Synthesis and antibacterial evaluation of novel 3-substituted ocotillol-type derivatives as leads

Article abstract of DOI:10.3390/molecules22040590

Due to the rapidly growing bacterial antibiotic-resistance and the scarcity of novel agents in development, bacterial infection is still a global problem. Therefore, new types of antibacterial agents, which are effective both alone and in combination with traditional antibiotics, are urgently needed. In this paper, a series of antibacterial ocotillol-type C-24 epimers modified from natural 20(S)-protopanaxadiol were synthesized and evaluated for their antibacterial activity. According to the screening results of Gram-positive bacteria (B. subtilis 168 and MRSA USA300) and Gram-negative bacteria (P. aer PAO1 and A. baum ATCC19606) in vitro, the derivatives exhibited good antibacterial activity, particularly against Gram-positive bacteria with an minimum inhibitory concentrations (MIC) value of 2-16 μg/mL. The subsequent synergistic antibacterial assay showed that derivatives 5c and 6c enhanced the susceptibility of B. subtilis 168 and MRSA USA300 to chloramphenicol (CHL) and kanamycin (KAN) (FICI < 0.5). Our data showed that ocotillol-type derivatives with long-chain amino acid substituents at C-3 were good leads against antibiotic-resistant pathogens MRSA USA300, which could improve the ability of KAN and CHL to exhibit antibacterial activity at much lower concentrations with reduced toxicity.

Full text of DOI:10.3390/molecules22040590

molecules
Communication
Synthesis and Antibacterial Evaluation of Novel
3-Substituted Ocotillol-Type Derivatives as Leads
Yi Bi 1,†, Xian-Xuan Liu 1,†, Heng-Yuan Zhang 2,†, Xiao Yang 3, Ze-Yun Liu 1, Jing Lu 1,
Peter John Lewis 4, Chong-Zhi Wang 5,6, Jin-Yi Xu 2,*, Qing-Guo Meng 1,*, Cong Ma 7,
and Chun-Su Yuan 5,6
*
1
School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University),
Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech
Drugs in Universities of Shandong, Yantai University, Yantai 264005, China; beeyee_413@163.com (Y.B.);
opendekevin@163.com (X.-X.L.); 18766565610@163.com (Z.-Y.L.); tainche@126.com (J.L.)
State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry,
China Pharmaceutical University, Nanjing 210009, China; panacea0928@163.com
Department of Microbiology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin,
Hong Kong, China; xiaoyang@cuhk.edu.hk
Biological Sciences, School of Environmental and Life Sciences, University of Newcastle,
Callaghan, NSW 2308, Australia; peter.lewis@newcastle.edu.au
Tang Center for Herbal Medicine Research, the University of Chicago, Chicago, IL 60637, USA;
czwang@dacc.uchicago.edu (C.-Z.W.); cyuan@dacc.uchicago.edu (C.-S.Y.)
Department of Anesthesia and Critical Care, The University of Chicago, Chicago, IL 60637, USA
Department of Applied Biology and Chemical Technology, and State Key Laboratory of Chirosciences,
The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China
Correspondence: jinyixu@china.com (J.-Y.X.); qinggmeng@163.com (Q.-G.M.);
cong.ma@polyu.edu.hk (C.M.); Tel.: +86-25-83271299 (J.-Y.X.); +86-535-6706022 (Q.-G.M.);
+852-3400-8810 (C.M.)
2
3
4
5
6
7
*
†
These authors contributed equally to this work.
Academic Editor: Daniela Barlocco
Received: 1 March 2017; Accepted: 3 April 2017; Published: 7 April 2017
Abstract: Due to the rapidly growing bacterial antibiotic-resistance and the scarcity of novel agents
in development, bacterial infection is still a global problem. Therefore, new types of antibacterial
agents, which are effective both alone and in combination with traditional antibiotics, are urgently
needed. In this paper, a series of antibacterial ocotillol-type C-24 epimers modified from natural
20(S)-protopanaxadiol were synthesized and evaluated for their antibacterial activity. According to
the screening results of Gram-positive bacteria (B. subtilis 168 and MRSA USA300) and Gram-negative
bacteria (P. aer PAO1 and A. baum ATCC19606) in vitro, the derivatives exhibited good antibacterial
activity, particularly against Gram-positive bacteria with an minimum inhibitory concentrations
(MIC) value of 2–16 µg/mL. The subsequent synergistic antibacterial assay showed that derivatives
5c and 6c enhanced the susceptibility of B. subtilis 168 and MRSA USA300 to chloramphenicol (CHL)
and kanamycin (KAN) (FICI < 0.5). Our data showed that ocotillol-type derivatives with long-chain
amino acid substituents at C-3 were good leads against antibiotic-resistant pathogens MRSA USA300,
which could improve the ability of KAN and CHL to exhibit antibacterial activity at much lower
concentrations with reduced toxicity.
Keywords: ocotillol; derivatives; synthesis; antibacterial activity; synergistic effect
Molecules 2017, 22, 590; doi:10.3390/molecules22040590
www.mdpi.com/journal/molecules

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1. Introduction
Therapeutic control of multidrug resistant bacteria has been emphasized in the area of global
public health [ ]. The generation of drug-resistant bacteria, such as MRSA USA300, has developed
1
much faster than the development of new antimicrobial drugs. As the most common bacteria in
blood-borne infection, MRSA USA 300 could cause lethal diseases, including severe sepsis, necrotizing
pneumonia, and necrotizing fasciitis, all of which pose a serious threat to human health [
first choice for the treatment of MRSA in clinic is vancomycin. However, the unreasonable use of
vancomycin has further led to the production of vancomycin resistant strains [ ]. Additionally, there
are many side effects associated with long-term drug use. For example, chloramphenicol (CHL)
can lead to aplastic anemia, granulocyte deficiency, double infection and gray baby syndrome [ ].
Kanamycin (KAN) has many side effects, including ototoxicity, renal toxicity, hematopoietic system
toxicity and neuromuscular blocking effects etc. [10 11]. Therefore, there is an urgent need to develop
2–6]. The
7
8,9
,
novel antibacterial agents to cure antibiotic-resistant bacteria infections, which can be used alone or in
combination with traditional antibiotics to enhance their effectiveness and reduce their side effects.
Natural products have been the most effective source of leading compounds for the development
of drugs, particularly anticancer and anti-infective agents [12]. Natural triterpenoids, including sterols,
steroids and saponins, form a wide family of compounds, biosynthesized by cyclization reactions from
2,3-epoxysqualene [13]. These compounds have been used in traditional medicine and therefore have
been studied for their anti-inflammatory, antimicrobial, anticancer and anti-HIV effects [14–17].
Ocotillol (Figure 1), isolated from Fouquieria splendens engelm, bears a characteristic tetrahydrofuran
ring at C-20 [18]. Studies have shown that ocotillone (Figure 1) isolated from the stem bark of
ailanthus altisshima has potent antibacterial activities against P. aeruginosa and S. typhimurium, without
hemolytic activity [19]. In our previous studies, ocotillol-type epimers OR and OS were synthesized
from 20(S)-protopanaxadiol (PPD) (Figure 1) [20]. Many ocotillol-type analogs, such as OS, D1, D2, D3
(Figure 1), showed antibacterial activity against Gram-positive bacteria and demonstrated outstanding
synergistic effects with CHL and KAN. For example, the minimum inhibitory concentrations (MIC) of
KAN combining with D1 against MRSA USA300 was reduced from 0.25 µg/mL to 0.0625 µg/mL [21–23].
Thus, ocotillol analogs with good synergistic activity against MRSA USA300 hold the potential to
become leads of novel antibiotic-resistant agents with unique mechanisms. The synthesis of new
ocotillol-type derivatives, followed by antibacterial evaluations, are described in this study.
Figure 1. The structures of ocotillol and analogs.
2. Results and Discussion
2.1. Synthesis of Ocotillol-Type Derivatives
As previously described [20], in the synthesis of epimers OS and OR, PPD was chosen as the
starting material. Firstly, PPD was protected with acetic anhydride under the catalysis of DMAP,

Molecules 2017, 22, 590
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then oxidized by m-CPBA, and finally deprotected by potassium hydroxide to obtain OS and OR with
the desired yields. OS was reacted with a series of carboxylic acids to give intermediates 3a , and
finally target compounds 5a were obtained by deprotection using trifluoroacetic acid. Referring to
–
e
–e
the synthesis of 5a–e, 6a–e were gained from OR as the starting material (Scheme 1).
O
O
O
Boc2O, (CH3)3COH
NaOH, H2O
H2N
OH
n
O
N
H
OH
n
1a-e
2a-e
HO
OH
H
HO
PPD
iii) KOH
i) Ac2O, DMAP;
ii) m-CPBA;
HO
O
HO
O
OH
OH
H
H
H
H
HO
HO
OS
H
H
OR
CH2Cl2,
rt,
10 h
2a-e,
DMAP,
EDCI
CH2Cl2,
rt,
10 h
2a-e,
DMAP,
EDCI
HO
O
HO
O
OH
OH
H
H
3a; n=1
3b; n=2
3c; n=4
3d; n=6
3e; n=9
4a; n=1
4b; n=2
4c; n=4
4d; n=6
4e; n=9
O
O
n
O
O
n
H
H
O
N
O
O
N
O
H
H
H
H
3a-e
4a-e
TFA,
CH2Cl2,
rt,
3 h
TFA,
CH2Cl2,
rt,
3 h
HO
O
HO
O
OH
OH
H
H
5a; n=1
5b; n=2
5c; n=4
5d; n=6
5e; n=9
6a; n=1
6b; n=2
6c; n=4
6d; n=6
6e; n=9
O
O
H
H
H2N
O
H2N
O
n
n
H
H
6a-e
5a-e
Scheme 1. Synthesis of ocotillol-type derivatives 5a–e and 6a–e.
2.2. Antibacterial Activity
Initial MIC screening results are shown in Figure 2. The results showed that most of the
ocotillol-type derivatives had moderate to good inhibitory activity against Gram-positive bacteria with
MIC values of about 2–32
that, with unprotected primary amine groups, all target compounds showed good inhibitory activity
against B. subtilis 168 with MIC values of about 2–16 g/mL, and against MRSA USA300 with MIC
values of about 4–16 g/mL, except 6e. The study found that, among the synthesized compounds,
5d was the most active compound against B. subtilis 168, with a MIC value of 2 g/mL, while 6c and
6d showed the most potent activity against MRSA USA300, with a MIC value of 4 g/mL. Among
µg/mL, but had no effect on Gram-negative bacteria. The data illustrated
µ
µ
µ
µ

Molecules 2017, 22, 590
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these derivatives, compound 6e had the weakest activity against Gram-positive bacteria but only a
moderate inhibitory activity against A. Baum ATCC19606, with a MIC value of 32 g/mL. Additionally,
a similar inhibitory activity against Gram-positive bacteria was observed between epimers 5a and
6a , but a dramatically dissimilar one was present between 5e and 6e, which suggested that the
µ
–d
–
d
spatial configuration of the isopropanol group at C-24 and the length of the C-3 side chain affected
both its conformation and bioactivity.
Figure 2. In vitro antibacterial activity of ocotillol-type derivatives against (A) B. subtilis 168; (B) MRSA
USA300; (C) P. aer PAO1 and (D) A. baum ATCC19606. “↑”: MIC > 128 µg/mL.
As shown in Figure 3, the minimum bactericidal concentration (MBC) was measured for
compounds 5a and 6a against B. subtilis 168 and MRSA USA300. The derivatives with bactericidal
activities in these strains warrant further studies as anti-bacterial leads. The results showed that
compounds 5a and 6a possess good bactericidal activity against B. subtilis 168 with MBC values of
4–32 g/mL, and compounds 5a and 6a possess good bacterial activity against MRSA USA300
with MBC values of 8–16 g/mL. Compounds 5b 5c and 6c could be candidates since they possess
–
e
–e
–
e
–e
µ
–
d
–d
µ
,
the most potent activities against the community-acquired meticillin-resistant strain MRSA USA300
and laboratory strain B. subtilis 168.
Figure 3. In vitro bactericidal activity of ocotillol-type derivatives against (A) B. subtilis 168 and (B)
MRSA USA300. “↑”: MIC > 128 µg/mL.

Molecules 2017, 22, 590
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2.3. Synergistic Antibacterial Activity
According to Figure 4, the MIC of kanamycin (KAN) and chloramphenicol (CHL) against MRSA
USA300 and B. subtilis 168 significantly decreased when combining compounds 5c and 6c at half-MIC,
and the synergistic effect was significant (FICI < 0.5, According to the literature [24]: FICI = FICA + FICB
= (MICA+B/MICA) + (MICA+B/MICB). When FICI value was less than or equal to 0.5, it has a synergistic
effect). Compounds 5c and 6c could decrease the MICs of KAN or CHL against MRSA USA300, and the
MICs were all less than 0.0078 µg/mL (FICI < 0.0088, 0.0098, 0.0029, 0.0039). Synergistic activity against
B. subtilis 168 was also observed during the combination of 5c and 6c with KAN or CHL (FICI = 0.2656,
0.2656, 0.0938, 0.3750). In contrast, when 5c (or 6c) was combined with KAN, the bactericidal activity
of KAN against MRSA USA300 was significantly enhanced from 4 µg/mL to 0.25 (or 1) µg/mL. For
B. subtilis 168, potent bactericidal effects were also observed during the combination of KAN with 5c
and 6c. Surprisingly, CHL alone was bacteriostatic, but possessed bactericidal effect when combined
with 6c, with MBC value of 2 µg/mL against MRSA USA 300 and 0.5 µg/mL against B. subtilis 168.
Figure 4. Synergistic effect of different antibiotics with compounds 5c and 6c against MRSA USA300
and B. subtilis 168. (
) MBC of different compounds against MRSA USA300 and (
compounds against MRSA USA300 and (F) B. subtilis 168.
A
) MIC of different compounds against MRSA USA300 and (
B) B. subtilis 168;
(
C
D
) B. subtilis 168; ( ) FICI of different
E
2.4. The Structure-Activity Relationships (SARs) of Ocotillol-Type Derivatives
Based on the previous and present data of ocotillol-type derivatives, preliminary SARs can
be summarized. The (24S)-configuration is preferred for pharmacological activity of compounds

Molecules 2017, 22, 590
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without substitution at 3-OH; substitution at 3-OH may cause changes of molecular conformation
resulting in bioactive (24R)-compounds [21 25]; hydrogen donors at C-3 positions are the effective
group and enhance the antibacterial activity against Gram-positive bacteria [21 23 25]; the length of
,
,
,
4–8 carbon atoms at the C-3 side chain is preferred if there is -NH2 on the side chain; however, the
substitution of 11 carbon atoms at the C-3 side chain resulted in mild activity against Gram-negative
bacteria, in particular compound 6e with (24R)-configuration showed specific activity against
Gram-negative bacteria.
3. Materials and Methods
3.1. Chemical Reagents and Instruments
The majority of chemicals and reagents used in the experiment was analytically pure and was
purified and dried with standard methods when necessary. 1H-NMR and 13C-NMR spectra were
measured using a Bruker AV-300 spectrometer in the specific solvents (Tetramethyl silane (TMS) as
internal standard): the chemical shifts are expressed with δ values (ppm) and the coupling constants
(J) with Hz. High-resolution mass spectra were measured using an Agilent QTOF 6520.
3.2. General Procedure for the Synthesis of (20S,24S)-Epoxy-dammarane-3β,12β,25-triol (OS) and
(20S,24R)–Epoxy-dammarane-3β,12β,25-triol (OR)
Compounds OR and OS were prepared from PPD according to the published procedures [18
,21].
3.3. General Procedure for the Synthesis of Compounds 5a–e and 6a–e
To a solution of sodium hydroxide (800 mg, 19.5 mmol) and di-tert-butyl dicarbonate (4.3 g,
19.5 mmol) in tertiary butyl alcohol (11 mL) and H2O (9 mL) the corresponding amino acids 1a
–
e
(19.5 mmol) were added. After the mixture was stirred at room temperature for 16 h, diluted with 10%
HCl (100 mL), and extracted with ethyl acetate, and the organic layer was washed with water and
brine successively, dried over anhydrous sodium sulfate, filtered and concentrated to give 2a–e.
To a solution of 2a–e (1.5 eq.), dry dichloromethane EDCI (1.0 eq.) and DMAP (1.0 eq.) were
added, and the mixture was stirred at room temperature for 1 h. Then, OS (or OR) (1.0 eq.) was added.
After stirring for 12 h, the mixture was removed in vacuo and diluted by ethyl acetate, washed with
water and brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column
chromatography over silica gel (8:1–6:1 petroleum ether: ethyl acetate) to give 3a–e (or 4a–e).
To a solution of intermediates 3a
ice bath. After stirring at room temperature for 3 h, the mixture was removed in vacuo and purified by
column chromatography over silica gel (30:1–10:1 dichloromethane: methanol) to give 5a (or 6a ).
–e (or 4a–e) (1 eq.), trifluoroacetic acid (2 eq.) was added under an
–
e
–e
(20S,24S)-Epoxy-3 -O-(3-aminopropionyl)-dammarane-12 ,25-diol (5a). Yellow oily matter, 72% yield.
1H-NMR (300 MHz, CDCl3)
4.51 (m, 1H, -OC -), 3.87 (dd, J = 10.3Hz, 5.1Hz, 1H, -OC -), 3.53
(td, J = 10.2 Hz, 4.6 Hz, 1H, -OC -), 3.05 (t, 2H, -NCH2-), 2.56 (t, J = 6.3 Hz, 2H, -CH2CO-), 1.27 (s,
3H, CH3), 1.23 (s, 3H, CH3), 1.11 (s, 3H, CH3), 1.01 (s, 3H, CH3), 0.91 (s, 6H, CH3), 0.85 (s, 6H, CH3);
13C-NMR (75 MHz, CDCl3)
171.9, 87.4, 87.1, 81.4, 70.5, 70.2, 56.0, 52.1, 50.1, 48.9, 48.8, 39.8, 38.6, 37.9,
β
β
δ
H
H
H
δ
37.1, 34.7, 32.2, 31.7, 29.3, 28.9, 28.5, 28.0, 27.8, 27.4, 26.1, 25.2, 24.1, 23.7, 18.2, 17.8, 16.4, 16.3, 15.4.
ESI-MS: m/z [M + H]+: 548.4.
(20S,24S)-Epoxy-3
1H-NMR (300 MHz, CDCl3)
J = 10.1 Hz, 4.4 Hz, 1H, -OC
(s, 3H, C 3), 1.22 (s, 3H, C
0.85 (s, 3H, CH3), 0.83 (s, 3H, CH3); 13C-NMR (75 MHz, CDCl3)
β
-O-(4-aminobutyryl)-dammarane-12
4.47 (m, 1H, -OC -), 3.87 (dd, J = 10.4 Hz, 5.0 Hz, 1H, -OC
-), 3.07 (t, J = 7.2 Hz, 2H, -NCH2-), 2.48 (t, J = 6.9 Hz, 2H, -CH2CO-), 1.27
3), 1.11 (s, 3H, C 3), 1.01 (s, 3H, C 3), 0.92 (s, 3H, C 3), 0.91 (s, 3H, C 3),
172.7, 87.4, 87.1, 81.5, 70.5, 70.2, 56.0,
β
,25-diol (5b). Yellow oily matter, 69% yield.
δ
H
H
-), 3.53 (td,
H
H
H
H
H
H
H
δ
52.1, 50.1, 48.9, 48.8, 39.8, 39.3, 38.6, 37.9, 37.1, 34.7, 32.2, 31.7, 31.5, 29.3, 28.9, 28.5, 28.1, 27.8, 25.2, 24.1,
23.7, 22.8, 18.2, 17.8, 16.5, 16.3, 15.5. ESI-MS: m/z [M + H]+: 562.4.

Molecules 2017, 22, 590
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(20S,24S)-Epoxy-3
β
-O-(4-aminohexanoyl)-dammarane-12
4.47 (m, 1H, -OC
-), 2.99 (t, J = 7.5 Hz, 2H, -NCH2-), 2.32 (t, J = 7.1 Hz, 2H, -CH2CO-), 1.27
3), 1.12 (s, 3H, C 3), 1.01 (s, 3H, C 3), 0.92 (s, 3H, C 3), 0.91 (s, 3H, C 3),
0.85 (s, 3H, CH3), 0.83 (s, 3H, CH3); 13C-NMR (75 MHz, CDCl3)
173.4, 87.4, 87.1, 80.8, 70.5, 70.2, 56.0,
β
,25-diol (5c). Yellow oily matter, 68% yield.
1H-NMR (300 MHz, CDCl3)
δ
H-), 3.87 (dd, J = 10.4 Hz, 5.0 Hz, 1H, -OCH-), 3.52 (td,
J = 10.1 Hz, 4.4 Hz, 1H, -OC
H
(s, 3H, C 3), 1.22 (s, 3H, C
H
H
H
H
H
H
δ
52.1, 50.1, 48.9, 48.8, 39.8, 38.6, 37.9, 37.1, 34.7, 34.4, 32.2, 31.7, 31.0, 28.9, 28.5, 28.0, 27.8, 27.7, 27.1, 26.0,
25.2, 24.4, 24.1, 23.7, 18.2, 17.8, 16.5, 16.3, 15.5. ESI-MS: m/z [M + H]+: 590.4.
(20S,24S)-Epoxy-3
1H-NMR (300 MHz, CDCl3)
J = 10.3 Hz, 4.4 Hz, 1H, -OC
1.82–2.09 (m, 6H, C 2), 1.27 (s, 3H, C
6H, CH3), 0.86 (s, 3H, CH3), 0.84 (s, 3H, CH3); 13C-NMR (75 MHz, CDCl3)
β
-O-(8-aminooctanoyl)-dammarane-12
4.47 (m, 1H, -OC -), 3.88 (dd, J = 8.3 Hz, 6.9 Hz, 1H, -OC
-), 2.96 (t, J = 7.2 Hz, 2H, -NCH2-), 2.29 (t, J = 7.4 Hz, 2H, -CH2CO-),
3), 1.26 (s, 3H, C 3), 1.10 (s, 3H, C 3), 1.01 (s, 3H, C 3), 0.91 (s,
173.5, 87.4, 87.1, 80.6, 70.5,
β
,25-diol (5d). Yellow oily matter, 66% yield.
δ
H
H
-), 3.53 (td,
H
H
H
H
H
H
δ
70.2, 56.0, 52.1, 50.2, 48.9, 48.8, 39.9, 39.8, 38.6, 37.9, 37.1, 34.7, 32.2, 31.7, 31.2, 28.9, 28.6, 28.5, 28.0, 27.9,
27.5, 27.4, 26.3, 26.2, 25.2, 24.9, 24.1, 23.7, 18.2, 17.8, 16.5, 16.3, 15.5. ESI-MS: m/z [M + H]+: 618.5.
(20S,24S)-Epoxy-3
1H-NMR (300 MHz, CDCl3)
5.0 Hz, 1H, -OC -), 3.51 (td, J = 10.3 Hz, 5.6 Hz, 1H, -OC
(t, J = 7.4 Hz, 2H, -CH2CO-), 1.78–2.06 (m, 6H, CH2), 1.27 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.10 (s, 3H,
H3), 1.01 (s, 3H, CH3), 0.91 (s, 6H, CH3), 0.86 (s, 3H, CH3), 0.85 (s, 3H, CH3); 13C-NMR (75 MHz,
CDCl3) 173.6, 87.4, 87.1, 80.4, 70.5, 70.2, 56.0, 52.1, 50.1, 48.9, 48.8, 48.0, 40.0, 39.8, 38.6, 37.9, 37.1, 34.8,
β
-O-(11-undecanoyl)-dammarane-12
4.48 (dd, J = 10.1 Hz, 5.9 Hz, 1H, -OC
-), 2.96 (t, J = 7.5 Hz, 2H, -NCH2-), 2.29
β
,25-diol (5e). Yellow oily matter, 61% yield.
δ
H
-), 3.88 (dd, J = 10.4 Hz,
H
H
C
δ
32.2, 31.7, 31.4, 29.3, 29.2, 29.1, 29.0, 28.9, 28.5, 28.0, 27.8, 27.6, 26.5, 26.2, 25.2, 25.1, 24.1, 23.7, 18.2, 17.8,
16.5, 16.3, 15.4. ESI-MS: m/z [M + H]+: 660.5.
(20S,24R)-Epoxy-3
1H-NMR (300 MHz, CDCl3)
J = 10.4 Hz, 4.4 Hz, 1H, -OC
3H, CH3), 1.10 (s, 3H, CH3), 0.98 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.87 (s, 3H, CH3), 0.84 (s, 6H, CH3);
13C-NMR (75 MHz, CDCl3)
171.1, 86.5, 85.3, 82.2, 70.9, 70.1, 56.0, 52.0, 50.4, 49.3, 48.0, 39.8, 38.6, 37.9,
β
-O-(3-aminopropionyl)-dammarane-12
4.51 (m, 1H, -OC -), 3.84 (dd, J = 8.3 Hz, 6.9 Hz, 1H, -OC
-), 3.28 (t, 2H, -NC 2-), 2.83 (t, 2H, -C 2CO-), 1.27 (s, 3H, C
β
,25-diol (6a). Yellow oily matter, 79% yield.
-), 3.51 (td,
3), 1.26 (s,
δ
H
H
H
H
H
H
δ
37.0, 35.8, 32.6, 31.7, 31.3, 31.1, 28.6, 28.1, 27.9, 27.5, 26.1, 25.0, 24.0, 23.6, 18.2, 17.5, 16.4, 16.3, 15.4.
ESI-MS: m/z [M + H]+: 548.4.
(20S,24R)-Epoxy-3
1H-NMR (300 MHz, CDCl3)
J = 10.1 Hz, 4.4 Hz, 1H, -OC
(s, 3H, C 3), 1.23 (s, 3H, C
0.85 (s, 3H, C 3), 0.83 (s, 3H, C
β
-O-(4-aminobutyryl)-dammarane-12
4.47 (m, 1H, -OC -), 3.85 (dd, J = 10.4 Hz, 5.0 Hz, 1H, -OC
-), 3.05 (t, J = 7.2 Hz, 2H, -NCH2-), 2.47 (t, J = 6.9 Hz, 2H, -CH2CO-), 1.27
3), 1.11 (s, 3H, C 3), 1.01 (s, 3H, C 3), 0.92 (s, 3H, C 3), 0.91 (s, 3H, C 3),
3). 13C-NMR (75 MHz, CDCl3)
172.7, 86.5, 85.3, 81.1, 70.5, 70.2, 56.0,
β
,25-diol (6b). Yellow oily matter, 72% yield.
δ
H
H
-), 3.52 (td,
H
H
H
H
H
H
H
H
H
δ
52.1, 50.1, 48.9, 48.8, 39.8, 39.3, 38.5, 37.9, 37.1, 34.6, 32.2, 31.7, 31.5, 29.3, 28.8, 28.5, 28.1, 27.9, 25.2, 24.1,
23.6, 22.8, 18.2, 17.9, 16.5, 16.3, 15.5. ESI-MS: m/z [M + H]+: 562.4.
(20S,24R)-Epoxy-3
1H-NMR (300 MHz, CDCl3)
J = 10.2 Hz, 4.2 Hz, 1H, -OC
1.89–2.11 (m, 2H, CH2), 1.27 (s, 3H, CH3), 1.27 (s, 3H, CH3), 1.10 (s, 3H, CH3), 0.98 (s, 3H, CH3), 0.90
(s, 3H, CH3), 0.88 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.83 (s, 3H, CH3); 13C-NMR (75 MHz, CDCl3)
β
-O-(4-aminohexanoyl)-dammarane-12
4.46 (m, 1H, -OC -), 3.85 (dd, J = 8.6 Hz, 6.8 Hz, 1H, -OC
-), 2.97 (t, J = 7.6 Hz, 2H, -NCH2-), 2.32 (t, J = 7.3 Hz, 2H, -CH2CO-),
β
,25-diol (6c). Yellow oily matter, 70% yield.
δ
H
H
-), 3.52 (td,
H
δ
173.4, 86.5, 85.3, 80.8, 70.5, 70.2, 56.0, 52.1, 50.1, 48.9, 48.8, 39.8, 38.6, 37.9, 37.1, 34.7, 34.3, 32.5, 31.3,
31.1, 29.1, 28.6, 28.0, 27.8, 27.5, 27.0, 25.8, 25.0, 24.3, 24.1, 23.7, 18.1, 17.8, 16.4, 16.3, 15.4. ESI-MS: m/z
[M + H]+: 590.4.
(20S,24R)-Epoxy-3
1H-NMR (300 MHz, CDCl3)
J = 10.3 Hz, 4.4 Hz, 1H, -OC
1.90–2.10 (m, 2H, C 2), 1.82-1.90 (m, 2H, C
β
-O-(8-aminooctanoyl)-dammarane-12
4.46 (m, 1H, -OC -), 3.84 (dd, J = 8.3 Hz, 6.9 Hz, 1H, -OC
-), 2.95 (t, J = 7.2 Hz, 2H, -NCH2-), 2.28 (t, J = 7.4 Hz, 2H, -CH2CO-),
2), 1.27 (s, 3H, C 3), 1.26 (s, 3H, C 3), 1.10 (s, 3H, C 3),
β
,25-diol (6d). Yellow oily matter, 70% yield.
δ
H
H-), 3.52 (td,
H
H
H
H
H
H

Molecules 2017, 22, 590
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0.98 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.83 (s, 3H, CH3); 13C-NMR
(75 MHz, CDCl3) 173.6, 86.5, 85.3, 80.6, 70.9, 70.1, 56.1, 52.0, 50.4, 49.3, 48.0, 39.9, 39.8, 38.6, 37.9, 37.1,
δ
34.8, 32.6, 31.3, 31.1, 28.8, 28.6, 28.5, 28.0, 27.9, 27.5, 27.4, 26.2, 26.1, 25.0, 24.9, 24.1, 23.7, 18.1, 17.7, 16.5,
16.4, 15.4. ESI-MS: m/z [M + H]+: 618.5.
(20S,24R)-Epoxy-3
1H-NMR (300 MHz, CDCl3)
Hz, 1H, -OC -), 3.51 (td, J = 10.2 Hz, 6.1 Hz, 1H, -OC
J = 7.4 Hz, 2H, -C 2CO-), 1.80–2.08 (m, 6H, C 2), 1.27 (s, 3H, C
0.98 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.84 (s, 3H, CH3); 13C-NMR
(75 MHz, CDCl3) 173.6, 86.5, 85.3, 80.4, 70.9, 70.1, 56.1, 52.0, 50.4, 49.3, 48.8, 48.0, 40.0, 39.8, 38.6, 37.9,
β
-O-(11-undecanoyl)-dammarane-12
4.47 (dd, J = 9.9 Hz, 5.6 Hz, 1H, -OC
-), 2.96 (t, J = 7.3 Hz, 2H, -NCH2-), 2.29 (t,
3), 1.26 (s, 3H, C 3), 1.10 (s, 3H, C 3),
β
,25-diol (6e). Yellow oily matter, 67% yield.
δ
H
-), 3.85 (dd, J = 8.6 Hz, 6.8
H
H
H
H
H
H
H
δ
37.1, 34.8, 32.6, 31.3, 31.1, 29.3, 29.2, 29.1, 29.0, 28.6, 28.5, 28.0, 27.9, 27.5, 26.5, 26.1, 25.1, 25.0, 24.0, 23.7,
18.1, 17.6, 16.5, 16.4, 15.4. ESI-MS: m/z [M + H]+: 660.5.
3.4. Pharmacology
The antibacterial and synergistic activity experiment was carried out as described previously [23,26].
The antibacterial activity was screened in vitro, and the minimum inhibitory concentrations (MIC) were
determined against Gram-positive bacteria (B. subtilis 168 and MRSA USA300) and Gram-negative
bacteria (P. aeruginosa PAO1 and A. baumannii ATCC19606). These pathogens have been commonly
used to screen antibacterial compounds. Compounds with good inhibitory activity were selected to
determine their bactericidal activity. The minimum bactericidal concentration (MBC) was determined
against B. subtilis 168 and MRSA USA300 using a standard LB medium dilution technique. B. subtilis 168
was the tool strain used to research the antibacterial mechanism. MRSA USA300 was the drug-resistant
strain used to evaluate anti-drug-resistant candidates [4,22]. Kanamycin was used as a positive control.
4. Conclusions
In summary, ten novel 3-substituted ocotillol-type derivatives from natural PPD were synthesized
and evaluated for antimicrobial activity. According to the results of antibacterial tests in vitro
derivatives with a primary amine at C-3 were found, and these compounds possessed good
antibacterial activity against Gram-positive bacteria, such as B. subtilis 168 and MRSA USA300.
The synergistic antibacterial assay showed that 5c and 6c could enhance the susceptibility of B. sub 168
and MRS USA300 to KAN and CHL (FICI < 0.5).
,
These results showed that ocotillol-type derivatives 5c and 6c are promising leads to develop
novel antibacterial agents against many community-associated and health care-associated infections
caused by MRSA USA300. Further studies will be conducted to determine the bactericidal functional
mechanisms of these compounds.
Acknowledgments: The authors are grateful to Shandong Provincial Natural Science Foundation, China
(No. ZR2015HQ007); Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources
(Guangxi Normal University), Ministry of Education of China (CMEMR2014-B07); Taishan Scholar Project to
Fenghua Fu and an Early Career Research Grant from the University of Newcastle (CM) for financial support.
Author Contributions: Yi Bi, Jin-Yi Xu and Cong Ma conceived and designed the experiments; Xian-Xuan Liu,
Heng-Yuan Zhang, Xiao Yang and Ze-Yun Liu performed the experiments; Yi Bi, Jing Lu, Peter John Lewis and
Qing-Guo Meng analyzed the data; Xian-Xuan Liu, Yi Bi, Cong-Zhi Wang and Chun-Su Yuan wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Not available.
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