Bis-Cyclic Guanidines as a Novel Class of Compounds Potent against Clostridium difficile

Article abstract of DOI:10.1002/cmdc.201800240

Clostridium difficile infection (CDI) symptoms range from diarrhea to severe toxic megacolon and even death. Due to its rapid acquisition of resistance, C. difficile is listed as an urgent antibiotic-resistant threat, and has surpassed methicillin-resista

Full text of DOI:10.1002/cmdc.201800240

Accepted Article
Title: Bis-Cyclic-Guanidine as a Novel Class of Compounds Potent
Against Clostridium Difficile
Authors: Chunhui Li, Peng Teng, Zhong Peng, Peng Sang, Xingmin
Sun, and Jianfeng Cai
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Bis-Cyclic-Guanidine as a Novel Class of Compounds Potent
Against Clostridium Difficile
Chunhui Li,†,§,a Peng Teng,‡,a Zhong Peng,†,ǁ,a Peng Sang,‡ and Xingmin Sun,†,* and Jianfeng Cai‡,*
[†]
Dr. C. Li, Mr. Z. Peng, Prof. X. Sun,* E-mail: sun5@health.usf.edu
Department of Molecular, Morsani College of Medicine
University of South Florida,
12901 Bruce B. Down Blvd, Tampa, FL 33612, USA
Dr. P. Teng, ORCID 0000-0002-7412-9646, P. Sang, Prof. J. Cai,* ORCID 0000-0003-3106-3306, E-mail: jianfengcai@usf.edu.
Department of Chemistry
[‡]
University of South Florida
4202 E. Fowler Avenue, Tampa, FL, 33620, USA
Dr. C. Li
Department of Infection Control Center of Xiangya Hospital
Central South University, Changsha, 410008, China
Dr. Z. Peng,
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine
Huazhong Agricultural University, Wuhan, Hubei, China
These authors contributed equally to this work.
[§]
[ǁ]
[a]
Supporting information for this article is given via a link at the end of the document.
Abstract: Clostridium difficile infection (CDI) symptoms range from
diarrhea to severe toxic megacolon and even death. Due to its rapid
acquisition of resistance, C. difficile is listed as an urgent antibiotic-
reliable but more expensive than metronidazole/vancomycin. The
Centers for Disease Control and Prevention has listed C. difficile
as an urgent antibiotic-resistant threat.[8] Although C. difficile has
not yet developed significant resistance to the antibiotics most
used for CDI treatment, it is highly likely that these resistance
phenotypes will emerge, as has occurred through the use of
clindamycin and the fluoroquinolones.[9]
Novel antibiotics are in urgent need to more effectively treat
CDI. Bis-guanidine related compounds have been reported to
bear antiseptic and antibacterial activities, such as hexamidine,[10]
norspermidine analogues,[11] teixobactin,[12] Brilacidin,[13] and
amphipathic xanthone derivatives[14] etc. This type of compounds
display antimicrobial activity against Gram-positive organisms
including Methicillin-resistant Staphylococcus aureus, Methicillin-
resistant Staphylococcus epidermidis, and Vancomycin-Resistant
Enterococci faecalis, and relatively weaker activity against Gram-
negative organisms such as K. pneumoniae and P. aeruginosa.[15]
Recently, we have discovered a new type of symmetric bis-cyclic
guanidine compounds[15] bearing amphipathic structures that
could mimic mechanism of action of host-defense peptides
(HDPs).[16] These membrane active, amphipathic compounds
showed potent and broad-spectrum activity against both Gram-
resistant
threat
and
has
surpassed
methicillin-resistant
Staphylococcus aureus (MRSA) as the most common hospital-
acquired infections in the USA. To combat the pathogen, the new
structural class of pseudo peptides that exhibit antimicrobial activities
could play an important role. Herein, we report that bis-cyclic
guanidine compounds that exhibit potent antibacterial activity against
C. difficile with decent selectivity. Eight compounds showed high in
vitro potency against C. difficile UK6 with MIC of 1.0 μg/mL, and
cytotoxic selectivity index (SI) up to 37. Moreover, the most selective
compound 13 is also effective upon the treatment of C. difficile-
induced diseases in the mouse model of CDI and appears to be a very
promising new candidate for the treatment of CDI.
Introduction
Clostridium difficile (C. difficile) is a Gram-positive, spore-
forming, anaerobic and toxigenic microbe. Symptoms of C.
difficile infection (CDI) range from uncomplicated diarrhea to
pseudomembranous colitis and even toxic megacolon.[1] C.
difficile is recognized as the most common cause of hospital-
associated diarrhea,[2] and may lead to more related
complications,[3] resulting increasingly infectious morbidity and
mortality. More alarmingly, the emergence of hypervirulent strains
NAP1/BI/027 has been associating with higher mortality rates in
North America and several countries in Europe.[4] Antimicrobial
therapeutic options with oral vancomycin and metronidazole are
effective for severe and mild-to-moderate CDI respectively.[4b, 5]
Treatment options for severe CDI include the use of the newly
developed antimicrobial agents such as fidaxomicin and fecal
microbiota transplantation (FMT), which was identified as an
effective treatment for CDI recurrence.[6] However, initial therapy
with metronidazole and vancomycin has been associated with
increased the rate of failure and recurrence.[7] Fidaxomicin is more
positive and Gram-negative bacteria. However, to the best of our
[17]
knowledge, compounds bearing with guanidine groups
have
been rarely explored for bactericidal activities against C.
difficile.[18] Herein, we report the antibacterial activity of these
dimeric cyclic guanidines against C. difficile in vitro and in vivo.
Results and Discussion
The bis-cyclic guanidine library was synthesized following
the same procedure reported previously.[15] Synthesis of
compound 13 was shown as an example of typical synthesis
process (Scheme 1). Intermediate R4 was obtained from easily
accessible reagent R1 through straightforward way with decent
yield. The linear intermediate R5 was obtained by coupling
between R4 and diamine (p-phenylenediamine) in the presence
1
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10.1002/cmdc.201800240
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of
hydroxybenzotriazole
(HOBt)
and
N,N'-
cyanogen bromide to furnish the final bis-cyclic guanidine
dicyclohexylcarbodiimide (DCC) followed by removing Boc
protecting group. R5 could be easily cyclized in the presence of
compound 13.
Scheme 1 Typical synthesis protocol for bis-cyclic guanidine derivative 13. (i) Hexanal, NaBH3CN, MeOH, AcOH, room temperature (rt), 3 h; (ii) Boc2O, NaHCO3,
THF, H2O, rt, 5 h; (iii) LiAlH4, THF, ‒20 °C, 30 min; (iv) Glycine benzyl ester, NaBH3CN, MeOH, AcOH, room temperature, 3 h; (v) Boc2O, NaHCO3, THF, H2O, rt, 5
h; (vi) H2, Pd-C, MeOH, 2 h; (vii) Benzene-1,4-diamine, HOBt, DIPEA, DCC, DMF, rt, 24 h; (viii) TFA/DCM (50:50, v/v), rt, 2 h; (ix) CNBr, MeCN, rt, 12 h.
Table 1 The structure of compounds 1‒16 and their antibacterial activity against C. difficile.
Cpd
#
MIC
(μg/mL)
Cpd
#
MIC
(μg/mL)
Structure
Log Pa
Structure
Log P
6.12
1
2
3
1.21
128
32
9
2.0
8.0
2.0
2.28
3.17
3.23
10
11
4.31
5.58
5.31
32
4
5
8.0
1.0
12
13
1.0
1.0
5.51
5.38
6
7
6.56
5.57
1.0
4.0
14
15
5.78
5.72
1.0
1.0
8
5.55
1.0
16
5.01
1.0
Vancomycin
MIC 0.5 μg/mL
aLog P, calculated by Alogps 2.1 program.
2
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The antibacterial potency of these cyclic guanidine dimers
with isobutyl group. Overall, the compound 13 has the best SI
among all the compounds tested.
on the hypervirulent C. difficile UK6 was assessed and described
as minimal inhibitory concentration (MIC). As shown in Table 1,
the majority of these quinoline compounds displayed potent in
vitro activity, with MICs in the range of 1.0‒4.0 μg/mL.
Compounds 1 and 2 that do not bear hydrophobic groups on the
guanidine residues displayed weak activity, especially compound
1 showed a MIC of 128 μg/mL. When ethyl group was installed on
the guanidine to furnish compounds, i.e. 3 and 4, they exhibited
activity against C. difficile, with MIC of 8.0 μg/mL for compound 4.
Compound 5 and 6 have 3-phenylpropyl group attached on the
nitrogen atoms of guanidine groups and showed potent
antimicrobial activity (MIC=1.0 μg/mL), due to the enhanced
ability of interaction with the bacterial membrane. When
hydrophobic cyclohexane propyl group was conjugated on the
guanidine position to give compounds 7‒9, the activities
increased as the change of the linker between two cyclic
To further investigate the impact of hydrophobicity on the
activity profile, we measured HPLC retention time (RT) (Table S1)
and determined log P values (Table 1) of all compounds.
Generally, the antibacterial activity of compounds increases with
longer RT value when RT is shorter than 27.25 min. When RT is
longer than 27.25 min, the activity of compounds did not decrease,
however, the cytotoxicity of compounds increased (Table 2).
Similar trend can be obtained from the correlation of activity with
log P value. Based on these results, we could conclude that the
antibacterial activity and the selectivity of this type of compounds
could be improved by carefully tuning the balance between
hydrophobicity and hydrophilicity, which is of great help on design
HDPs mimics in the future.
Efficacy of compound 13 was further evaluated in mouse
model of CDI. As shown in Figure 1a, the C. difficile UK6
guanidine rings from p-phenylenediamine to m-phenylenediamine, challenged control group lead to 90% of diarrhea, while the
and then 1,6-hexamethylene, with MICs of 4.0, 1.0, and 2.0 μg/mL
respectively. Compound 10 that has aliphatic chain C6H13 on the
guanidine rings only showed MIC of 8 μg/mL, where the linker
was kept as 1,4-butylene group. However, the activity bounced
back when the linker was replaced with 1,8-octamethylene (11,
MIC=2.0 μg/mL), m-phenylenediamine (12, MIC=1.0 μg/mL), p-
phenylenediamine (13, MIC=1.0 μg/mL).
Interestingly, replacement of the aliphatic chain on the
guanidine rings with increased chains (C8H17) did not compromise
the activity, with the same MIC values of 1.0 μg/mL in the afforded
compounds 14 and 15. Replacing the benzyl group (initially
starting from α-Phenylalanine) with isobutyl group (initially starting
from α-Leucine) produced the compound 16, which was also
potent, with MIC vale of 1.0 μg/mL, very close to that of the
positive control Vancomycin under the same assay condition.
administration of compound 13 displayed significant improvement
for overcoming CDI over the entire experimental period (five days).
After 3 days, 90% of survival was observed with the administration
of compound 13 compared to the control group where only 40%
of mouse survived (Figure 1b). Five days later, 60% of the mice
were still alive with the treatment of compound 13, while only 40%
of survival was exhibited in the C. difficile UK6 challenged control
group. These data indicated that compound 13 could improve the
diarrhea and survival of mice challenged with C. difficile UK6, a
hypervirulent strain.
Table 2 The cytotoxicity assessment of active compounds (MIC < 4 μg/mL).
MIC
(μg/mL)
aCC50 (μg/mL)
HEK293T HepG2
*SI (CC50/MIC)
Cpd
HEK293T
HepG2
36.8
31.7
30.1
20.15
12
5
6
8
1
1
1
2
2
1
1
1
1
1
20.6
26.5
33.9
42.7
25.1
32.3
31.8
29.3
25.7
24.3
36.8
31.7
30.1
40.3
24.0
33.1
37.3
26.8
25.9
18.1
20.6
26.5
33.9
21.35
12.55
32.3
31.8
29.3
25.7
24.3
9
Figure 1. In vivo efficacy of the compound 13 in mouse model of C.
difficile infection. Two groups of mice (UK6 and UK6+cpd 13, n = 10 per group)
were challenged with C. difficile spores at 106 /mouse in the absence or
presence of compound 13, after pretreatment of antibiotics. The third group
mice (n = 5) were administered compound 13 only as controls. (a) Percent of
diarrhea with or without treatment of compound 13. (b) Survival rates of mice
with or without treatment of compound 13. Results were analyzed by the two-
way ANOVA method. The differences between the UK6 group and the treatment
group (UK6 + compound 13) are statistically significant, p < 0.05.
11
12
13
14
15
16
33.1
37.3
26.8
25.9
18.1
aCC50, the 50% cytotoxic centration; SI: selective index, SI = CC50/MIC.
To determinate the cytotoxicity of ten active compounds
(MIC < 4 μg/mL) out of sixteen compounds, MTT assays were
performed on human liver cancer cell line HepG2 and human
embryonic kidney cell line HEK293T. As shown in Table 2, 3-
phenylpropyl modified compounds 5 and 6, and cyclohexane
propyl modified compounds 8 and 9 showed about 20‒30-fold
selectivity index (SI) (CC50 on human cells/MIC on C. difficile
cells). Aliphatic side chain bearing compound 11 only displayed
ten-fold SI, however, compounds 12‒15 all had low cytotoxicity
against both cell lines, especially compound 13 had 37-fold SI
against HEK293T cells. Compound 12 and 13 were also not
hemolytic even at the concentration of 250 μg/mL.[15] The
selectivity decreased slightly when the benzyl group was replaced
The amount of C. difficile in fecal samples after treatment
were also determined. As shown in Figure 2, one day after the
infection, the amount of C. difficile in feces from mice treated with
compound 13 was 50% less than that of C. difficile UK6
challenged group. After 5 days, the amount of C. difficile in fecal
samples from control group continued to increase, while mice
treated with compound 13 were observed with significant
decrease of C. difficile in fecal samples, down by 80% compared
to control levels. This result demonstrated that compound 13 had
good efficacy on the inhibition of C. difficile in mice.
3
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for 5 h, after which EtOAc was added and the organic layer was collected.
The solvent was removed in reduced pressure to give the colorless crude,
which was purified by flash column chromatography to give 9.1 g of
compound R2. Next, compounds R2 was taken in THF and reduced by
LiAlH4 (926 mg, 23.2 mol) for 30 min at -20 oC, then water was added to
quench the reaction. The mixture was extracted with EtOAc, and the
organic layer was separated, the solvent was removed in vacuo to give the
crude R3 (7.2 g), which was used in the next reaction without any further
purification. BOC protecting group was attached as the same procedure
for attaching BOC onto compound R2, followed by hydrogenation to
remove benzyl protecting group in MeOH to give the building block R4 (7.5
g) as a white solid after filtration and concentration.
Building block R4 (400 mg, 0.81 mmol), HOBt (249 mg, 1.6 mmol), DIPEA
(284 μL, 1.6 mmol), and p-Phenylenediamine (53 mg, 0.49 mmol) was
dissolved in DMF (3 mL) and then DCC (335 mg, 1.6 mmol) was added.
The reaction mixture was stirred at room temperature for 24 h. The
afforded byproduct DCU was filtered off and the filtration was added into
water and extracted with EtOAc (×3). The organic phase was combined
and washed with 1M HCl (×2), dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure. The crude oil compound was
treated with TFA in DCM (1:1, v/v) for 2 h to completely remove BOC
protecting groups to yield crude compound R5. Subsequently, R5 was
dissolved in acetonitrile (3 mL), to which CNBr (4 eq.) was added carefully
(caution: very toxic). The reaction was stirred for 12 h at room temperature.
1M NaOH solution was added carefully, followed by proper amount of
bleach to deactivate excessive CNBr. The mixture was filtered through a
millpore filter and purified by HPLC purification on Waters HPLC system,
and the desired fraction was lyophilized to give the pure product 13.
Figure 2. C. difficile in fecal samples from compound 13-treated (UK6+cpd 13)
or control (UK6) mice. Two groups of mice (UK6 and UK6+cpd 13, n = 10 per
group) were challenged with C. difficile spores at 106 /mouse in the absence or
presence of compound 13, after pretreatment of antibiotics. Fecal samples were
collected, and C. difficile spores were determined as described in methods. C.
difficile isolated in fecal from mouse treated with compound 13 showed
significantly decrease compared with that of UK6 challenged group (Two-way
ANOVA, P<0.001)
Conclusions
We have reported a series of membrane active bis-cyclic
guanidines (molecular mass 600‒900) which displayed potency
against C. difficile UK6, an emerging hypervirulent bacteria. In
vitro studies demonstrated that eight out of sixteen cyclic
guanidine dimeric compounds showed MIC value of 1.0 μg/mL
against C. difficile, very close to that of Vancomycin (MIC=0.5
μg/mL). Moreover, the cyclic guanidine dimers also revealed
significant efficacy in mouse model of CDI. Further modifications
of these compounds may lead to novel potent antibiotics against
C. difficile.
The other compounds were synthesized according to the same procedure
as compound 13. Different aldehydes were used at the first step to give
different compounds with various side chains.
The NMR data of compounds 1‒5, 12, 13, and 16 are consist with the data
in the previous paper.[15] The NMR and HRMS of other compounds are
shown below:
Compound 6: 1H NMR (500 MHz, CD3OD) δ 7.64 (d, J = 7.5 Hz, 4H), 7.57
(d, J = 7.5 Hz, 4H), 7.18‒7.33 (m, 20H), 4.22‒4.27 (m, 2H), 4.14 (s, 4H),
3.63 (t, J = 9.5 Hz, 2H), 3.51‒3.57 (m, 2H), 3.42 (dd, J = 9.5, 5.0 Hz, 2H),
3.34 (dd, J = 9.0, 5.5 Hz, 2H), 3.09 (dd, J = 13.5, 4.5 Hz, 2H), 2.85 (dd, J
= 14.0, 8.5 Hz, 2H), 2.62‒2.74 (m, 4H), 1.92‒2.06 (m, 4H). 13C NMR (125
MHz, CD3OD) δ 165.1, 158.0, 140.9, 137.2, 136.2, 135.7, 134.3, 129.0
(2C), 128.5 (2C), 128.2 (2C), 128.0 (2C), 126.8, 126.6 (2C), 125.8, 119.8,
58.0, 51.7, 42.3, 37.4, 32.1 (2C), 28.3. HRMS (ESI) C54H59N8O2 [M+H]+
calcd = 851.4755; found = 851.4742.
Experimental Section
General information.
The starting material to synthesize R1 was purchased from Chem-Impex
International, Inc. Solvents and other reagents were purchased from either
Sigma-Aldrich or Fisher Scientific and were used without further
purification. The final products were purified on a Waters Breeze 2 HPLC
system and lyophilized on a Labconco lyophilizer. The purity of the
compounds was determined to be >95% by analytical HPLC (1 mL/min
flow, 5% to 100% linear gradient of solvent B (0.1% TFA in acetonitrile) in
A (0.1% TFA in water) over 50 min was used) and the data is shown in the
Supporting Information. The NMR spectra were obtained on a Varian Inova
500 instrument.
Compound 7: 1H NMR (500 MHz, CD3OD) δ 7.53 (s, 4H), 7.31‒7.34 (m,
4H), 7.24‒7.28 (m, 6H), 4.27‒4.32 (m, 2H), 4.13, 4.10 (ABq, J = 18.0 Hz,
4H), 3.68 (t, J = 9.0 Hz, 2H), 3.41‒3.48 (m, 4H), 3.25 (ddd, J = 15.0, 9.5,
5.5 Hz, 2H), 3.15 (dd, J = 13.5, 5.0 Hz, 2H), 2.89 (dd, J = 13.5, 8.0 Hz, 2H),
1.58‒1.76 (m, 14H), 1.15‒1.32 (m, 12H), 0.90‒0.97 (m, 4H). 13C NMR (125
MHz, CD3OD) δ 165.1, 158.0, 135.8, 134.3, 128.9 (2C), 128.5 (2C), 126.8,
120.1 (2C), 57.9, 51.8, 47.0, 43.1, 37.6, 37.5, 37.3, 33.6, 33.0, 32.9, 26.3,
26.0, 24.0. HRMS (ESI) C48H67N8O2 [M+H]+ calcd = 787.5381; found =
787.5374.
Synthesis of the intermediate build block R4.
Compound R1 (TFA salt, 13.6 g, 42.3 mmol) was dissolved in MeOH and
treated with TEA (5.8 mL, 42.3 mmol) before adding to a solution of
hexanal (5.2 mL, 42.3 mmol) in MeOH and acetic acid (5.1 mL, 82.6 mmol).
After stirring for 10 min under ice/H2O bath, NaBH3CN (5.6 g, 82.6 mmol)
was added portion wise. The reaction was stirred for 3 h at room
temperature before solvent was removed. The crude mixture was treated
with NaHCO3 (aq.) and extracted with EtOAc, and the organic layer was
separated and evaporated to give an oil crude, which was purified by silica
gel column chromatography to give 8.9 g of the desired secondary amine.
Boc2O (8 g, 36.6 mmol) was added in the THF/H2O (1:1, v/v) solution of
this intermediate containing NaHCO3 (5.1 g, 61 mmol) and allowed to react
Compound 8: 1H NMR (500 MHz, CD3OD) δ 8.04 (s, 1H), 7.31‒7.34 (m,
4H), 7.24‒7.28 (m, 9H), 4.26‒4.32 (m, 2H), 4.14, 4.10 (ABq, J = 18.0 Hz,
4H), 3.68 (t, J = 9.5 Hz, 2H), 3.41‒3.47 (m, 4H), 3.25 (ddd, J = 14.5, 9.0,
5.5 Hz, 2H), 3.15 (dd, J = 13.5, 5.0 Hz, 2H), 2.89 (dd, J = 13.5, 8.0 Hz, 2H),
1.60‒1.75 (m, 14H), 1.14‒1.32 (m, 12H), 0.90‒0.96 (m, 4H). 13C NMR (125
MHz, CD3OD) δ 165.2, 158.0, 138.6, 135.8, 128.9 (2C), 128.5 (2C), 126.8,
120.1 115.3, 58.0, 51.7, 47.0, 43.1, 37.6, 37.3, 33.7, 33.0, 32.9, 26.3, 26.0
(2C), 24.0. HRMS (ESI) C48H67N8O2 [M+H]+ calcd = 787.5381; found =
787.5357.
4
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Compound 9: 1H NMR (500 MHz, CD3OD) δ 7.31‒7.34 (m, 4H), 7.25‒7.27
(m, 6H), 4.24‒4.30 (m, 2H), 3.93, 3.89 (ABq, J = 17.5 Hz, 4H), 3.60 (t, J =
9.5 Hz, 2H), 3.42 (ddd, J = 14.5, 9.0, 6.0 Hz, 2H), 3.33 (dd, J = 9.5, 6.0 Hz,
2H), 3.21‒3.26 (m, 2H), 3.19 (t, J = 7.0 Hz, 4H), 3.13 (dd, J = 13.5, 5.0 Hz,
2H), 2.85 (dd, J = 13.5, 8.0 Hz, 2H), 1.65‒1.74 (m, 12H), 1.58‒1.62 (m,
2H), 1.50 (p, J = 6.5 Hz, 4H), 1.32‒1.35 (m, 4H), 1.14‒1.31 (m, 12H), 0.85‒
0.95 (m, 4H). 13C NMR (125 MHz, CD3OD) δ 166.9, 158.0, 135.8, 128.9
(2C), 128.5 (2C), 126.8, 57.9, 51.6, 46.6, 43.1, 39.1, 37.5, 37.3, 33.7, 33.0,
32.9, 28.8, 26.3, 26.1, 26.0 (2C), 24.0. HRMS (ESI) C48H74N8O2 [M+H]+
calcd = 795.6006; found = 795.5998.
MTT
(3-(4,5-dimethylthiazol-2yl)-2,5-dipheynyltetrazolium
bromide;
Sigma–Aldrich, St. Louis, MO) cell viability assay was performed to
evaluate the cytotoxicity of the compounds on human HepG2 and
HEK293T cell lines. HepG2 is an immortalized cell line consisting of
human liver carcinoma cells. HEK293T is a specific cell line originally
derived from human embryonic kidney cells grown in tissue culture. Both
cells were maintained in a Dulbecco’s Modified Eagle Medium (DMEM with,
4.5 g/L Glucose, L-Glutamine and Sodium Pyruvate, Corning; Corning,
Manassas, VA) containing 10% FBS (Thermo Scientific) and 1%
penicillin/streptomycin at 37°C in 5% CO2. Cells (104 cells/ well) were
plated in 96-well plates. After incubation overnight, cells were treated with
the compounds at concentrations from 128 µg/ml to 0.125 µg/ml or 1%
DMSO (as a control reagent) for 24 h at 37°C. Then 10 µl of MTT stock
solution (5 mg/ml) were added to cells in each well, and further incubated
for 4 h at 37°C. After careful removal of media from each well without
disturbing cells, 100 µl of DMSO was added to each well, and incubated
for 15 min at 37°C. Absorbance at 540 nm was read in a Synergy HTX
multi-mode reader (Bio Tek Instruments, Inc. Winooski VT). Data were
analyzed using Graphpad PRISM 6 (GraphPad Software, Inc., La Jolla,
CA), and the 50% cytotoxic concentration (IC50) was reported as a
concentration of compound that reduced the cell viability by 50% when
compared to untreated controls. Then CC50 was determined to establish a
selectivity index (SI) (SI = CC50/MIC).
Compound 10: 1H NMR (500 MHz, CD3OD) δ 7.30‒7.34 (m, 4H), 7.24‒
7.26 (m, 6H), 4.24‒4.30 (m, 2H), 3.94, 3.90 (ABq, J = 18.0 Hz, 4H), 3.59
(t, J = 9.5 Hz, 2H), 3.45 (ddd, J = 15.0, 9.0, 6.5 Hz, 2H), 3.34 (dd, J = 9.5,
6.0 Hz, 2H), 3.23‒3.29 (m, 2H), 3.19‒3.23 (m, 4H), 3.13 (dd, J = 13.5, 5.0
Hz, 2H), 2.87 (dd, J = 14.0, 8.0 Hz, 2H), 1.51‒1.54 (m, 4H), 1.28‒1.36 (m,
12H), 0.92 (t, J = 6.5 Hz, 6H). 13C NMR (125 MHz, CD3OD) δ 167.0, 157.9,
135.8, 129.0 (2C), 128.5 (2C), 126.8, 57.9, 51.6, 46.7, 42.9, 38.7, 37.5,
31.1, 26.6, 26.2, 25.8, 22.1, 12.9. HRMS (ESI) C40H63N8O2 [M+H]+ calcd =
687.5068; found = 687.5056.
Compound 11: 1H NMR (500 MHz, CD3OD) δ 7.31‒7.34 (m, 4H), 7.24‒
7.27 (m, 6H), 4.24‒4.30 (m, 2H), 3.94, 3.90 (ABq, J = 18.0 Hz, 4H), 3.59
(t, J = 9.0 Hz, 2H), 3.45 (ddd, J = 15.0, 9.0, 6.5 Hz, 2H), 3.34 (dd, J = 9.5,
5.5 Hz, 2H), 3.25 (ddd, J = 15.0, 9.0, 5.5 Hz, 2H), 3.18 (t, J = 7.0 Hz, 4H),
3.14 (dd, J = 13.5, 5.0 Hz, 2H), 2.86 (dd, J = 13.5, 8.0 Hz, 2H), 1.54‒1.69
(m, 4H),1.50 (t, J = 6.0 Hz, 4H), 1.30‒1.36 (m, 20H), 0.92 (t, J = 7.0 Hz,
6H). 13C NMR (125 MHz, CD3OD) δ 166.9, 157.9, 135.8, 128.9 (2C), 128.5
(2C), 126.8, 57.9, 51.5, 46.7, 42.8, 39.2, 37.5, 31.2, 28.9, 26.6, 26.5, 25.8,
22.2, 12.9. HRMS (ESI) C44H71N8O2 [M+H]+ calcd = 743.5694; found =
743.5675.
Evaluation of compounds in mouse model of C. difficile infection
(CDI).
C57BL/6 female mice (6-week old) were purchased from Charles River
Laboratories, MA. During the experiment, the mice were housed in groups
of 5 animals per cage under the same conditions. All animal experiments
were approved by the institutional committee for animal care and use at
the University of South Florida. The experimental design is illustrated in
Figure S1. Twenty-five mice were divided into three groups (group 1‒3).
Group 1 (n = 10) were challenged with spores of C. difficile UK6. Groups
2 (n = 10) were challenged with spores of C. difficile UK6, and treated by
compound 13. Group 3 (n = 5) were only treated with compound 13
without infection. The mice were given drinking water containing a mixture
of six antibiotics including ampicillin (200 mg/kg), kanamycin (40 mg/kg),
gentamycin (3.5 mg/kg), colistin (4.2 mg/kg), metronidazole (21.5 mg/kg)
and vancomycin (4.5 mg/kg) for 5 days, and then received autoclaved
water for 2 days, followed by a single dose of clindamycin (10 mg/kg)
intraperitoneally 1 day before (day-1) challenge day. In the challenge day
(day 0), mice in groups 1‒2 were challenged with C. difficile UK6 spores
at 106 colony-forming unit (CFU) by gavage. At 4 hours post challenge, the
mice in groups 2 were given one dose of compound 13 (50 mg/kg) via oral
routine. From the first day post challenge (day 1), mice in group 2 received
one dose of compound 13 twice a day (50 mg/kg/day) for five days.
Meanwhile, the mice in group 3 were also given compound 13 at the same
time with the same dose to determine the toxicity of the compound to the
mice. After C. difficile challenge and/or compound treatment, mice were
monitored twice a day during the experiment for weight changes, diarrhea
(defined as soft or watery feces) and other symptoms of the disease.
Compound 14: 1H NMR (500 MHz, CD3OD) δ 8.02 (d, J = 1.0 Hz, 1H),
7.31‒7.34 (m, 4H), 7.24‒7.28 (m, 9H), 4.27‒4.32 (m, 2H), 4.14, 4.11 (ABq,
J = 18.0 Hz, 4H), 3.67 (t, J = 9.5 Hz, 2H), 3.47 (ddd, J = 15.5, 9.5, 7.0 Hz,
2H), 3.42 (dd, J = 9.5, 5.5 Hz, 2H), 3.27 (ddd, J = 15.0, 9.5, 5.5 Hz, 2H),
3.16 (dd, J = 13.5, 5.0 Hz, 2H), 2.89 (dd, J = 14.9, 8.5 Hz, 2H), 1.59‒1.70
(m, 4H), 1.30‒1.34 (m, 20H), 0.91 (t, J = 7.0 Hz, 6H). 13C NMR (125 MHz,
CD3OD) δ 165.2, 158.0, 138.6, 135.8, 129.0 (2C), 128.5 (2C), 126.8, 115.3,
111.2, 58.0, 51.7, 42.8, 37.5, 31.5, 28.9 (2C), 26.7, 26.1, 22.3, 13.0. HRMS
(ESI) C46H67N8O2 [M+H]+ calcd = 763.5381; found = 763.5359.
Compound 15: 1H NMR (500 MHz, CD3OD) δ 7.52 (s, 4H), 7.31‒7.34 (m,
4H), 7.24‒7.28 (m, 6H), 4.27‒4.33 (m, 2H), 4.12, 4.09 (ABq, J = 18.5 Hz,
4H), 3.67 (t, J = 9.5 Hz, 2H), 3.48 (ddd, J = 15.5, 9.0, 7.0 Hz, 2H), 3.42 (dd,
J = 9.5, 5.5 Hz, 2H), 3.25‒3.29 (m, 2H), 3.16 (dd, J = 14.0, 5.0 Hz, 2H),
2.89 (dd, J = 13.5, 8.5 Hz, 2H), 1.58‒1.71 (m, 4H), 1.27‒1.35 (m, 20H),
0.91 (t, J = 6.5 Hz, 6H). 13C NMR (125 MHz, CD3OD) δ 165.0, 158.0, 135.8,
134.3, 129.0 (2C), 128.5 (2C), 126.7, 120.0 (2C), 57.9, 51.7, 47.0, 42.8,
37.5, 31.5, 28.9 (2C), 26.6, 26.0, 22.3, 13.0. HRMS (ESI) C46H67N8O2
[M+H]+ calcd = 763.5381; found = 763.5358.
Minimum inhibitory concentrations (MICs) against bacteria.
Fecal samples were collected at the 1st, 3rd, and 5th day post challenge
for C. difficile spore enumeration. Fecal samples were weight and shocked
in 95% ethanol (0.1 g/ml) for 1 hour followed by serial dilution in PBS,
spreading on BHI plates supplemented with 10% taurocholic acid, and
incubation in an anaerobic chamber. After incubation for 48 hours, the
colonies on plates in three duplicates for the selected dilutions were
counted.
The antimicrobial activities of the cyclic guanidine dimers against C.
difficile UK6 were tested using media and methods recommended by the
Clinical and Laboratory Standards Institute for susceptibility testing of
anaerobes.[19] Compounds at 5 mg/ml were added to wells of 96-well
microplates containing UK6 culture at a density of 0.5 McFarland (100
µL/well) in BHIS medium to make final concentrations of extracts ranging
from 128 µg/ml to 0.5 µg/ml at a two-fold reduction. The plates were
incubated at 37 °C for 24 h. The MICs were determined as the lowest
concentration that completely inhibits the bacteria growth in the wells.
Vancomycin was included as positive controls.
Statistical analysis.
Data were analyzed using GraphPad Prism 6.0 (GraphPad Software Inc.).
Statistical analyses were performed using the Kaplan-Meier survival
analysis (survival rate) and the two-way ANOVA method (results of
diarrhea rate and the amount of C. difficile in fecal samples after treatment
MTT assay.
5
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10.1002/cmdc.201800240
ChemMedChem
FULL PAPER
were expressed as means ± standard errors). P values less than or equal
to 0.05 were considered significant.
K. P. C., W. W. M., Chembiochem 2014, 15, 2211‒2215; c) S. E. Rossiter, M.
H. Fletcher, W. M. Wuest, Chem. Rev. 2017, 117, 12415‒12474.
[17] K. Tanaka, H. Mikamo, K. Nakao, T. Ichiishi, T. Goto, Y. Yamagishi, K.
Watanabe, Antimicrob. Agents Chemother. 2009, 53, 319‒322.
[18] L. S. Tsutsumi, Y. B. Owusu, J. G. Hurdle, D. Sun, Curr. Top. Med. Chem.
2014, 14, 152‒175.
Acknowledgements
[19] D. H. Hecht, O. A. Onderdonk, D. M. Citron, D. Roe-Carpenter, M. Cox,
J. E. Rosenblatt, N. Jacobus, H. M. Wexler, S. G. Jenkins, Methods for
Antimicrobial Susceptibility Testing of Anaerobic Bacteria—Seventh Edition:
Wayne, PA, USA, 2007.
This work was in part supported by NSF CAREER 1351265, NIH
1R01GM112652-01A1, NIH K01-DK092352, NIH R21-AI113470,
NIH R03-DK112004, and NIH R01-AI132711.
Keywords: Clostridium difficile • bis-cyclic guanidines • C. difficile
infection • mouse study
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6
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Entry for the Table of Contents
Clostridium difficile is listed as an urgent antibiotic-resistant threat and has surpassed MRSA as the most common hospital-acquired
infections. Here we reported a series of small molecular antibacterial agents based on the dimeric cyclic guanidine scaffold, which
display remarkable antibacterial activity toward C. difficile. The antibacterial efficacy was further evaluated in mouse model of C. difficile
infection.
7
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