Synthesis, biological evaluation and anti-proliferative mechanism of fluorine-containing proguanil derivatives

Article abstract of DOI:10.1016/j.bmc.2019.115258

Proguanil, a member of biguanide family, has excellent anti-proliferative activities. Fluorine-containing compounds have been demonstrated to have super biological activities including enhanced binding interactions, metabolic stability, and reduced toxici

Full text of DOI:10.1016/j.bmc.2019.115258

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and anti-proliferative mechanism of
fluorine-containing proguanil derivatives
Di Xiaoa, Zhicheng Lub, Zhiren Wanga, Sichun Zhoua, Mengru Caoc, Jun Denga, Xin Hua,
Mei Penga, Caimei Hea, Jingtao Wua, Simeng Xua, Huihui Zhanga, Cangcang Xua, Wei Wangc,
⁎
,
⁎
⁎
Aiying Guanb, , Xiaoping Yanga,
a Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Pharmacy, School of Medicine, Hunan Normal University,
Changsha, Hunan, China
b State Key Laboratory of the Discovery and Development of Novel Pesticide, Shenyang Sinochem Agrochemicals R&D Company Ltd., Shenyang, China
c TCM and Ethnomedicine Innovation & Development International Laboratory, Innovative Materia Medical Research Institute, School of Pharmacy, Hunan University of
Chinese Medicine, Changsha, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Proguanil, a member of biguanide family, has excellent anti-proliferative activities. Fluorine-containing com-
pounds have been demonstrated to have super biological activities including enhanced binding interactions,
metabolic stability, and reduced toxicity. In this study, based on the intermediate derivatization methods, we
synthesized 13 new fluorine-containing proguanil derivatives, and found that 7a,7d and 8e had much lower IC50
than proguanil in 5 human cancerous cell lines. The results of clonogenic and scratch wound healing assays
revealed that the inhibitory effects of derivatives 7a,7d and 8e on proliferation and migration of human cancer
cell lines were much better than proguanil as well. Mechanistic study based on representative derivative 7a
indicated that this compound up-regulates AMPK signal pathway and downregulates mTOR/4EBP1/p70S6K. In
conclusion, these new fluorine-containing derivatives show potential for the development of cancer che-
motherapeutic drugs.
Biguanides
Fluorine-containing compounds
Anti-proliferative
AMPK
1. Introduction
the strongest inhibitory effect on nine cancer cell lines.8 Based on the
intermediate derivatization method, further chemical modifications to
Around the world, it is estimated that there were 9.6 million cancer
deaths in 2018, and to search high efficacy and safe anti-proliferative
drugs is still a tough challenge.1 Biguanides have been used clinically to
treat diabetes (metformin) or malaria (proguanil).2–4 Interestingly, anti-
proliferative activities of biguanide drugs, especially metformin have
attracted considerable attention in recent years.5,6 However, preclinical
studies have shown that effective metformin concentration to kill
cancer cells is always in the milli molar range, making it difficult to
translate into clinical administration since it is practically arduous to
achieve this high concentration via conventional administration
routes.7 In contrast, we noticed that proguanil (Fig.1), a member of the
biguanide family, has been demonstrated to have excellent anti-pro-
liferative effects. Lea et al reported that among the biguanides including
buformin, phenformin, proguanil and phenyl biguanide, proguanil has
proguanil will have a great potential to obtain superior anti-pro-
liferative compounds.9
Fluorine-containing compounds have been demonstrated to have
super biological activities including enhanced binding interactions,
metabolic stability, and reduced toxicity.10–12 The introduction of
fluorine into drugs has become common, and best-performing drugs on
the present market contain fluorine atom.13 Particularly, in the anti-
tumor drug market, most of compounds, particularly targeted drugs
including Gefitinib and Olaparib, have fluorine atom.14,15 Previous
studies in our laboratory have also demonstrated that fluorinated
quercetin plays a better role in the treatment of bladder cancer than
quercetin.16 In present study, we introduced a fluorine or fluoroalkyl
into a specific position of the biguanide backbone, and found that de-
rivatives 7a,7d and 8e have a stronger inhibitory effect on the
Abbreviations: THF, Tetrahydrofuran; SAR, Structure activity relationship; HRMS, High resolution mass spectrometry; ESI, Electrospray ionization; HPLC, High-
performance liquid chromatography; PBS, Phosphatebuffered saline; SDS-PAGE, Sodium dodecyl sulphate polyacrylamide gel electrophoresis; PVDF, Polyvinylidene
fluoride; TBST, Tris-buffered saline with 0.1% Tween
⁎ Corresponding authors.
E-mail addresses: wangwei402@hotmail.com (W. Wang), guanaiying@sinochem.com (A. Guan), xiaoping.yang@hunnu.edu.cn (X. Yang).
https://doi.org/10.1016/j.bmc.2019.115258
Received 22 August 2019; Received in revised form 28 November 2019; Accepted 7 December 2019
0968-0896/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:DiXiao,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2019.115258

D. Xiao, et al.
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compounds were completely reacted. Finally, after HCl solution was
added and stirred for 30 min, ammonia ethylenediaminetetraacetic acid
(EDTA) solution was dripped to the reaction mixture and filtered to
obtain the target derivatives 7a-7f,8a-8e and 9a-9b.17,18
Fig. 1. Chemical structure of proguanil.
2.2. In vitro inhibitory effect on cell proliferation of fluorine-containing
derivatives
proliferation and invasive phenotype of cancer cells than proguanil.
Anti-proliferative activity of these derivatives to human cancer cell
lines were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) assay, as each compound’s IC50 values illu-
strated in Table 1. Fig 2 illustrated the concentration-response curves for
proguanil, 7a,7d and 8e. As shown in Table 1, most derivatives de-
monstrated significant anti-proliferative activity to these tested cancer
cell lines, especially to lung cancer cell line A549. Furthermore, anti-
proliferative effects of derivatives 7a, 7d and 8e in all cancer cell lines
were even greatly stronger than proguanil (Table 1 and Fig.2). Most
interestingly, derivative 7a showed best anti-proliferative activities with
IC50 value of 2.0 µM for UMUC3, 3.1 µM for T24, 1.3 µM for A549 among
13 tested derivatives (Table 1). In contrast, anti-proliferative activity of
2. Results and discussion
2.1. Chemistry
The target derivatives 7a-7f,8a-8e and 9a-9b were synthesized ac-
cording to the method as shown in Scheme 1. The commercially
available compounds 4-(trifluoromethyl) aniline, 4-fluoroaniline, 3,4-
difluoroaniline, and 2,4-difluoroaniline were firstly reacted respectively
with sodium dicyandiamide at 80 °C to obtain corresponding inter-
mediates. They were then separately reacted with alkylamines or cy-
cloalkylamines at 40 °C in tetrahydrofuran (THF) until the intermediate
Scheme 1. Synthesis of derivatives 7a-7f, 8a-8e, 9a-9b. Reagents and conditions: (a) NaN (CN) 2, HCl, 80 °C, 1h. (b) THF, CuSO4·5H2O, alkylamines or cy-
cloalkylamines, HCl, EDTA, 40 °C.
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Table 1
The anti-proliferative activities of proguanil and its derivatives 7a-f, 8a-e, 9a-b against five human cancer cell lines.
Compounds
R
R1
R2
H
IC50(µM)
UMUC3
16.3
SD
T24
A2780
A549
HCT116
proguanil
7a
1.3
18.4
3.1
1.2
13
1.6
3.8
1.3
0.66
0.21
8.4
3.5
0.98
CF3
CF3
CF3
2.0
4.9
5.2
0.45
0.86
0.96
0.38
2.1
0.55
0.29
0.45
0.78
7b
7c
7d
6.2
4.8
3.7
0.54
0.77
0.62
3.8
3.2
2.5
0.29
0.31
0.26
3.7
3.6
1.8
0.31
0.55
0.33
4.3
4.8
3.8
H
H
CF3
3.3
0.21
0.92
0.35
0.41
7e
7f
CF3
F
CH3
H
6.9
5.7
0.48
5.1
28
0.83
3.6
4.1
7.5
0.81
1.3
4.0
> 50
> 50
> 50
8a
8b
H
7.1
0.45
1.7
42.9
44.8
4.9
2.2
6.7
0.83
2.8
2.4
7.8
0.53
1.1
29.7
39.4
2.5
2.9
22.1
20.4
8c
8d
H
H
25.0
7.2
2.5
1.1
45.0
24.9
4.2
1.7
26
3.2
1.2
5.2
3.5
0.49
0.38
> 50
16.8
9.3
0.53
8e
9a
H
H
2.1
0.37
2.3
4.3
0.35
4.3
1.5
0.37
1.3
1.5
3.6
0.17
0.41
1.8
0.24
2.1
17.3
40.8
19.8
21.1
9b
H
18.4
1.7
37.8
2.9
14.7
1.1
4.0
0.44
29.5
2.9
aCell lines were treated with derivatives for 72 h.
bIC50 values are the mean of triplicate measurements.
derivative 8e with an IC50 value of 1.5 µM for A2780, 1.8 µM for HCT116
was stronger than derivative 7a (Table 1), indicating existence of cellular
specificities of these derivatives. The detailed information and mechan-
isms of these cellular specificities, this is, why and how different deri-
vatives have different activities in different cell lines warrant further
study. However, we would like to highlight several observations to dis-
cuss preliminary structure activity relationship (SAR). For substituents
on phenyl ring, derivatives with trifluoromethylphenyl substitutions (7a,
7c, 7d) showed obviously stronger activity than those with 3,4-di-
fluorophenyl substitutions (8a, 8c, 8d), 2,4-difluorophenyl substitutions
(9a, 9b) or 4-fluorophenyl substitution (7f) targeting all tested cell lines.
For R1 substituent, when the phenyl ring was replaced with 4-tri-
fluoromethy, the activity of derivative with R1 of isobutyl displayed some
better than that with R1 of cyclopentyl (7a Vs 7d); when the phenyl ring
was substituted with 3,4-difluoro, derivative 8e (R1 = heptyl) inhibited
the best activity among four compounds of 8a, 8c-8d, it looked like the
longer the alkyl chain, the higher the activity; while when the phenyl
ring was 2,4-difluorophenyl, these two compounds 9a (R1 = isobutyl)
and 9d (R1 = cyclopentyl) performed almost equal activity. For the re-
placement of R1 and R2 happened simultaneously, (7b, 7e) Vs (7a, 7d),
the activity decreased dramatically to some extent. From the results
obtained above, derivatives with trifluoromethylphenyl or R1 of heptyl
may make a positive contribution to anti-proliferative activity improve-
ment. Subsequent experiments revealed that the cytotoxicity of deriva-
tives 7a, 7d, and 8e on normal cell line HUVEC was significantly lower
than these cancer cell lines (Table 1, Table 2 and Fig.2), suggesting that
derivatives 7a and 8e could be further developed as novel biguanide
candidates for treating cancer.
2.3. Derivatives 7a, 7d and 8e inhibit colony formation in five cancer cell
lines
Due to the excellent inhibitory activity of derivatives 7a, 7d and 8e
on 5 kinds of cancer cell lines in proliferation assay, their abilities to
inhibit cellular colony formation were evaluated through standard
colony formation assay (Fig. 3). The results showed that derivatives 7a,
7d and 8e had more effective inhibition to all cell lines than proguanil.
When 2 µM derivatives 7a, 7d and 8e were applied, colony forming
ability of four cancer cell lines (UMUC3, T24, A2780, HCT116) was
dramatically inhibited, much lower than proguanil. The more inter-
esting was that colony formation of A549 was totally inhibited at this
concentration, implying that these derivatives may be more suitable for
treating lung cancer. Consistent with MTT assay, derivative 7a showed
highest activity in bladder (UMUC3, T24) and lung (A549) cancer cell
lines, while ovarian(A2780) and colorectal (HCT116) cancer cell lines
were most sensitive to derivative 8e.
2.4. Derivatives 7a,7d and 8e inhibit cell migration in three cancer cell lines
The effects of derivatives (7a, 7d and 8e) and proguanil on the
migration of cancer cells were determined by cellular scratch assay. As
shown in Fig.4, after treatment with different concentrations of deri-
vatives 7a,7d and 8e, cell migration was significantly inhibited at 24 h
and 48 h compared with the control group. In contrast, proguanil
treatment at this concentration did not cause significant change. These
results demonstrated that derivatives 7a,7d and 8e have stronger in-
hibitory effect than proguanil on migration.
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Fig. 2. Concentration-response curves for proguanil, derivatives 7a,7d and 8e. UMUC3, T24, A2780, HCT116 cell lines were treated with these Compounds (0–8 µM)
for 72 h. A549 cell line was treated with these Compounds (0–4 µM) for 72 h. HUVEC cell line was treated with 7a,7d and 8e (0–8 µM) for 72 h. The results were
showed as the average of 3 independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001).
29
group, which was consistent with the results of Bridges et al.
This
Table 2
The anti-proliferative activities of derivatives 7a, 7d
and 8e against normal cell line HUVEC.
means that fluorine-containing derivatives may enhance permeability
into mitochondria. These results indicated that derivative 7a strongly
activated AMPK and down-regulated the mTOR signal pathway,
thereby inhibiting p70S6K and 4EBP1 phosphorylation to block the
tumor cell growth (Fig.6).
Compounds
IC50(µM)
SD
HUVEC
7a
7d
8e
8.5
7.3
6.4
0.56
3. Conclusions
0.68
0.63
In summary, we have designed and synthesized a series of proguanil
derivatives by introducing trifluoromethyl and fluorine atoms into the
benzene ring to discover promising anti-cancer drug candidates. The
inhibitory effect of most derivatives of these newly synthesized com-
pounds on the proliferation and migration of cancer cells is more pro-
found than proguanil. Among them, derivative 7a exhibited the highest
anti-proliferative activities in bladder cancer cell lines (UMUC3 T24)
and Lung cancer cell line (A549) while derivative 8e shows the stron-
gest activity to ovarian cancer cell line (A2780) and colorectal carci-
noma cell line (HCT116). To further explore the anti-tumor mechanism
of derivatives, western blot test showed that the derivative 7a inhibits
tumor proliferation and influencing AMPK-mTOR signal pathway.
Overall, derivatives 7a and 8e are worthy of further study as new po-
tential anti-proliferative drugs.
2.5. Derivative 7a regulates AMPK and its downstream signaling pathway
Studies have shown that AMPK as an energy regulator counts a great
deal for cell proliferation.19,20 Previously, our laboratory demonstrated
that biguanides exerted anti-tumor effects mainly through the AMPK
signal pathway.21–24 For further understanding the mechanism of anti-
proliferative activities of these newly synthesized derivatives, we ex-
plored the effect of derivative 7a on AMPK signal transduction
pathway. As shown in Fig.5, phosphorylation of AMPK was significantly
up-regulated with concentration gradient (0–2 µM) or time gradient
(0–12 h) after treatment with derivative 7a, indicating that derivative
7a induced activation of AMPK. Studies have shown that activation of
mTOR, one of the downstream targets of AMPK,25,26 can phosphorylate
its downstream p70S6K and 4EBP1, which both played a key role in
protein synthesis in cancer cells. These two downstream proteins pro-
duce translational proteins required for cells growth, which closely
related with cancer progression.27,28 Thus, we further investigated the
effects of derivative 7a on mTOR, p70S6K and 4EBP1.In Fig 5, we can
see that the phosphorylation of mTOR, 4EBP1 and p70S6K were de-
creased in derivative 7a treatment group. Further, we found that de-
rivative 7a regulated p-AMPK, p-mTOR, p-p70S6K, and p-4EBP1 much
more strongly than proguanil at a concentration of 2 µM (Fig.5i).
However, there was no significant difference in the regulatory effect of
proguanil treatment group on p-AMPK compared with 0 µM treatment
4. Experimental
4.1. Chemistry
All solvents and materials were commercial grade and no further
purification was required unless otherwise stated. Infrared spectra were
recorded by the Thermo Nicolet iS5 spectrometer. Proton nuclear
magnetic resonance (1H-NMR) spectra and 13C NMR were recorded on a
Brucker DRX spectrometer (600 MHz) in dimethylsulfoxide (DMSO)
solvent by using TMS as an internal standard. High resolution mass
spectrometry (HRMS) analysis was performed on an Agilent 1290
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Fig. 3. Inhibitory effect of derivatives 7a, 7d and 8e on colony suppression to five human cancer cell lines, comparing to proguanil. (a, c, e, g and i) UMUC3, T24 and
HCT116 cell lines were treated with 0–8 µM of proguanil or derivatives 7a, 7d and 8e; A2780 cell line was treated with these compounds at a concentration of
0–4 µM. A549 cell line was treated with these compounds at a concentration of 0–2 µM. After7 days, the quantity of colonies was examined by counting after staining
with crystal violet. (b, d, f, h and j) Bar charts showed the average quantitative data of 3 independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001).
Quantitative data were measured by microplate area scan (OD = 550 nm). The results were showed as the average of 3 independent experiments. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.)
HPLC-6540-Q-TOF instrument. Low resolution mass spectra were ac-
quired with an Accurate-Mass-Q-TOF MS 6520 system (Agilent Tech-
nologies, Milford, MA) equipped with an electrospray ionization (ESI)
source. The melting point of the derivatives was obtained by a micro
melting point apparatus XT3A. Column chromatographic separations
were carried out with silica gel (200–300 mesh), and the samples were
eluted with a 1:4 Petroleum ether/ethyl acetate mixture, Fractions were
monitored by TLC (Qingdao Marine Chemical Inc., P. R. China) and
spots were visualized by using a UV lamp. The purity of the derivatives
was determined by high-performance liquid chromatography (HPLC),
conducted on an agilent 1260 HPLC system with a TC-C18 column
(4.6 mm × 250 mm, 5 μm), and the samples were eluted with a 45:55
methanol/H2O mixture, at a flow rate of 1 ml/min.
4.1.1. Method for synthesizing compounds 4,5 and 6
NaN (CN) (50 g, 0.56 mol) was dissolved in 430 ml of water to
2
form a solution, and substituted anilines solution (45 g, 0.3 mol of
substituted anilines dissolved in water and concentrated HCl (132 ml /
23.5 ml)) was added at 80 °C, and then maintaining the reaction at
80 °C for about 1 h. TLC detected the reaction solution until no sub-
stituted anilines was present. Finally, the white powdery solids were
filtered and dried in vacuum to obtain intermediate products.
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Fig. 4. Inhibitory effect of derivatives 7a, 7d and 8e on migration to three human cancer cell lines, comparing to proguanil. UMUC3, T24 cell lines were treated with
3 µM proguanil or derivative7a, 7d and 8e. A549 cell line was treated with these compounds at a concentration of 2 µM. (a-c) showed the gap of scratch areas and bar
graphs of each cell line after drug treatment or without drug treatment (Ctrl) for 24 h or 48 h. The average wound closure distance was obtained by Image J software.
The results were showed as the average of 3 independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001).
4.1.1.1. 1-(4-trifluoromethyl)
phenyl-3-cyanoguanidine
(4a). Yield:
disodium salt) was dropped to the reactive mixture, maintaining the
temperature in the range of 15–20 °C. After that, the mixture was
stirred at the same temperature for 30 min. The products were sepa-
rated by filtration and repeatedly washed in cold water, and then dried
at 90–95 °C. Finally, derivatives were purified by column chromato-
graphy on silica gel.
95.5%. 1H NMR (600 MHz, DMSO-d6) δ 9.45 (s, 1H), 7.66 (d,
J = 8.5 Hz, 2H), 7.60 (d, J = 8.5 Hz, 2H). ESI-MS: m/z 229.0[M+1]+
.
4.1.1.2. 1-(4-fluoro) phenyl-3-cyanoguanidine (4b). Yield: 90.7%. 1H
NMR (600 MHz, DMSO-d6) δ 9.02 (s, 1H), 7.37–7.32 (m, 2H),
7.17–7.12 (m, 2H). ESI-MS: m/z176.8[M-1]−
.
4.1.2.1. 1-isobutyl-5-(4-trifluoromethylphenyl)
biguanide(7a). Yield:
56.9%. Mp: 213–215 °C. IR: n/cm−1: 3306, 3155 (NH), 1634(C]N).
1H NMR (600 MHz, DMSO) δ 10.22 (s, 1H), 7.65 (d, J = 13.6 Hz, 2H),
7.62 (d, J = 9.4 Hz, 2H), 2.93 (d, J = 31.9 Hz, 2H), 1.31–1.19 (m, 1H),
0.89 (d, J = 6.2 Hz, 6H). 13C NMR (151 MHz, Methanol-d4) δ 160.04,
153 99, 142.19, 126.98, 125.02, 123.65, 119.95, 53.61, 32.36, 23.28.
ESI-MS: m/z 302.1[M+1]+. HRMS (ESI) (m/z) [M+H]+ calcd for
4.1.1.3. 1-(3,4-difluoro) phenyl-3-cyanoguanidine (5). Yield: 95.2%. 1H
NMR (600 MHz, DMSO-d6) δ 7.50 (d, J = 7.5 Hz, 1H), 7.34 (dd,
J = 18.0, 8.9 Hz, 1H), 7.09 (d, J = 5.8 Hz, 1H). ESI-MS: m/z197.0[M
+1]+
.
4.1.1.4. 1-(2,4-difluoro) phenyl-3-cyanoguanidine (6). Yield: 94.9%.1H
NMR (600 MHz, DMSO-d6) δ 8.87 (s, 1H), 7.59 (d, J = 6.2 Hz, 1H),
C13H18F3N5, 302.1548; found, 302.1594. Purity: 99.9% (by HPLC).
7.35–7.29 (m, 1H), 7.07 (d, J = 8.6 Hz, 1H). ESI-MS: m/z194.8[M-1]−
.
4.1.2.2. 1-diethyl-5-(4-trifluoromethylphenyl)
biguanide(7b). Yield:
39.5%. Mp: 215–217 °C. IR: n/cm−1: 3310, 3162 (NH), 1637(C]N).
1H NMR (600 MHz, DMSO-d6) δ 9.93 (s, 1H), 7.72 (s, 2H), 7.65 (d,
J = 8.7 Hz, 2H), 7.60 (d, J = 8.7 Hz, 2H), 3.40–3.34 (m, 4H),
1.18–1.04 (m, 6H). 13C NMR (151 MHz, Methanol-d4) δ 159.39, 153.99,
142.49, 126.98, 125.52, 123.65, 119.95, 42.80, 11.78. ESI-MS: m/
z302.1[M+1]+. HRMS (ESI) (m/z) [M+H]+ calcd for C13H18F3N5,
302.1548; found, 302.1605.
4.1.2. Method for synthesizing derivatives 7a-7e
9.12 g (0.04 mol) of 1-(4-trifluoromethyl) phenyl-3-cyanoguanidine
was added to 48 ml of THF and 40 ml of water while stirring at
25–35 °C, followed by addition of 6.8 g (0.085 mol) of copper sulfate
pentahydrate and 0.16 mol of alkylamines or cycloalkylamines. The
mixed solution was heated to 40 °C and continuously stirred. After the
TLC was used to check the absence of 1-(4-trifluoromethyl) phenyl-3-
cyanoguanidine, the THF was evaporated to dryness under reduced
pressure. When the products was cooled to 25–30 °C, HCl solution
(20 ml of concentrated hydrochloric acid in 32 ml of water) was added
and stirred for 30 min, and cooled ammonia EDTA solution (32 ml of
water, 15 ml of ammonia water (25%) and 14 g EDTA disodium
4.1.2.3. 1-isopropyl-5-(4-trifluoromethylphenyl) biguanide(7c). Yield:46.3%.
1H NMR (600 MHz, DMSO-d6) δ 7.51 (d, J = 8.2 Hz, 3H), 7.16–7.00 (m,
2H), 3.85 (s, 1H), 1.10 (d, J = 6.4 Hz, 6H). 13C NMR (151 MHz,
Methanol‑d4) δ 158.93, 156.33, 147.56, 127.36, 125.32, 123.65, 121.61,
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Fig. 5. Effect of derivative 7a on the expression of AMPK and its downstream pathway in UMUC3 and T24 Cells. (a and c) Effect of derivative 7a in protein levels (p-
AMPK, p-mTOR, p-p70S6K, p-4EBP1, t-AMPK, t-mTOR, t-p70S6K and t-4EBP1) of bladder cancer cell lines treated with derivative 7a for 12 h. (e and g) Effect of
derivative 7a in protein levels (p-AMPK, p-mTOR, p-p70S6K, p-4EBP1, t-AMPK, t-mTOR, t-p70S6K and t-4EBP1) of bladder cancer cell lines treated with derivative
7a at a concentration of 2 µM. (i) Effect of derivative 7a and proguanil in protein levels (p-AMPK, p-mTOR, p-p70S6K, p-4EBP1) of UMUC3 treated with compounds
at a concentration of 2 µM. Equal loading was established by β-actin. The density values of each protein were obtained by imagine J software, (b, d, f, h and j) show
the ratio of optical density values of these proteins to β-actin. Results were expressed as the mean of 3 independent experiments (*P
***P < 0.001).
< 0.05, **P < 0.01,
43.10, 20.94. HRMS (ESI) (m/z) [M+H]+ calcd for C12H16F3N5, 288.1391;
4.1.2.5. 1-methyl-1-ethyl-5-(4-trifluoromethylphenyl) biguanide(7e). Yield:
59.2%. Mp: 201–203 °C. IR: n/cm−1: 3362 (NH), 1649(C]N). 1H NMR
(600 MHz, DMSO-d6) δ 9.81 (s, 1H), 7.74 (s, 2H), 7.65 (d, J = 8.0 Hz,
2H), 7.61 (d, J = 7.6 Hz, 2H), 3.37 (q, J = 7.0 Hz, 2H), 2.97 (s, 3H),
1.18–0.96 (m, 3H). 13C NMR (151 MHz, Methanol‑d4) δ 160.04, 153.31,
142.19, 126.98, 125.62, 123.65, 119.57, 45.52, 33.72, 11.10. ESI-MS: m/
z 288.0[M+1] +. HRMS (ESI) (m/z) [M+H]+ calcd for C12H16F3N5,
288.1396; found, 288.1450.
found, 288.1444.
4.1.2.4. 1-cyclopentyl-5-(4-trifluoromethylphenyl) biguanide(7d). Yield:
51.7%. Mp:225–227 °C. IR: n/cm−1: 3307,3186 (NH), 1626(C]N).
1H NMR (600 MHz, DMSO) δ 9.54 (s, 1H), 8.19 (s, 1H), 7.64 (d,
J = 4.1 Hz, 2H), 7.58 (d, J = 7.5 Hz, 2H), 3.93–3.72 (m, 1H),
+
1.96–1.60 (m, 4H), 1.56–1.40 (m, 4H). ESI-MS: m/z 314.1[M+1]
.
13C NMR (151 MHz, Methanol‑d4) δ 160.34, 154.59, 142.81, 126.98,
125.32, 123.28, 120.25, 53.61, 32.36, 23.28. HRMS (ESI) (m/z) [M
+H]+ calcd for C14H18F3N5, 314.1548; found, 314.1603. Purity: 96.6%
(by HPLC).
4.1.3. Method for synthesizing derivatives 7f
7.12 g (0.04 mol) of 1-(4-fluoro) phenyl-3-cyanoguanidine was
added to 48 ml of THF and 40 ml of water while stirring at 25–35 °C,
7

D. Xiao, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
was used to check the absence of 1-(3,4-difluoro) phenyl-3-cyanogua-
nidine, the THF was evaporated to dryness under reduced pressure.
When the products was cooled to 25–30 °C, HCl solution (20 ml of
concentrated hydrochloric acid in 32 ml of water) was added and
stirred for 30 min, and cooled ammonia EDTA solution (32 ml of water,
15 ml of ammonia water (25%) and 14 g EDTA disodium disodium salt)
was dropped to the reactive mixture, maintaining the temperature in
the range of 15–20 °C. After that, the mixture was stirred at the same
temperature for 30 min. The products were separated by filtration and
repeatedly washed in cold water, and then dried at 90-95℃. Finally,
derivatives were purified by column chromatography on silica gel.
4.1.4.1. 1-isobutyl-5-(3,4-difluorophenyl) biguanide(8a). Yield:65.6%.
Mp:179–181 °C. IR: n/cm−1
:
3313 (NH), 1641(C]N). 1H NMR
(600 MHz, DMSO-d6) δ 9.63 (s, 1H), 8.12 (s, 1H), 7.88–7.67 (m, 1H),
7.64–7.48 (m, 1H), 7.36 (d,
J = 7.6 Hz, 1H), 3.02–2.85 (m,
2H),1.87–1.66 (m, 1H) ,0.86 (d, J = 33.4 Hz, 6H). ESI-MS: m/z
270.0[M+1]+
.
13C NMR (151 MHz, Methanol‑d4) δ 160.72, 155.65,
154.29, 150.58, 148.54, 117.60, 116.54, 110.49, 48.85, 27.67, 18.89.
HRMS (ESI) (m/z) [M+H]+ calcd for C12H17F2N5, 270.1486; found,
270.1535.
4.1.4.2. 1,1-diethyl-5-(3,4-difluorophenyl) biguanide(8b). Yield:42.3%.
Mp:178–180 °C. IR: n/cm−1: 3306, 3189 (NH), 1628(C]N). 1H NMR
(600 MHz, DMSO-d6) δ 9.72 (s, 1H), 7.55 (d, J = 10.2 Hz, 1H), 7.37
(dd, J = 19.2, 9.4 Hz, 1H),7.11 (d, J = 6.4 Hz, 1H), 3.41–3.33 (m, 4H),
1.28–0.98 (m, 6H). 13C NMR (151 MHz, DMSO-d6) δ 159.14, 154.02,
150.16, 148.46, 146.22, 117.84, 116.60, 109.42, 42.50, 13.50. ESI-MS:
m/z 270.1[M+1]+. HRMS (ESI) (m/z) [M+H]+ calcd for C12H17F2N5,
270.1486; found, 270.1537. Purity: 99.0% (by HPLC).
4.1.4.3. 1-isopropyl-5-(3,4-difluorophenyl) biguanide(8c). Yield:46.5%.
Mp:223–225 °C. IR: n/cm−1: 3305, 3187 (NH), 1647(C]N). 1H NMR
(600 MHz, DMSO-d6) δ 9.63 (s, 1H), 8.02 (s, 1H), 7.74 (d, J = 35.8 Hz,
1H), 7.63–7.49 (m, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.10 (d, J = 8.0 Hz,
2H), 6.75 (s, 1H), 3.86–3.58 (m, 1H), 1.12 (d, J = 6.5 Hz, 6H). ESI-MS:
Fig. 6. Schematic diagram of derivative 7a on AMPK cell signaling pathway.
m/z256.1[M+1]+
.
13C NMR (151 MHz, Methanol‑d4)
δ 161.70,
159.36, 155.27, 150.89, 148.92, 117.91, 116.54, 110.87, 43.78,
20.94. HRMS (ESI) (m/z) [M+H]+ calcd for C11H15F2N5, 256.1329;
found, 256.1376.
followed by addition of 6.8 g (0.085 mol) of copper sulfate pentahy-
drate and 0.16 mol of isopropylamine. The mixed solution was heated
to 40 °C and continuously stirred. After the TLC was used to check the
absence of 1-(4-fluoro) phenyl-3-cyanoguanidine, the THF was evapo-
rated to dryness under reduced pressure. When the products was cooled
to 25–30 °C, HCl solution (20 ml of concentrated hydrochloric acid in
32 ml of water) was added and stirred for 30 min, and cooled ammonia
EDTA solution (32 ml of water, 15 ml of ammonia water (25%) and
14 g EDTA disodium disodium salt) was dropped to the reactive mix-
ture, maintaining the temperature in the range of 15–20 °C. After that,
the mixture was stirred at the same temperature for 30 min. The pro-
ducts were separated by filtration and repeatedly washed in cold water,
and then dried at 90-95℃. Finally, derivatives were purified by column
chromatography on silica gel.
4.1.4.4. 1-cyclopentyl-5-(3,4-difluorophenyl) biguanide(8d). Yield:71.3%.
Mp:215–217 °C. IR: n/cm−1
:
3317 (NH), 1603(C]N). 1H NMR
(600 MHz, DMSO-d6)
δ 9.85 (s, 1H), 8.12 (s, 1H), 7.74 (d,
J = 30.2 Hz, 1H), 7.43–7.27 (m, 1H), 7.23–7.15 (m, 1H), 3.96–3.75
(m, 1H), 1.98–1.61 (m, 4H), 1.59–1.39 (m, 4H). ESI-MS: m/z 282.1[M
+1]+
.
13C NMR (151 MHz, Methanol‑d4) δ 160.34, 155.95, 153.61,
150.58, 148.92, 117.60, 116.75, 140.74, 53.23, 32.05, 22.90. HRMS
(ESI) (m/z) [M+H]+ calcd for C13H17F2N5, 282.1486; found, 282.1538.
Purity: 98.3% (by HPLC).
4.1.4.5. 1-n-heptyl-5-(3,4-difluorophenyl) biguanide(8e). Yield:60.3%.
Mp:119–121 °C. IR: n/cm−1: 3308, 3192 (NH), 1644(C]N). 1H NMR
(600 MHz, DMSO-d6) δ 9.98–9.78 (m, 1H), 8.09 (s, 1H), 7.74 (d,
J = 22.0 Hz, 1H), 7.42–7.32 (m, 1H), 7.10 (d, J = 8.9 Hz, 1H),
3.14–3.05 (m, 2H), 1.58–1.37 (m, 2H), 1.33–1.16 (m, 8H), 0.90–0.81
(m, 3H). 13C NMR (151 MHz, DMSO-d6) δ 161.02, 154.97, 153.81,
150.28, 148.24, 117.91, 116.83, 109.51, 42.11, 31.68, 29.63, 28.65,
26.61, 22.90, 13.82. ESI-MS: m/z 309.9[M-1]−. HRMS (ESI) (m/z) [M
+H]+ calcd for C15H23F2N5, 312.1955; found, 312.2008.
4.1.3.1. 1-isopropyl-5-(4-fluorophenyl) biguanide (7f). Yield:49.5%. 1H
NMR (600 MHz, DMSO-d6) δ 9.85 (s, 1H), 9.54 (s, 1H), 7.44–7.33 (m,
2H), 7.18–7.12 (m, 2H), 3.69 (s, 1H), 1.11 (d, J = 6.5 Hz, 6H). 13C
NMR (151 MHz, Methanol-d4) δ 160.34, 158.68, 133.71, 123.96,
115.18, 43.48, 21.24. HRMS (ESI) (m/z) [M+H]+ calcd for
C
11H16FN5, 238.1423; found, 238.1485. Purity: 99.4% (by HPLC).
4.1.4. Method for synthesizing derivatives 8a-8e
7.84 g (0.04 mol) of 1-(3,4-difluoro) phenyl-3-cyanoguanidine was
added to 48 ml of THF and 40 ml of water while stirring at 25–35 °C,
followed by addition of 6.8 g (0.085 mol) of copper sulfate pentahy-
drate and 0.16 mol of alkylamines or cycloalkylamines. The mixed
solution was heated to 40 °C and continuously stirred. After the TLC
4.1.5. Method for synthesizing derivatives 9a-9b
7.84 g (0.04 mol) of 1-(2,4-difluoro) phenyl-3-cyanoguanidine was
added to 48 ml of THF and 40 ml of water while stirring at 25–35 °C,
followed by addition of 6.8 g (0.085 mol) of copper sulfate pentahy-
drate and 0.16 mol of alkylamines or cycloalkylamines. The mixed
8

D. Xiao, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
solution was heated to 40 °C and continuously stirred. After the TLC
was used to check the absence of 1-(2,4-difluoro) phenyl-3-cyanogua-
nidine, the THF was evaporated to dryness under reduced pressure.
When the products was cooled to 25–30 °C, HCl solution (20 ml of
concentrated hydrochloric acid in 32 ml of water) was added and
stirred for 30 min, and cooled ammonia EDTA solution (32 ml of water,
15 ml of ammonia water (25%) and 14 g EDTA disodium disodium salt)
was dropped to the reactive mixture, maintaining the temperature in
the range of 15–20 °C. After that, the mixture was stirred at the same
temperature for 30 min. The products were separated by filtration and
repeatedly washed in cold water, and then dried at 90-95℃. Finally,
derivatives were purified by column chromatography on silica gel.
4.5. Measurement of cell migration
Several cell lines (3.0 × 103 cells per well) were plated in 12-well
plates and allowed to reach confluence. Then use a 10 μL sterile pipette
tip to scratch a straight line at the center of the plate. After washing
twice with PBS, serum-free medium in which compounds were dis-
solved was added to each well. Finally, photographs were taken at 0,
24, and 48 h using a DFC450C microscope (Leica). The experiments
were repeated three times.
4.6. Protein characterization
Protein extracts of cells were loaded and run on 8% or 10% sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).
Later, proteins were transferred onto polyvinylidene fluoride (PVDF)
membranes and blocked in 5% milk in TBST for 1 h. the membranes
were blotted with the specific primary antibodies at 4 °C overnight,
then washed three times with Tris-buffered saline with 0.1% Tween
(TBST) the next day. After incubating with secondary antibodies for 1 h,
the membrane was washed three times with TBST again. Protein signals
were immediately detected with a Chemi Doc (Bio-Rad, Hercules, CA,
USA) after adding Pierce Super Signal chemiluminescent substrate
(Rockford, IL, USA).
4.1.5.1. 1-isobutyl-5-(2,4-difluorophenyl) biguanide(9a). Yield:54.8%.
Mp:189–181 °C. IR: n/cm−1: 3305, 3192 (NH), 1604(C]N). 1H NMR
(600 MHz, DMSO-d6) δ 9.09 (s, 1H), 7.89 (s, 1H), 7.29 (d, J = 17.6 Hz,
1H), 7.11–7.03 (m, 1H), 6.97 (d, J
= 20.3 Hz, 1H), 2.92 (d,
J = 17.0 Hz, 2H), 1.83–1.61 (m, 1H), 0.87 (d, J = 19.6 Hz, 6H).
ESI-MS: m/z 267.9[M-1]−
.
13C NMR (151 MHz, Methanol-d4) δ 161.40
160.72 156.63 154.97 127.36 122.29 110.49 103.76 48.85 28.35
18.89. HRMS (ESI) (m/z) [M+H]+ calcd for C12H17F2N5, 270.1486;
found, 270.1529.
4.1.5.2. 1-cyclopentyl-5-(2,4-difluorophenyl) biguanide(9b). Yield:81.2%.
Mp:217–219 °C. IR: n/cm−1
:
3360 (NH), 1603(C]N). 1H NMR
Acknowledgements
(600 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.10–7.93 (m, 1H), 7.65 (s, 2H),
7.31 (t, J = 8.7 Hz, 1H), 7.10–7.05 (m, 1H), 6.84 (s, 1H), 4.00–3.64 (m,
This study was supported by the National Natural Science
Foundation of China, China (81874212); the Key Project of Hunan
Province, China (2016JC2036); the Key Laboratory of Study and
Discovery of Small Targeted Molecules of Hunan Province, Hunan
Normal University, China (2017TP1020); Huxiang High-Level Talent
Innovation Team, China (2018RS3072)
1H), 1.95–1.57 (m, 4H), 1.57–1.34 (m, 4H). ESI-MS: m/z 282.1[M+1]+
.
13C NMR (151 MHz, DMSO-d6) δ 160.10, 159.64, 156.27, 154.73,
126.98, 122.97, 111.48, 104.44, 52.93, 32.66, 23.58. HRMS (ESI) (m/
z) [M+H]+ calcd for C13H17F2N5, 282.1486; found, 282.1533. Purity:
98.9% (by HPLC).
4.2. Cell lines and culture conditions
Declaration of Competing Interest
Two human bladder cancer cell lines (T24, UMUC3) were obtained
from Dr. P Guo (Xi’an Jiaotong University, China). Human ovarian
cancer cell line (A2780) was a gift from Dr. Y Zhang (Xiangya Hospital,
China). Human lung cancer cell line (A549) and colorectal cancer cell
line (HCT116) were purchased from ATCC. All cells were grown in
DMEM (Hyclone, Logan, UT, USA) or RPMI-1640 (Hyclone, Logan, UT,
USA) supplied with 10% FBS (Hyclone, Logan, UT, USA) and 1% pe-
nicillin/streptomycin mixture. Besides, cells were maintained in a 37
℃, 5% CO2 humidified incubator.
The authors declare that there are no conflicts of interests.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2019.115258.
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