Evolution and Antibacterial Evaluation of 8-Hydroxy-cycloberberine Derivatives as a Novel Family of Antibacterial Agents Against MRSA

Article abstract of DOI:10.3390/molecules24050984

Twenty-five new derivatives of 8-hydroxycycloberberine (1) were synthesized and evaluated for their activities against Gram-positive bacteria, taking 1 as the lead. Part of them displayed satisfactory antibacterial activities against methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA), as well as vancomycin-intermediate Staphylococcus aureus (VISA). Especially, compound 15a displayed an excellent anti-MRSA activity with MICs (minimum inhibitory concentrations) of 0.25–0.5 μg/mL, better than that of 1. It also displayed high stability in liver microsomes and whole blood, and the LD₅₀ value of over 65.6 mg·kg^?1 in mice via intravenous route, suggesting a good druglike feature. The mode of action showed that 15a could effectively suppress topo IV-mediated decatenation activity at the concentration of 7.5 μg/mL, through binding a different active pocket of bacterial topo IV from quinolones. Taken together, the derivatives of 1 constituted a promising kind of anti-MRSA agents with a unique chemical scaffold and a specific biological mechanism, and compound 15a has been chosen for the next investigation.

Full text of DOI:10.3390/molecules24050984

molecules
Article
Evolution and Antibacterial Evaluation of
8-Hydroxy-cycloberberine Derivatives as a Novel
Family of Antibacterial Agents Against MRSA
Yuan-Shuai Yang †, Wei Wei †, Xin-Xin Hu, Sheng Tang, Jing Pang , Xue-Fu You, Tian-Yun Fan *,
Yan-Xiang Wang * and Dan-Qing Song
Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, China; yangmarshal88@163.com (Y.-S.Y.);
weiwei082695@163.com (W.W.); huxinxin1985@163.com (X.-X.H.); tang13874204108@163.com (S.T.);
pangjing.pangjing@163.com (J.P.); 13311123098@163.com (X.-F.Y.); songdanqingsdq@hotmail.com (D.-Q.S.)
* Correspondence: fty1668@163.com (T.-Y.F.); wangyanxiang@imb.pumc.edu.cn (Y.-X.W.);
Tel.: +86-10-6316-5284 (T.-Y.F.); +86-10-6303-3012 (Y.-X.W.)
† These authors made equal contribution to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 14 February 2019; Accepted: 8 March 2019; Published: 11 March 2019
Abstract: Twenty-five new derivatives of 8-hydroxycycloberberine (
1) were synthesized and
evaluated for their activities against Gram-positive bacteria, taking as the lead. Part of them
1
displayed satisfactory antibacterial activities against methicillin-susceptible Staphylococcus aureus
(MSSA) and methicillin-resistant Staphylococcus aureus (MRSA), as well as vancomycin-intermediate
Staphylococcus aureus (VISA). Especially, compound 15a displayed an excellent anti-MRSA activity
with MICs (minimum inhibitory concentrations) of 0.25–0.5
displayed high stability in liver microsomes and whole blood, and the LD50 value of over 65.6
mg
kg−1 in mice via intravenous route, suggesting a good druglike feature. The mode of
action showed that 15a could effectively suppress topo IV-mediated decatenation activity at the
concentration of 7.5 g/mL, through binding a different active pocket of bacterial topo IV from
quinolones. Taken together, the derivatives of constituted a promising kind of anti-MRSA agents
µg/mL, better than that of 1. It also
·
µ
1
with a unique chemical scaffold and a specific biological mechanism, and compound 15a has been
chosen for the next investigation.
Keywords: cycloberberine; anti-MRSA; structure–activity relationship; topoisomerase IV
1. Introduction
Methicillin-resistant Staphylococcus aureus (MRSA), classified as serious threat pathogen, has
become a main cause of hospital and community-acquired infections across the world [1–4]. In the
US alone, at least two million illnesses and 23,000 deaths are caused by multidrug-resistant bacterial
infections every year according to data released by the Centers for Disease Control and Prevention
(CDC) [1]. Thereinto, around 19,000 deaths and 360,000 hospitalizations resulted from infections of
MRSA along with $3–4 billion in healthcare costs [
5]. Vancomycin, linezolid and daptomycin have been
used as the last resort for MRSA infections in the clinic. Unfortunately, vancomycin-intermediate S.
aureus (VISA), heteroresistant VISA (hVISA), vancomycin-resistant S. aureus (VRSA), linezolid-resistant
S. aureus and daptomycin-resistant S. aureus have been successively reported [6–10]. Especially,
MRSA/VISA have been listed as global priority pathogens of antibiotic-resistant bacteria released
by the World Health Organization (WHO) on Feb 27th 2017 [11], and the treatment of MRSA/VISA
infections has become severe concerns across the world. Therefore, great efforts must be made to
Molecules 2019, 24, 984; doi:10.3390/molecules24050984
www.mdpi.com/journal/molecules

Molecules 2019, 24, 984
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explore alternative agents witH-Novel structure scaffold or mechanism of action for the treatment of
invasive life-threatening infections arising from MRSA/VISA.
In the past several years, our team has been committed to the discovery and development of novel
antibacterial agents against multidrug-resistant pathogens, witH-Novel chemical entities and biological
mode of action [12
berberine (BBR) in our lab [14], was a special molecular scaffold against both methicillin-susceptible
S. aureus (MSSA) and MRSA, as well as VISA [15 16]. The primary structure–activity relationships
(SAR) demonstrated that introducing a suitable mono-substituent at the 8- or 13-position of CBBR
could significantly enhance the antibacterial activity against MSSA/MRSA [15 16], though CBBR itself
had no bactericidal activity at all, as shown in Figure 1. The top compound 8-hydroxycycloberberine
, Figure 1) displayed a promising effect against the tested strains such as MRSA with MIC values of
,13]. We first discovered that the cycloberberine (CBBR, Figure 1), generated from
,
,
(1
0.5−>64 µg/mL, which is much higher than that of levofloxacin (Lev) widely used in the clinic.
O
O
A
B
Cl
methylenedioxyring
wasopened
8-OH wasretained
as an active group
A
B
Cl
N
C
O
1
N
C
O
1
E
OCH3
OCH3
E
8
13
OH
8
varioussubstituents
wasintroduced
13
D
D
CBBR
MICs: >64 ug/mL
1
OCH3
MICs:0.5->64 ug/mL
Figure 1. Structures of CBBR and 1 as well as modification strategies.
Interestingly, compared with CBBR possessing a 8-OCH3 group, compound
1
with 8-OH showed
a greatly significant improvement in activity against both MSSA and MRSA, indicating that the 8-OH
function might play a critical role in enhancing the antibacterial effects. The unique chemical scaffold
and biological activity of
at developing these compounds into a new class of drug candidates against MRSA. Therefore, in the
present study, as depicted in Figure 1, taking compound as the lead, the 8-OH in CBBR was retained,
and various substituents were respectively attached on the 13-position, by which a series of new
1 encouraged us to continue a new round of SAR of its derivatives, aiming
1
1
derivatives were designed, synthesized and then evaluated for their antibacterial effect against MSSA
and MRSA. Additionally, the stability in liver microsomes and whole blood, toxicity in vivo as well as
the bactericidal mechanism studies of the representative compounds were carried out as well.
2. Results and Discussion
2.1. Chemistry
The synthetic routes to all twenty-five target compounds are displayed in Schemes 1 and 2
respectively. As shown in Scheme 1, palmatine was selectively reduced to dihydropalmatine (
89% yield using NaBH4 as a reducing agent in the presence of 5% NaOH/K2CO3 using a previous
procedure [14]. Compound was then reacted with 40% glyoxal in refluxing HOAc/CH3CN to give the
intermediate 13-acetaldehyde palmatine ( ), to which CH3OH/HCl (2/1 by vol.) was added directly to
complete the cyclization reaction and the desired product cyclopalmatine ( ) was obtained in an overall
yield of 62%. Then, compound
was heated at 195–210 ◦C under vacuum (20–30 mmHg) to get the
key intermediate . Finally, product was acquired with a combined 90% conversion via acidification
2) in
2
3
4
4
5
6
of compound 5 with concentrated HCl/EtOH (5/95 by vol.) by a classic keto-enol tautomerism.
As described in Scheme 2, CBBR was prepared from BBR using a previously reported
procedure [14]. Compound
60% H2SO4 in a yield of 62%. The demethylation of
8
was prepared via demethylenation of CBBR with benzene-1,3,5-triol and
yielded intermediate , which was converted
8
9
quickly to 1,2,8-trihydroxyl 10 in the presence of concentrated HCl/EtOH (5/95) in a combined 39%
yield. Dihydrocycloberberine (11) was prepared in 83% yield via reduction of CBBR witH-NaBH4 as a

Molecules 2019, 24, 984
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reducing agent in CH3OH in the presence of K2CO3 [15]. The compounds in series 12 were generated
in 24–47% yields from 11 and diverse substituted benzyl bromides at reflux temperature.
Scheme 1. Reagents and conditions: (a) NaBH4, 5% NaOH/K2CO3, CH3OH, room temperature, 3 h;
(
b
) 40% glyoxal, HOAc/CH3CN, reflux, 6 h; (
c
) CH3OH/HCl (2/1 by vol.), room temperature, 24 h,
◦
62% (over two steps); (
two steps).
d
) 20–30 mmHg, 195–210 C, 40 min; (
e
) HCl/EtOH (5/95 by vol.), 90% (over
Scheme 2. Reagents and conditions: (
HOAc, CH3CN, reflux, 6 h; (ii) CH3OH/HCl (2/1 by vol.), room temperature, 24 h, 67% (over two
steps); ( ) benzene-1,3,5-triol, 60% H2SO4; ( ) HCl/EtOH (5/95
) 20–30 mmHg, 195–210 oC, 40 min; (
by vol.), 39% (over two steps); ( ) NaBH4, K2CO3, CH3OH, room temperature, 12 h; ( ) substituted
) aliphatic aldehyde, HOAc, 80% EtOH,
a) NaBH4, 5% NaOH, K2CO3, CH3OH, r.t., 3 h; (b) (i) 40% glyoxal,
c
d
e
f
g
benzyl bromide, K2CO3 (2.0 eq.), CH3CN, reflux, 3–24 h; (
reflux, 8 h.
h
Similarly, compounds 12a
and acidified using concentrated HCl/EtOH (5/95) to produce the target compounds 13a
yields. Meanwhile, the products in series 14 were obtained in good yields from 11 and diverse
aliphatic aldehydes in HOAc and 80% EtOH at reflux temperature. The products 15a were generated
from 14a using a similar synthetic method as for compounds 13a . All the desired products were
–t
were heated at 195–210 ◦C for 40 min under vacuum (20–30 mmHg)
in good
–t
–
c
–c
–t
purified with flash column chromatography on silica gel using CH2Cl2 and CH3OH as eluents or
washed with 95% EtOH. The 1H-NMR, 13C-NMR and HRMS data of compound 15a is provided in
Supplementary Materials.

Molecules 2019, 24, 984
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2.2. Pharmacological Evaluation
2.2.1. SAR for Anti-Bacterial Activity
SAR study for antibacterial activity against drug-susceptible Gram-positive bacteria was initially
carried out, taking Lev and compound as reference drugs. The chemical structures of 25 target
1
compounds and their MIC values against a panel of drug-susceptible strains [17], including MSSA,
methicillin-susceptible S. epidermidis (MSSE), S. saprophyticus (S. s) and S. hominis (S. h), were
summarized in Table 1. Bacteria strains used in this study were from the ATCC collection and
clinical isolates in Chinese hospitals. The influence of the methylenedioxy ring was explored first, and
compounds
6 and 10 were created and tested. As shown in Table 1, both of them lost the antimicrobial
activities partially or completely, suggesting that methylenedioxy ring might be essential for the
activity against Gram-positive strains.
Table 1. Antimicrobial activities of the target compounds against drug-susceptible Gram-positive
strains (MIC (µg/mL) a).
MSSA c
15 b
MSSE c
S.s c
S.h c
R3
R1/R2
Code
ATCC
29213
ATCC
ATCC
15305
ATCC
35982
16-1 b
16-5 b
12228
CBBR
1
6
-
-
-
-
H
H
H
H
H
H
H
H
H
H
H
H
-
-
-
-
H
>64
0.5
>64
32
8
>64
8
>64
8
>64
4
>64
1
>64
32
8
>64
8
>64
4
>64
0.5
>64
8
16
>64
8
>64
8
4
>64
4
8
32
16
32
>64
16
8
>64
0.5
>64
16
8
>64
8
>64
4
2
>64
2
4
16
8
32
>64
32
16
4
32
4
16
32
0.25
1
>64
>64
>64
32
8
>64
16
>64
16
4
>64
16
16
64
32
64
>64
>64
16
16
>64
32
>64
1
>64
32
8
>64
8
>64
8
>64
16
16
>64
8
>64
8
4
>64
4
8
32
32
32
>64
>64
4
10
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
13p
13q
13r
13s
13t
15a
15b
15c
Lev
p-F
0
0
2 ,4 -(F)2
0
0
2 ,6 -(F)2
p-Cl
p-Br
4
>64
4
2
>64
4
4
>64
8
o-CH3
m-CH3
p-CH3
8
4
4
0
0
3 ,5 -(CH3)2 32
32
16
32
>64
>64
8
32
32
64
>64
64
16
8
64
8
32
64
0.5
2
p-tBu
p-OCF3
o-CN
m-CN
p-CN
o-NO2
m-NO2
p-NO2
H
H
-
-
-
32
32
>64
>64
8
8
>64
16
16
64
0.5
2
8
H
H
H
H
H
H
CH3
CO2CH3
CH2CH3
(CH2)2CH3
(CH2)3CH3
8
>64
8
4
8
64
4
32
64
0.5
2
64
16
16
>64
0.5
2
8
64
>64
1
8
16
64
0.5
2
4
4
0.25
8
8
4
0.125
0.125
0.126
0.125
1
0.125
a
b
c
MIC (
µ
g/mL), minimum inhibitory concentration; Strains isolated from patients in China. Methicillin-
susceptible S. aureus (MSSA), methicillin-susceptible S. epidermidis (MSSE), S. saprophyticus (S. s) and S. hominis (S. h).
Then, we moved SAR analysis to the influence of substituent at the 13-position in compound 1.
Our previous SAR results on CBBR indicated that the introduction of a 13-benzyl motif was
beneficial for the activity [16], therefore, various benzyl moieties with electron-donating and
electron-withdrawing groups on the benzene ring were respectively introduced into position 13,
and 20 new analogs 13a–t were constructed and tested. As described in Table 1, compound 13a
with a 13-benzyl displayed moderate antimicrobial activity with MICs of 8–16 µg/mL, while the

Molecules 2019, 24, 984
5 of 16
compounds 13b
–d
with fluoro atoms as an isostere of the hydrogen atom lost their potency to varying
degrees. However, the compounds 13e and 13f with p-chloro or p-bromo groups showed preferable
antimicrobial activities with MIC values of 2–8 g/mL. Compounds 13h and 13i bearing m-methyl and
p-methyl gave potent activities with MICs of 2–16 g/mL, less than that of lead . Next, compounds
13o and 13p with p-CN and o-NO2 respectively displayed moderate antimicrobial activities with most
MIC values in the 4–16 g/mL range, while other compounds with electron-withdrawing groups
µ
µ
1
µ
showed partially or completely diminished antimicrobial activities.
Furthermore, a methyl or methyloxycarbonyl group was introduced to the α-C atom of ring E to
generate 13s and 13t, respectively, and both of them showed decreased activity to different degrees.
Finally, an ethyl, n-propyl or n-butyl were respectively introduced at the 13-position of , whereby
three new analogs 15a were prepared and investigated. All of them exhibited good activities against
the tested strains with MIC values of 0.25–16 g/mL, and compound 15a showed especially potent
activities, with MIC values of 0.25–1.0 g/mL, superior or similar to those of the lead compound
1
–
c
µ
µ
1.
The results thus indicate that introducing a small-sized substituent at the 13-position might enhance
the activity.
All target compounds were then measured for their antibacterial activities against drug-resistant
organisms, such as MRSA/VISA and methicillin-resistant S. epidermidis strains (MRSE). As listed
in Table 2, their potencies against MRSE/MRSA/VISA strains were basically consistent with those
against drug-susceptible ones. Most of compounds showed potential anti-MRSA and anti-VISA effects
with MIC values ranging from 1 to 64
promising effects, with MIC values ranging from 0.25 to 8
an excellent effect against MRSA and VISA with MIC values of 0.25–0.5
µ
g/mL. Compounds 13e
g/mL. Especially, compound 15a displayed
g/mL, better than that of 1,
–f, 13h–i, 13o–p and 15a–c displayed
µ
µ
and then it was selected as the representative compound for the next investigation.
Table 2. Antibacterial activities of the target compounds against drug-resistant Gram-positive strains
(MIC (µg/mL)).
MRSA
ATCC
VISA
MRSE
Code
ATCC-B
ATCC
ATCC ATCC
700698 700699
13-18 13-23 16-34
13-3
16-5
43300 AA-1708 a
-BAA-976 b
CBBR
1
6
>64
1
>64
1
>64
0.5
>64
16
8
>64
8
>64
4
>64
0.5
>64
32
16
>64
8
>64
8
4
>64
0.5
>64
16
8
>64
4
>64
8
1
>64
4
2
16
8
32
>64
>64
8
4
>64
4
16
64
0.25
2
8
16
>64
1
>64
1
>64
32
8
>64
8
>64
4
>64
0.5
>64
32
8
>64
8
>64
8
>64
>64
>64
32
8
>64
0.5
>64
16
16
>64
8
32
4
2
64
4
2
16
8
32
>64
32
8
>64
32
16
>64
16
>64
8
4
>64
8
>64
64
16
>64
8
>64
8
4
>64
8
8
32
32
32
>64
>64
8
>64
64
16
>64
8
>64
4
2
10
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
13p
13q
13r
13s
13t
15a
15b
15c
Lev
>64
8
>64
64
16
64
4
2
16
8
32
>64
64
16
32
64
>64
32
64
1
4
>64
4
2
>64
4
1
>64
4
>64
8
>64
8
4
8
8
16
32
32
64
>64
>64
4
4
4
32
16
32
>64
>64
8
16
>64
32
16
64
2
16
16
32
>64
>64
8
4
>64
8
16
>64
0.25
2
8
64
32
16
64
>64
>64
16
8
64
32
32
64
0.5
2
32
32
64
>64
>64
4
8
>64
4
32
64
0.5
4
4
32
32
32
32
>64
>64
8
4
64
4
32
64
0.25
2
4
8
8
>64
8
8
8
>64
64
16
64
0.5
4
32
16
32
32
0.25
2
8
64
0.5
4
8
64
4
4
32
4
16
32
4
32
4
8
8
0.125
a
b
mupA positive (isolates with mupirocin resistance). clindamycin-resistant S. aureus.

Molecules 2019, 24, 984
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2.2.2. Time-kill Curve Study of 15a
To further confirm the antibacterial activities of this kind of compounds, the time–kill curve assay
of compound 15a for MRSA strain isolated from Chinese patients was carried out subsequently with
DMSO as a negative control. As shown in Figure 2, distinct reductions of the viable cell counts were
observed after 4 h exposure of the bacteria to 15a at concentrations of 2
it exhibited significant time-dependent action and the maximum reduction in viable counts was
over 6 log10CFU/mL in 12 h for the 8 MIC, therefore, it was determined that 15a was a marginal
×
and 8 MIC. Especially,
×
×
bactericide [18] at the concentration of 8× MIC.
10
9
8
1/2× MIC
2 × MIC
7
8 × MIC
control
6
5
4
3
2
0
2
4
8
12
Time(h)
◦
Figure 2. Time-kill curve assay of compound 15a over a 12 h incubation period at 37 C against MRSA
18-4 (MIC = 0.5 µg/mL). Samples were taken at 2, 4, 8 and 12 h to determine viable bacterial numbers.
2.2.3. Evaluation of Metabolic Stability and Acute Toxicity Assay of Compound 15a
The metabolic stability of compound 15a in liver microsomes was conducted in human and rat as
well as mouse with the clinical reagent testosterone (Tes) as reference. As depicted in Table 3, compared
to Tes, compound 15a showed an obviously longer half-life (T1/2) and residual ratio, as well as lower
microsomal intrinsic clearance in all three species. Especially, the T1/2 of compound 15a was over
105 min and the remaining ratio was still 70.6% after 60 min in human liver microsomes, indicating a
good characteristic of metabolic stability.
Table 3. Metabolic stability of tested compound in human, rat and mouse liver microsomes a.
Code
15a
Species
T1/2 (min)
CL Int (mic) (µL/min/mg)
Remaining Ratio (%) (T = 60 min)
Human
Rat
Mouse
105.3
50.9
18.9
13.2
27.3
73.4
70.6
47.9
10.4
Human
Rat
Mouse
12.9
0.6
5.5
107.2
2349.8
253.9
3.9
0
0
Tes
a
T1/2 = half-life. CL int (mic) = microsomal intrinsic clearance. Liver weight: 20 g/kg for humans.
Next, a stability assay of compound 15a was carried out in whole blood in vitro, taking enalapril
(Ems) [15 16] as the positive control. As indicated in Table 4, as expected, compound 15a gave an
,
exciting stability profile and the remaining ratio was still over 85.4% after 24 h in blood, while Ems
was completely decomposed within three hours. The results suggest that 15a might possess an ideal
in vivo metabolic stability.

Molecules 2019, 24, 984
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Table 4. Metabolic stability of 15a in whole blood.
Time (h)
0
0.5
1
3
7
24
15a Rr (%)
Ems Rr (%)
100
100
98.9
37.5
98.0
9.1
95.8
0
93.1
0
85.4
0
Rr = Remaining ratio.
Then, acute toxicity test on compound 15a was performed in Kunming mice through intravenous
route. The results indicated that compound 15a gave a half-lethal dose (LD50) value of 65.6 mg ,
kg−1
·
suggesting a good safety profile.
2.2.4. Preliminary Mechanism of Compound 15a
Taking into account the planar rigid structure similarity between 15a and quinolones, we initially
examined its effects on gyrase and topoisomerase (topo) IV activities of S. aureus by gyrase-mediated
supercoiling of relaxed DNA assay and topo IV-mediated decatenation of kinetoplast DNA (kDNA)
assay [19,20], respectively. As shown in Figure 3, topo IV-mediated decatenation activity was
significantly inhibited in a concentration-dependent manner after treatment of 15a. It could partially
inhibit topo IV activity at the concentration of 7.5
decatenation reaction at the concentration of 15
µ
g/mL (lane 5), and block almost completely the
µg/mL (lane 7). However, inhibition activity on
gyrase-mediated supercoiling was not distinctly observed after treatment with 15a at even upped
to 100 g/mL. These results indicated that 15a could effectively suppress the topo IV-mediated
µ
decatenation activity, but a different mode of action from quinolones.
Figure 3. Inhibition of S. aureus topo IV. In all panels, enzyme and 15a are absent (-) in lane 1, enzyme
present (+) and 15a absent in lane 2, and enzyme (arrow) and 15a present in lanes 3–10 with different
concentration. Decatenation activity of topo IV was partially blocked by 15a (lanes 4, 5, 6) and
completely blocked by 15a (lanes 7, 8, 9, 10).
2.2.5. Molecular Docking Study of Key Compound 15a
It was reported that topo IV enzyme of Gram-positive bacteria possesses at least two active
pockets, one is the well-known quinolone-binding cavity and the other is kibdelomycin-binding
site [21–23]. In view of the good activity of compound 15a against quinolone-resistant MRSA or VISA,
we performed molecular docking between the topo IV binding site of kibdelomycin and compound
15a interactions using Discovery Studio 4.5 software (Edition 4.5; BIOVIA: San Diego, CA, USA,
2015) [17,24]. As expected, compound 15a fit well in the active hydrophobic pocket of the binding
site (Figure 4A, brown area). One conventional hydrogen bond with GLU53, two attractive charges
formed by nitrogen ions with ASP76 and GLU53, van der Waals forces and hydrophobic interactions
(Figure 4B) contributed together to the strong interactions. The results suggested that 15a might bind
to the kibdelomycin-pocket site on topo IV, a different mode of action from the quinolones widely
used in the clinic.

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Figure 4.
colored regions correspond to negatively charged, positively charged, and neutral areas, respectively.
) Binding modes within the receptor S. aureus topo IV active pocket. These figures were produced
(A) Solid surface map of the interaction pocket with compound 15a. Red, blue, and white
(
B
using the Discovery Studio 4.5 software. The receptor structure is shown in surface form. Key bonds
are indicated by dashed lines between the atoms involved, and the colors of key bonds and residues
are shown according to the interaction mode (van der Waals, light green; attractive charge, orange;
hydrogen bond, green; carbon hydrogen bond, celadon; amide-π stacked, amaranth; alkyl and π-alkyl,
mauve pink).
3. Experimental Section
3.1. Apparatus, Materials, and Analysis Reagents
Melting points (m.p.) were obtained with a CXM-300 melting point apparatus (Shanghai
Changfang Optical Instrument Co., Ltd., Shanghai, China) and are uncorrected. The 1H-NMR spectra
was recorded on an Inova 500 or 600 MHz spectrometer (Varian, San Francisco, CA, USA) and
13C-NMR on an Avance III 400, 500 or 600 spectrometer (Bruker, Zürich, Switzerland), with Me4Si as
the internal standard. High-resolution mass spectra (HRMS-ESI) data was recorded on an Autospec
UItima-TOF mass spectrometer (Micromass UK Ltd., Manchester, UK). Flash chromatography (particle
size 0.038 mm) was performed on a CombiflashRf 200 system (Teledyne, Lincoln, NE, USA).
3.2. Chemistry
3.2.1. Synthesis of 1,2-Dimethoxy-8-hydroxy-9-methoxycycloberberine Chloride (6)
Palmatine (7.7 g, 20 mmol) and K2CO3 (8.3 g, 60 mmol) were dissolved in CH3OH (250 mL),
and then 5% NaOH (10 mL) solution containing NaBH4 (0.83 g, 22 mmol) was added dropwise.
The reaction mixture was stirred at room temperature for 3 h. After the reaction was complete, the
precipitated solid was filtered, washed with distilled water (100 mL) and 80% EtOH (100 mL) to give
yellow green solid
stirred solvent mixture of CH3CN (160 mL) and HOAc (40 mL), which was refluxed for 6 h to prepare
compound . The solvent was evaporated under vacuum, and 2 N HCl/CH3OH (100 mL) was added
to the residue. The reaction mixture was stirred at room temperature for 24 h, then evaporated under
vacuum and recrystallized from 95% EtOH to obtain scarlet solid
; Yield: 62%, m.p.: 180–182 ◦C. 1H-
NMR (DMSO-d6) 10.15 (s, 1H), 8.87 (d, J = 9.2 Hz, 1H), 8.83 (d, J = 9.2 Hz, 1H), 8.40 (d, J = 9.2 Hz, 1H),
8.27 (d, J = 9.2 Hz, 1H), 7.70 (s, 1H), 5.26 (t, J = 6.8 Hz, 2H), 4.15 (s, 3H), 4.09 (s, 3H), 4.03 (s, 3H), 3.94 (s,
3H), 3.65 (t, J = 6.8 Hz, 2H). ESI-MS m/z: 376.15. Then,
(3.7 g, 9.1 mmol) was heated at 195–210 ◦C
in a dry oven under vacuum (20–30 mmHg) for 40 min. After completion of the reaction, the crude
material was obtained, which was used without further purification. Next was acidified with
2. Intermediate 2 (5.6 g, 16 mmol) was then reacted with 40% glyoxal (3 mL) in a
3
4
δ
4
5
5
concentrated HCl/EtOH (5:95 by vol.) for 10 min. Then the mixture was evaporated under vacuum
and purified with flash column chromatography on silica gel using a CH2Cl2 and CH3OH gradient as

Molecules 2019, 24, 984
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eluent to obtain the red solid compound
6
; yield: 90%; m.p.: 176–178 ◦C; 1H-NMR (DMSO-d6)
δ
10.07
(s, 1H), 8.78 (d, J = 9.6 Hz, 1H), 8.40–8.32 (m, 2H), 8.06 (d, J = 8.4 Hz, 1H), 7.67 (s, 1H), 5.18 (t, J = 6.6 Hz,
2H), 4.04 (s, 6H), 3.95 (s, 3H), 3.62 (t, J = 6.6 Hz, 2H); 13C-NMR (DMSO-d6)
δ
151.0, 146.9, 146.1, 141.3,
128.4, 128.0 (2), 127.4, 127.3, 123.8, 123.7, 121.8, 121.0, 116.0, 115.8, 114.5, 112.6, 61.2, 56.8, 56.6, 54.9, 26.3;
HRMS: calcd for C22H20NO4Cl [M − Cl]+: 362.1387, found: 362.1390.
3.2.2. Synthesis of 1,2,8-Trihydroxy-9-methoxycycloberberine Chloride (10)
CBBR (3.95 g, 10 mmol) and phloroglucin (5.0 g, 40 mmol) were added into 60% H2SO4 (20 mL),
◦
and the reaction mixture was stirred at 90 C for 10 h. Then the mixture was cooled, poured into
saturated sodium chloride solution (10 mL) and filtered. The residue was washed with water and
redissolved in 1 N NaOH (60 mL). After that, 2 N HCl (60 mL) was added and the mixture filtered.
The residue was purified with flash column chromatography on silica gel using CH2Cl2 and CH3OH
◦
to obtain the intermediate
8
in 62% yield. Next
8
was heated at 195–210 C in a dry oven under
vacuum (20–30 mmHg) for 40 min to create the crude material
9
, which was acidified in concentrated
HCl/EtOH (5:95 by vol.) for 10 min. Then the mixture was filtered and the residue was washed with
water, CH2Cl2 and EtOH to afford the title compound 10 as a black-purple solid; yield: 39%; m.p:
191–193 ◦C; 1H-NMR (DMSO-d6)
δ
10.30 (s, 1H), 10.00 (s, 1H), 9.55 (s, 1H), 8.67 (d, J = 9.6 Hz, 1H),
8.44–8.35 (m, 2H), 8.08 (d, J = 9.0 Hz, 1H), 7.29 (s, 1H), 5.11 (t, J = 6.6 Hz, 2H), 4.03 (s, 3H), 3.48 (t,
J = 6.6 Hz, 2H); 13C-NMR (DMSO-d6)
146.2, 145.5, 144.1, 137.9, 128.6, 127.6, 124.2, 123.9 (3), 122.1,
δ
121.2, 118.8, 117.3, 115.7, 114.8, 56.7, 55.9, 55.2, 25.6; HRMS: calcd for C20H16NO4Cl [M
found: 334.1079.
−
Cl]+: 334.1074,
3.2.3. General Procedure for the Synthesis of 13a–t
To a stirred solution of compound 11 (361 mg, 1.0 mmol, 1.0 equiv) and K2CO3 (2.0 equiv)
in anhydrous CH3CN (20 mL) were added various benzyl bromides (15.0 equiv). The reaction
mixture was refluxed for 3–24 h, cooled, the mixture was filtered and the filtrate was purified by flash
column chromatography on silica gel using a gradient of CH2Cl2 and CH3OH as eluent to obtain the
compounds 12a–t. Then 12a–t
were heated at 195–210 ◦C in a dry oven under vacuum (20–30 mmHg)
for 40 min to create the crude materials, which were acidified in concentrated HCl/EtOH (5:95 by
vol.) for 10 min. Then the mixture was evaporated under vacuum and purified by flash column
chromatography on silica gel using a CH2Cl2 and CH3OH gradient as eluent to obtain the desired
compounds 13a–t.
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-benzylcycloberberine chloride (13a): red solid; yield: 78%; m.p.:
◦
248–250 C; 1H-NMR (CDCl3/CD3OD)
δ
9.91 (s, 1H), 8.20 (s, 1H), 8.11 (d, J = 9.0 Hz, 1H), 7.99 (d,
J = 9.0 Hz, 1H), 7.39 (s, 1H), 7.36–7.31 (m, 2H), 7.29–7.24 (m, 3H), 6.22 (s, 2H), 5.14 (t, J = 6.6 Hz, 2H),
4.78 (s, 2H), 4.11 (s, 3H), 3.66 (t, J = 6.6 Hz, 2H); 13C-NMR (CDCl3/CD3OD)
148.6, 147.2, 146.7, 143.3,
δ
140.5, 140.0, 129.6 (2), 129.2 (2), 128.8, 128.3, 127.1 (2), 126.8, 124.7, 123.6, 121.5, 119.4, 118.1, 116.8, 114.2,
111.3, 102.9, 57.2, 56.7, 41.1, 28.1; HRMS: calcd for C28H22NO4Cl [M – Cl]+: 436.1543, found: 436.1535.
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-p-fluorobenzylcycloberberine chloride (13b): red solid; yield:
◦
1
83%; m.p.: 203–205 C; H-NMR (CDCl3/CD3OD)
1H), 8.04 (d, J = 9.0 Hz, 1H), 7.41 (s, 1H), 7.30–7.24 (m, 2H), 7.04–6.98 (m, 2H), 6.19 (s, 2H), 5.12 (t,
J = 6.6 Hz, 2H), 4.74 (s, 2H), 4.11 (s, 3H), 3.64 (t, J = 6.7 Hz, 2H); 13C-NMR (CDCl3/CD3OD)
162.8,
δ 9.93 (s, 1H), 8.33 (s, 1H), 8.22 (d, J = 9.0 Hz,
δ
149.0, 147.6, 147.2, 146.7, 143.3, 140.3, 136.9, 131.5, 131.4, 129.4, 128.7, 127.9, 125.1, 123.9, 122.2, 119.5,
118.7, 117.2, 116.1, 116.0, 114.7, 111.6, 103.4, 57.4, 57.0, 40.5, 28.3; HRMS: calcd for C28H21NO4FCl
[M − Cl]+: 454.1449, found: 454.1447.
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-20,40-difluorobenzylcycloberberine chloride (13c): red solid;
yield: 78%; m.p.: 218–220 ◦C; 1H-NMR (DMSO-d6)
δ
11.56 (s, 1H), 10.09 (s, 1H), 8.54 (s, 1H), 8.29 (d,
J = 9.0 Hz, 1H), 8.15 (d, J = 9.0 Hz, 1H), 7.56 (s, 1H), 7.30–7.26 (m, 1H), 7.00–6.93 (m, 2H), 6.19 (s, 2H),
5.17 (t, J = 6.6 Hz, 2H), 4.67 (s, 2H), 4.07 (s, 3H), 3.59 (t, J = 6.6 Hz, 2H); 13C-NMR (DMSO-d6)
161.7,
δ

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160.1, 147.3, 147.0, 146.3, 146.2, 141.7, 136.0, 131.2, 128.6, 127.7, 127.4, 124.9, 123.7, 122.4, 122.3, 118.1,
117.6, 116.3, 114.0, 111.8, 111.0, 104.2, 102.5, 57.4, 55.4, 32.7, 26.9; HRMS: calcd for C28H20NO4F2Cl
[M − Cl]+: 472.1355, found: 472.1346.
1,2-Methylenedioxy-8-hydr◦oxy-9-methoxy-13-20,60-difluorobenzylcycloberberine chloride (13d): red solid;
yield: 73%; m.p.: 214–216 C; 1H-NMR (DMSO-d6)
δ 11.57 (s, 1H), 10.07 (s, 1H), 8.11 (d, J = 9.0 Hz, 1H),
7.90 (s, 1H), 7.84 (d, J = 9.0 Hz, 1H), 7.60 (s, 1H), 7.56–7.50 (m, 1H), 7.27–7.20 (m, 2H), 6.33 (s, 2H), 5.15
(t, J = 6.6 Hz, 2H), 4.77 (s, 2H), 4.02 (s, 3H), 3.59 (t, J = 6.6 Hz, 2H); 13C-NMR (DMSO-d6)
161.5 (2),
147.5, 147.0, 146.5, 146.2, 142.1, 136.3, 130.3, 128.5, 127.7, 127.1, 125.0, 121.8, 118.7, 117.9, 117.4, 116.3,
δ
114.3, 113.1, 112.4, 112.3, 111.1, 102.6, 57.3, 55.4, 27.6, 26.9; HRMS: calcd for C28H20NO4F2Cl [M
472.1355, found: 472.1342.
−
Cl]+:
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-p-chlorobenzylcycloberberine chloride (13e): red solid; yield:
88%; m.p.: 199–201 ◦C; 1H-NMR (CDCl3/CD3OD)
9.93 (s, 1H), 8.27 (s, 1H), 8.19 (d, J = 8.4 Hz, 1H),
8.02 (d, J = 8.4 Hz, 1H), 7.39 (s, 1H), 7.29 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H), 6.21 (s, 2H), 5.14 (t,
J = 6.6 Hz, 2H), 4.75 (s, 2H), 4.12 (s, 3H), 3.67 (t, J = 6.6 Hz, 2H); 13C-NMR (CDCl3/CD3OD)
148.6,
147.3, 146.8, 146.1, 143.1, 139.7, 138.9, 132.9, 131.0 (2), 129.2 (2), 128.9, 128.3, 127.0, 124.9, 123.6, 121.9,
δ
δ
119.2, 118.2, 116.8, 114.4, 111.4, 103.0, 57.3, 56.8, 40.5, 28.1; HRMS: calcd for C28H21NO4Cl2 [M
470.1154, found: 470.1144.
−
Cl]+:
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-p-bromobenzylcycloberberine chloride (13f): red solid; yield:
88%; m.p.: 207–209 ◦C; 1H-NMR (CDCl3/CD3OD)
9.93 (s, 1H), 8.22 (s, 1H), 8.15 (d, J = 8.4 Hz, 1H),
7.99 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 7.8 Hz, 2H), 7.38 (s, 1H), 7.15 (d, J = 8.4 Hz, 2H), 6.21 (s, 2H), 5.15 (t,
J = 6.6 Hz, 2H), 4.73 (s, 2H), 4.12 (s, 3H), 3.68 (t, J = 6.6 Hz, 2H); 13C-NMR (CDCl3/CD3OD)
148.4,
147.1, 146.6, 145.7, 142.9, 139.4, 139.0, 132.1 (2), 131.1 (2), 128.6, 128.0, 126.6, 124.6, 123.4, 121.6, 120.8,
δ
δ
119.0, 117.9, 116.5, 114.1, 111.3, 102.8, 57.2, 56.6, 40.4, 28.0; HRMS: calcd for C28H21BrNO4Cl [M
514.0648, found: 514.0653.
−
Cl]+:
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-o-methylbenzylcycloberberine chloride (13g): red solid; yield:
83%; m.p.: 203–205 ◦C; 1H-NMR (DMSO-d6)
11.54 (s, 1H), 10.09 (s, 1H), 8.34 (s, 1H), 8.15 (s, 2H),
δ
7.57 (s, 1H), 7.29 (d, J = 7.8 Hz, 1H), 7.16 (t, J = 7.2 Hz, 1H), 7.05 (t, J = 7.2 Hz, 1H), 6.73 (d, J = 7.5 Hz,
1H), 6.16 (s, 2H), 5.18 (t, J = 6.6 Hz, 2H), 4.66 (s, 2H), 4.06 (s, 3H), 3.34 (s, 2H), 2.38 (s, 3H); 13C-NMR
(DMSO-d6) δ 146.8, 146.2, 145.8, 145.7, 141.4, 138.2, 137.2, 135.8, 129.9, 129.5, 127.9, 127.7, 126.9, 126.1,
125.9, 124.4, 121.8, 121.1, 118.1, 117.1, 115.8, 113.2, 110.4, 101.9, 56.8, 54.9, 37.1, 26.4, 19.2; HRMS: calcd
for C29H24NO4Cl [M − Cl]+: 450.1700, found: 450.1687.
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-m-methylbenzylcycloberberine chloride (13h): red solid; yield:
◦
79%; m.p.: 203–205 C; 1H-NMR (DMSO-d6)
δ 11.52 (s, 1H), 10.07 (s, 1H), 8.68 (s, 1H), 8.44 (d, J = 9.0 Hz,
1H), 8.17 (d, J = 9.0 Hz, 1H), 7.55 (s, 1H), 7.20–7.10 (m, 2H), 7.06 (d, J = 7.8 Hz, 1H), 6.99 (d, J = 7.2 Hz,
1H), 6.27 (s, 2H), 5.15 (t, J = 6.6 Hz, 2H), 4.68 (s, 2H), 4.08 (s, 3H), 3.58 (t, J = 6.6 Hz, 2H), 2.23 (s, 3H);
13C-NMR (DMSO-d6)
δ 146.7, 146.2, 145.7, 145.7, 141.2, 140.0, 137.8, 137.3, 129.5, 129.0, 128.2, 127.8,
127.0, 126.7, 125.5, 124.3, 122.0, 121.8, 117.7, 117.1, 115.8, 113.7, 110.4, 101.8, 56.9, 54.9, 39.7, 26.4, 21.0;
HRMS: calcd for C29H24NO4Cl [M − Cl]+: 450.1700, found: 450.1687.
1,2-Methylenedioxy-◦8-hydroxy-9-methoxy-13-p-methylbenzylcycloberberine chloride (13i): red solid; yield:
81%; m.p.: 229–231 C; 1H-NMR (DMSO-d6)
δ 11.51 (s, 1H), 10.06 (s, 1H), 8.67 (s, 1H), 8.43 (d, J = 9.0 Hz,
1H), 8.16 (d, J = 9.0 Hz, 1H), 7.55 (s, 1H), 7.17 (d, J = 7.8 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H), 6.26 (s, 2H), 5.15
(t, J = 6.6 Hz, 2H), 4.66 (s, 2H), 4.08 (s, 3H), 3.57 (t, J = 6.6 Hz, 2H), 2.22 (s, 3H); 13C-NMR (DMSO-d6)
δ
147.3, 146.7, 146.2, 146.2, 141.7, 138.6, 137.6, 135.5, 129.4 (2), 128.8 (2), 128.4, 127.6, 127.6, 124.8, 122.5,
122.2, 118.2, 117.7, 116.3, 114.3, 110.9, 102.4, 57.4, 55.4, 39.3, 27.0, 21.0; HRMS: calcd for C29H24NO4Cl
[M − Cl]+: 450.1700, found: 450.1704.
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-30,50-dimethylbenzylcycloberber ine chloride (13j): red solid;
yield: 83%; m.p.: 218–220 ◦C; 1H-NMR (DMSO-d6)
δ 11.48 (s, 1H), 10.03 (s, 1H), 8.64 (s, 1H), 8.42 (d,

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J = 9.0 Hz, 1H), 8.15 (d, J = 9.0 Hz, 1H), 7.53 (s, 1H), 6.87 (s, 2H), 6.78 (s, 1H), 6.25 (s, 2H), 5.12 (t,
J = 6.6 Hz, 2H), 4.61 (s, 2H), 4.06 (s, 3H), 3.55 (t, J = 6.6 Hz, 2H), 2.15 (s, 6H); 13C-NMR (DMSO-d6)
δ
146.7, 146.1, 145.7 (2), 141.2, 139.9, 137.9, 137.1, 129.5, 127.8, 127.5, 127.1, 127.0, 126.2 (2), 124.2, 122.0,
121.8, 117.7, 117.1, 115.8, 113.8, 110.4, 101.8, 56.9, 54.9, 39.5, 26.4, 20.9 (2); HRMS: calcd for C30H26NO4Cl
[M − Cl]+: 464.1856, found: 464.1848.
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-p-tert-butylmethylbenzylcyclo berberine chloride (13k): red solid;
yield: 77%; m.p.: 212–214 ◦C; 1H-NMR (DMSO-d6)
δ
11.51 (s, 1H), 10.06 (s, 1H), 8.73 (s, 1H), 8.48 (d,
J = 9.0 Hz, 1H), 8.17 (d, J = 9.0 Hz, 1H), 7.56 (s, 1H), 7.30–7.21 (m, 4H), 6.29 (s, 2H), 5.14 (t, J = 6.6 Hz,
2H), 4.68 (s, 2H), 4.09 (s, 3H), 3.54 (t, J = 6.6 Hz, 2H), 1.21 (s, 9H); 13C-NMR (DMSO-d6)
148.3, 146.7,
δ
146.1, 145.7, 141.1, 138.1, 137.2, 132.5, 128.0 (2), 127.8, 127.2, 127.0, 125.0 (2), 124.2, 122.0, 121.8, 117.6,
117.2, 115.8, 113.8, 110.3, 101.8, 56.9, 54.9, 48.5, 38.5, 31.0 (3), 26.4; HRMS: calcd for C32H30NO4Cl
[M − Cl]+: 492.2169, found: 492.2158.
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-p-trifluoromethoxybenzylcyclo berberine chloride (13l): red solid;
yield: 77%; m.p.: 185–187 ◦C; 1H-NMR (DMSO-d6)
δ
11.53 (s, 1H), 10.08 (s, 1H), 8.78 (s, 1H), 8.48 (d,
J = 9.0 Hz, 1H), 8.17 (d, J = 9.0 Hz, 1H), 7.56 (s, 1H), 7.42 (d, J = 7.8 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H), 6.26
(s, 2H), 5.15 (t, J = 6.6 Hz, 2H), 4.73 (s, 2H), 4.09 (s, 3H), 3.58 (t, J = 6.6 Hz, 2H); 13C-NMR (DMSO-d6)
δ
146.8, 146.6, 146.3, 145.7 (2), 141.0, 139.8, 137.1, 130.1 (2), 129.5, 127.9, 127.2, 127.0, 124.3, 122.2, 122.0,
120.9 (2), 117.4, 117.2, 115.8, 113.8, 110.4, 101.9, 56.9, 54.9, 38.4, 26.4; HRMS: calcd for C29H21NO5F3Cl
[M − Cl]+: 520.1366, found: 520.1349.
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-o-cyanbenzylcycloberberine chloride (13m): red solid; yield:
83%; m.p.: 209–211 ◦C; 1H-NMR (CDCl3/CD3OD)
δ 9.96 (s, 1H), 8.27 (s, 1H), 8.14 (d, J = 9.0 Hz, 1H),
8.01 (d, J = 9.0 Hz, 1H), 7.80 (dd, J = 7.8, 1.2 Hz, 1H), 7.54 (td, J = 7.8, 1.2 Hz, 1H), 7.44 (t, J = 7.8 Hz, 1H),
7.40 (s, 1H), 7.19 (d, J = 7.8 Hz, 1H), 6.19 (s, 2H), 5.16 (t, J = 6.6 Hz, 2H), 4.98 (s, 2H), 4.12 (s, 3H), 3.68 (t,
J = 6.6 Hz, 2H); 13C-NMR (CDCl3/CD3OD)
δ 148.6, 147.4, 146.8, 146.3, 144.0, 142.9, 137.5, 133.9, 133.8,
130.1, 129.0, 128.2, 128.0, 127.0, 124.9, 123.4, 122.2, 118.9, 118.6, 118.1, 116.7, 114.2, 113.2, 111.5, 103.0,
57.2, 56.7, 39.5, 28.0; HRMS: calcd for C29H21N2O4Cl [M − Cl]+: 461.1496, found: 461.1502.
1,2-Methylenedioxy-8-hydroxy-9-dimethoxy-13-m-cyanbenzylcycloberberine chloride (13n): red solid; yield:
87%; m.p.: 189–191 ◦C; 1H-NMR (CD3OD)
δ
9.95 (s, 1H), 8.50 (s, 1H), 8.33 (d, J = 9.0 Hz, 1H), 8.07 (d,
J = 9.0 Hz, 1H), 7.58 (td, J = 9.0, 1.2 Hz, 2H), 7.55 (s, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.42 (s, 1H), 6.15 (s,
2H), 5.13 (t, J = 6.6 Hz, 2H), 4.81 (s, 2H), 4.11 (s, 3H), 3.64 (t, J = 6.6 Hz, 2H); 13C-NMR (CD3OD)
151.0,
δ
148.4, 146.9, 145.8, 142.5, 141.3, 138.2, 133.8, 132.3, 130.6, 129.8, 129.2, 128.5, 126.9, 126.3, 123.2, 121.7,
120.7, 119.0, 118.7, 118.5, 117.5, 112.6, 111.4, 102.5, 57.2, 56.8, 40.3, 27.6; HRMS: calcd for C29H21N2O4Cl
[M − Cl]+: 461.1496, found: 461.1495.
1,2-Methylenedioxy-8-hydroxy-9-dimethoxy-13-p-cyanbenzylcycloberberine chloride (13o): red solid; yield:
◦
83%; m.p.:197–199 C; 1H-NMR (DMSO-d6)
δ
11.54 (s, 1H), 10.09 (s, 1H), 8.78 (s, 1H), 8.46 (d, J = 9.0 Hz,
1H), 8.16 (d, J = 9.0 Hz, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.55 (s, 1H), 7.46 (d, J = 7.8 Hz, 2H), 6.21 (s, 2H),
5.16 (t, J = 6.6 Hz, 2H), 4.78 (s, 2H), 4.09 (s, 3H), 3.58 (t, J = 6.6 Hz, 2H); 13C-NMR (DMSO-d6)
146.8,
146.4, 146.2, 145.8, 145.7, 140.9, 136.0, 132.2 (2), 129.31 (2), 128.0, 127.2, 127.0, 124.3, 122.5, 121.9, 118.8,
δ
117.4, 117.1, 115.8, 113.8, 110.5, 108.9, 101.9, 56.9, 54.9, 39.2, 26.4; HRMS: calcd for C29H21N2O4Cl [M
Cl]+: 461.1496, found: 461.1500.
−
1,2-Methylenedioxy-◦8-hydroxy-9-methoxy-13-o-nitrobenzylcycloberberine chloride (13p): red solid; yield:
82%; m.p.: 199–201 C; 1H-NMR (DMSO-d6)
δ 11.57 (s, 1H), 10.11 (s, 1H), 8.69 (s, 1H), 8.35 (d, J = 9.0 Hz,
1H), 8.18 (d, J = 9.0 Hz, 1H), 8.05 (dd, J = 7.8, 1.2 Hz, 1H), 7.56–7.46 (m, 3H), 7.07 (dd, J = 7.8, 1.2 Hz,
1H), 6.04 (s, 2H), 5.17 (t, J = 6.6 Hz, 2H), 4.88 (s, 2H), 4.08 (s, 3H), 3.58 (t, J = 6.6 Hz, 2H); 13C-NMR
(DMSO-d6) δ 149.7, 147.3, 147.1, 146.4, 146.3, 141.4, 135.9, 134.8, 133.7, 131.0, 130.1, 128.6, 128.1, 127.8,
127.5, 124.9, 123.1, 122.3, 118.0, 117.6, 116.4, 114.2, 111.0, 102.3, 57.4, 55.4, 36.7, 26.9; HRMS: calcd for
C28H21N2O6Cl [M − Cl]+: 481.1394, found: 481.1381.

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1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-m-nitrobenzylcycloberberine chloride (13q): red solid; yield:
89%; m.p.: 186–188 ◦C; 1H-NMR (CDCl3/CD3OD)
9.97 (s, 1H), 8.47 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H),
δ
8.12–8.08 (m, 2H), 8.05 (d, J = 9.0 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.54 (t, J = 8.4 Hz, 1H), 7.40 (s,
1H), 6.20 (s, 2H), 5.16 (t, J = 6.6 Hz, 2H), 4.90 (s, 2H), 4.13 (s, 3H), 3.68 (t, J = 6.6 Hz, 2H); 13C-NMR
(CDCl3/CD3OD) δ 149.2, 148.7, 147.5, 146.9, 146.4, 142.8 (2), 138.2, 135.9, 130.2, 129.2, 128.4, 127.2, 125.0,
124.0, 123.7, 122.7, 122.2, 119.0, 118.3, 116.9, 114.5, 111.6, 103.0, 57.3, 56.8, 40.7, 28.1; HRMS: calcd for
C28H21N2O6Cl [M − Cl]+: 481.1394, found: 481.1392.
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-p-nitrobenzylcycloberberine chloride (13r): red solid; yield:
85%; m.p.: 186–188 ◦C; 1H-NMR (CDCl3/CD3OD)
δ
9.98 (s, 1H), 8.49 (s, 1H), 8.33 (d, J = 9.0 Hz, 1H),
8.16 (s, 1H), 8.15 (s, 1H), 8.07 (d, J = 9.0 Hz, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.41 (s, 1H), 6.17 (s, 2H),
5.15 (t, J = 6.6 Hz, 2H), 4.91 (s, 2H), 4.13 (s, 3H), 3.66 (t, J = 6.6 Hz, 2H); 13C-NMR (CDCl3/CD3OD)
148.9, 148.8, 147.6 (2), 147.1, 146.7, 143.0, 138.2, 130.4 (2), 129.4, 128.6, 127.6, 125.1, 124.4 (2), 123.8, 123.0,
δ
119.2, 118.5, 117.1, 114.7, 111.7, 103.2, 57.4, 56.9, 41.1, 28.2; HRMS: calcd for C28H21N2O6Cl [M
481.1394, found: 481.1390.
−
Cl]+:
0
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-1 -phenylethylcycloberberine chloride (13s): orange solid; yield:
82%; m.p.: 213–215 C; 1H-NMR (DMSO-d6)
δ 11.53 (s, 1H), 10.07 (s, 1H), 8.49 (s, 1H), 8.43 (d, J = 9.0 Hz,
◦
1H), 8.13 (d, J = 9.0 Hz, 1H), 7.56 (s, 1H), 7.41–7.37 (m, 2H), 7.30 (t, J = 7.8 Hz, 2H), 7.19 (tt, J = 7.2,
1.2 Hz, 1H), 6.35 (s, 1H), 6.23–6.20 (m, 1H), 5.48 (q, J = 6.6 Hz, 1H), 5.20–5.06 (m, 2H), 4.08 (s, 3H), 3.57
(t, J = 6.6 Hz, 2H), 1.87 (d, J = 7.2 Hz, 3H); 13C-NMR (DMSO-d6)
δ
147.4, 146.8, 146.3, 146.2, 145.7, 144.0,
141.6, 128.8 (2), 128.3, 128.2 (2), 127.8, 127.5, 126.7, 124.7, 122.2, 119.0, 118.2, 117.7, 116.3, 114.3, 111.0,
102.2, 57.4, 55.4, 41.0, 27.0, 22.7; HRMS: calcd for C29H24NO4Cl [M
Cl]+: 450.1700, found: 450.1706.
−
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-20-methoxy-20-oxo-10-phenylethylcycloberberine chloride (13t):
orange solid; yield: 84%; m.p.: 173–175 ◦C; 1H-NMR (DMSO-d6)
11.62 (s, 1H), 10.10 (s, 1H), 8.14 (d,
J = 9.0 Hz, 1H), 7.97 (s, 1H), 7.76 (s, 1H), 7.61 (s, 1H), 7.53–7.41 (m, 5H), 6.35 (s, 1H), 6.32 (s, 1H), 6.24
(s, 1H), 5.20–5.09 (m, 2H), 4.04 (s, 3H), 3.73 (s, 3H), 3.63–3.57 (m, 2H); 13C-NMR (DMSO-d6)
171.9,
δ
δ
147.0, 146.9, 146.0, 145.7, 141.0, 137.0, 136.2, 129.5, 129.1 (2), 128.9 (2), 128.1, 127.7, 127.4, 126.7, 124.6,
120.9, 117.6, 117.0, 115.8, 112.3, 110.6, 102.1, 56.8, 54.8, 54.1, 52.4, 26.4; HRMS: calcd for C30H24NO6Cl
[M − Cl]+: 494.1598, found: 494.1591.
3.2.4. General Procedure for the Synthesis of 15a–c
To a stirred solution of intermediate 11 [15] (361 mg, 1.0 mmol, 1.0 equiv) in 80% EtOH (20 mL),
various aliphatic aldehydes (5.0 equiv) and HOAc (4 mL) were added. The reaction mixture was
heated to reflux for 8 h. The solvent was removed by evaporation to afford a red oil. The red oil
was acidified by 2% HCl, stirred at room temperature for 0.5 h. The solvent was removed and the
residue was purified by flash chromatography over silica gel using CH2Cl2/CH3OH (5:1) as eluent to
obtain the intermediate compounds 14a–c. Next 14a–c were heated at 195–210 oC in a dry oven under
vacuum (20–30 mmHg) for 40 min to create the crude materials, to were acidified in concentrated
HCl/EtOH (5:95 by vol.) for 10 min. Then the mixture was evaporated under vacuum and purified by
flash column chromatography on silica gel using a gradient of CH2Cl2 and CH3OH as eluent to obtain
the desired compounds 15a–c.
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-ethylcycloberberine chloride (15a): orange solid; yield: 82%;
◦
m.p.: 166–168 C; 1H-NMR (DMSO-d6)
δ
11.49 (s, 1H), 10.05 (s, 1H), 8.54 (s, 1H), 8.49 (d, J = 9.0 Hz, 1H),
8.12 (d, J = 9.0 Hz, 1H), 7.55 (s, 1H), 6.34 (s, 2H), 5.14 (t, J = 6.6 Hz, 2H), 4.08 (s, 3H), 3.57 (t, J = 6.6 Hz,
2H), 3.30 (q, J = 7.2 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H); 13C-NMR (DMSO-d6)
146.8, 145.9, 145.7 (2), 141.5,
δ
141.2, 127.6, 127.1 (2), 124.2, 122.2, 119.8, 117.5, 117.1, 115.9, 114.1, 110.3, 102.0, 57.0, 55.0, 28.0, 26.6, 16.1;
HRMS: calcd for C23H20NO4Cl [M − Cl]+: 374.1387, found: 374.1396.
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-propylcycloberberine chloride (15b): orange solid; yield: 85%;
◦
m.p.: 165–167 C; 1H-NMR (DMSO-d6)
δ 11.49 (s, 1H), 10.04 (s, 1H), 8.58 (s, 1H), 8.53 (d, J = 9.0 Hz, 1H),

Molecules 2019, 24, 984
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8.15 (d, J = 9.0 Hz, 1H), 7.55 (s, 1H), 6.34 (s, 2H), 5.15 (t, J = 6.6 Hz, 2H), 4.09 (s, 3H), 3.58 (t, J = 6.6 Hz,
2H), 3.28 (t, J = 7.8 Hz, 2H), 1.80–1.72 (m, 2H), 1.02 (t, J = 7.2 Hz, 3H); 13C-NMR (DMSO-d6)
146.6,
145.8, 145.6 (2), 141.2, 139.7, 127.6, 127.1, 127.0, 124.1, 122.0, 120.5, 117.6, 117.1, 115.8, 114.0, 110.2, 101.9,
56.9, 54.9, 36.5, 26.4, 24.3, 13.72; HRMS: calcd for C24H22NO4Cl [M
Cl]+: 388.1543, found: 388.1553.
δ
−
1,2-Methylenedioxy-8-hydroxy-9-methoxy-13-butylcycloberberine chloride (15c): orange solid; yield: 88%;
◦
m.p.: 162–164 C; 1H-NMR (DMSO-d6)
δ 11.45 (s, 1H), 10.02 (s, 1H), 8.55 (s, 1H), 8.50 (d, J = 9.0 Hz, 1H),
8.12 (d, J = 9.0 Hz, 1H), 7.53 (s, 1H), 6.31 (s, 2H), 5.12 (t, J = 6.6 Hz, 2H), 4.06 (s, 3H), 3.55 (t, J = 6.6 Hz,
2H), 3.27 (t, J = 7.8 Hz, 2H), 1.68 (t, J = 7.8 Hz, 2H), 1.46–1.38 (m, 2H), 0.93 (t, J = 7.2 Hz, 3H); 13C-NMR
(DMSO-d6)
δ 146.7, 145.8, 145.6 (2), 141.2, 140.0, 127.6, 127.1, 127.0, 124.1, 122.1, 120.4, 117.6, 117.1,
115.8, 114.0, 110.2, 101.9, 56.9, 54.9, 34.3, 33.3, 25.4, 22.0, 13.7; HRMS: calcd for C25H24NO4Cl [M
402.1700, found: 402.1692.
−
Cl]+:
3.3. Antimicrobial Assay
Minimum inhibitory concentrations (MICs) of the target compounds were determined by using
the agar dilution assay at various concentrations of 64, 32, 16, 8.0, 4.0, 2.0, 1.0, 0.5, 0.25, 0.125, 0.06
and 0.03 mg/mL described by the Clinical Laboratory Standards Institute [17]. Bacterial strains
were purchased from the ATCC collection or isolated from Chinese hospitals. The medium was
Mueller-Hinton◦agar, and the inoculum was 104 colony forming units (cfu)/spot. Culture plates were
incubated at 35 C for 18 h, and MICs were defined as the lowest concentrations that prevented visible
growth of the bacteria.
3.4. Time-kill Curves of 15a
Kill kinetics of 15a was determined by time-kill experiments for MRSA strain (clinical isolate
MRSA 18-4) according to the method described by Verma et al. with slight modifications [25].
An overnight culture was diluted with CAMH broth in a total volume of 30 mL containing an
inoculum of 2
concentrations of 0
×
106 CFU/mL in a 250 mL flask for 18-4. Distilled water or 15a was added to yield
, 1/2 , 2 and 8 MIC in the broth at standard inocula. Viability counts were
×
×
×
×
performed at 0, 2, 4, 8 and 12 h of incubation at 37 ◦C by plating 0.1 mL undiluted and 10-fold serial
diluted samples onto TSA plates in duplicate. Drug carryover effect was eliminated by saline and
agar dilution. The experiments were performed three times on different days and the results were
presented as mean and standard deviation.
3.5. Stability Assay of Key Compound in Liver Microsomes
The assay was performed with liver microsomes from CD-1 mouse, SD rat and human.
Microsomes in 100 mM potassium phosphate buffer (0.5 mg/mL microsomal protein), cofactor MgCl2
(10 mM), tested compound (1
µ
M, cosolvent (0.01% DMSO), 0.99% CH3OH), and then NADPH (1 mM)
◦
at 37 C for 60 min. The reaction can be started by the addition of liver microsomes or the tested
compound or NADPH. Aliquots were sampled at 0, 5, 10, 20, 30 and 60 min incubation, and enzymatic
reaction was stopped by protein precipitation in cold acetonitrile including 100 ng/mL tolbutamide
and 100 ng/mL labetalol as internal standard. After centrifugation, samples were then analyzed by
LC/MS/MS [26].
3.6. Stability Assay of Key Compound in SD Rat Whole Blood
Fresh blood was collected on the day of experiment from SD rats and pre-warmed in a water bath
at 37 ◦C. 10 mM test compound or Ems stock solutions were prepared in DMSO, and then diluted
with 45% MeOH/H2O to achieve 100
with 98
L of blank blood at 37 ◦C in water bath for 0, 0.5, 1, 3, 7 and 24 h, respectively. At the end
of incubation, for each sample, 100 L water and 800 L of stop solution (200 ng/mL tolbutamide
plus 20 ng/mL buspirone in ACN) were immediately added to to precipitate protein and centrifuge at
µM dosing solutions. Each dosing solution (2 µL) was incubated
µ
µ
µ

Molecules 2019, 24, 984
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4,000 rpm for 20 min. An aliquot of supernatant (100
µL) was then extracted, mixed with 200
µL H2O
and then shook at 800 rpm for about 10 min before submitting to LC-MS/MS analysis. The experiment
was repeated two times [18].
3.7. Acute Toxicity Assay
Female Kunming mice with weight of 20.0 ( 1.0 g) were fed with regular rodent chow and
±
housed in an air conditioned room. The mice were randomly divided into different groups with 6 mice
−1
each. Each compound was given intravenously in a single-dosing experiment at 40, 60, or 80 mg kg
·
(ddH2O as control), respectively. The mice were closely monitored for 7 days. Body weight as well as
survival was monitored [18].
3.8. Enzymatic Assay
The gyrase supercoling assay kit and topo IV decatenation kit were the tools for detection the
target compound inhibited the activity of DNA gyrase and topo IV from MRSA (both kits obtained
from Inspiralis, Norwich, United Kingdom). Briefly, supercoiled pBR322 plasmid DNA (0.5 mg) and
13a of varying concentrations (2.5–100
µ
g/mL) were incubated with 1 U gyrase in the dedicated
◦
supercoiling assay buffer supplied by the manufacturer. Reactions were carried out at 37 C for 30 min
and then terminated by the addition of equal volume of STEB buffer (40% sucrose, 100 mM Tris-HCl
pH 7.5, 1 mM EDTA, 0.5 mg/ml bromophenol blue) and chloroform/isoamyl alcohol. Samples were
vortexed, centrifuged and run through a 15 cm 1% agarose gel in TAE buffer (40 mM Tris-acetate,
2 mM EDTA) for 3 h at 50 V. Gels were stained with ethidium bromide and visualized under UV
light. The decatenation assay was performed using MRSA topo IV decatenation kit. The operational
approach was similar to above. Bands were visualized by UV light and photographed [27].
3.9. Molecular Docking Assay
The topo IV crystal structure in complex with kibdelomycin (PDB code 4URL, resolution: 2.29 Å)
was selected as the template to generate the binding modes [24]. Discovery Studio 4.5 was used to
create structures of the ligands and to perform energy minimization [16]. The LibDock software was
employed. Before docking, protein structure and ligands were processed [28]. In the course of protein
preparation, ligands and water molecules were removed from the structure. The docking conformation
with the highest docking score was finally chosen to analyze the receptor ligand interaction in the
Discovery Studio 4.5 software.
4. Conclusions
A total of 25 new derivatives of
anti-Gram-positive microbes activities with
1
were designed, synthesized and evaluated for their
as the lead. SAR studies revealed that introducing
1
a small-sized substituent at the 13-position might be beneficial for activity. Part of them showed
good activities against MRSA and VISA. Compound 15a bearing a 13-ethyl group exhibited the most
potent antibacterial activities, with MIC values of 0.25–0.5
. Time-kill curve analysis of 15a further confirmed its potential bactericidal activity. Meanwhile,
compound 15a displayed high liver microsomal metabolic stability and reasonable whole blood
stability. It also showed a good in vivo safety profile with an LD50 value over 65.6 mg
kg−1 in mice
µg/mL, superior to those of the lead
1
·
via the intravenous route. A preliminary mechanism study showed that compound 15a could inhibit
topo IV activity through binding with the active site of kibdelomycin, a different mechanism of action
from the quinolone antibiotics currently used in hospitals. Taken together, these results provide
powerful information for developing these compounds into promising anti-MRSA candidates with
novel chemical scaffolds and a specific biological mechanism of action, and compound 15a has been
chosen for further investigation.

Molecules 2019, 24, 984
15 of 16
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/5/984/s1,
the 1H-NMR, 13C-NMR and HRMS data of compound 15a.
Author Contributions: Y.-S.Y., W.W. and S.T. performed part of synthetic experiments and wrote the paper,
X.-X.H. and J.P. performed the biological assay, D.-Q.S. conceived and designed the chemistry experiments,
X.-F.Y. conceived and designed the biology experiments, T.-Y.F. and Y.-X.W. designed the target compounds and
chemistry experiments.
Funding: This work was supported by the National Natural Science Foundation of China (81621064 and
81361138020), Beijing Natural Science Foundation (7172136), and the CAMS Innovation Fund for Medical Sciences
(2017-12M-1-012).
Acknowledgments: The authors thank center for analysis and testing of Institute of Materia Medica and Institute
of Medicinal Biotechnology, Chinese Academy of Medical Sciences for their contributions to the determination of
HR-MS, 1H-NMR, and 13C-NMR.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 6, 10, 13a–t and 15a–c are available from the authors.
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