesis of 4-((4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl) Methyl)-
benzamidines 4a–e
A suspension of 3 (3.0 g, 14.0 mmol) in absolute ethanol (50 mL) was cooled to below 5 °C and
saturated with dry HCl gas. Then the mixture was sealed and stirred at ambient temperature for 48 h.
White crystalline imino ester was obtained after evaporation of the solvent under anhydrous conditions
and washing with dry ether (3 × 15 mL). The resulting imino ester was dissolved in absolute methanol
(20 mL). An exothermic reaction took place and the mixture became clear when it was treated with the
corresponding amines (60 mmol). Keeping the reaction mixture stirring overnight in sealed flasks
resulted in the formation of the corresponding amidines. After evaporation of the solvent in anhydrous
conditions, the residue was dissolved in a mixture of ethanol (5 mL) and 2 N aqueous NaOH (5 mL).
The target product was precipitated by the addition of ether (40 mL) to the organic phase and then
purified by column chromatography on silica gel (CH2Cl2/MeOH = 6:1). Compounds 4a–d were
prepared by the same method.
N-Butyl-4-((4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl)benzamidine (4a): Yield, 42.1%; white
crystals; m.p. 111–112 °C. 1H-NMR (400 MHz, MeOD) δ: 8.05 (d, J = 12.1 Hz, 1H, triazole-H), 7.75 (d,
J = 8.2 Hz, 2H, benzene-H), 7.53 (d, J = 8.2 Hz, 2H, benzene-H), 5.75 (s, 2H, CH2), 5.69 (d, J = 6.0 Hz,
2H, CH2), 3.42 (t, J = 7.3 Hz, 2H, N-CH2), 1.83–1.73 (m, 2H, CH2), 1.05 (t, J = 7.4 Hz, 3H, CH3).
13C-NMR (100 MHz, MeOD) δ: 166.56, 141.31, 129.99, 129.68, 129.16, 128.23, 127.76, 60.92, 54.98,
44.61, 20.73, 10.25. MS (ESI+) m/z: 288 [M+H]+. Anal. Calc. for C15H21N5O·HCl (323.15): C, 55.64; H,
6.85; N, 21.63; found: C, 55.11; H, 6.62; N, 21.56.
N-Sec-butyl-4-((4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl)benzamidine (4b): Yield, 47.4%;
1
white crystals; m.p. 118–120 °C. H-NMR (400 MHz, MeOD) δ: 8.02 (s, 1H, triazole-H), 7.71 (d,
J = 8.2 Hz, 2H), 7.52 (d, J = 8.2 Hz, 2H, benzene-H), 5.72 (s, 2H, CH2), 4.65 (s, 2H, CH2), 3.81 (dd,
J = 13.2, 6.6 Hz, 1H, CH), 3.28 (m, 2H, N-CH2), 1.62–1.48 (m, 3H, CH3), 1.31 (d, J = 6.5 Hz, 3H,
CH3). MS (ESI+) m/z: 288 [M+H]+. Anal. Calc. for C15H21N5O·HCl (323.15): C, 55.64; H, 6.85; N,
21.63; found: C, 55.23; H, 6.71; N, 21.75.
N,N-Diethyl-4-((4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl)benzamidine (4c): Yield, 42.1%; yellow
crystals; m.p. > 300 °C. 1H-NMR (500 MHz, DMSO) δ: 8.13 (s, 1H, triazole-H), 8.06 (d, J = 8.3 Hz, 2H,
benzene-H), 7.46 (d, J = 8.3 Hz, 2H, benzene-H), 5.71 (s, 2H, CH2), 4.51 (d, J = 5.1 Hz, 2H, CH2), 3.89 (s,
13
4H, N-CH2). C-NMR (125 MHz, DMSO) δ: 164.49, 148.93, 142.23, 129.28, 128.63, 124.19, 123.73,
55.46, 52.57, 45.94. MS (ESI+) m/z: 288 [M+H]+. Anal. Calc. for C15H21N5O·HCl (323.15): C, 55.64; H,
6.85; N, 21.63; found: C, 55.37; H, 6.65; N, 21.73.

Molecules 2014, 19
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N-Cyclohexyl-4-((4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl)benzamidine (4d): Yield, 42.1%;
1
white crystals; m.p. 171–173 °C. H-NMR (400 MHz, MeOD) δ: 8.21 (s, 1H, triazole-H), 7.79 (d,
J = 8.1, 1.5 Hz, 2H, benzene-H), 7.73 (d, J = 8.3 Hz, 2H, benzene-H), 5.77 (s, 2H, CH2), 5.35 (s, 2H,
CH2), 3.82 (d, J = 6.7 Hz, 1H, N-CH), 1.79–1.65 (m, 4H, CH2), 1.65–1.52 (m, 2H, CH2), 1.34 (d,
J = 6.5 Hz, 3H, CH2), 1.04 (t, J = 7.3 Hz, 3H, CH2). MS (ESI+) m/z: 314 [M+H]+. Anal. Calc. for
C17H23N5O·HCl (349.17): C, 58.36; H, 6.91; N, 20.02; found: C, 57.97; H, 6.85; N, 20.34.
N,N-Dibutyl-4-((4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl)benzamidine (4e): Yield, 42.1%;
1
white crystals; m.p. 111–112 °C. H-NMR (400 MHz, MeOD) δ: 7.99 (s, 1H, triazole-H), 7.87–7.82
(m, 2H, benzene-H), 7.47–7.43 (m, 2H, benzene-H), 5.69 (s, 2H, CH2), 5.65 (s, 2H, CH2), 2.81–2.74
(m, 4H, N-CH2), 1.80 (d, J = 7.5 Hz, 4H, CH2), 1.24 (m, 4H, CH2), 0.80 (t, J = 7.3 Hz, 6H, CH3). MS
(ESI+) m/z: 344 [M+H]+. Anal. Calc. for C19H29N5O·HCl (379.21): C, 60.06; H, 7.96; N, 18.43; found:
C, 59.78; H, 7.81; N, 18.71.
3.5. Synthesis of 1-Nitro-2-(prop-2-ynyloxy) Benzene (7)
2-Nitrophenol (5, 9.10 g, 65 mmol) and K2CO3 (9.0 g, 65 mmol) were dissolved in acetone (60 mL) in a
three necked flask equipped with a thermometer. Then a solution of propargyl bromide (16.03 g,
300 mmol) in acetone (30 mL) was added dropwise at ambient temperature in about 15 min while
stirring. Then the reaction mixture was heated to reflux for 3 h after being stirred for 30 min when
TLC (petroleum ether/ethyl acetate = 6:1) indicated no 5 remained. The mixture was allowed to cool to
ambient temperature and the resulting white solid was recrystallized in ethanol, to afford yellowish
needle-shaped crystals of 7 (8.11 g, 70%); m.p. 115–116 °C. 1H-NMR (400 MHz, CDCl3) δ: 7.85 (dd,
J = 8.1, 1H, benzene-H), 7.64–7.51 (m, 1H, benzene-H), 7.27 (t, J = 8.8 Hz, 1H, benzene-H), 7.10 (t,
13
J = 7.8 Hz, 1H, benzene-H), 4.85 (d, J = 2.4 Hz, 2H, CH2), 2.59 (t, J = 2.4 Hz, 1H CH). C-NMR
(100 MHz, CDCl3) δ: 150.76, 140.35, 134.02, 125.76, 121.45, 115.49, 77.42, 76.78, 57.20.
3.6. Synthesis of 4-((4-((2-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl) Benzonitrile (8)
A solution (260 mL) of 3 prepared from 4-cyanobenzyl bromide (39.20 g, 200 mmol) was added to a
500-mL round bottom flask, followed by the evaporation of the solvent under reduced pressure.
1-Nitro-2-(prop-2-ynyloxy) benzene (7, 35.4 g, 200 mmol) was dissolved in t-BuOH (300 mL). The
solution of 7 and distilled water (100 mL) was mixed with the residue of 3 in the flask and followed by the
addition of sodium ascorbate (250 mg, 1.26 mmol, in 3 mL of water) and cupric sulfate (CuSO4·5H2O,
85 mg, 0.34 mmol, in 2 mL of water). After stirring at 60 °C for 24 h, the mixture was allowed to cool to
room temperature. Compound 8 (58.21 g, 82%) was obtained as a white solid after the evaporation of
about half of the solvent, filtration and drying at 110 °C, m.p. 136–138 °C. 1H-NMR (500 MHz, DMSO) δ:
8.36 (s, 1H, triazole-H), 7.86 (m, 2H, benzene-H), 7.67 (t, J = 7.4 Hz, 1H, benzene-H), 7.58 (d, J = 8.4 Hz,
1H, benzene-H), 7.46 (d, J = 8.1 Hz, 2H, benzene-H), 7.15 (t, J = 7.7 Hz, 1H, benzene-H), 5.77 (s, 2H,
CH2), 5.38 (s, 2H, CH2). 13C-NMR (125 MHz, DMSO) δ: 151.01, 142.73, 141.86, 140.31, 134.76, 133.21,
129.16, 125.85, 125.40, 121.50, 118.99, 116.21, 111.45, 63.08, 52.70.

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3.7. General Procedure for the Synthesis of 4-((4-((2-Nitrophenoxy)methyl)-1H-1,2,3 -triazol-1-yl)-
methyl)-benzamidines 9a–e
A suspension of 8 (3.55 g, 10 mmol), in absolute ethanol (30 mL) was saturated with anhydrous
HCl under water-ice bath cooling; the solution was then stirred at room temperature for 4–5 days.
After evaporating most of the solvent, the residue was diluted with dry ether, and the resultant solid
obtained by filtration was dissolved in dry methanol. The corresponding amines (40 mmol) were added
to the solution and stirred for 24 h under anhydrous conditions. Then amidines 9a–d were treated with
the same manner described for compound 4. Compounds 9 were purified by column chromatography
on silica gel, using dichloromethane/methanol (7:1, v/v) as the eluent.
4-((4-((2-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-N-butylbenzamidine (9a): Yield, 51.2%;
1
yellow crystals; m.p. 119–121 °C. H-NMR (400 MHz, MeOD) δ: 8.25 (s, 1H, triazole-H), 7.77 (d,
J = 8.3 Hz, 1H, benzene-H), 7.75 (d, J = 8.3 Hz, 2H, benzene-H), 7.65–7.60 (m, 1H, benzene-H),
7.60–7.56 (m, 1H, benzene-H), 7.53 (d, J = 8.2 Hz, 2H, benzene-H), 7.11 (t, J = 7.7 Hz, 1H, benzene-H) ,
5.77 (s, 2H, CH2), 5.34 (s, 2H, CH2), 3.46 (t, J = 7.2 Hz, 2H, N-CH2), 1.74 (m, 2H, CH2), 1.48 (m, 2H,
13
CH2), 1.01 (t, J = 7.3 Hz, 3H, CH2). C-NMR (100 MHz, MeOD) δ: 166.02, 153.22, 143.27, 142.65,
131.41, 130.87, 130.73, 130.68, 130.49, 127.22, 127.06, 123.17, 117.67, 64.74, 55.05, 41.37, 31.38,
21.51, 14.75. MS (ESI+) m/z: 409 [M+H]+. Anal. Calc. for C21H24N6O3·HCl (444.17): C, 56.69; H, 5.66;
N, 18.89; found: C, 56.78; H, 5.81; N, 18.81.
4-((4-((2-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-N-isobutylbenzamidine (9b): Yield, 36.5%;
white solid; m.p. 109–110 °C. 1H-NMR (400 MHz, MeOD) δ: 8.22 (s, 1H, triazole-H), 7.79–7.74 (m, 1H,
benzene-H), 7.73 (d, J = 8.2 Hz, 2H, benzene-H), 7.60 (t, J = 7.3 Hz, 1H, benzene-H), 7.52 (d,
J = 8.2 Hz, 2H, benzene-H), 7.09 (t, J = 7.6 Hz, 1H, benzene-H), 5.75 (s, 2H, CH2), 5.32 (s, 2H, CH2),
13
3.30–3.22 (d, 2H, N-CH2), 2.06 (m, 1H, CH), 1.02 (d, J = 6.9 Hz, 6H, CH3). C-NMR (100 MHz,
MeOD) δ: 166.36, 153.23, 143.28, 143.31, 136.12, 131.53, 130.88, 130.72, 130.48, 127.15, 127.06,
123.17, 117.66, 64.76, 55.06, 52.30, 29.43, 21.25. MS (ESI+) m/z: 409 [M+H]+. Anal. Calc. for
C21H24N6O3·HCl (444.17): C, 56.69; H, 5.66; N, 18.89; found: C, 56.73; H, 5.71; N, 18.82.
4-((4-((2-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-N,N-diethylbenzamidine (9c): Yield, 45.2%;
white solid; m.p. 135–137 °C. 1H-NMR (400 MHz, MeOD) δ: 8.21 (s, 1H, triazole-H), 7.76–7.73 (m, 1H,
benzene-H), 7.71 (d, J = 8.2 Hz, 2H, benzene-H), 7.60 (t, J = 7.3 Hz, 1H, benzene-H), 7.52 (d, J = 8.2 Hz,
2H, benzene-H), 7.45 (d, J = 8.4 Hz, 1H, benzene-H), 7.10 (t, J = 7.6 Hz, 1H, benzene-H), 5.74 (s, 2H,
13
CH2), 5.31 (s, 2H, CH2), 3.09 (q, J = 7.3 Hz, 4H, N-CH2), 1.31 (t, J = 7.3 Hz, 6H, CH3). C-NMR
(100 MHz, MeOD) δ: 166.33, 153.20, 143.99, 142.71, 136.11, 130.71, 130.52, 130.18, 127.04, 123.18,
117.75, 64.76, 55.23, 44.41, 12.47. MS (ESI+) m/z: 409 [M+H]+. Anal. Calc. for C21H24N6O3·HCl (444.17):
C, 56.69; H, 5.66; N, 18.89; found: C, 56.81; H, 5.69; N, 18.78.
4-((4-((2-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-N-cyclohexylbenzamidine (9d): Yield,
1
57.0%; white crystals; m.p. 110–113 °C. H-NMR (400 MHz, MeOD) δ: 8.17 (s, 1H, triazole-H),
7.77–7.71 (m, 1H, benzene-H), 7.67 (d, J = 8.2 Hz, 2H, benzene-H), 7.60–7.53 (m, 1H, benzene-H), 7.48
(d, J = 8.2 Hz, 2H, benzene-H), 7.42 (d, J = 8.4 Hz, 1H, benzene-H), 7.07 (t, J = 7.6 Hz, 1H, benzene-H),

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5.72 (s, 2H, CH2), 5.30 (s, 2H, CH2), 3.70–3.59 (m, 1H, N-CH), 1.51–1.35 (m, 5H, CH2), 1.16 (m, 5H,
CH2). 13C-NMR (100 MHz, MeOD) δ: 165.09, 153.26, 145.33, 143.21, 136.09, 131.79, 131.45, 130.63,
130.59, 127.07, 123.18, 117.64, 64.79, 55.07, 52.41, 32.76, 26.62, 26.21. MS (ESI+) m/z: 435 [M+H]+.
Anal. Calc. for C19H29N5O·HCl (470.18): C, 58.66; H, 5.78; Cl, 7.53; N, 17.84; found: C, 58.78; H,
5.80; N, 17.91.
4-((4-((2-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-N,N-dibutylbenzamidine (9e): Yield, 54.6%;
light yellow crystals; m.p. 119–121 °C. 1H-NMR (400 MHz, MeOD) δ: 8.10 (s, 1H, triazole-H), 7.82 (d,
J = 8.2 Hz, 2H, benzene-H), 7.77 (m, 1H, benzene-H), 7.57 (t, J = 7.4 Hz, 1H, benzene-H), 7.41 (d, J = 8.5
Hz, 1H, benzene-H), 7.34 (d, J = 8.2 Hz, 2H, benzene-H), 7.08 (t, J = 7.7 Hz, 1H, benzene-H), 5.67 (s, 2H,
CH2), 5.32 (s, 2H, CH2), 4.23 (t, J = 6.9 Hz, 4H, N-CH2), 3.59 (m, 4H, CH2), 1.48–1.29 (m, 4H, CH2), 0.97
(t, J = 7.4 Hz, 6H, CH3). 13C-NMR (100 MHz, MeOD) δ:165.09, 153.01, 145.11, 143.44, 135.75, 130.70,
129.83, 129.58, 126.91, 122.98, 117.44, 64.60, 55.01, 52.25, 29.98, 21.43, 19.98. MS (ESI+) m/z: 465
[M+H]+. Anal. Calc. for C19H29N5O·HCl (500.23): C, 59.93; H, 6.64; N, 16.77; found: C, 59. 87; H,
6.73; N, 16.72.
3.8. Synthesis of 1-Nitro-4-(prop-2-ynyloxy)benzene (12) [51]
Compound 12 was prepared following the same procedure used for 7. Yield, 76%; m.p. 118–119 °C;
1H-NMR (400 MHz, CDCl3) δ: 8.22 (d, J = 9.3 Hz, 2H, benzene-H), 7.06 (d, J = 9.3 Hz, 2H, benzene-H),
4.81 (d, J = 2.4 Hz, 2H, CH2), 2.60 (s, 1H, CH). 13C-NMR (100 MHz, CDCl3) δ: 162.35, 125.85, 114.99,
77.42, 77.10, 76.80, 56.31.
3.9. Synthesis of 3-(Azidomethyl)benzonitrile (14)
3-(Bromomethyl)benzonitrile (13, 39.2 g, 200 mmol) was dissolved in DMF (180 mL) in an
Erlenmeyer flask equipped with a thermometer, then NaN3 (16.50 g, 250 mmol, in 90 mL of distilled
water) was added in portions under water-ice bath cooling while stirring, keeping the mixture under
10 °C. After the addition of the azide, the mixture was stirred for 1.5 h at 30 °C when the color of the
reaction mixture turned colorless and TLC indicated no 13 remained. The mixture was diluted with
toluene (180 mL) and distilled water (500 mL) in turn. The aqueous phase was extracted with toluene
(2 × 60 mL) and the organic phase was combined and dried with MgSO4. After removing of the solvent,
1
14 (39.6 g, 97%) was obtained as a white solid; m.p. 30–31 °C. H-NMR (500 MHz, CDCl3) δ:
7.62–7.57 (m, 2H, benzene-H), 7.57–7.54 (m, 1H, benzene-H), 7.51–7.45 (m, 1H, benzene-H), 4.41 (s,
2H, CH2). 13C-NMR (125 MHz, CDCl3) δ: 137.25, 132.41, 131.80, 131.38, 129.73, 118.49,
112.85, 53.62.
3.10. General Procedure for the Synthesis of 3-(Azidomethyl)benzamidines 15a–d [42]
Absolute ethanol (80 mL) was added to the solution (25 mL) of 14. The mixture was sealed and
stirred for 7 days at room temprature after being saturated with anhydrous HCl at ice-water bath. The
residue was recrystallized from ether, and then dissolved in absolute methanol (30 mL). The solution
was stirred at 30 °C overnight after the corresponding amines (43 mmol) were added. After
evaporating most of the solvent, the residue was mixed with ethanol (5 mL) and 2 N aqueous NaOH

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(5 mL). Ether (50 mL) was added after the aqueous phase being removed, and the target amidines
15a–d was obtained by precipitation.
1
3-(Azidomethyl)-N-butylbenzamidine (15a): Yield, 75.3%; yellow crystals; m.p. 106–109 °C. H-NMR
(400 MHz, MeOD) δ: 7.67 (s, 1H, benzene-H), 7.64 (d, J = 3.9 Hz, 2H, benzene-H), 7.57 (t, J = 7.6 Hz,
1H, benzene-H), 4.48 (s, J = 6.3 Hz, 2H, CH2), 3.41 (t, J = 7.3 Hz, 2H, N-CH2), 1.75–1.52 (m, 2H, CH2),
13
1.50–1.30 (m, 2H, CH2), 0.94 (t, 7.4 Hz, 3H, CH3). C-NMR (100 MHz, MeOD) δ: 164.17, 137.64,
132.78, 129.80, 129.48, 127.23, 127.17, 53.38, 46.98, 29.36, 19.75, 12.50. MS (ESI+) m/z: 232 [M+H]+.
Anal. Calc. for C12H17N5·HCl (267.13): C, 53.83; H, 6.78; N, 26.16; found: C, 53. 87; H, 6.71; N, 26.18.
N-Sec-butyl-3-(azidomethyl)benzamidine (15b): Yield, 71.2%; yellow crystals; m.p. 108–110 °C.
1H-NMR (400 MHz, DMSO) δ: 9.64 (s, 2H, N-H), 8.07 (s, 1H, benzene-H), 7.98 (d, J = 7.7 Hz, 2H,
benzene-H), 7.89 (t, J = 7.7 Hz, 1H, benzene-H), 4.38 (s, 2H, CH2), 3.28 (m, 1H, N-CH), 1.85 (m, 2H,
13
CH2), 1.45 (d, J = 6.6 Hz, 3H, CH3), 1.21 (t, J = 7.4 Hz, 3H, CH3). C-NMR (100 MHz, DMSO) δ:
162.39, 136.98, 133.17, 130.16, 129.62, 128.65, 128.53, 53.38, 51.12, 28.59, 19.65, 10.83. MS (ESI+)
m/z: 232 [M+H]+. Anal. Calc. for C12H17N5·HCl (267.13): C, 53.83; H, 6.78; N, 26.16; found: C, 53.
85; H, 6.82; N, 26.19.
3-(Azidomethyl)-N-cyclohexylbenzamidine (15c): Yield, 78.0%; white crystals; m.p. 110–111 °C.
1H-NMR (400 MHz, MeOD) δ: 7.77 (s, 1H, benzene-H), 7.59 (d, J = 7.7 Hz, 2H, benzene-H), 7.39 (t,
J = 7.7 Hz, 1H, benzene-H), 4.42 (s, 2H, CH2), 2.94–2.79 (m, 1H, N-CH), 1.91 (m, 5H, CH3),
13
1.80–1.65 (m, 5H, CH3). C-NMR (100 MHz, MeOD) δ: 163.08, 137.52, 132.67, 131.37, 130.09,
129.41, 127.31, 127.22, 53.39, 48.28, 31.29, 24.80. MS (ESI+) m/z: 258 [M+H]+. Anal. Calc. for
C14H19N5·HCl (293.14): C, 57.23; H, 6.86; Cl, 12.07; N, 23.84; found: C, 57. 35; H, 6.81; N, 26.20.
1
3-(Azidomethyl)-N,N-diethylbenzamidine (15d): Yield, 55.4%; white crystals; m.p. 99–101 °C. H-NMR
(400 MHz, DMSO) δ: 9.44 (s, 3H, N-H), 8.10 (s, 1H, benzene-H), 7.93 (d, J = 7.7 Hz, 2H,
benzene-H), 7.87 (t, J = 7.7 Hz, 1H, benzene-H), 4.45 (s, 2H, CH2), 2.82–2.75 (m, 4H, N-CH2), 1.16
(t, J = 7.4 Hz, 6H, CH3). MS (ESI+) m/z: 232 [M+H]+. Anal. Calc. for C12H17N5·HCl (267.13): C,
53.83; H, 6.78; N, 26.16; found: C, 53. 86; H, 6.75; N, 26.14.
3.11. General Procedure for the Synthesis of 3-((4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl)-
benzamidines 16a–d
Compound 15 (4.5 mmol) and propargyl alcohol (28 mg, 5 mmol) was dissolved in a mixture of
t-butanol (30 mL) and distilled water (12 mL), and then, ascorbate sodium(45 mg, 0.23 mmol, in 1 mL
water) and CuSO4·5H2O (10 mg, 0.04 mmol, in 1 mL water) were added to the mixture. After stirring
at 60 °C for 48 h, half of the solvent was evaporated and the target molecule was obtained by
recrystallization from ether (16a) or column chromatography on silica gel (16b, 16c, 16d), using
dichloromethane/methanol (5:1, v/v) as the eluent.
N-Butyl-3-((4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl)benzamidine (16a): Yield, 68.8%; light
yellow crystals; m.p. 106–109 °C. 1H-NMR (400 MHz, MeOD) δ: 8.07 (s, 1H, triazole-H), 7.80 (s, 1H,
benzene-H), 7.66 (d, J = 7.7 Hz, 2H, benzene-H), 7.59 (t, J = 7.7 Hz, 1H, benzene-H), 5.72 (s, 2H,

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CH2), 4.65 (s, 2H, CH2), 3.46 (t, 2H, N-CH2), 1.79–1.68 (m, 2H, CH2), 1.48 (m, 2H, CH2), 0.99 (m,
13
3H, CH3). C-NMR (100 MHz, DMSO) δ: 163.18, 136.16, 132.01, 129.11, 128.93, 126.94, 126.64,
122.58, 54.22, 51.94, 42.09, 38.35, 18.50, 11.92. MS (ESI+) m/z: 288 [M+H]+. Anal. Calc. for
C15H21N5O·HCl (323.15): C, 55.64; H, 6.85; N, 21.63; found: C, 55.41; H, 6.72; N, 21.57.
N-Sec-butyl-3-((4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl)benzamidine (16b): Yield, 71.1%; light
1
yellow crystals; m.p. 108–110 °C. H-NMR (400 MHz, MeOD) δ: 8.05 (s, 1H, triazole-H), 7.72 (s, 1H,
benzene-H), 7.67 (d, J = 7.7 Hz, 2H, benzene-H), 7.59 (t, J = 7.6 Hz, 1H, benzene-H), 5.71 (s, 2H, CH2),
4.64 (s, 2H, CH2), 3.81 (t, J = 7.4 Hz, 1H, N-H), 1.75–1.64 (m, 2H, CH2), 1.32 (m, 2H, CH2), 1.01 (t,
J = 7.4 Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO) δ: 162.68, 136.11, 131.94, 129.40, 128.89, 126.94,
126.63, 122.54, 54.22, 51.92, 50.56, 26.58, 17.26, 16.06. MS (ESI+) m/z: 288 [M+H]+. Anal. Calc. for
C15H21N5O·HCl (323.15): C, 55.64; H, 6.85; N, 21.63; found: C, 55.42; H, 6.78; N, 21.71.
N-Cyclohexyl-3-((4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl)benzamidine (16c): Yield, 76.2%;
1
light yellow crystals; m.p. 110–111 °C. H-NMR (400 MHz, DMSO) δ: 7.95 (s, 1H, triazole-H), 7.63
(s, 1H, benzene-H), 7.57 (d, J = 7.8 Hz, 2H, benzene-H), 7.49 (t, J = 7.6 Hz, 1H, benzene-H), 5.61 (s,
2H, CH2), 4.55 (s, 2H, CH2), 3.59 (m, 1H, N-H), 1.44–1.29 (m, 5H, CH2), 1.24 (m, 5H, CH2).
13C-NMR (100 MHz, DMSO) δ: 162.12, 136.04, 131.92, 129.39, 128.84, 126.73, 126.55, 122.46,
54.22, 52.34, 51.92, 51.88, 46.20, 30.30, 23.98. MS (ESI+) m/z: 314 [M+H]+. Anal. Calc. for
C17H23N5O·HCl (349.17): C, 58.36; H, 6.91; N, 20.02; found: C, 58.47; H, 6.89; N, 20.13.
N,N-Diethyl-3-((4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl)benzamidine (16d): Yield, 70.3%;
light yellow crystals; m.p. 99–101 °C. 1H-NMR (400 MHz, MeOD) δ: 8.09 (s, 1H, triazole-H), 7.65 (d,
J = 7.4 Hz, 1H, benzene-H), 7.62 (d, J = 8.1 Hz, 1H, benzene-H), 7.55 (m, 2H, benzene-H), 5.74 (s,
2H, CH2), 4.66 (s, 2H, CH2), 3.33–3.27 (m, 4H, N-CH2), 1.37 (t, J = 7.2 Hz, 6H, CH3). 13C-NMR (100
MHz, DMSO) δ: 163.75, 162.39, 136.43, 130.50, 129.51, 129.04, 126.35, 125.91, 54.24, 51.92, 42.31,
11.63. MS (ESI+) m/z: 288 [M+H]+. Anal. Calc. for C15H21N5O·HCl (323.15): C, 55.64; H, 6.85; N,
21.63; found: C, 55.42; H, 6.78; N, 21.71.
3.12. General Procedure for the Synthesis of 3-((4-((4-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)
methyl)benzamidines 17a–d
Compounds 17a–d were prepared using the same procedure as for compounds 16a–d, by replacing
the propargyl alcohol with 1-nitro-4-(prop-2-ynyloxy)benzene (12).
3-((4-((4-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-N-butylbenzamidine (17a): Yield, 80.1%;
1
light yellow crystals; m.p. 164–166 °C. H-NMR (400 MHz, MeOD) δ: 8.29 (s, 1H, triazole-H), 8.13
(s, J = 9.1 Hz, 2H, benzene-H), 7.74 (s, 1H, benzene-H), 7.67 (d, J = 7.1 Hz, 1H, benzene-H), 7.65 (d,
J = 7.2 Hz, 1H, benzene-H), 7.61 (s, 1H, benzene-H), 7.11 (d, J = 9.1 Hz, 2H, benzene-H), 5.71 (s, 2H,
CH2), 5.24 (s, 2H, CH2), 2.93–2.81 (m, 2H, N-CH2), 1.74–1.63 (m, 2H, CH2), 1.34 (m, 2H, CH2), 0.93
13
(d, J = 2.3 Hz, 3H, CH3). C-NMR (100 MHz, DMSO) δ: 163.11, 162.58, 140.85, 135.89, 132.13,
132.02, 129.12, 129.00, 128.73, 127.04, 126.77, 126.48, 126.40, 124.18, 114.04, 60.80, 52.10, 42.11,

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28.56, 18.95, 11.92. MS (ESI+) m/z: 409 [M+H]+. Anal. Calc. for C21H24N6O3·HCl (444.17): C, 56.69;
H, 5.66; N, 18.89; found: C, 56.71; H, 5.72; N, 18.84.
3-((4-((4-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-N-sec-butylbenzamidine (17b): Yield,
69.6%; yellow solid; m.p. 92–94 °C. 1H-NMR (400 MHz, MeOD) δ: 8.31 (s, 1H, triazole-H), 8.17 (d,
J = 9.1 Hz, 2H, benzene-H), 7.76 (s, 1H, benzene-H), 7.68 (d, J = 7.1 Hz, 1H, benzene-H), 7.66 (d,
J = 7.2 Hz, 1H, benzene-H), 7.60 (m, 1H, benzene-H), 7.15 (d, J = 9.2 Hz, 2H, benzene-H), 5.75 (s,
2H, CH2), 5.28 (s, 2H, CH2), 3.89–3.75 (m, 1H, N-CH), 1.67 (m, 2H, CH2), 1.27 (d, J = 6.6 Hz, 3H,
CH3), 1.02–0.98 (m, 3H, CH3). 13C-NMR (100 MHz, DMSO) δ: 162.64, 162.57, 140.89, 135.89,
132.04, 131.92, 129.43, 128.94, 127.03, 126.77, 126.48, 126.40, 124.08, 113.99, 60.79, 52.05, 50.57,
28.98, 17.25, 8.61. MS (ESI+) m/z: 409 [M+H]+. Anal. Calc. for C21H24N6O3·HCl (444.17): C, 56.69;
H, 5.66; N, 18.89; found: C, 56.63; H, 5.61; N, 18.93.
3-((4-((4-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-N-cyclohexylbenzamidine (17c): Yield,
1
71.5%; brown solid; m.p. 101–103 °C. H-NMR (400 MHz, MeOD) δ: 8.19 (s, 1H, triazole-H), 8.05
(d, J = 7.8 Hz, 2H, benzene-H), 7.65 (s, 1H, benzene-H), 7.58 (d, J = 7.1 Hz, 1H, benzene-H), 7.56 (d,
J = 7.1 Hz, 1H, benzene-H), 7.48 (d, J = 7.8 Hz, 1H, benzene-H), 7.04 (d, J = 7.8 Hz, 2H, benzene-H),
5.63 (s, 2H, CH2), 5.17 (s, 2H, CH2), 3.18 (m, 1H, N-CH), 1.55 (m, 5H, CH2), 1.32 (d, J = 10.0 Hz,
5H, CH2). MS (ESI+) m/z: 435 [M+H]+. Anal. Calc. for C19H29N5O·HCl (470.18): C, 58.66; H, 5.78;
Cl, 7.53; N, 17.84; found: C, 58.78; H, 5.82; N, 17.81.
3-((4-((4-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-N,N-diethylbenzamidine (17d): Yield,
1
68.9%; brown solid; m.p. 159–161 °C. H-NMR (400 MHz, MeOD) δ: 8.36 (s, 1H, triazole-H), 8.21
(d, J = 8.9 Hz, 2H, benzene-H), 7.68 (s, 1H, benzene-H), 7.64 (m, 2H, benzene-H), 7.54 (d, J = 7.7 Hz,
1H, benzene-H), 7.20 (d, J = 8.9 Hz, 2H, benzene-H), 5.79 (s, 2H, CH2), 5.32 (s, 2H, CH2), 3.40–3.25
(m, 4H, N-CH2), 1.38 (t, J = 7.2 Hz, 6H, CH3). MS (ESI+) m/z: 409 [M+H]+. Anal. Calc. for
C21H24N6O3·HCl (444.17): C, 56.69; H, 5.66; N, 18.89; found: C, 56.72; H, 5.61; N, 19.02.
3.13. Fungicidal Activity Bioassay: Effect of the New Compounds on the Mycelial Growth of
C. lagenarium and B. cinerea in Solid Media
The compounds 4a–e, 9a–e, 15a–d, 16a–d, 17a–d were dissolved in ethanol (5,000 μg/mL) and
mixed with sterile molten potato dextrose agar (PDA) to obtain final concentrations of 200 μg/mL. The
commercial fungicide carbendazim SC (with a 50% composition) was used as the positive control.
Fungicidal activities of the compounds against B. cinerea were evaluated in vitro using the method
given in [52]. The inhibition rate was calculated according to Equation (1):
I1 = (D1−D0)/D1 × 100%
(1)
where I1 is the inhibition rate, D1 is the average diameter of mycelia in the blank test, and D0 is the
average diameter of mycelia in the presence of compounds. The results are given in Table 1.

Molecules 2014, 19
5687
3.14. Effect on C. lagenarium and B. cinerea Activity on Cucumber Leaves [53]
Fungicidal activity against Colletotrichum lagenarium and Botrytis cinerea was tested on cucumber
(Cucumis sarivus L.) seedlings at three leaf stages. The commercial fungicide carbendazim SC (with a
50% composition) was adopted as the positive control. Methanol solutions of the compounds and
carbendazim (5,000 μg/mL) were diluted to concentrations at 300 μg/mL with 0.1% PEG400 solution of
water to obtain solutions that were spread on the surface of the cucumber leaves. After air drying for
24 h, the upper sides of the leaves were inoculated with 4 mm plugs of C. lagenarium or B. cinerea
maintained on PDA. Nine replicates were performed. The inhibition rate was calculated according to
Equation (1), and the results are shown in Table 1.
4. Conclusions
Eighteen novel benzamidines with 1,2,3-triazole moieties connected to the benzene ring and four
benzamidines containing azide groups were synthesized and characterized. Bioassays showed that the
synthesized benzamidine compounds had weak antifungal activities against the tested fungi in vitro,
however, the activities in vivo of some compounds to the same strains were excellent. Compounds 4c,
9a, 9b, 16d and 15b exhibited significant activities toward C. lagenarium in vivo. Compounds 4b, 4e,
and 17a and 15b showed excellent inhibitory activities toward B. cinerea.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/5/5674/s1.
Acknowledgments
We appreciate the kind help of Hong-li Zhang during the NMR spectroscopic analysis of the
compounds synthesized.
Author Contributions
The work presented was carried out in collaboration between all authors. Guangyou Chen and Xing
Zhang defined the research theme. Guangyou Chen and Chonglin Cai designed methods and
experiments, carried out the laboratory experiments, analyzed the data, interpreted the results and
wrote the paper. Jia Lu and Yiwan Zhou co-designed the synthesis experiments, and co-worked on
associated data collection and their interpretation. All authors have contributed to, seen and approved
the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 2, 3, 7, 8, 4a–c, 9a–b, 12, 14, 15a–b, 16a–b, 17a–b,
18, 19 are available from the authors.
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