Synthesis and biological evaluation of ursolic acid derivatives containing an aminoguanidine moiety

Article abstract of DOI:10.1007/s00044-019-02349-x

Three series of ursolic acid derivatives containing an aminoguanidine moiety were designed, synthesized, and evaluated for anti-bacterial and anti-inflammatory activity. Some compounds displayed potent anti-bacterial activity against Gram-positive bacterial strains (including multidrug-resistant clinical isolates) and Gram-negative bacterial strains, with minimum inhibitory concentration (MIC) values in the range of 2–64 μg/mL. Compounds 3a, 5a, and 7l showed significant inhibitory activity against the Gram-positive bacterial strain Staphylococcus aureus RN 4220, the Gram-negative bacterial strain Escherichia coli 1924, and four multidrug-resistant Gram-positive bacterial strains, with MIC values of 2 and 4 μg/mL. In anti-inflammatory tests, most of the compounds exhibited potent activity, in particular compound 3a displayed the most potent activity with 81.61% inhibition after intraperitoneal administration, which was more potent than ursolic acid and the reference drugs (ibuprofen and indomethacin). The cytotoxic activity of compound 3a was assessed in HeLa, Hep3B, and A549 cells.

Full text of DOI:10.1007/s00044-019-02349-x

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02349-x
ORIGINAL RESEARCH
Synthesis and biological evaluation of ursolic acid derivatives
containing an aminoguanidine moiety
Jie Wu1 Song Ma Tian-Yi Zhang Zhi-Yu Wei Hui-Min Wang Fang-Yan Guo Chang-Ji Zheng Hu-Ri Piao1
1 ●
1 ●
2 ●
1 ●
1 ●
1 ●
●
Received: 10 December 2018 / Accepted: 20 April 2019
©
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Three series of ursolic acid derivatives containing an aminoguanidine moiety were designed, synthesized, and evaluated for
anti-bacterial and anti-inflammatory activity. Some compounds displayed potent anti-bacterial activity against Gram-positive
bacterial strains (including multidrug-resistant clinical isolates) and Gram-negative bacterial strains, with minimum
inhibitory concentration (MIC) values in the range of 2–64 µg/mL. Compounds 3a, 5a, and 7l showed significant inhibitory
activity against the Gram-positive bacterial strain Staphylococcus aureus RN 4220, the Gram-negative bacterial strain
Escherichia coli 1924, and four multidrug-resistant Gram-positive bacterial strains, with MIC values of 2 and 4 µg/mL. In
anti-inflammatory tests, most of the compounds exhibited potent activity, in particular compound 3a displayed the most
potent activity with 81.61% inhibition after intraperitoneal administration, which was more potent than ursolic acid and the
reference drugs (ibuprofen and indomethacin). The cytotoxic activity of compound 3a was assessed in HeLa, Hep3B, and
A549 cells.
●
●
●
●
Keywords Ursolic acid Aminoguanidine Antibacterial activity Anti-inflammatory activity Cytotoxicity
Introduction
common and important basic pathological process, but
exaggerated inflammation can cause a physiological imbal-
ance in tissues and organs (Serhan and Savill 2005). Cur-
rently, nonsteroidal anti-inflammatory drugs, such as
indomethacin and ibuprofen, are used to treat acute inflam-
mation, but their long-term use can cause adverse reactions,
including bone tissue damage, gastrointestinal injury, liver
damage, and toxic kidney injury. As a result, there is a need
for novel anti-bacterial and anti-inflammatory agents
Emerging infectious diseases and the increasing number of
multidrug-resistant microbial pathogens make bacterial
infections a challenging therapeutic problem. Infectious dis-
eases are responsible for a large number of deaths in the
world population (Zafer et al. 2008; Nelson et al. 2014). In
addition, there is a relationship between inflammation and
bacterial infection (Sy et al. 2011). Inflammation is a very
(Damodar et al. 2016; Tanaka et al. 2014).
Ursolic acid (UA) is a natural pentacyclic triterpene iso-
lated from many foods and herbs, including apples, cranber-
ries, rosemary, and oregano (Ikeda et al. 2008). UA has been
reported to have antiviral (Chiang et al. 2005), anti-bacterial
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02349-x) contains supplementary
material, which is available to authorized users.
(
Nascimento et al. 2014), anti-HIV (Ma et al. 2000), anti-
cancer (Shanmugam et al. 2013), and anti-inflammatory
Chattopadhyay et al. 2002) properties. Key functionalities,
including C -OH and C -COOH, make UA amenable to a
*
*
Chang-Ji Zheng
zhengcj@ybu.edu.cn
(
Hu-Ri Piao
3
17
piaohr@ybu.edu.cn
variety of chemical transformations (Pathak et al. 2002). The
guanidine moiety plays an important biological role in many
compounds, and aminoguanidine derivatives have a diverse
range of biological properties, including anti-bacterial, anti-
fungal, and anti-inflammatory activities (Gadad et al. 2000;
Xu and Wang 2010). In our previous work, we reported the
development of a series of aminoguanidine derivatives that
1
Key Laboratory of Natural Resources and Functional Molecules of
the Changbai Mountain, Affiliated Ministry of Education, Yanbian
University College of Pharmacy, Jilin Province, Yanji 133002,
China
2
Medical College of Dalian University, Liaoning Province,
Dalian 116622, P. R. China

Medicinal Chemistry Research
Fig. 1 The structures of the
target compounds
exhibited good anti-bacterial and anti-inflammatory activities
color persists. The reaction was monitored by TLC till its
completion in 2 h. After quenching the reaction with cold
water, there action mixture was filtered to remove insoluble
residue. The filtrate was extracted with ethyl acetate
(3 × 20 ml). The combined organic layer was dried over
sodium sulfate, concentrated under vacuum and purified by
column chromatography to give pure compound 2 (Dar
et al. 2016).
(Wei et al. 2016; Li et al. 2015), and Wei et al. (2018)
reported that UA derivatives showed potent anti-inflammatory
activitiy. Thus, as part of our ongoing research, we designed
and synthesized three novel series of UA derivatives con-
taining an aminoguanidine moiety via a structure-based
design strategy (Fig. 1). The compounds synthesized were
characterized and screened for anti-bacterial activity in vitro
and anti-inflammatory activity in vivo.
General procedure for the preparation of target
compounds 3a–c
Materials and methods
To a solution of compound 2 (1 mmol) and aminoguanidine
bicarbonate (semicarbazide hydrochloride or thiosemi-
carbazide) (1 mmol) in absolute ethanol (10 mL) was added
drops of Con.HCl. The reaction mixture was refluxed for
6 h and further purified by the silica gel column chroma-
tography (methanol /dichloromethane, 1: 30) to afford white
compounds 3a–c. The yield, melting point and spectral data
of each compound are given below.
Chemistry
All commercially available reagents were obtained from
Sigma-Aldrich (Saint Louis, MO, USA) and Fluka (Mil-
waukee, WI, USA). Melting points were determined in open
capillary tubes and are uncorrected. Reaction courses were
monitored by TLC on silica gel precoated F254 Merck
plates and the developed plates were examined under UV
lamps (254 nm). Column chromatography was undertaken
with 200-mesh silica gel (Merck). H NMR and C NMR
spectra were measured on Bruker AV-300 or 500 spectro-
meter, with TMS as internal standard. Mass spectra were
obtained on an HP1100LC (Agilent Technologies, USA).
(1S,2R,4aS,6aS,6bR,10E,12aR)-10-guanidinoimino-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosa-
hydro-1,2,6a,6b,9,9,12a-heptamethylpicene-4a-carboxylic
1
13
acid (3a) R = 0.30, methanol/dichloromethane (v/v 1:8);
f
1
White powder; Yield: 82%; m.p. 262–264 °C; H NMR
(
DMSO-d , 300 MHz, ppm) δ 11.96 (s, 1H, COOH), 10.92
6
Synthesis
(s, 1H, C=NH), 7.45 (s, 3H, NH , C-NH), 5.16 (s, 1H),
2
3
.50–3.40 (m, 1H), 2.58 (s, 1H), 2.37 (s, 1H), 2.12 (d,
J = 10.8 Hz, 1H), 1.90 (s, 3H), 1.78 (d, J = 13.6 Hz, 2H),
.47 (d, J = 25.1 Hz, 8H), 1.33 (s, 2H), 1.24 (s, 2H), 1.17 (s,
4H), 1.05 (s, 8H), 0.93 (d, J = 7.6 Hz, 6H), 0.81 (d,
General procedure for the synthesis of the intermediates 2
1
To a solution of UA (10 mmol) in acetone (20 mL) at room
temperature was added Jones reagent drop wise till yellow
1
3
J = 8.6 Hz, 5H). C NMR (DMSO-d , 126 MHz, ppm)
6

Medicinal Chemistry Research
δ 178.75 (C=O), 166.11 (C=NH), 156.51 (C=N), 138.71
(1 mmol), the resulting mixture was stirred with heating at
room temperature for 6 h. The mixture was extracted with
ethyl acetate, then combined the organic phases, dried
(
CH=C), 125.00 (CH=C), 56.49, 55.05, 52.94, 47.36,
4
3
2
6.58, 42.26, 41.61, 38.96, 38.14, 36.78, 36.66, 32.76,
0.67, 28.93, 27.98, 24.29, 23.99, 23.62, 23.43, 21.52,
1.36, 19.56, 19.02, 17.49, 17.35, 15.24. HRMS (ESI) m/z
(MgSO ) and concentrated under pressure to give the crude
4
product. The crude product was purified by the silica gel
column chromatography (petroleum ether /ethyl acetate,
5:1) to give the desired product 4a.
+
+
calcd for C H N O
2
(M+H) 511.40065, found
3
1
51
4
5
11.40033. Purity: 99.37% by HPLC.
General procedure for the synthesis of the intermediates
(
1
1S,2R,4aS,6aS,6bR,10E,12aR)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,
0,11,12,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-
4b–g
heptamethyl-10-thiosemicarbazidopicene-4a-carboxylic
A mixture of compound 2 (1 mmol), catalytic amount of
acid (3b) R = 0.57, methanol/dichloromethane (v/v 1:20);
White powder; Yield: 47%; m.p. 195–197 °C; H NMR
K CO (1 mmol) and the different substituted benzyl
f
2
3
1
chlorides(1 mmol) was stirred in refluxing acetone (10 mL)
at 56 °C for 12 h. Upon completion of the reaction, as
determined by TLC, the solvent was removed under
reduced pressure to give a solid. The solid was dissolved in
(
(
DMSO-d , 300 MHz, ppm) δ 11.94 (s, 1H, COOH), 10.01
s, 1H, C=NH), 8.06 (s, 1H, NH), 7.33 (s, 1H, NH), 5.15 (s,
6
1
H), 3.44 (s, 1H), 2.63 (s, 1H), 2.32 (s, 1H), 2.12 (d, J =
9
.5 Hz, 1H), 1.88 (s, 4H), 1.67 (s, 1H), 1.51 (s, 8H), 1.27 (d,
ethyl ether, dried (MgSO ) and concentrated to give the
4
J = 20.7 Hz, 5H), 1.13 (s, 3H), 1.04 (s, 8H), 0.93 (s, 6H),
crude product. The crude product was purified by the silica
gel column chromatography (petroleum ether /ethyl acetate,
30:1) to give the desired products 4b–g.
1
3
0
.80 (s, 5H). C NMR (DMSO-d , 126 MHz, ppm) δ
6
1
79.20 (C=S), 178.74 (C=O), 163.05 (C=N), 138.69
(
CH=C), 125.02 (CH=C), 56.48, 55.18, 52.93, 47.35,
4
3
2
6.70, 42.24, 41.59, 38.96, 38.47, 36.77, 36.65, 32.80,
0.66, 28.76, 27.98, 24.28, 23.97, 23.62, 23.44, 21.52,
0.58, 19.60, 19.02, 17.49, 17.35, 15.30. HRMS (ESI) m/z
General procedure for the synthesis of the intermediates
6a–p
+
+
calcd for C H N O S (M+H) 528.36182, found
5
To a mixture of compound 2 (1 mmol) and sodium hydro-
xide (1 mmol) in absolute ethanol (10 mL) was added dif-
ferent aldehydes (1 mmol) at room temperature. Upon
completion of the reaction, as determined by TLC, added
water to the mixture, adjusted PH = 7 with 5% aqueous
hydrochloric acid and filtered to give the crude product. The
crude product was purified by the silica gel column chro-
matography (dichloromethane) to give the desired products
6a–p.
3
1
50
3
2
28.36163.
(
8
1S,2R,4aS,6aS,6bR,10E,12aR)-1,2,3,4,4a,5,6,6a,6b,7,8,
a,9,10,11,12,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-
heptamethyl-10-semicarbazidopicene-4a-carboxylic acid
3c) R = 0.41, methanol/dichloromethane (v/v 1:20);
(
f
1
White powder; Yield: 41%; 286–288 °C; H NMR (DMSO-
d6, 300 MHz, ppm) δ 11.96 (s, 1H, COOH), 8.97 (s, 1H,
C=NH), 6.14 (s, 2H, NH ), 5.15 (s, 1H), 3.53–3.40 (m, 1H),
General procedure for the preparation of target
2
2
4
.58 (s, 1H), 2.12 (d, J = 10.6 Hz, 2H), 1.85 (d, J = 26.7 Hz,
H), 1.66 (s, 1H), 1.50 (dd, J = 24.4, 15.9 Hz, 6H), 1.28 (d,
compounds 5a–g and 7a–p
J = 24.4 Hz, 5H), 1.11 (s, 3H), 1.04 (s, 4H), 0.99 (s, 4H),
To a solution of compounds 4a–g or 7a–p (1 mmol) and
aminoguanidine bicarbonate (1 mmol) in absolute ethanol
(10 mL) was added drops of Con.HCl. The reaction mixture
was refluxed for 6 h and further purified by the silica gel
column chromatography (methanol/dichloromethane, 1: 30)
to afford white compounds 5a–g and 7a–p. The yield,
melting point and spectral data of each compound are
given below.
13
0
.93 (d, J = 6.4 Hz, 6H), 0.81 (d, J = 9.3 Hz, 6H). C NMR
(
(
DMSO-d , 126 MHz, ppm) δ 178.77 (COOH), 158.17
C=O), 157.71 (C=N), 138.70 (CH=C), 125.05 (CH=C),
6
5
3
2
6.50, 55.32, 52.93, 47.36, 46.68, 42.24, 41.03, 38.97, 38.29,
6.78, 36.68, 32.84, 30.67, 28.91, 27.98, 24.29, 24.20, 23.62,
3.42, 21.53, 19.84, 19.63, 19.03, 17.51, 17.39, 15.10.
+
(M+H)+
HRMS (ESI) m/z calcd for C H N O
1 50 3 3
3
5
12.38467, found 512.38440.
(
1S,2R,4aS,6aS,6bR,10E,12aR)-methyl 10-guanidinoimino-
General Procedure for the synthesis of the target
compound 4a
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosa-
hydro-1,2,6a,6b,9,9,12a-heptamethylpicene-4a-carboxylate
(
5a) R = 0.40, methanol/dichloromethane (v/v 1:10);
f
1
To a solution of compound 2 (1 mmol) and K CO
White powder; Yield: 57%; 152–154 °C; H NMR (DMSO-
d6, 500 MHz, ppm) δ 10.80 (s, 1H, C=NH), 7.38 (s, 3H,
2
3
(
1 mmol) in DMF (10 mL) was added methyl iodide

Medicinal Chemistry Research
NH , C-NH), 5.18 (s, 1H), 3.52 (s, 3H, O-CH ), 2.59 (dt,
J = 9.5, 5.6 Hz, 1H), 2.41–2.32 (m, 1H), 2.16 (d, J =
1.65 (dd, J = 14.3, 7.2 Hz, 4H), 1.58 (d, J = 6.4 Hz, 2H), 1.45
(d, J = 10.3 Hz, 4H), 1.39–1.33 (m, 3H), 1.23 (s, 2H), 1.16
(s, 3H), 1.08 (s, 1H), 1.04 (d, J = 6.5 Hz, 8H), 0.91 (s, 7H),
0.81 (d, J = 6.0 Hz, 3H), 0.49 (s, 3H). C NMR (DMSO-d6,
75 MHz, ppm) δ 176.43 (C=O), 166.07 (C=NH), 156.50
(C=N), 138.22 (CH=C), 135.70 (Ar-C), 133.10 (Ar-C),
131.96 (Ar-C), 130.52 (Ar-C), 129.11 (Ar-C), 128.80 (Ar-C),
2
3
1
2
1
1
1
6
6
1.3 Hz, 1H), 2.00 (d, J = 4.2 Hz, 1H), 1.88 (dd, J = 10.2,
.9 Hz, 2H), 1.76–1.67 (m, 2H), 1.58 (d, J = 13.0 Hz, 2H),
.54–1.51 (m, 1H), 1.49 (s, 1H), 1.47 (s, 1H), 1.45–1.42 (m,
H), 1.36 (dd, J = 14.2, 9.5 Hz, 2H), 1.32–1.27 (m, 2H),
.24 (s, 1H), 1.17 (s, 4H), 1.09 (d, J = 11.2 Hz, 1H), 1.05 (s,
H), 0.94 (s, 3H), 0.91 (d, J = 5.1 Hz, 3H), 0.82 (d, J =
1
3
125.55 (CH=C), 65.47 (O-CH ), 56.48, 55.00, 53.09, 47.93,
2
.4 Hz, 3H), 0.72 (s, 3H). 13C NMR (DMSO-d , 126 MHz,
46.53, 42.15, 41.59, 38.86, 38.79, 38.14, 36.58, 30.47, 28.87,
27.73, 24.26, 23.94, 23.59, 23.37, 21.39, 19.45, 19.11, 19.01,
17.35, 17.04, 15.18, 14.00. HRMS (ESI) m/z calcd for
6
ppm) δ 177.32 (C=O), 166.13 (C=NH), 156.46 (C=N),
1
5
3
2
1
5
38.45 (CH=C), 125.32 (CH=C), 56.49, 55.04, 53.04,
+
+
1.78 (O-CH ), 47.94, 46.54, 42.15, 41.62, 38.89, 38.76,
C H ClN O (M+H) 635.40863, found 635.40869.
38 56 4 2
3
8.12, 36.64, 32.64, 30.48, 28.88, 27.91, 24.24, 23.97,
3.69, 23.44, 21.42, 21.29, 19.51, 19.03, 17.42, 17.03,
5.20. HRMS (ESI) m/z calcd for C H N O (M+H)+
(1S,2R,4aS,6aS,6bR,10E,12aR)-2,4-dichlorobenzyl
10-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,
11,12,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-
+
32
53
4
2
25.41630, found 525.41638.
heptamethylpicene-4a-carboxylate (5d) R = 0.54, metha-
f
(
1
1
1S,2R,4aS,6aS,6bR,10E,12aR)-3-chlorobenzyl
0-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,
2a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-heptamethyl-
nol/dichloromethane (v/v 1:8); White powder; Yield: 47%;
1
167–169 °C; H NMR (DMSO-d , 300 MHz, ppm) δ 10.90
6
(s, 1H, C=NH), 7.65 (d, J = 2.0 Hz, 1H, Ar-H), 7.57–7.28
picene-4a-carboxylate (5b) R = 0.50, methanol/dichlor-
(m, 5H, NH , C-NH, Ar-H), 5.16 (dd, J = 8.1, 4.4 Hz, 2H,
f
2
omethane (v/v 1:10); White powder; Yield: 45%; 160–162 °
O-CH ), 4.98 (d, J = 12.7 Hz, 1H), 2.60 (dd, J = 10.7,
2
1
C; H NMR (DMSO-d , 300 MHz, ppm) δ 10.74 (s, 1H,
4.1 Hz, 1H), 2.42–2.34 (m, 1H), 2.18 (d, J = 11.2 Hz, 1H),
1.99 (td, J = 13.2, 4.2 Hz, 1H), 1.90–1.82 (m, 1H), 1.71
(dd, J = 11.5, 6.7 Hz, 3H), 1.58 (d, J = 11.7 Hz, 3H),
1.50–1.42 (m, 4H), 1.36–1.27 (m, 3H), 1.22 (d, J =
11.3 Hz, 2H), 1.15 (s, 4H), 1.07 (s, 1H), 1.04 (dd, J = 11.6,
7.0 Hz, 7H), 0.93–0.88 (m, 6H), 0.81 (d, J = 6.4 Hz, 3H),
6
C=NH), 7.41 (s, 2H, Ar-H), 7.39 (s, 3H, C-NH, NH ), 7.33
2
(
d, J = 6.7 Hz, 2H, Ar-H), 5.25–5.05 (m, 2H, O-CH ), 4.94
2
(
d, J = 12.7 Hz, 1H), 2.57 (s, 1H), 2.37 (d, J = 5.8 Hz, 1H),
2
.20 (d, J = 11.1 Hz, 1H), 2.01 (s, 1H), 1.84 (d, J =
1
2.0 Hz, 1H), 1.70 (s, 2H), 1.63 (s, 1H), 1.59 (s, 1H), 1.46
1
3
(
d, J = 7.1 Hz, 3H), 1.39 (s, 1H), 1.34 (s, 1H), 1.24 (s, 5H),
0.44 (s, 3H). C NMR (DMSO-d , 75 MHz, ppm) δ 177.13
6
1
2
1
.16 (s, 4H), 1.04 (s, 7H), 0.91 (d, J = 4.7 Hz, 5H), 0.86 (s,
H), 0.84–0.80 (m, 4H), 0.53 (s, 3H). 1 C NMR (DMSO-d6,
26 MHz, ppm) δ 176.50 (C=O), 166.16 (C=NH), 156.44
(C=O), 166.83 (C=NH), 157.34 (C=N), 138.86 (CH=C),
135.48 (Ar-C), 135.11 (Ar-C), 133.98 (Ar-C), 135.77
(Ar-C), 130.12 (Ar-C), 128.70 (Ar-C), 126.43 (CH=C),
3
(
1
(
4
3
2
C=N), 139.18 (Ar-C), 138.34 (CH=C), 133.47 (Ar-C),
30.75 (Ar-C), 128.32 (Ar-C), 128.30 (Ar-C), 127.15
Ar-C), 125.55 (CH=C), 64.97 (CH ), 56.49, 55.00, 53.03,
63.59 (O-CH ), 57.29, 55.81, 53.87, 48.91, 42.93,
42.39, 39.68, 37.37, 31.28, 29.68, 24.75, 24.39,
2
24.18, 22.19, 20.27, 19.82, 18.16, 15.97. HRMS (ESI) m/z
2
+
(M+H)+ 669.36966, found
8.02, 46.51, 42.19, 41.61, 38.85, 38.82, 38.11, 36.61,
2.71, 30.49, 28.87, 27.74, 24.30, 23.97, 23.60, 23.35,
1.41, 21.27, 19.47, 19.03, 17.37, 17.06, 15.16. HRMS
calcd for C H Cl N O
2
3
8
55
2
4
669.36945.
+
+
(
ESI) m/z calcd for C H ClN O (M+H) 635.40863,
(1S,2R,4aS,6aS,6bR,10E,12aR)-2,6-dichlorobenzyl
38
56
4
2
found 635.40894.
10-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,
12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-heptamethyl-
(
1S,2R,4aS,6aS,6bR,10E,12aR)-3-chlorobenzyl
picene-4a-carboxylate (5e) R = 0.68, methanol/dichlor-
f
1
1
0-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,
2a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-heptamethyl-
omethane (v/v 1:8); White powder; Yield: 46%; 158–160 °
1
C; H NMR (DMSO-d , 300 MHz, ppm) δ 10.89 (s, 1H,
6
picene-4a-carboxylate (5c) R = 0.36, methanol/dichlor-
C=NH), 7.53 (d, J = 7.9 Hz, 3H, NH , C-NH), 7.49–7.35
f
2
omethane (v/v 1:8); White powder; Yield: 40%; 150–152 °C;
(m, 3H, Ar-H), 5.19 (dd, J = 24.3, 12.1 Hz, 3H, O-CH2,
CH=C), 2.60 (d, J = 16.1 Hz, 1H), 2.38 (d, J = 10.0 Hz,
1H), 2.13 (d, J = 11.6 Hz, 1H), 1.96 (d, J = 11.8 Hz, 1H),
1.84 (s, 1H), 1.75 (s, 2H), 1.57 (d, J = 9.5 Hz, 3H), 1.49 (d,
J = 10.7 Hz, 2H), 1.42 (s, 2H), 1.30 (dd, J = 24.9, 13.6 Hz,
5H), 1.16 (s, 3H), 1.08 (s, 1H), 1.04 (d, J = 4.6 Hz, 8H),
1
H NMR (DMSO-d , 300 MHz, ppm) δ 10.93 (s, 1H,
6
C=NH), 7.74–7.64 (m, 2H, Ar-H), 7.43 (d, J = 8.1 Hz, 3H,
C-NH, NH ), 7.35 (d, J = 8.2 Hz, 2H, Ar-H), 5.23–5.03
2
(
m, 2H, O-CH ), 4.93 (d, J = 12.6 Hz, 1H), 2.61 (d, J =
2
1
1
6.0 Hz, 1H), 2.38 (d, J = 10.8 Hz, 1H), 2.18 (d, J = 11.0 Hz,
H), 1.98 (d, J = 11.7 Hz, 1H), 1.82 (d, J = 10.5 Hz, 1H),
13
0.91 (s, 7H), 0.79 (d, J = 6.1 Hz, 3H), 0.67 (s, 3H).
C

Medicinal Chemistry Research
NMR (DMSO-d , 75 MHz, ppm) δ 176.35 (C=O), 166.08
38.79, 38.14, 36.60, 32.72, 30.47, 28.88, 27.76, 24.25,
23.95, 23.62, 23.38, 21.40, 21.22, 19.47, 19.02, 17.37,
6
(
C=NH), 156.49 (C=N), 138.06 (CH=C), 136.48 (Ar-C),
36.48 (Ar-C), 131.95 (Ar-C), 131.63 (Ar-C), 129.19
Ar-C), 129.19 (Ar-C), 125.60 (CH=C), 61.15 (O-CH2),
+
1
(
17.10, 15.17. HRMS (ESI) m/z calcd for C H N O
2
3
9
59
4
+
(M+H) 615.46325, found 615.46301.
5
3
2
1
6.48, 55.03, 53.10, 48.30, 46.50, 42.17, 41.59, 38.89,
8.72, 38.11, 36.59, 32.76, 30.44, 28.89, 27.83, 24.23,
3.98, 23.61, 23.45, 21.35, 21.35, 19.49, 19.02, 17.34,
(1S,2R,4aS,6aS,6bR,10Z,11E,12aR)-11-benzylidene-10-gua-
nidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,
13,14b-icosahydro-1,2,6a,6b,9,9,12a-heptamethylpicene-
+
7.20, 15.19. HRMS (ESI) m/z calcd for C H Cl N O
2
38
55
2
4
+
(
M+H) 669.36966, found 669.36951.
4
a-carboxylic acid (7a) R = 0.34, methanol/dichlor-
f
(
1
1
1S,2R,4aS,6aS,6bR,10E,12aR)-4-fluorobenzyl
0-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,
2,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-hepta-
omethane (v/v 1:8); White powder; Yield: 54%; 143–145 °
1
C; H NMR (DMSO-d , 300 MHz, ppm) δ 11.95 (s, 1H,
6
COOH), 10.19 (s, 1H, C=NH), 7.69 (d, J = 8.6 Hz, 5H, Ar-
methylpicene-4a-carboxylate (5f) R = 0.54, methanol/
H, C-NH), 7.45 (d, J = 7.6 Hz, 3H, NH , Ar-H), 6.52 (s,
f
2
dichloromethane (v/v 1:8); White powder; Yield: 48%;
1H, C=CH), 5.17 (s, 1H), 2.89 (d, J = 15.9 Hz, 1H), 2.10
(t, J = 14.2 Hz, 1H), 1.90 (s, 2H), 1.79 (s, 1H), 1.68–1.61
(m, 5H), 1.55 (s, 2H), 1.37 (dd, J = 14.8, 7.3 Hz, 7H), 1.23
(s, 1H), 1.15 (d, J = 5.0 Hz, 3H), 1.09–1.03 (m, 3H), 1.00
(s, 2H), 0.91 (t, J = 7.3 Hz, 11H), 0.81 (d, J = 5.4 Hz, 2 H),
1
1
38–140 °C; H NMR (DMSO-d , 300 MHz, ppm) δ 10.93
6
(
s, 1H, C=NH), 7.45 (s, 3H, NH , C-NH), 7.41–7.34
2
(
m, 2H, Ar-H), 7.19 (t, J = 8.8 Hz, 2H, Ar-H), 5.23–4.90 (m,
3
6
1
1
6
H, O-CH , CH=C), 2.61 (d, J = 17.4 Hz, 1H), 2.38 (d, J =
2
1
3
.4 Hz, 1H), 2.18 (d, J = 11.2 Hz, 1H), 1.98 (d, J = 12.2 Hz,
H), 1.84 (s, 1H), 1.70 (s, 2H), 1.57 (s, 3H), 1.48 (s, 4H),
.33 (s, 2H), 1.23 (s, 3H), 1.16 (s, 3H), 1.08 (s, 1H), 1.03 (s,
0.75 (s, 1H). C NMR (DMSO-d , 126 MHz, ppm)
6
δ 178.74 (C=O), 167.42 (C=NH), 156.32 (C=N), 138.77
(CH=C), 135.90 (C=C), 133.12 (Ar-C), 132.18 (Ar-C),
131.98 (Ar-C), 130.18 (Ar-C), 129.13 (Ar-C), 128.77
(Ar-C), 128.21 (C=C), 124.66 (CH=C), 65.48, 56.49,
52.94, 47.35, 46.77, 42.93, 42.23, 39.05, 38.95,
36.78, 32.21, 30.67, 30.48, 27.98, 24.26, 23.61,
23.43, 22.18, 21.49, 19.12, 19.03, 17.56, 16.88, 16.37,
1
3
H), 0.90 (s, 7H), 0.81 (d, J = 6.2 Hz, 5H), 0.53 (s, 3H).
C
NMR (DMSO-d , 75 MHz, ppm) δ 176.47 (C=O), 166.06
6
(
1
(
C=NH), 163.92 (Ar-C), 156.51 (C=N), 138.25 (CH=C),
32.98 (Ar-C), 130.91 (Ar-C), 130.80 (Ar-C), 125.50
CH=C), 115.76 (Ar-C), 115.47 (Ar-C), 65.07 (O-CH2),
+
5
3
2
1
6.47, 55.02, 53.08, 47.91, 46.53, 42.15, 41.59, 38.87,
8.79, 38.14, 36.59, 32.70, 30.46, 28.87, 27.74, 24.24,
3.96, 23.59, 23.38, 21.39, 21.32, 19.48, 19.02, 17.36,
14.01. HRMS (ESI) m/z calcd for C H N O
2
(M+H) 599.43195, found 599.43202.
3
8
55
4
+
+
7.06, 15.15. HRMS (ESI) m/z calcd for C H FN O
2
38
56
4
+
(
M+H) 619.43818, found 619.43811.
(1S,2R,4aS,6aS,6bR,10Z,11E,12aR)-11-(4-chlorobenzyli-
dene)-10-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,
11,12,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-
(
1S,2R,4aS,6aS,6bR,10E,12aR)-4-methylbenzyl
1
1
0-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,
2a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-heptamethyl-
heptamethylpicene-4a-carboxylic acid (7b) R = 0.24,
f
methanol/dichloromethane (v/v 1:8); White powder; Yield:
1
picene-4a-carboxylate (5g) R = 0.43, methanol/dichlor-
43%; 190–192 °C; H NMR (DMSO-d , 300 MHz, ppm)
f
6
omethane (v/v 1:8); White powder; Yield: 47%; 153–155 °C;
δ 11.95 (s, 1H, COOH), 10.37 (s, 1H, C=NH), 7.69 (d, J =
1
H NMR (DMSO-d , 300 MHz, ppm) δ 10.92 (s, 1H,
8.6 Hz, 5H, C-NH, Ar-H), 7.48 (s, 2H, NH ), 6.52 (s, 1H,
6
2
C=NH), 7.69 (d, J = 8.5 Hz, 1H, Ar-H), 7.46 (s, 3H, C-NH,
C=CH), 5.16 (s, 1H), 2.83 (d, J = 14.9 Hz, 1H), 2.12 (d,
J = 10.3 Hz, 1H), 2.03 (d, J = 16.1 Hz, 1H), 1.89 (s, 2H),
1.79 (s, 1H), 1.69–1.61 (m, 4H), 1.54 (s, 2H), 1.44–1.35
(m, 5H), 1.23 (s, 1H), 1.14 (d, J = 5.9 Hz, 5H), 1.08 (s, 1H),
1.00 (s, 3H), 0.90 (dd, J = 13.8, 6.8 Hz, 10H), 0.82 (s, 3H),
NH ), 7.19 (q, J = 8.0 Hz, 3H, Ar-H), 5.22–4.84 (m, 3H, O-
2
CH , CH=C), 2.61 (d, J = 15.2 Hz, 1H), 2.37 (s, 1H), 2.29
2
(
s, 3H), 2.18 (d, J = 11.3 Hz, 1H), 1.97 (d, J = 9.5 Hz, 1H),
1
1
3
0
.82 (d, J = 12.4 Hz, 2H), 1.72 (s, 1H), 1.66 (d, J = 7.0 Hz,
H), 1.58 (d, J = 10.1 Hz, 2H), 1.47 (s, 2H), 1.41–1.32 (m,
H), 1.23 (s, 3H), 1.16 (s, 4H), 1.08 (s, 1H), 1.04 (s, 7H),
1
3
0.75 (s, 2H). C NMR (DMSO-d , 126 MHz, ppm) δ
6
178.72 (C=O), 167.42 (C=NH), 165.34 (Ar-C), 156.36
(C=N), 138.74 (CH=C), 134.82 (C=C), 132.65 (Ar-C),
132.18 (Ar-C), 131.98 (Ar-C), 131.59 (Ar-C), 129.13
(Ar-C), 128.71 (C=C), 124.67 (CH=C), 65.48, 56.49,
52.93, 47.34, 46.72, 42.93, 42.23, 39.11, 38.95, 36.78,
32.20, 30.66, 30.48, 27.98, 24.25, 23.61, 23.42, 22.18,
21.50, 19.12, 19.03, 17.58, 16.88, 16.33, 14.01. HRMS
1
3
.91 (s, 7H), 0.81 (d, J = 6.2 Hz, 4H), 0.57 (s, 3H).
C
NMR (DMSO-d , 126 MHz, ppm) δ 176.52 (C=O), 166.08
6
(
1
(
5
C=NH), 156.50 (C=N), 138.27 (CH=C), 137.67 (Ar-C),
33.65 (Ar-C), 131.97 (Ar-C), 129.35 (Ar-C), 129.12
Ar-C), 128.62 (Ar-C), 125.50 (CH=C), 65.73 (O-CH2),
6.48, 55.03, 53.09, 47.90, 46.56, 42.15, 41.60, 38.88,

Medicinal Chemistry Research
ESI) m/z calcd for C H ClN O (M+H)+ 633.39298,
+
56.47, 52.89, 47.32, 46.73, 43.05, 42.21, 39.23, 38.93,
36.75, 32.23, 30.47, 27.97, 24.22, 23.60, 22.15, 21.50,
(
38
54
4
2
found 633.39325.
1
(
9.66, 19.11, 19.02, 17.53, 16.89, 16.33, 14.00. HRMS
ESI) m/z calcd for C H ClN O (M+H)+ 633.39298,
+
38 54 4 2
(
1S,2R,4aS,6aS,6bR,10Z,11E,12aR)-11-(2-chlorobenzyli-
dene)-10-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,
0,11,12,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-hep-
found 633.39331.
1
(1S,2R,4aS,6aS,6bR,10Z,11E,12aR)-11-(4-fluorobenzyli-
dene)-10-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,
11,12,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-hepta-
tamethylpicene-4a-carboxylic acid (7c) R = 0.34, metha-
nol/dichloromethane (v/v 1:8); White powder; Yield: 57%;
2
f
1
07–209 °C; H NMR (DMSO-d , 300 MHz, ppm) δ 11.93
methylpicene-4a-carboxylic acid (7e) R = 0.41, methanol/
6
f
(
s, 1H, COOH), 10.44 (s, 1H, C=NH), 7.75–7.70 (m, 3H,
dichloromethane (v/v 1:8); White powder; Yield: 41%;
1
Ar-H), 7.68 (s, 1H, C-NH), 7.52 (s, 1H, NH), 7.40 (dt, J =
279–281 °C; H NMR (DMSO-d , 300 MHz, ppm) δ 11.95
6
1
1
1
1
7
1
6
1
5.0, 7.3 Hz, 2H, NH, Ar-H), 6.52 (s, 1H, C=CH), 5.10 (s,
H), 2.73 (d, J = 15.4 Hz, 1H), 2.09 (d, J = 11.1 Hz, 1H),
.88 (d, J = 13.8 Hz, 2H), 1.73 (s, 1H), 1.70–1.59 (m, 5H),
.55 (s, 1H), 1.50 (s, 1H), 1.45 (s, 1H), 1.37 (dd, J = 15.1,
.5 Hz, 5H), 1.22 (s, 2H), 1.14 (s, 2H), 1.08 (s, 1H), 1.06 (s,
H), 1.03 (s, 1H), 1.00 (s, 2H), 0.94 (s, 1H), 0.92–0.88 (m,
H), 0.82–0.76 (m, 6H), 0.72 (s, 2H). 1 C NMR (DMSO-d6,
26 MHz, ppm) δ 178.67 (C=O), 167.42 (C=NH), 165.13
(s, 1H, COOH), 10.36 (s, 1H, C = NH), 7.53 (dd, J = 8.3,
5.7 Hz, 5H, C-NH, NH , Ar-H), 7.26 (t, J = 8.8 Hz, 2H,
2
Ar-H), 6.52 (s, 1H, C=CH), 5.16 (s, 1H), 2.83 (d, J =
14.8 Hz, 1H), 2.12 (d, J = 11.1 Hz, 1H), 2.03 (d, J =
16.1 Hz, 1H), 1.89 (s, 3H), 1.79 (s, 1H), 1.52 (d, J = 9.1 Hz,
5H), 1.46 (s, 1H), 1.41 (s, 2H), 1.30 (s, 3H), 1.23 (s, 1H),
1.15 (d, J = 5.6 Hz, 6H), 1.05 (s, 1H), 1.00 (s, 3H), 0.94 (s,
1H), 0.89 (d, J = 11.7 Hz, 6H), 0.81 (d, J = 6.3 Hz, 3H),
3
1
3
(
Ar-C), 156.52 (C=N), 138.76 (CH=C), 133.75 (C=C),
33.20 (Ar-C), 132.99 (Ar-C), 132.18 (Ar-C), 131.98 (Ar-
C), 129.13 (Ar-C), 127.24 (C=C), 124.51 (CH=C), 65.48,
0.75 (s, 3H). C NMR (DMSO-d , 126 MHz, ppm) δ
6
1
178.71 (C=O), 165.51 (C=NH), 160.87 (Ar-C), 156.45
(C=N), 138.74 (CH=C), 132.47 (Ar-C), 132.35 (C=C),
132.29 (Ar-C), 132.04 (Ar-C), 130.68 (C=C), 124.68
(CH=C), 115.66 (Ar-C), 115.49 (Ar-C), 56.48, 55.39,
52.93, 47.34, 46.75, 45.32, 42.89, 42.22, 39.05, 38.95,
36.77, 32.21, 30.66, 29.09, 27.98, 24.26, 23.62, 23.42,
5
3
2
6.49, 52.89, 47.32, 46.72, 43.37, 42.23, 38.93, 38.90,
6.75, 32.39, 30.65, 30.48, 27.96, 24.23, 23.60, 23.37,
2.15, 21.49, 19.12, 19.03, 17.50, 16.97, 15.88, 14.01.
+
(M+H)+
HRMS (ESI) m/z calcd for C H ClN O
2
38
54
4
6
33.39298, found 633.39355.
22.18, 21.51, 19.77, 19.03, 17.58, 16.88, 16.34. HRMS
ESI) m/z calcd for C H FN O (M+H)+ 617.42253,
+
(
3
8
54
4
2
found 617.42285.
(
1S,2R,4aS,6aS,6bR,10Z,11E,12aR)-11-(3-chlorobenzyli-
dene)-10-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,
0,11,12,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-hep-
1
(1S,2R,4aS,6aS,6bR,10Z,11E,12aR)-11-(2-fluorobenzyli-
dene)-10-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,
11,12,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-hepta-
tamethylpicene-4a-carboxylic acid (7d) R = 0.24, metha-
f
nol/dichloromethane (v/v 1:8); White powder; Yield: 46%;
1
2
21–223 °C; H NMR (DMSO-d , 300 MHz, ppm) δ 11.95
methylpicene-4a-carboxylic acid (7f) R = 0.41, methanol/
6
f
(
s, 1H, COOH), 10.42 (s, 1H, C=NH), 7.78–7.61 (m, 3H,
dichloromethane (v/v 1:8); White powder; Yield: 51%;
1
Ar-H, C-NH), 7.52 (s, 1H, NH), 7.46 (d, J = 4.8 Hz, 2H,
NH, Ar-H), 7.39 (d, J = 4.8 Hz, 1H, Ar-H), 6.54 (s, 1H,
C=CH), 5.16 (s, 1H), 2.82 (d, J = 14.5 Hz, 1H), 2.12
274–276 °C; H NMR (DMSO-d , 300 MHz, ppm) δ 11.94
6
(s, 1H, COOH), 10.46 (s, 1H, C=NH), 7.91–7.58 (m, 4H,
C-NH, NH , Ar-H), 7.47–7.36 (m, 1H, Ar-H), 7.26
2
(
d, J = 11.4 Hz, 1H), 2.02 (d, J = 15.1 Hz, 1H), 1.93 (s,
(dd, J = 16.6, 8.5 Hz, 2H, Ar-H), 6.49 (s, 1H, C=CH), 5.13
(s, 1H), 2.76 (d, J = 15.0 Hz, 1H), 2.10 (d, J = 11.2 Hz,
1H), 1.96 (d, J = 14.7 Hz, 2H), 1.87 (s, 1H), 1.80 (d, J =
8.4 Hz, 2H), 1.52 (d, J = 10.9 Hz, 5H), 1.44 (d, J =
12.7 Hz, 3H), 1.33 (s, 1H), 1.28 (d, J = 9.7 Hz, 2H), 1.23
(s, 1H), 1.18 (s, 3H), 1.13 (s, 3H), 1.07 (d, J = 7.1 Hz, 1H),
1.00 (s, 3H), 0.90 (s, 4H), 0.84 (s, 3H), 0.79 (d, J = 6.2 Hz,
1
1
1
H), 1.86 (d, J = 9.3 Hz, 2H), 1.78 (s, 1H), 1.65 (dd, J =
4.0, 7.1 Hz, 2H), 1.53 (d, J = 12.4 Hz, 4H), 1.46 (s, 1H),
.40 (d, J = 7.7 Hz, 2H), 1.35 (d, J = 7.2 Hz, 1H), 1.29 (d,
J = 8.0 Hz, 2H), 1.23 (s, 1H), 1.17 (s, 3H), 1.13 (s, 2H),
1
6
.01 (s, 4H), 0.94 (s, 1H), 0.91–0.86 (m, 6H), 0.80 (d, J =
.1 Hz, 3H), 0.75 (s, 3H). 13C NMR (DMSO-d , 75 MHz,
6
1
3
ppm) δ 178.68 (C=O), 165.14 (C=NH), 156.46 (C=N),
38.75 (CH=C), 133.29 (C=C), 132.45 (Ar-C), 131.96
Ar-C), 130.49 (Ar-C), 129.89 (Ar-C), 129.11 (Ar-C),
28.56 (Ar-C), 127.84 (C=C), 124.61 (CH=C), 65.46,
3H), 0.73 (s, 3H). C NMR (DMSO-d , 75 MHz, ppm) δ
6
1
(
1
178.67 (C=O), 165.07 (C=NH), 156.55 (C=N), 138.74
(CH=C), 133.32 (C=C), 132.22 (Ar-C), 130.33 (C=C),
125.46 (Ar-C), 124.56 (CH=C), 123.51 (Ar-C), 123.33

Medicinal Chemistry Research
(
4
3
2
Ar-C), 115.78 (Ar-C), 115.50 (Ar-C), 56.47, 54.47, 52.89,
(CH=C), 123.95 (Ar-C), 56.49, 55.38, 52.89, 47.33, 46.72,
44.57, 43.48, 42.23, 38.93, 38.90, 36.75, 32.41, 30.65,
28.13, 27.96, 24.23, 23.61, 23.36, 22.22, 21.50, 19.50,
19.03, 17.50, 16.99, 15.87. HRMS (ESI) m/z calcd for
7.32, 46.72, 45.23, 43.10, 42.22, 38.92, 38.92, 36.75,
2.29, 30.64, 28.58, 27.97, 24.25, 23.60, 23.39, 22.12,
1.49, 19.66, 19.02, 17.51, 16.91, 16.10. HRMS (ESI) m/z
+
+
+
+
calcd for C H FN O
(M+H) 617.42253, found
C H BrN O (M+H) 677.34247, found 677.34249.
38 54 4 2
3
8
54
4
2
6
17.42212.
(
1S,2R,4aS,6aS,6bR,10Z,11E,12aR)-11-(3-bromobenzyli-
(
1S,2R,4aS,6aS,6bR,10Z,11E,12aR)-11-(3-fluorobenzyli-
dene)-10-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,
0,11,12,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-hep-
dene)-10-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,
10,11,12,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-hep-
1
tamethylpicene-4a-carboxylic acid (7i) R = 0.30, metha-
f
tamethylpicene-4a-carboxylic acid (7g) R = 0.48, metha-
nol/dichloromethane (v/v 1:8); White powder; Yield: 43%;
f
1
nol/dichloromethane (v/v 1:8); White powder; Yield: 52%;
279–281 °C; H NMR (DMSO-d , 300 MHz, ppm) δ 11.96
6
1
2
79–281 °C; H NMR (DMSO-d , 300 MHz, ppm) δ 11.95
(s, 1H, COOH), 10.42 (s, 1H, C=NH), 7.65 (s, 3H, C-NH,
6
(
s, 1H, COOH), 10.44 (s, 1H, C=NH), 7.60 (s, 3H, C-NH,
NH ), 7.49 (s, 3H, Ar-H), 7.39 (s, 1H, Ar-H), 6.53 (s, 1H,
2
NH ), 7.46 (dd, J = 14.4, 7.4 Hz, 1H, Ar-H), 7.31 (d, J =
C=CH), 5.15 (s, 1H), 2.81 (d, J = 14.4 Hz, 1H), 2.12
(d, J = 11.2 Hz, 1H), 2.02 (d, J = 16.1 Hz, 1H), 1.85
(dd, J = 23.4, 15.8 Hz, 3H), 1.52 (d, J = 12.3 Hz, 5H), 1.43
(d, J = 13.3 Hz, 2H), 1.36–1.27 (m, 3H), 1.23 (s, 2H), 1.15
(d, J = 11.7 Hz, 6H), 1.09–1.03 (m, 2H), 1.00 (s, 3H), 0.88
(d, J = 10.4 Hz, 7H), 0.80 (d, J = 6.2 Hz, 3H), 0.74 (s, 3H).
2
8
1
.0 Hz, 2H, Ar-H), 7.16 (t, J = 8.1 Hz, 1H, Ar-H), 6.54 (s,
H, C=CH), 5.16 (s, 1H), 2.84 (d, J = 15.6 Hz, 1H), 2.12
(
d, J = 11.5 Hz, 1H), 2.03 (d, J = 15.6 Hz, 1H), 1.88 (s,
3
1
H), 1.79 (s, 1H), 1.52 (d, J = 11.6 Hz, 5H), 1.43 (d, J =
2.7 Hz, 3H), 1.29 (d, J = 8.6 Hz, 2H), 1.23 (s, 1H), 1.15
1
3
(
d, J = 9.5 Hz, 7H), 1.07 (d, J = 7.1 Hz, 1H), 1.00 (s, 3H),
C NMR (DMSO-d , 126 MHz, ppm) δ 178.70 (C=O),
6
0
3
1
.88 (d, J = 11.5 Hz, 7H), 0.80 (d, J = 6.0 Hz, 3H), 0.75 (s,
165.16 (C=NH), 156.37 (C=N), 138.78 (CH=C), 138.38
(C=C), 132.77 (Ar-C), 132.40 (Ar-C), 131.65 (Ar-C),
130.82 (Ar-C), 130.76 (Ar-C), 128.88 (C=C), 124.61
(CH=C), 121.93 (Ar-C), 56.49, 55.39, 52.89, 47.34, 46.72,
45.12, 43.07, 42.23, 39.25, 38.94, 36.76, 32.24, 30.65,
28.88, 27.98, 24.25, 23.62, 23.39, 22.17, 21.51, 19.69,
19.03, 17.55, 16.90, 16.35. HRMS (ESI) m/z calcd for
1
3
H). C NMR (DMSO-d , 75 MHz, ppm) δ 178.68 (C=O),
6
65.24 (C=NH), 160.71 (Ar-C), 156.52 (C=N), 138.70
(
1
(
CH=C), 138.43 (C=C), 132.30 (Ar-C), 131.90 (Ar-C),
30.59 (Ar-C), 126.24 (C=C), 124.66 (CH=C), 116.91
Ar-C), 116.63 (Ar-C), 56.46, 54.31, 52.90, 47.32, 46.73,
4
2
1
5.26, 43.00, 42.21, 38.93, 38.93,36.75, 32.23, 30.65,
8.91, 27.97, 24.25, 23.60, 23.41, 22.15, 21.49, 19.71,
9.01, 17.54, 16.88, 16.31. HRMS (ESI) m/z calcd for
+
+
C H BrN O (M+H) 677.34247, found 677.34271.
3
8
54
4
2
+
+
C H FN O (M+H) 617.42253, found 617.42224.
3
8
54
4
2
(
1S,2R,4aS,6aS,6bR,10Z,11E,12aR)-11-(2,4-dichlorobenzyli-
dene)-10-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,
11,12,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-hepta-
(
1S,2R,4aS,6aS,6bR,10Z,11E,12aR)-11-(2-bromobenzyli-
dene)-10-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,
0,11,12,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-hep-
methylpicene-4a-carboxylic acid (7j) R = 0.33, methanol/
f
1
dichloromethane (v/v 1:8); White powder; Yield: 41%;
1
tamethylpicene-4a-carboxylic acid (7h) R = 0.40, metha-
285–287 °C; H NMR (DMSO-d , 300 MHz, ppm) δ 11.95
f
6
nol/dichloromethane (v/v 1:8); White powder; Yield: 44%;
(s, 1H, COOH), 10.62 (s, 1H, C=NH), 7.79 (d, J = 8.4 Hz,
1H, Ar-H), 7.69 (d, J = 2.0 Hz, 4H, Ar-H, C-NH, NH2),
7.51 (d, J = 8.3 Hz, 1H, Ar-H), 6.47 (s, 1H, C=CH), 5.11
(s, 1H), 2.67 (d, J = 14.7 Hz, 1H), 2.09 (d, J = 11.1 Hz,
1H), 1.95–1.81 (m, 2H), 1.76 (s, 2H), 1.52 (d, J = 10.1 Hz,
5H), 1.43 (d, J = 11.9 Hz, 2H), 1.22 (d, J = 7.3 Hz, 9H),
1.13 (s, 3H), 1.00 (s, 4H), 0.90 (s, 4H), 0.85 (s, 1H), 0.79 (d,
J = 9.7 Hz, 5H), 0.72 (s, 3H). C NMR (DMSO-d6,
126 MHz, ppm) δ 178.68 (C=O), 164.86 (C=NH), 156.48
(C=N), 138.75 (CH=C), 134.16 (C=C), 133.91 (Ar-C),
133.85 (Ar-C), 133.52 (Ar-C), 132.81 (Ar-C), 129.04 (Ar-
C), 128.99 (Ar-C), 127.51 (C=C), 124.52 (CH=C), 56.49,
52.89, 47.32, 46.68, 44.71, 43.37, 42.24, 38.93, 38.91,
1
2
78–279 °C; H NMR (DMSO-d , 300 MHz, ppm) δ 11.93
6
(
s, 1H, COOH), 10.51 (s, 1H, C=NH), 7.88–7.60 (m, 5H,
C-NH, NH , Ar-H), 7.45 (s, 1H, Ar-H), 7.29 (s, 1H, Ar-H),
2
6
1
1
3
1
.46 (s, 1H, C=CH), 5.10 (s, 1H), 2.71 (d, J = 13.8 Hz,
H), 2.09 (d, J = 11.1 Hz, 1H), 1.85 (d, J = 14.9 Hz, 2H),
.72 (s, 3H), 1.53 (d, J = 8.1 Hz, 5H), 1.44 (d, J = 8.3 Hz,
H), 1.23 (s, 7H), 1.15 (s, 3H), 1.07 (d, J = 7.1 Hz, 1H),
.00 (s, 3H), 0.90 (s, 4H), 0.83–0.75 (m, 6H), 0.72 (s, 3H).
1
3
1
3
C NMR (DMSO-d , 126 MHz, ppm) δ 178.67 (C=O),
6
1
65.06 (C=NH), 156.47 (C=N), 138.77 (CH=C), 135.45
(
1
C=C), 132.82 (Ar-C), 132.67 (Ar-C), 132.61 (Ar-C),
32.36 (Ar-C), 130.21 (Ar-C), 127.79 (C=C), 124.50

Medicinal Chemistry Research
3
2
6.75, 32.36, 30.65, 29.50, 27.97, 24.23, 23.61, 23.37,
39.32, 38.95, 36.77, 32.20, 30.67, 28.93, 27.98, 24.26,
23.62,23.42, 22.18, 21.51, 21.35, 19.56, 19.03, 17.59,
2.14, 21.50, 19.54, 19.03, 17.53, 16.97, 15.88, 14.42.
+
(M+H)+
+
HRMS (ESI) m/z calcd for C H Cl N O
6
16.87, 16.39, 14.42. HRMS (ESI) m/z calcd for C H N O
39 57 4 2
38
53
2
4
2
+
67.35401, found 667.35425.
(M+H) 613.44760, found 613.44745.
(
1S,2R,4aS,6aS,6bR,10Z,11E,12aR)-11-(2,6-dichlorobenzyli-
dene)-10-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,
0,11,12,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-hep-
(1S,2R,4aS,6aS,6bR,10Z,11E,12aR)-11-(4-nitrobenzylidene)-
10-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,
11,12,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-hepta-
1
tamethylpicene-4a-carboxylic acid (7k) R = 0.53, metha-
methylpicene-4a-carboxylic acid (7m) R = 0.30, metha-
f
f
nol/dichloromethane (v/v 1:8); White powder; Yield: 57%;
nol/dichloromethane (v/v 1:8); Yellow powder; Yield: 55%;
1
1
2
97–299 °C; H NMR (DMSO-d , 300 MHz, ppm) δ 11.93
257–259 °C; H NMR (DMSO-d , 300 MHz, ppm) δ 11.95
6
6
(
s, 1H, COOH), 9.53 (s, 1H, C=NH), 7.73 (s, 3H, C-NH,
(s, 1H, COOH), 10.54 (s, 1H, C=NH), 8.27 (d, J = 8.7 Hz,
2H, Ar-H), 7.75 (d, J = 8.7 Hz, 2H, Ar-H), 7.60 (s, 3H, C-
NH ), 7.59 (d, J = 8.1 Hz, 2H, Ar-H), 7.48–7.41 (m, 1H,
2
Ar-H), 6.46 (s, 1H, C=CH), 5.06 (s, 1H), 2.24–2.15 (m,
NH, NH ), 6.69 (s, 1H, C=CH), 5.15 (s, 1H), 2.87 (d, J =
2
2
1
2
H), 2.08 (d, J = 11.1 Hz, 1H), 1.90 (d, J = 12.8 Hz, 1H),
.78 (d, J = 13.1 Hz, 3H), 1.60 (s, 4H), 1.55 (s, 3H), 1.49 (s,
H), 1.45 (s, 1H), 1.39 (s, 1H), 1.33 (s, 2H), 1.30
15.0 Hz, 1H), 2.11 (d, J = 10.9 Hz, 1H), 2.05 (s, 1H), 1.90
(d, J = 7.7 Hz, 2H), 1.84 (s, 1H), 1.79 (s, 1H), 1.52 (d, J =
13.2 Hz, 5H), 1.45 (s, 1H), 1.41 (s, 1H), 1.35–1.27 (m, 3H),
1.23 (s, 2H), 1.16 (d, J = 9.9 Hz, 6H), 1.09–1.03 (m, 2H),
(
s, 2H), 1.12 (s, 3H), 1.02 (s, 3H), 0.90 (s, 4H), 0.85 (s, 1H),
0
.81 (s, 3H), 0.77 (d, J = 6.1 Hz, 3H), 0.71 (s, 3H).
1.00 (s, 2H), 0.88 (d, J = 11.3 Hz, 6H), 0.80 (d, J = 6.2 Hz,
1
3
13
C NMR (DMSO-d , 126 MHz, ppm) δ 178.64 (C=O),
3H), 0.74 (s, 3H). C NMR (DMSO-d , 126 MHz, ppm) δ
6
6
1
1
63.76 (C=NH), 156.44 (C=N), 138.82 (CH=C),
34.57 (C=C), 134.52 (Ar-C), 132.96 (Ar-C), 132.96
178.71 (C=O), 164.87 (C=NH), 156.39 (C=N), 146.70
(Ar-C), 142.78 (Ar-C), 138.74 (CH=C), 134.76 (C=C),
131.46 (Ar-C), 131.36 (Ar-C), 131.36 (C=C), 124.66
(CH=C), 123.83 (Ar-C), 123.83 (Ar-C), 56.49, 52.91,
47.34, 46.69, 45.39, 43.10, 42.24, 39.34, 38.94, 36.77,
32.19, 30.64, 28.93, 27.99, 24.24, 23.62, 23.38, 22.20,
(
Ar-C), 130.95 (Ar-C), 128.86 (Ar-C), 128.86 (Ar-C),
1
4
3
1
27.51 (C=C), 124.35 (CH=C), 56.48, 55.48, 52.84,
7.29, 46.62, 45.84, 44.06, 42.22, 39.34, 38.91, 36.74,
2.57, 30.64, 29.51, 27.97, 24.21, 23.58, 22.57, 21.50,
9.48, 19.03, 17.51, 17.07, 15.61, 14.42. HRMS (ESI) m/z
21.50, 19.74, 19.03, 17.60, 16.89, 16.32. HRMS (ESI) m/z
+
+
+
(M+H)+ 644.41703, found
calcd for C H Cl N O
(M+H) 667.35401, found
calcd for C H N O
4
3
8
53
2
4
2
38 54
5
6
67.35413.
644.41724.
(
1S,2R,4aS,6aS,6bR,10Z,11E,12aR)-10-guanidinoimino-
(
1S,2R,4aS,6aS,6bR,10Z,11E,12aR)-11-(4-methylbenzyli-
dene)-10-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,
0,11,12,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-hep-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosa-
hydro-1,2,6a,6b,9,9,12a-heptamethyl-11-((naphthalen-1-yl)
1
methylene)picene-4a-carboxylic
acid
(7n) R = 0.40,
f
tamethylpicene-4a-carboxylic acid (7l) R = 0.33, metha-
methanol/dichloromethane (v/v 1:8); White powder; Yield:
f
1
nol/dichloromethane (v/v 1:8); White powder; Yield: 45%;
40%; 293–295 °C; H NMR (DMSO-d , 300 MHz, ppm) δ
6
1
3
02–304 °C; H NMR (DMSO-d , 300 MHz, ppm) δ 11.96
11.95 (s, 1H, COOH), 10.35 (s, 1H, C=NH), 7.94 (s, 4H,
6
(
s, 1H, COOH), 10.24 (s, 1H, C=NH), 7.52 (s, 3H, C-NH,
Ar-H), 7.55 (s, 6H, C-NH, NH , Ar-H), 6.70 (s, 1H,
2
NH ), 7.36 (d, J = 8.0 Hz, 2H, Ar-H), 7.26–7.17 (m, 2H,
Ar-H), 6.47 (s, 1H, C=CH), 5.17 (s, 1H), 2.87 (d, J =
C=CH), 5.15 (s, 1H), 3.01 (d, J = 14.6 Hz, 1H), 2.09 (s,
2H), 1.90 (s, 5H), 1.55 (s, 3H), 1.43 (d, J = 15.5 Hz, 3H),
1.22 (d, J = 6.8 Hz, 7H), 1.16 (s, 3H), 1.05 (dd, J = 7.9,
6.0 Hz, 4H), 0.99 (s, 3H), 0.94 (s, 1H), 0.89 (s, 4H), 0.83 (s,
2
1
5.6 Hz, 1H), 2.32 (s, 3H), 2.28 (s, 1H), 2.14 (s, 1H), 2.10
(
s, 1H), 2.03 (s, 1H), 1.90 (s, 3H), 1.52 (s, 3H), 1.42 (s, 2H),
1
3
1
3
7
.33 (s, 2H), 1.23 (s, 3H), 1.14 (d, J = 3.0 Hz, 3H), 1.05 (s,
1H), 0.78 (d, J = 6.7 Hz, 3H), 0.74 (s, 2H). C NMR
H), 1.00 (s, 2H), 0.89 (d, J = 10.8 Hz, 7H), 0.81 (d, J =
(DMSO-d , 126 MHz, ppm) δ 178.70 (C=O), 166.10 (Ar-
6
13
.7 Hz, 5H), 0.75 (s, 3H), 0.65 (s, 1H). C NMR (DMSO-
C), 165.57 (C=NH), 156.53 (Ar-C), 156.33 (C=N), 138.73
(CH=C), 133.55 (C=C), 133.20 (Ar-C), 132.62 (Ar-C),
131.17 (Ar-C), 129.27 (Ar-C), 128.57 (Ar-C), 128.08 (Ar-
C), 128.02 (Ar-C), 126.93 (C=C), 124.68 (CH=C), 56.49,
55.38, 52.89, 47.33, 46.76, 43.12, 42.21, 41.61, 39.27,
38.93, 36.77, 32.26, 30.63, 28.94, 27.98, 24.24, 23.61,
d6, 126 MHz, ppm) δ 178.71 (C=O), 165.99 (C=NH),
65.74 (Ar-C), 156.64 (Ar-C), 156.37 (C=N), 138.74
CH=C), 137.60 (C=C), 133.11 (Ar-C), 130.16 (Ar-C),
29.81 (Ar-C), 129.33 (Ar-C), 125.00 (C=C), 124.69
CH=C), 56.48, 55.06, 52.93, 47.35, 46.78, 42.85, 42.24,
1
(
1
(

Medicinal Chemistry Research
2
3.41, 22.26, 21.49, 19.74, 19.03, 17.56, 16.91, 16.35.
Evaluation of anti-bacterial and anti-fungal
activities in vitro
+
(M+H)+
HRMS (ESI) m/z calcd for C H N O
2
42
57
4
6
49.44760, found 649.44733.
The microbes used in the present study were S. aureus RN
4220, S.mutans KTCT 3289, Escherichia coli KTCT 1924,
(
1S,2R,4aS,6aS,6bR,10Z,11E,12aR)-11-((furan-2-yl)methy-
lene)-10-guanidinoimino-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,
1,12,12a,12b,13,14b-icosahydro-1,2,6a,6b,9,9,12a-hepta-
C. albicans 7535. The strains of multidrug-resistant clinical
isolates were methicillin-resistant Staphylococcus aureus
(MRSA CCARM 3167 and MRSA CCARM 3506) and
quinolone-resistant Staphylococcus aureus (QRSA CCARM
3505 and QRSA CCARM 3519). Clinical isolates were
collected from various patients hospitalized in several clin-
ics. In order to obtain final concentrations of 64-1 µg/mL, a
twofold serial dilution technique was used. Test bacteria
were diluted 1000-fold in Mueller-Hinton broth (MHB)
when they grew to mid-log phase in the same medium. The
1
methylpicene-4a-carboxylic acid (7o) R = 0.45, methanol/
dichloromethane (v/v 1:8); Yellow powder; Yield: 34%;
2
(
7
6
1
9
1
1
3
1
(
1
(
4
2
1
f
1
90–292 °C; H NMR (DMSO-d , 300 MHz, ppm) δ 11.93
6
s, 1H, COOH), 10.10 (s, 1H, C=NH), 7.82 (s, 1H, Ar-H),
.39 (s, 3H, C-NH, NH ), 6.66 (d, J = 18.5 Hz, 2H, Ar-H),
2
.39 (s, 1H, C=CH), 5.22 (s, 1H), 2.98 (d, J = 17.1 Hz,
H), 2.17 (d, J = 7.2 Hz, 1H), 2.09 (s, 1H), 1.98 (d, J =
.4 Hz, 2H), 1.91 (s, 1H), 1.82 (s, 1H), 1.53 (s, 1H), 1.48 (s,
H), 1.44 (s, 1H), 1.39 (s, 1H), 1.24 (s, 7H), 1.14 (s, 2H),
.12 (s, 2H), 1.08 (s, 1H), 1.06 (s, 3H), 1.03 (s, 1H), 0.91 (s,
H), 0.86 (s, 3H), 0.79 (s, 3H). 1 C NMR (DMSO-d6,
26 MHz, ppm) δ 178.74 (C=O), 164.69 (C=NH), 156.18
5
bacteria of 10 CFU/mL were vaccinated into MHB and
distributed at 0.2 mL/well in a 96-well microtiter plate.
Oxacillin, norfloxacin, gatifloxacin, moxifloxacin, flucona-
zole and itraconazole were used as positive controls. The
compounds which were tested were dissolved in DMSO, the
final concentration did not surpass 0.05%. The MIC value
was defined as the concentration of a compound used in test
that entirely inhibited bacteria growth during 24 h cultivation
at 37 °C. The absorption at 650 nm by which the Bacteria
growth was determined was measured by using a microtiter
enzyme-linked immunosorbent assay (ELISA) reader. All
experiments were tested three times.
3
C=N), 151.39 (C-O), 144.14 (C-O), 138.78 (CH=C),
27.59 (C=C), 124.78 (CH=C), 120.98 (C=C), 113.72
C=C), 112.68 (C=C), 56.49, 52.95, 47.37, 46.57, 45.89,
3.08, 42.26, 38.98, 38.81, 36.79, 34.14, 32.28, 30.67,
9.48, 24.28, 23.62, 22.56, 22.39, 21.52, 19.89, 19.03,
7.61, 16.93, 15.97, 14.42. HRMS (ESI) m/z calcd for
+
+
C H N O (M+H) 589.41122, found 589.41144.
3
6
53
4
3
(
1
1S,2R,4aS,6aS,6bR,10Z,11E,12aR)-10-guanidinoimino-
,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosa-
hydro-1,2,6a,6b,9,9,12a-heptamethyl-11-((thiophen-2-yl)
methylene)picene-4a-carboxylic acid (7p) R = 0.45,
methanol/dichloromethane (v/v 1:8); White powder; Yield:
Evaluation of anti-inflammatory activity in vivo
Anti-inflammatory assay: Assay in the xylene-induced ear
edema method via the intraperitoneal route: The anti-
inflammatory activity was evaluated by an in vivo inhibition
assay by monitoring xylene-induced ear edema in mice. In
the primary screening, all tested compounds, ibuprofen and
indomethacin were freshly prepared (dissolved with
DMSO) prior to administered i.p. at a dose of 100 mg/kg to
mice and at a concentration of 0.2 mL/20 g body weight.
Control mice received the vehicle only (DMSO, 0.2 mL/
20 g of body weight). Thirty minutes after administration
i.p., animals were used in the xylene-induced ear edema
test, 20 μL xylene was applied to the surface of the right ear
of each mouse by a micropipette. After keeping them from
struggling for 30 min, a cylindrical plug (diameter, 7 mm)
was excised from each of the treated and untreated ears.
Edema was quantified by the difference in weight between
the two plugs. Anti-inflammatory activity was expressed as
percent reduction in edema compared with the DMSO-
administered control group. The NSAID ibuprofen and
indomethacin were tested in parallel as reference. Assay in
the xylene-induced ear edema method via the oral route:
In thelatter evaluation, tested compounds and indometacin
f
1
3
1
6%; 287–289 °C; H NMR (DMSO-d , 300 MHz, ppm) δ
6
1.90 (s, 1H, COOH), 7.56 (d, J = 4.7 Hz, 4H, C-NH, NH2,
Ar-H), 7.14–7.05 (m, 1H, Ar-H), 6.87 (d, J = 3.0 Hz, 1H,
Ar-H), 5.97 (d, J = 11.1 Hz, 1H), 5.08 (s, 1H, CH=C), 2.73
(
1
(
5
s, 1H), 2.18 (t, J = 7.1 Hz, 1H), 2.09 (d, J = 11.2 Hz, 1H),
.90 (d, J = 13.9 Hz, 1H), 1.78 (d, J = 12.2 Hz, 1H), 1.52
s, 3H), 1.45 (s, 1H), 1.40 (d, J = 6.0 Hz, 2H), 1.29 (d, J =
.1 Hz, 3H), 1.24 (s, 7H), 1.20 (s, 2H), 1.08 (s, 2H), 1.05 (d,
J = 6.7 Hz, 5H), 0.91 (s, 3H), 0.84 (d, J = 6.8 Hz, 2H), 0.78
13
(
(
1
1
d, J = 6.1 Hz, 2H), 0.68 (s, 3H), 0.41 (s, 2H). C NMR
DMSO-d , 126 MHz, ppm) δ 178.68 (C=O), 174.95 (C-S),
6
72.82 (C=NH), 153.24 (C=N), 138.64 (CH=C),
38.57 (C-S), 127.79 (C=C), 127.56 (C=C), 126.69
C=C), 126.69 (C=C), 124.68 (CH=C), 56.48, 53.01,
(
5
3
2
1.08, 47.42, 46.37, 45.42, 42.39, 39.37, 38.95, 36.73,
4.16, 31.76, 31.34, 29.49, 24.26, 23.44, 22.74, 22.56,
1.49, 19.25, 19.03, 17.36, 16.83, 16.63, 14.42. HRMS
+
+
(
ESI) m/z calcd for C H N O S (M+H) 605.38837,
36 53 4 2
found 605.38812.

Medicinal Chemistry Research
Scheme 1 Synthetic scheme for the synthesis of compounds 3a–c, 5a–g,
a–p. Reagents and conditions: a Jones reagent, acetone, 0 °C, 5 h;
acetone, reflux, 8 h; d R-CHO, 5 % NaOH, absolute ethanol, r.t 2 h;
e Concentrated HCl, absolute ethanol, reflux, 8 h
7
b Methyl iodide, K2CO3, DMF, r.t., 6 h; c Benzyl chlorides, K2CO3,
were homogenized with 0.5% sodium carbox-
ymethylcellulose (CMC-Na) and administered via the oral
route to mice at a concentration of 0.2 mL/20 g mice weight.
Control mice received the vehicle only (0.5% CMC-Na,
104 cells/well. A variety of concentrations of the test
compounds (200, 100, 50, 25, 12.5, 6.25, 3.125,
1.625 μmol/L) dissolved by distilled 10% DMSO was
added to each well. Incubation for 24 h at 37 °C under 5%
0
.2 mL/20 g). To explore the peak activity of the compound,
CO , 2.5 mg/mL of MTT solution was added to each well.
2
edema was quantified at different intervals (1, 2, 3, 4, 5, and
Further the plate was incubated for 4 h. Then, the incubation
medium was aspirated, added DMSO (100 μL) to solubilize
the MTT formazan product. The absorbance at 570 and
630 nm were measured after mixing. The difference
between absorbance of 570 and 630 nm was used as an
index of the cell viability. The morphology of the cells was
observed by Giemsa stain under Phase contrast microscope.
The selected compounds were used as positive control,
whereas untreated cells were used as negative controls. The
IC50 values were defined as the concentrations inhibiting
50% of cell growth. All experiments were performed in
triplicate.
2
4 h). Compounds 3a and ibuprofen homogenized with
0
.5% CMC-Na were administered orally to mice (lower
doses of 50 and 25 mg/kg and 0.2 mL/20 g mice body
weight). Control mice received 0.5% CMC-Na (0.2 mL/20 g
body weight) and edema quantified at the peak interval of
3
h. Edema was quantified by the difference in weight
between the two plugs. Edema values, expressed as mean
standard deviation, were compared statistically using one-
way-ANOVA followed by Dunnet’s test. A level of
p < 0.05 was adopted as the test of significance.
Evaluation of cytotoxicity in vitro
Cytotoxicity on human cancer cells: The cytotoxicity test of
selected compounds was measured through the colorimetric
MTT assay. Human cervical cancer cells (HeLa), liver
cancer cells (Hep3B) and lung cancer cells (A549) sus-
pension in DMEM medium supplemented with 10% FBS
and antimycotic was added in 96-well microplates at 1.8 ×
Results and discussion
Chemistry
The synthetic pathway for the target compounds 3a–c,
5a–g, and 7a–p is presented in Scheme 1. Intermediate 2

Medicinal Chemistry Research
Table 1 Inhibitory activity of compounds 3a–c, 5a–g, and 7a–p
S. aureus RN 4220 is a genotype of S. aureus. KCTC Korean
Collection for Type Cultures, CCARM Culture Collection of
Antimicrobial Resistant Microbes, n.d. not determined
expressed as MICa (µg/mL) against strains of Gram-positive
(
Staphylococcus aureus RN 4220, Streptococcus mutans 3289)
bacteria, Gram-negative (Escherichia coli KCTC 1924) bacteria and
Candida albicans 7535
aThe antibacterial tests was carried out three times, and the average
values were taken as the MICs
bStaphylococcus aureus 4220
cStreptococcus mutans 3289
dEscherichia coli 1924
eCandida albicans7535
Compound
R
Gram-
positive
strains
Gram-
negative
strains
Fungus
4
220b 3289c 1924d
7535e
UA
–
32
32
>64
2
>64
64
3
3
3
5
5
a
b
c
NH
S
2
16
was synthesized by reacting UA with Jones reagent in
acetone. Compounds 3a–c were prepared by reacting
intermediate 2 with aminoguanidine bicarbonate, semi-
carbazide hydrochloride, and thiosemicarbazide in refluxing
ethanol. Compound 2 was reacted with methyl iodide and
different substituents of benzyl chloride to provide com-
pounds 4a–g. Compounds 6a–p were prepared by
Claisen–Schmidt condensations between compound 2 and
different aldehydes. Finally, compounds 4a–g and 6a–p
were reacted with aminoguanidine bicarbonate in refluxing
ethanol to afford compounds 5a–g and 7a–p, respectively.
The structures of the desired compounds were characterized
>64
>64
8
>64
>64
16
>64
>64
2
>64
>64
16
O
a
b
CH3
Phenyl(3-
Cl)
>64
>64
>64
>64
5
5
5
c
d
e
Phenyl(4-
Cl)
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
Phenyl(2,4- >64
Cl)
Phenyl(2,6- >64
Cl)
Phenyl(4-F) >64
2
2
5
5
f
>64
>64
>64
>64
>64
>64
1
13
by H NMR, C NMR, and HRMS spectra.
g
Phenyl(4-
CH3)
>64
Anti-bacterial and anti-fungal activities
7
7
a
Phenyl
>64
>64
>64
>64
>64
>64
>64
>64
b
Phenyl(4-
Cl)
The minimum inhibitory concentration (MIC) is defined as
the lowest concentration of anti-bacterial agent required to
visibly inhibit the growth of bacteria. In vitro anti-bacterial
activity was evaluated using a 96-well microtiter plate and
the serial dilution method to obtain the MIC values for
several different pathogenic strains, including four
multidrug-resistant Gram-positive strains (methicillin-resis-
tant Staphylococcus aureus CCARM 3167 and 3506, and
quinolone-resistant S. aureus CCARM 3505 and 3519), two
Gram-positive strains (S. aureus RN 4220 and S. mutans
KTCT 3289), one Gram-negative strain (Escherichia coli
KCTC 1924), and one fungus (Candida albicans 7535).
The anti-bacterial agents gatifloxacin, moxifloxacin, nor-
floxacin, and oxacillin, and the antifungal agents flucona-
zole and itraconazole were included as positive controls.
The results for the anti-bacterial and antifungal activities
are presented in Tables 1 and 2. As shown in Table 1, most
of the compounds (except 5b–g) showed potent inhibitory
activities against the different bacteria, with MIC values in
the range of 2–64 µg/mL. Compounds 3a and 5a, which
showed high levels of inhibitory activity against E. coli
KCTC 1924 with MIC values of 2 µg/mL, had the same
potency as the standard drugs gatifloxacin (MIC = 2 µg/mL)
and moxifloxacin (MIC = 2 µg/mL). Compounds 3a, 5a, and
7
c
Phenyl(2-
Cl)
64
64
>64
>64
7
7
7
7
7
d
e
f
Phenyl(3-Cl 64
Phenyl(4-F) 16
Phenyl(2-F) 64
Phenyl(3-F) 16
64
64
64
64
64
>64
8
>64
>64
>64
>64
>64
32
g
h
32
Phenyl(2-
Br)
64
>64
7
7
7
7
7
i
Phenyl(3-
Br)
32
64
32
>64
8
>64
>64
>64
16
>64
>64
>64
>64
>64
j
Phenyl(2,4- 64
Cl)
Phenyl(2,6- 64
Cl)
2
k
l
2
Phenyl(4-
CH3)
4
m
Phenyl(4-
NO2)
32
32
16
7
7
7
n
o
p
Naphthyl
Furyl
32
32
16
32
64
32
>64
32
16
2
>64
>64
16
Thienyl
Gatifloxacin
Moxifloxacin
Fluconazole
Itraconazole
0.25 0.25
0.25 0.25
0.5
0.5
1
2
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
7
l exhibited the highest activity of all the synthesized com-
pounds against S. aureus RN 4220 and S. mutans KTCT
289 with MIC values of 2–16 µg/mL, which was lower
0.6
3

Medicinal Chemistry Research
a
Table 2 Inhibitory activity (MIC , µg/mL) of compounds 3a–c, 5a–g,
Table 3 Inhibitory activities (MIC50 and MIC90, µg/mL) of compounds
3a, 5a, and 7l against test bacteria
and 7a-p against clinical isolates of multidrug-resistant Gram-positive
strains
Compound
Gram-positive
strains
Gram-negative
strains
Compound
R
Multidrug-resistant Gram-
positive strains
4
220a
1924b
MIC50
MRSA
QRSA
MIC50
MIC90
MIC90
3
167b 3506c 3505d 3519e
3
5
7
a
a
l
1.7
3.3
10.8
5.2
1.4
1.6
5.5
1.4
2.7
2.8
UA
–
32
32
32
2
32
5.4
3
3
3
5
5
5
5
5
5
5
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
a
b
c
d
e
f
NH
2
2
4
2.3
13.9
2.7
S
>64
>64
4
>64
64
>64
64
4
>64
>64
4
Gatifloxacin
0.02
0.38
O
aStaphylococcus aureus 4220
bEscherichia coli 1924
CH3
4
Phenyl(3-Cl)
Phenyl(4-Cl)
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
32
>64
>64
64
64
64
>64
>64
>64
32
32
16
32
32
32
16
16
64
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
32
Phenyl(2,4-2Cl) >64
Phenyl(2,6-2Cl) >64
activity with MIC values of 16 µg/mL. As shown in Table 2,
against the four multidrug-resistant Gram-positive strains
(methicillin-resistant S. aureus CCARM 3167 and 3506 and
quinolone-resistant S. aureus CCARM 3505 and 3519),
compounds 3a, 5a, and 7l exhibited good activity with MIC
values in the range of 2–8 µg/mL. Compounds 3a and 7l
were the most potent against methicillin-resistant S. aureus
CCARM 3167 with MIC values of 2 µg/mL, which was four
times more potent than norfloxacin (MIC = 8 µg/mL) and
equipotent with gatifloxacin (MIC = 2 µg/mL). Compound
Phenyl(4-F)
Phenyl(4-CH3)
Phenyl
64
>64
16
>64
>64
64
8
g
a
b
c
d
e
f
Phenyl(4-Cl)
Phenyl(2-Cl)
Phenyl(3-Cl
Phenyl(4-F)
Phenyl(2-F)
Phenyl(3-F)
Phenyl(2-Br)
Phenyl(3-Br)
8
64
>64
>64
>64
>64
>64
>64
8
3a was equipotent or more potent than the standard drugs
g
h
i
8
16
gatifloxacin and moxifloxacin against quinolone-resistant S.
aureus CCARM 3505 and 3519 with MIC values of 2 and
16
64
>64
64
4
µg/mL, respectively.
j
Phenyl(2,4-2Cl) >64
Phenyl(2,6-2Cl) 32
>64
>64
8
Compound 3a, 5a and 7l were chosen to evaluate their
MIC50 and MIC , gatifloxacin was used as positive control.
k
l
90
Phenyl(4-CH3)
Phenyl(4-NO2)
Naphthyl
2
As indicated in Tables 3 and 4, compound 3a displayed
great activity (MIC = 1.4 µg/mL, MIC = 2.7 µg/mL),
equivalent to gatifloxacin against E. coli KCTC 1924, and
exhibited more potent activity than gatifloxacin against S.
aureus CCARM 3505.
m
n
o
p
8
32
16
32
32
16
8
32
5
0
90
16
8
32
32
Furyl
32
32
Thienyl
8
8
16
Gatifloxacin
Moxifloxacin
Norfloxacin
Oxacillin
2
2
4
From the analysis of these results, we obtained several
structure–activity relationships (SAR). First, it was clear that
the aminoguanidine moiety is critical for anti-bacterial activ-
ity, as determined from the activity results of compounds 3a–
c. Second, except for compound 5a, compounds 5b–g did not
have any anti-bacterial activity, suggesting that the presence
of a benzene ring on the ester group was detrimental for
activity. Third, the introduction of different substituents (dif-
ferent substituted benzene rings, a naphthalene ring, and a
heterocycle) to the C-2 of UA increased the anti-bacterial
activity. For the compounds in series 7, the naphthalene ring
and heterocycle substituted derivatives (7n, 7o and 7p) the
activity was in the order thienyl > furyl > naphthyl. In addi-
tion, the order of activity for the benzene rings substituted
1
1
4
4
8
4
>64
1
>64
1
>64
>64
aThe antibacterial tests were carried out three times, and the average
values were taken as the MICs
bMethicillin-resistant Staphylococcus aureus3167
cMethicillin-resistant Staphylococcus aureus 3506
dQuinolone-resistant Staphylococcus aureus 3505
eQuinolone-resistant Staphylococcus aureus 3519
activity than observed for the standard drugs gatifloxacin
(
MIC = 0.25 µg/mL) and moxifloxacin (MIC = 0.25 µg/mL).
Furthermore, UA did not exhibit any inhibitory activity
against the one fungus tested (C. albicans 7535), while
compounds 5a and 7p showed moderate to good levels of
derivatives was 4-CH > -F > -Br > -Cl. Overall, compound
3a showed the most potent anti-bacterial activity, and is a
promising lead compound for further investigation.
3

Medicinal Chemistry Research
Table 4 Inhibitory activities
Compound
Multidrug-resistant Gram-positive strains
(
MIC50 and MIC , µg/mL) of
90
3167a
3506b
3505c
3519d
MIC50
compounds 3a, 5a, and 7l
against the clinical isolates of
multidrug-resistant Gram-
positive bacterial strains
MIC50
MIC90
MIC50
MIC90
MIC50
MIC90
MIC90
3
5
7
a
a
l
1.5
3.0
1.5
1.1
3.0
7.1
2.8
2.6
1.4
3.3
5.4
0.7
2.7
6.2
1.4
2.9
1.4
5.1
2.7
5.4
1.1
2.9
6.6
1.4
4.6
5.7
10.7
2.4
8.1
11.4
3.1
Gatifloxacin
10.7
aMethicillin-resistant Staphylococcus aureus3167
bMethicillin-resistant Staphylococcus aureus 3506
cQuinolone-resistant Staphylococcus aureus 3505
dQuinolone-resistant Staphylococcus aureus 3519
Table 5 Anti-inflammatory
activity of compounds 3a–c,
Compound
DMSO
R
Dose (mg/kg) Number
Edema mean ± S.
D. (mg)
Inhibition
rate (%)
of mice
5a–g, and 7a–p administrated i.p.
–
100
100
100
100
100
100
100
100
100
100
100
10
10
10
10
10
10
10
10
10
10
10
6.19 ± 0.66
–
Indometacin –
3.56 ± 0.89*
3.35 ± 0.55*
2.76 ± 0.41**
1.14 ± 0.31***
2.03 ± 0.49***
4.63 ± 0.86
42.42
45.86
55.35
81.61
67.28
25.26
68.28
42.42
43.63
50.31
Ibuprfen
–
UA
–
3
3
3
5
5
5
5
a
b
c
NH
S
O
a
b
c
CH3
1.96 ± 0.64***
3.56 ± 0.55*
3.49 ± 0.56*
3.08 ± 0.55**
Phenyl(3-Cl)
Phenyl(4-Cl)
Phenyl(2,4-
d
2
Cl)
Phenyl(2,6-
Cl)
Phenyl(4-F)
5
e
100
100
10
4.28 ± 0.79
30.91
2
5
5
7
7
7
7
7
7
7
7
7
7
7
f
10
10
10
10
10
10
10
10
10
10
10
10
10
5.00 ± 0.85
19.2
g
a
b
c
Phenyl(4-CH3) 100
3.11 ± 0.45**
1.55 ± 0.36***
2.14 ± 0.43***
2.59 ± 0.67***
2.36 ± 0.29***
3.63 ± 0.59*
3.58 ± 0.79*
1.63 ± 0.51***
1.89 ± 0.66***
5.76 ± 1.22
49.69
74.95
65.45
58.18
61.81
41.42
42.23
73.67
69.49
6.87
Phenyl
100
100
100
100
100
100
Phenyl(4-Cl)
Phenyl(2-Cl)
Phenyl(3-Cl)
Phenyl(4-F)
Phenyl(2-F)
d
e
f
l
Phenyl(4-CH3) 100
Phenyl(4-NO2) 100
m
n
o
p
Naphthyl
Furyl
100
100
100
3.41 ± 0.72**
3.95 ± 1.22
44.84
36.17
Thienyl
*
: p < 0.05, **p < 0.01, ***p < 0.001 compared with vehicle group
: no anti-inflammatory activity
–
Anti-inflammatory activity
determined relative to the ability to prevent edema. Dime-
thyl sulfoxide was used as the vehicle and ibuprofen and
indomethacin were used as reference drugs. As shown in
Table 5, most of the test compounds exhibited good anti-
inflammatory activity at 100 mg/kg. Compounds 3a, 3b, 5a,
Some of the synthesized compounds were screened for their
anti-inflammatory activity using the xylene-induced ear-
edema test in mice. The potency of the test compounds was

Medicinal Chemistry Research
Table 6 Anti-inflammatory activity of compound 3a administered
orally at different times before xylene application
xylene application. As shown in Table 6, the activity of
compound 3a showed a regular increase as the time interval
lengthened until a peak was reached at 3 h (38.06%), and
compound 3a displayed more potent activity than ibuprofen
Time (h) Dose (mg/kg)
Number of mice Inhibition (%)
3
a
Ibuprfen
(
34.44%). The activity of compound 3a was also screened
1
2
3
4
5
2
100
100
100
100
100
100
10
10
10
10
10
10
25.45
31.04*
38.06** 34.44**
33.23** 25.45*
28.96*
19.49
23.80*
at concentrations of 100, 50, and 25 mg/kg at 3 h after oral
administration. As shown in Table 7, compound 3a showed
the highest anti-inflammatory activity at 100 mg/kg with
47.94% inhibition.
To determine whether the synthesized compounds were
selectively toxic toward bacteria, we evaluated the cyto-
toxicity of compound 3a using a standard technique. As
shown in Table 8, compound 3a did not show any appre-
ciable cytotoxic activity (IC > 100 µmol/L) against HeLa,
16.35
13.35
4
15.50
*
0.01 < p < 0.05 compared with vehicle group, **p < 0.01 compared
with vehicle group
5
0
Table 7 Anti-inflammatory activity of compound 3a administered
orally at different doses
Hep3B, and A549 cells. This result indicated that the pro-
mising anti-bacterial activity of these compounds is not
because of cytotoxicity and compound 3a is a promising
anti-bacterial inhibitor for use in further work.
Time (h) Dose (mg/kg) Number of mice Inhibition (%)
3
a
Ibuprfen
The mechanism of action for the antimicrobial and anti-
inflammatory activities of these compounds is unknown.
Wei et al. (2018) reported that the UA derivatives exhibited
high affinity for the COX-2 active site and possibly exhibit
their anti-inflammatory potency via inhibiting COX-2. We
infer that a similar mechanism might be involved for our
synthesized compounds. Further study on the possible
mechanism of action is currently ongoing in our laboratory.
3
3
3
100
50
10
10
10
47.94*** 33.42**
29.39*
23.48
21.72
13.58
25
*
p < 0.05, **p < 0.01, ***p < 0.001 compared with vehicle group
Table 8 Cytotoxic activity (IC50a, µg/mL) of compound 3a against
human cell lines
a
Compound Substituents
R
In vitro cytotoxicity IC50 (µg/mL)
HeLab
Hep3Bc
A549d
Conclusion
3
a
NH
>100
>100
>100
In summary, three series of compounds, a total of 26 UA
derivatives containing an aminoguanidine moiety, were
synthesized, characterized, and evaluated for anti-bacterial
and anti-inflammatory activity. Some of these compounds
exhibited high levels of anti-bacterial and anti-inflammatory
activity. Compound 3a showed the most potent anti-bacterial
activity against the different bacteria tested with MIC values
in the range of 2–16 µg/mL, and also exhibited the highest
anti-inflammatory activity with inhibition of 81.61%. Com-
pound 3a was also evaluated for cytotoxicity and did not
exhibit any appreciable cytotoxic activity in human cervical
cancer HeLa, liver cancer Hep3B, and lung cancer A549 cell
lines. This work provides an experimental basis for the
development of UA derivatives with potent anti-bacterial and
anti-inflammatory activities. Further work toward the design
of more potent compounds and investigating the mechanism
of action are currently underway in our laboratory.
aIC50 is the concentration required to inhibit the cell growth by 50%.
Data represent the average of three independent experiments running
in triplicate. Variation was generally between 5–10%
bHuman cervical cancer cells
cHuman liver cancer cells
dHuman lung cancer cells
7
a, 7b, 7c, 7d, 7l, and 7m showed significant activity with
inhibition in the range of 58.18% to 81.61%, which was
higher than for the reference drugs and UA. Compounds 3c,
5
e, 5f, 7n, and 7p did not display any anti-inflammatory
activity at the same dose. SAR analysis revealed that the
aminoguanidine moiety was important for anti-
inflammatory activity (3a > 3b and 3c). Compound 3a and
the compounds in series 7 were more active than the
compounds in series 5, indicating that retaining the car-
boxylic acid at C-17 was beneficial for the activity.
According to the anti-inflammatory activity of the
designed compounds, compound 3a was chosen for further
evaluation. A dose of 100 mg/kg was administered via the
oral route at different intervals (1, 2, 3, 4, 5, and 24 h) after
Acknowledgements This work was supported by the National Natural
Science Foundation of China (No. 81260468, 81460524 and
8
2
1060257) and the Natural Science Foundation of Jilin Province (No.
0160101218JC). We thank Victoria Muir, PhD, from Liwen Bianji,
Edanz Group China (www.liwenbianji.cn/ac), for editing the English
text of a draft of this manuscript.

Medicinal Chemistry Research
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Conflict of interest The authors declare that they have no conflict of
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