European Journal of Medicinal Chemistry 101 (2015) 71e80

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Research paper

Design, synthesis, nitric oxide release and antibacterial evaluation of novel nitrated ocotillol-type derivatives Yi Bi a, 1, Xiao Yang b, 1, Tingting Zhang a, c, Zeyun Liu a, Xiaochen Zhang a, Jing Lu a, Keguang Cheng c, Jinyi Xu d, Hongbo Wang a, Guangyao Lv a, Peter John Lewis b, Qingguo Meng a, *, Cong Ma b, * a

School of Pharmacy, Yantai University, Yantai 264005, China Biological Sciences, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia Key Laboratory for The Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University, Guilin 541004, China d State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 February 2015 Received in revised form 6 June 2015 Accepted 8 June 2015 Available online 17 June 2015

Nitric oxide (NO) and its auto-oxidation products are known to disrupt normal bacterial function and NO releasing molecules have the potential to be developed as antibacterial leads in drug discovery. We have designed and synthesized a series of novel nitrated compounds by combining NO releasing groups with ocotillol-type triterpenoids, which have previously demonstrated activity only against Gram-positive bacteria. The in vitro NO release capacity and antibacterial activity were sequentially evaluated and the data showed that most of the synthesized compounds could release nitric oxide. Compound 16a, 17a and 17c, with nitrated aliphatic esters at C-3 position, displayed higher NO release than other analogues, correlating to their good antibacterial activity, in which 17c demonstrated broad-spectrum activity against both Gram positive and -negative bacteria, as well as excellent synergism at sub-minimum inhibitory concentration when using with kanamycin and chloramphenicol. Furthermore, the epifluorescent microscopic study indicated that the ocotillol-type triterpenoid core may induce NO release on the bacterial membrane. Our results demonstrate that nitrated substitutions at C-3 of ocotillol-type derivatives could provide an approach to expand their antibacterial spectrum, and that ocotillol-type triterpenoids may also be developed as appropriate carriers for NO donors in antibacterial agent discovery with low cytotoxicity. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: Triterpenoid Ocotillol NO release Antibacterial activity Synergistic effect

1. Introduction As an important cellular signalling molecule in mammals, nitric oxide (NO) is involved in numerous physiological and pathological processes [1]. In addition to serving as a powerful vasodilator and neurotransmitter, NO also takes part in the human immune system, in which phagocytes generate NO through inducible nitric oxide synthase (iNOS) [2]. As an immunological response, the resulting free radicals together with the auto-oxidation products are toxic to bacteria damaging their lipid membrane and DNA [3], as well as affecting biofilm formation [4].

* Corresponding authors. E-mail addresses: [email protected] (Q. Meng), [email protected] (C. Ma). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ejmech.2015.06.021 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

Exogenous gaseous NO (gNO) was demonstrated to be bacteriostatic at 1.9 ppm [5], and bactericidal at relatively high concentration (120e200 ppm) [6,7], which suggested a high level of NO production close to a membrane is critical for its therapeutic potential for use against bacterial infections [3]. It has also been shown that protein bound and nanoparticle fixed NO donors displayed antibacterial activity [8,9]. These results encouraged us to explore the feasibility of hybrid compounds by linking NO releasing agents to membrane targeting antibacterial agents, which may exhibit improved membrane penetration and greater activity than using solely NO or one membrane targeting antibacterial agent. In fact, some reports already showed that NO hybrid drugs could exhibit dual-action effects, the hybrid drugs of NO with NSAIDs such as nitroaspirins could be used to inhibit inflammation without causing gastric ulcer through protective functions of NO [10]. Ocotillol is a triterpenoid saponin isolated from Fouquieria

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splendens engelm [11] (Fig. 1). It can be easily synthesized from 20(S)-protopanaxadiol (PPD), one of the main components of Panax ginseng, which can also be isolated from other natural sources [12]. Research showed that plant derived saponins commonly participated in the plant defence system by perturbing bacterial and viral membranes [13]. We recently synthesized a series of non-cytotoxic ocotillol-type derivatives, such as 3, D1 and D2, which displayed excellent antibacterial activity against Grampositive bacteria including community-associated methicillin resistant Staphylococcus aureus (CA-MRSA), and demonstrated outstanding synergism with chloramphenicol (CHL) and kanamycin (KAN) [14,15]. In this study, we used the core of ocotillol to combine with NO releasing agents, then tested their in vitro NO release capacity and antibacterial activity. 2. Synthetic chemistry As previously described [14], PPD was transformed to epimeric triols 3 and 4 (24R-epimer) by a straightforward three-step procedure (Fig. 1): protection as diacetate by addition of acetic anhydride in the presence of DMAP; epoxidation followed by intramolecular nucleophilic addition in situ through mCPBA treatment; and deprotection of acetate with aqueous KOH solution furnished the desired products. To test our hypothesis on hybrid antibacterials, we decided to start with the synthesis of a series of uncomplicated nitrates as NO donors. 4-(2-hydroxyethyl)phenol was protected using ethyl

chlorocarbonate in the presence of NaOH to furnish carbonate 5 (Scheme 1), which was subjected to the reaction of nitration to give nitrate 6. After deprotection of carbonate group, nitrate 7 was obtained with excellent overall yield (Scheme 1). With the same starting material, substitution of phenol with dibromoethane provided bromide 8, which was transformed to nitrate 9 in the presence of silver nitrate (Scheme 1). As shown in Scheme 2, after the esterification of 3 and 4 with succinic anhydride, the resulting carboxylic acid 10 and 11 were obtained with excellent yields. 10 and 11 then reacted with aromatic nitrates 7 and 9 in the presence of DMAP and EDCI to provide hybrid ocotillol-type nitrate 12aeb and 13aeb, respectively. Alternatively, esterification of 3 and 4 with bromoacetic acid, 5bromopentanoic acid or 6-bromohexanoic acid in the presence of Et3N, DMAP and EDCI respectively furnished 14aec and 15aec (Scheme 3), which were subjected to nitration with silver nitrate to give 16aec and 17aec accordingly. To study the mechanism of action of ocotillol-type compounds, a fluorescent chemical probe was also synthesized through esterification using amine-diprotected lysine in the presence of DMAP and EDCI followed by deprotection of the Boc group. The desired compound 18 was obtained with 86% yield for 2 steps (Scheme 4). 3. Results and discussion The in vitro release of nitric oxide was measured using Griess reagent as nitrite (NO2-) and nitrate (NO3-) produced by the

Fig. 1. Ocotillol, PPD, 3, D1 and D2.

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Scheme 1. Synthesis of nitrate 7 and 9.

reaction of nitric oxide with oxygen and water could be detected by the colourimetric azo coupling reaction with a sulphonamide and an azo dye agent. Since discovery, Griess reaction has been widely used in chemical and biological systems [16]. As typical NO donors, all of the ocotillol-type nitrate derivatives displayed NO releasing capacity in a dose-dependent manner. As shown in Fig. 2, NO release could be visualized by UV absorbance after reaction with sulfanilamide and N-1-napthylethylenediamine under acidic conditions. As a control, sodium nitroprusside (SNP) released a significant quantity of NO after 30 min of reaction, which is comprehensible as this inorganic compound is commonly used in hospital for acute hypertension. In comparison, 6-nitrohexanoic acid (6-NHA, SigmaeAldrich) can also release NO, but at a lower level. In our case, most of the ocotillol-type compounds showed similar NO release capability at 100 mM, however 16a, 17a and 17c demonstrated superior NO release quantity (OD > 0.2) than other analogues at 500 mM after 30 min and in a time-course experiment (Fig. 2A and B). Furthermore, 17c demonstrated a substantial augmentation on NO release to 6-NHA as the side chain of 17c (Fig. 2A), suggesting that the ocotillol-type triterpenoid core may facilitate NO release of NO donor groups. Fig. 2B also showed that 17c could maximize the NO release quantity of 6-NHA, as well as accelerate the NO release rate converting 17c to a quick-acting NO donor agent than 6-NHA, which has a stabilized NO release rate after 6 h of reaction. The ocotillol-type nitrates were then tested for antibacterial activity. Five representative bacteria were used: Gram-positive S. aureus USA300, Bacillus subtilis 168 and Gram-negative Escherichia coli DH5a, Acinetobacter baumannii ATCC 19606, Pseudomonas aeruginosa PAO1. As shown in Table 1, compound 12a,b and 13a,b only displayed mild activity at 128 mg/ml against some of the bacteria being tested (12a against P. aeruginosa; 12b against E. coli, 13a against B. subtilis and 13b against both B. subtilis and E. coli), while compounds with an aliphatic nitrate bound at the C-3 ester showed improved activity: 16a inhibited the cell growth of Gram-positive S. aureus and B. subtilis at 16 mg/ml, while the epimeric compound 17a could only inhibit B. subtilis at 32 mg/ml, but gained activity against Gramnegative E. coli at 64 mg/ml. Although epimeric 16b and 17b only showed antibacterial activity against Gram-negative E. coli and A. baumannii at 64e128 mg/ml, the best activity obtained in the antibacterial assay was with nitrate 17c, which showed broad-

spectrum activity against Gram-positive and -negative bacteria at 16e32 mg/ml, despite the fact its epimer 16c only had activity against E. coli at 64 mg/ml. The result suggested that the stereochemistry at C-24 of epimeric 16c and 17c was critical for both NO release capacity and antibacterial activity. In our previous studies [14,15], we demonstrated that these ocotillol-type triterpenoids were narrow-spectrum antibacterials that only possessed activity against Gram-positive bacteria. The structure-activity relationship at present showed that hydrogen donors at C-3, C-12 and C-24 are required for activity as shown in Fig. 1 (3, D1 and D2), whereas non-hydrogen donor at C-3 and C-12 only led to loss of antibacterial activity. Our results again proved the necessity of a hydrogen donor at C-3 which was lacking in our nitrate derivatives, as a result, 12ae13b and 17b didn't demonstrate remarkable antibacterial activity. However, 16a maintained the activity against Gram positive bacteria with a NO releasing group substitution. Moreover, 16b, 16c, 17a and especially 17c surprisingly acquired activity against Gram-negative bacteria, including naturally drug-resistant species, A. baumannii and P. aeruginosa, which are two particularly difficult organisms to treat in a clinical setting. The activity of 17c broadly against Gram negative bacteria highlighted the promise for antibacterial development of its derivatives. Comparing the in vitro antibacterial results, the antibacterial activity of these compounds positively correlated with their in vitro NO release capacity (Fig. 2): compound 16a, 17a and 17c demonstrating greater NO release capacity displayed superior antibacterial activity than other analogues, the resulting relationship of NO concentration and antibacterial activity confirmed the previous experiments [5e7]. Furthermore, the NO releasing curve (Fig. 2B) showed that high NO concentration at early time was critical for antibacterial activity (16a, 17a vs. 12a). The NO release capacity of 17c was significantly higher all along than other compounds, which may explain its specific activity against A. baumannii and P. aeruginosa. As an analogue of 17c, 15c with a bromide group rather than nitrate didn't show any antibacterial activity, signifying the activity of 17c was due to NO release. Lack of activity of 6-NHA as the nitro side chain of 17c suggested that the ocotillol-type core of 17c was also important to induce the antibacterial effect of NO. To confirm the antibacterial activity of 17c that was triggered by the ocotillol core as shown previously [14,15], we decided to test these ocotillol-type nitrates in a synergistic antibacterial assay to study the mechanism of action. Ocotillol-type nitrate 17c was added

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Scheme 2. Synthesis of 12aeb and 13aeb.

to the bacterial cell culture at a sub-MIC level in the presence of different concentrations of antibiotics (chloramphenicol or kanamycin). As shown in Table 2, 17c possessed synergistic activity when used at sub-MIC levels in the presence of kanamycin or chloramphenicol, similar to the antibacterial derivatives described previously with H-donor at C-3 and C-12 [14,15]. When used in combination with 17c, kanamycin and chloramphenicol prevented growth of CAMRSA USA300 at 0.5 and 0.125 mg/ml, respectively. Chloramphenicol, when used with 17c, inhibited the growth of B. subtilis (bacteriostatic) at only 0.0078 mg/ml, and became bactericidal at 0.0625 mg/ml. The FIC index [14] illustrated that 17c showed excellent synergistic effects with FICIs of 0.03 and 0.004 when used with chloramphenicol against CA-MRSA and B. subtilis, respectively (Table 2). Furthermore, when 17c was used together with kanamycin or chloramphenicol against Gram-negative E. coli, synergism was also observed with an FIC index of 0.063e0.13. As we proposed in

previous studies [14,15], ocotillo-type triterpenoids may affect the membrane of Gram-positive bacteria, however they appeared to be ineffective against the lipopolysaccharides (LPSs) specific to the Gram-negative bacterial outer membrane. The activity gained against Gram-negative bacteria may be due to the membrane damaging effect of nitric oxide on LPS. Furthermore, it is known that NO donors are more efficient if they can be targeted to the bacterial membrane [17]. As triterpenoid saponins function as membrane perturbing agents, ocotillol-type nitrates containing the ocotillol core structure probably bind at the same target of the antibacterial derivatives described previously with H-donor at C-3 and C-12 [14,15], which places the NO donor groups in close juxtaposition with the bacterial membrane helping maximize the function of NO on inhibiting bacterial growth. We haven't tested 17c against A. baumannii and P. aeruginosa for synergism due to its relatively high MIC, but the synergistic effect of an analogue with

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Scheme 3. Synthesis of 16aec and 17aec.

Scheme 4. Synthesis of 18.

improved activity against these two bacteria would be more compelling in the future work. Epifluorescent microscopy was then used to test our hypothesis on the mechanism of action in live bacteria. B. subtilis strain BS125 comprises the green fluorescent protein (GFP) tagged p16.7 protein,

which is a membrane integrated protein [18], while B. subtilis strain BS3 contains GFP tagged NusA protein, which is a transcription factor distributed within the bacterial nucleoid [19]. As shown in Fig. 3, GFP distribution reflected the membrane of B. subtilis in BS125 and the nucleoids in BS3, respectively.

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As NO is known to affect bacterial and mammalian membranes [3], the cytotoxicity of NO donor lead compounds should be considered in drug discovery. We have tested the cytotoxicity of 17c against human breast cancer (MCF-7), gastric cancer (MKN-45) and proximal tubule epithelial (HK-2) cell lines as shown in Table 3. Compared to doxorubicin as an anti-cancer drug, 17c didn't demonstrated significant cytotoxicity. The selectivity index has also been calculated using the half toxicity concentration (TC50) to human cell lines and half inhibitory concentration (IC50) to B. subtilis or S. aureus, which indicated that 17c was relatively safe when solely using against bacteria. Moreover, if 17c was synergistically used at sub-MIC concentration with kanamycin or chloramphenicol, the selectivity index could be further improved. 4. Conclusions

Fig. 2. A) NO release of 12ae17c, 6-NHA and sodium nitroprusside (SNP) at 100 and 500 mM after 30 min; B) Time course of NO release of 12ae17c at 500 mM.

We synthesized compound 18 composing the ocotillol-type triterpenoid core, an H-donor at C-3 and a fluorescent Fmoc group and measured the MIC against Gram positive bacteria at 8 mg/ ml (Scheme 4). When normal B. subtilis strain 168 cells were treated with compound 18 at sub-MIC concentration, as shown in Fig. 3 (middle), after 15 min of treatment, the fluorescence could be observed with similar distribution to BS125, which indicated that compound 18 was evenly distributed on the bacterial membrane rather than within the nucleoid. This result suggested that the NO release of 17c should be induced on the bacterial membrane due to affinity of the ocotillol-type triterpenoid core to the membrane to exhibit its antibacterial activity. Table 1 In vitro antibacterial activity of ocotillol-type derivatives 12ae17c (MIC: mg/ml). Strain

S. aureus

B. subtilis

E. coli

A. baumannii

P. aeruginosa

12a 12b 13a 13b 16a 16b 16c 17a 17b 17c 15c 6-NHAa KANb

>128 >128 >128 >128 16 >128 >128 >128 >128 16 >128 >128 1

>128 >128 128 128 16 >128 >128 32 >128 16 >128 >128 0.25

>128 128 >128 128 >128 64 64 64 128 16 >128 >128 1

>128 >128 >128 >128 >128 >128 >128 >128 >128 32 >128 >128 1

128 >128 >128 >128 >128 >128 >128 >128 >128 32 >128 >128 8

a b

6-NHA: 6-nitrohexanoic acid. KAN: kanamycin.

We synthesized a series of ocotillol-type triperpenoid nitrates from the natural plant extract PPD. These new derivatives have been investigated for their in vitro NO release capacity, and the results showed that all the compounds can release nitric oxide, with compound 16a, 17a and 17c demonstrating more efficient releasing capability than other compounds. Furthermore, the in vitro antibacterial activity was tested and correlated to the NO release capacity of compounds. 17c releasing most NO displayed good activity against both Gram positive and -negative bacteria and low cytotoxicity against three different human cell lines, whereas 15c lacking nitrate substituent and 6-NHA without the ocotilloltype triterpenoid core didn't show any antibacterial activity, indicating that the nitrate substituent and the triterpenoid core were both required for activity. The epifluorescent microscopic study also showed that fluorescent ocotillol-type triterpenoid 18c had affinity to the bacterial membrane rather than the nucleoid, which suggested the antibacterial activity of nitrated ocotillol-type triperpenoids were due to the ocotillol-type triperpenoid core directed nitric oxide release on the bacterial membrane. Furthermore, the synergistic study showed that ocotillol-type triperpenoids in combination with kanamycin or chloramphenicol maintained the synergistic activity against Gram positive bacteria as the ocotillol-type compounds reported previously [14,15], suggesting they shared the same mechanism of synergism. Additionally, the newly gained activity of 17c against clinically relevant Gram negative bacteria demonstrated the potential of nitrate substitution in our ocotillol-type antibacterial discovery. Although we discovered that the hydrogen donor substituents at C-3 and C-12 positions of the ocotillol-type triterpenoid core were essential for antibacterial activity against Gram positive bacteria [14,15], our study in this chapter demonstrated an exception that some nitrated derivatives with non-hydrogen donor at C-3 could also exhibit antibacterial activity with broader spectrum including Gram negative bacteria due to directed NO release on the bacterial membrane. Based on the data shown above, an ideal ocotillol-type antibacterial agent should possess hydrogen donors at C-3 and C-12 positions and also a NO releasing group for superior antibacterial activity and expansion of the antibacterial spectrum, respectively. In the near future, the synthesis of novel nitrated ocotillol-type tritepenoids containing both hydrogen donors and nitrated substituents at C-3 position as new antibacterial leads could be expected. 5. Experimental 5.1. Chemistry Most chemicals and solvents were analytical grade and, when necessary, were purified and dried with standard methods. Melting

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Table 2 Synergistic effect of kanamycin and chloramphenicol with compounds 17c against S. aureus USA 300, B. subtilis 168 and E. coli DH5a.

Fig. 3. Epifluorescent microscopic images of B. subtilis strain BS125 (left), strain 168 with compound 18 treatment (middle) and strain BS3 (right). Scale bar: 4 mm.

points were taken on a WRS-13 micro melting point apparatus and uncorrected. 1H NMR spectra were recorded with a Bruker AVANCE-400 spectrometer in the indicated solvents (TMS as internal standard): the values of the chemical shifts are expressed in d values (ppm) and the coupling constants (J) in Hz. High-resolution mass spectra were recorded using an Agilent QTOF 6520. 5.1.1. (20S, 24S)-Epoxy-dammarane-3b, 12b, 25-triol (3) and (20S, 24R)-Epoxy-dammarane-3b, 12b, 25-triol (4) Compound 3 and 4 were prepared from PPD according to the published procedures [14]. 5.1.2. 4-[(2-nitroxy)ethyl]phenol (7) A solution of 2-(4-Hydroxyphenyl)ethanol (10 mmol) in sodium hydroxide solution (10 ml, 1 M) was cooled to
5  C. Then ethyl chlorocarbonate (12 mmol) was added slowly and stirred for 5 h at room temperature. After the pH of mixture was adjusted to 2 with 10% HCl, the solution was extracted by ethyl acetate and the organic layer was washed with brine (3  10 ml), dried over anhydrous Na2SO4 and the solvent was filtered and evaporated in vacuo. The oily residue was purified by column chromatography (50:1

petroleum ether: ethyl acetate) to provide 5 as a yellow oil (1.93 g, 9.2 mmol, 92%) and the spectrometric data of 5 are identical to the literature [20]. A solution of 5 (10 mmol) and acetic anhydride (1 ml) in CH2Cl2 was cooled to 0  C, nitrosonitric acid (1 ml) was added slowly and stirred for 4 h at room temperature. The solution was washed with water (3  20 ml) followed by brine (3  10 ml), then dried over anhydrous Na2SO4, filtered and evaporated in vacuo. Purification by column chromatography (60:1 petroleum ether: ethyl acetate) gave 6 as a yellow oil (2.30 g, 9.0 mmol, 90%). A solution of 6 (10 mmol) and cholamine (1 ml) in 95% ethanol (25 ml) was stirred for 6 h at room temperature. The mixture was neutralized by 10% HCl, and was added ethyl acetate. The organic layer was washed with water and brine, dried over anhydrous Na2SO4, filtered and evaporated. Purification by column chromatography (50:1 petroleum ether: ethyl acetate) furnished 7 as a yellow oil (1.57 g, 8.6 mmol, 86%) and the spectrometric data are identical to the literature [21].

5.1.3. 4-[O-(2-nitro)ethyl]phenylethanol (9) To a solution of 2-(4-Hydroxyphenyl)ethanol (10 mmol) and potassium carbonate (20.00 mmol) in THF (5 ml) was added 1,2dibromoethane (15 mmol) slowly. The resulting mixture was stirred for 5 h at room temperature, then washed with water and brine, dried over anhydrous Na2SO4, filtered and evaporated. Purification by column chromatography (30:1 petroleum ether: acetone) gave 8 as a yellow solid (2.17 g, 8.9 mmol, 89%). A solution of 8 (10 mmol), silver nitrate (20 mmol) in acetonitrile (12 ml) was stirred for 6 h at 70  C under protection from light. The resulting mixture was washed with 10% HCl, water and brine successively, dried over anhydrous Na2SO4, filtered and evaporated. Purification by column chromatography (50:1 petroleum ether: acetone) gave 9 as a yellow solid (1.93 g, 8.5 mmol, 85%). mp: 41e43  C. 1H NMR (CDCl3, d, 400 MHz): 2.80 (2H, t, J ¼ 6.6 Hz), 3.81

Table 3 Cytotoxicity and selectivity index of 17c against human cell line MCF-7, MKN-45 and HK-2. TC50 (mM)

17c Doxorubicin Selectivity Index of 17c (TC50/IC50)

MCF-7 (breast cancer)

MKN-45 (gastric cancer)

HK-2 (human proximal tubule epithelial cell)

45.43 ± 0.86 9.99 ± 1.21 3.60

52.00 ± 0.54 15.83 ± 2.32 4.12

97.74 ± 5.82 18.47 ± 3.41 9.05

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(2H, t, J ¼ 6.6 Hz), 4.22 (2H, t, J ¼ 4.6 Hz), 4.79 (2H, t, J ¼ 4.6 Hz), 6.85 (2H, d, J ¼ 8.5 Hz), 7.15 (2H, d, J ¼ 8.5 Hz). 13C NMR (CDCl3, d, 100 MHz): 38.1, 63.6, 64.0, 71.0, 114.6, 130.1, 131.5, 156.5. HR-MS (ESI, m/z) [M þ Na]þ calcd. for C10H13NO5, 250.0686, found: 250.0693. 5.1.4. (20S, 24S)-Epoxy-3b-O-(3-carboxy propionyl)-dammarane12b, 25-diol (10) Butanedioic anhydride (60 mg, 0.60 mmol) was added to a solution of 3 (143 mg, 0.30 mmol) and DMAP (20 mg, 0.16 mmol) in dry CH2Cl2 (8 ml). The reaction mixture was stirred at 35  C for 5 h, then washed with 10% HCl, water and brine successively, dried over anhydrous Na2SO4, concentrated and purified by silica gel column chromatography (20:1 dichloromethane: methanol) to afford the compound as a white solid (159 mg, 0.28 mmol, 92%). 5.1.5. (20S, 24R)-Epoxy-3b-O-(3-carboxy propionyl)-dammarane12b, 25-diol (11) Butanedioic anhydride (63 mg, 0.63 mmol) was added to a solution of 4 (151 mg, 0.32 mmol) and DMAP (17 mg, 0.14 mmol) in dry CH2Cl2 (10 ml). The reaction mixture was stirred at 35  C for 5 h, then washed with 10% HCl, water and brine successively, dried over anhydrous Na2SO4, concentrated and purified by silica gel column chromatography (30:1 dichloromethane: methanol) to afford the compound as a white solid (164 mg, 0.28 mmol, 89%). 5.1.6. General procedure for the synthesis of 12aeb and 13aeb To a solution of the appropriate nitrate (7 or 9) (0.15 mmol), DMAP (0.12 mmol) and EDCI (0.084 mmol) in dry CHCl3 (8 ml), compound 10 or 11 (0.1 mmol) was added. The mixture was stirred for 6 h at 35  C. The organic solution was washed with 10% HCl, water and brine successively, dried over anhydrous Na2SO4, concentrated and purified by silica gel column chromatography to provide compound 12aeb and 13aeb. 5.1.7. 4-oxo-[(20S,24S)-Epoxy-dammarane-12b-hydroxy-3b-O]-1[4-(2-nitro)ethyl]benzyl butyrate (12a) Yellow solid, 83% yield, mp: 112e113  C. 1H NMR (CDCl3, d, 400 MHz): 0.84 (3H, s), 0.85 (3H, s), 0.91 (3H, s), 1.01 (3H, s), 1.10 (3H, s), 1.23 (3H, s), 1.25 (3H, s), 1.27 (3H, s), 2.73 (2H, t, J ¼ 6.3 Hz), 2.88 (2H, t, J ¼ 6.3 Hz), 3.01 (2H, t, J ¼ 7.0 Hz), 3.51 (1H, dt, J ¼ 10.5 Hz, J ¼ 4.4 Hz), 3.84 (1H, dd, J ¼ 8.6 Hz, J ¼ 6.8 Hz), 4.52 (1H, dd, J ¼ 10.3 Hz, J ¼ 5.9 Hz), 4.62 (2H, t, J ¼ 7.0 Hz), 7.05 (2H, d, J ¼ 8.4 Hz), 7.22 (2H, d, J ¼ 8.4 Hz). 13C NMR (CDCl3, d, 100 MHz): 15.3, 16.3, 16.4, 18.1, 23.6, 24.9, 26.1, 27.5, 27.8, 27.9, 28.5, 29.3, 29.4, 29.6, 31.1, 31.2, 32.5, 32.6, 34.6, 37.0, 37.8, 38.5, 39.7, 47.8, 49.3, 50.3, 51.9, 56.0, 70.0, 70.8, 73.1, 81.3, 87.1, 87.4, 121.8, 129.8, 133.5, 149.6, 170.9, 171.7. MS (ESI, m/z) [MþH]þ: 742.5, HR-MS (ESI, m/z) [MþH]þ calcd. for C42H63NO10, 742.4525, found: 742.4550. 5.1.8. 4-oxo-[(20S,24R)-Epoxy-dammarane-12b-hydroxy-3b-O]-1[4-O-(2-nitro)ethyl]phenethyl butyrate (12b) Yellow solid, 87% yield, mp: 114e115  C. 1H NMR (CDCl3, d, 400 MHz): 0.83 (3H, s), 0.85 (3H, s), 0.91 (3H, s), 1.02 (3H, s), 1.10 (3H, s), 1.22 (3H, s), 1.25 (3H, s), 1.26 (3H, s), 2.61 (4H, s), 2.87 (2H, t, J ¼ 7.1 Hz), 3.52 (1H, dt, J ¼ 10.2 Hz, J ¼ 4.7 Hz), 3.89 (1H, dd, J ¼ 10.8 Hz, J ¼ 5.4 Hz), 4.23 (2H, t, J ¼ 4.6 Hz), 4.25 (2H, t, J ¼ 7.2 Hz), 4.49 (1H, dd, J ¼ 10.4 Hz, J ¼ 6.0 Hz), 4.62 (2H, t, J ¼ 4.7 Hz), 5.74 (1H, s), 7.05 (2H, d, J ¼ 8.4 Hz), 7.23 (2H, d, J ¼ 8.4 Hz). 13C NMR (CDCl3, d, 100 MHz): 15.4, 16.3, 16.4, 17.7, 18.1, 23.6, 24.2, 25.0, 27.9, 28.0, 28.5, 29.2, 29.5, 31.6, 31.7, 32.2, 34.1, 34.6, 37.0, 37.8, 38.5, 39.7, 48.8, 48.9, 50.1, 52.1, 55.9, 64.0, 65.2, 69.9, 70.1, 70.4, 71.0, 81.1, 87.1, 87.3, 114.4, 130.0, 130.8, 156.6, 171.8, 172.2. MS (ESI, m/z) [M þ H]þ: 786.5, HRMS (ESI, m/z) [M þ H]þ calcd. for C44H67NO11, 786.4787, found: 786.4826.

5.1.9. 4-oxo-[(20S,24R)-Epoxy-dammarane-12b-hydroxy-3b-O]-1[4-(2-nitro)ethyl]benzyl butyrate (13a) Yellow solid, 90% yield, mp: 110e111  C. 1H NMR (CDCl3, d, 400 MHz): 0.84 (3H, s), 0.86 (3H, s), 0.90 (3H, s), 1.01 (3H, s), 1.09 (3H, s), 1.22 (3H, s), 1.26 (3H, s), 1.28 (3H, s), 2.61 (4H, s), 2.87 (3H, t, J ¼ 7.1 Hz), 3.51 (1H, dt, J ¼ 10.4 Hz, J ¼ 4.5 Hz), 3.85 (1H, dd, J ¼ 8.7 Hz, J ¼ 6.7 Hz), 4.23 (2H, t, J ¼ 4.7 Hz), 4.25 (2H, t, J ¼ 7.2 Hz), 4.48 (1H, dd, J ¼ 10.3 Hz, J ¼ 5.9 Hz), 4.81 (2H, t, J ¼ 4.6 Hz), 6.83 (2H, d, J ¼ 8.6 Hz), 7.13 (2H, d, J ¼ 8.5 Hz). 13C NMR (CDCl3, d, 100 MHz): 15.3, 16.3, 16.4, 18.1, 23.6, 24.9, 26.1, 27.6, 27.8, 27.9, 28.5, 29.3, 29.4, 29.7, 31.2, 31.3, 32.6, 32.7, 34.7, 37.0, 37.9, 38.5, 39.7, 47.9, 49.3, 50.4, 51.9, 56.0, 70.1, 70.9, 73.1, 81.4, 85.4, 86.5, 121.8, 129.9, 133.6, 149.7, 170.9, 171.7. MS (ESI, m/z) [M þ H]þ: 742.5, HR-MS (ESI, m/z) [M þ H]þ calcd. for C42H63NO10, 742.4525, found: 742.4536. 5.1.10. 4-oxo-[(20S,24R)-Epoxy-dammarane-12b-hydroxy-3b-O]1-[4-O-(2-nitro)ethyl]phenethyl butyrate (13b) Yellow solid, 89% yield, mp: 116e117  C. 1H NMR (CDCl3, d, 400 MHz): 0.84 (3H, s), 0.85 (3H, s), 0.91 (3H, s), 1.01 (3H, s), 1.10 (3H, s), 1.23 (3H, s), 1.25 (3H, s), 1.27 (3H, s), 2.73 (2H, t, J ¼ 6.3 Hz), 2.88 (2H, t, J ¼ 6.3 Hz), 3.01 (2H, t, J ¼ 7.0 Hz), 3.52 (1H, dt, J ¼ 10.4 Hz, J ¼ 4.4 Hz), 3.84 (1H, dd, J ¼ 8.6 Hz, J ¼ 6.8 Hz), 4.52 (1H, dd, J ¼ 10.4 Hz, J ¼ 6.0 Hz), 4.81 (2H, t, J ¼ 7.0 Hz), 6.83 (2H, d, J ¼ 8.6 Hz), 7.13 (2H, d, J ¼ 8.5 Hz). 13C NMR (CDCl3, d, 100 MHz): 15.3, 16.3, 16.4, 18.1, 23.6, 24.2, 24.9, 26.1, 27.6, 27.9, 28.5, 29.2, 29.5, 31.2, 31.3, 32.6, 34.1, 34.7, 37.0, 37.9, 38.6, 39.7, 47.9, 49.4, 50.4, 52.0, 56.0, 64.0, 65.2, 70.1, 70.9, 71.0, 73.2, 81.2, 85.4, 86.5, 114.6, 130.0, 130.8, 156.6, 171.9, 172.2. MS (ESI, m/z) [M þ H]þ: 786.5, HR-MS (ESI, m/z) [M þ H]þ calcd. for C44H67NO11, 786.4787, found: 786.4809. 5.1.11. General procedure for the synthesis of 16aec and 17aec To a solution of bromoacetic acid, 5-bromopentanoic acid or 6bromohexanoic acid (0.2 mmol), triethylamine (3 drops), DMAP (0.12 mmol) and EDCI (0.084 mmol) in dry CHCl3 (8 ml), compound 3 or 4 (0.1 mmol) was added. The mixture was stirred for 4 h at room temperature, then washed with 10% HCl, water and brine successively, dried over anhydrous Na2SO4, concentrated and purified by silica gel column chromatography to provide the compounds 14aec and 15aec. 15c was characterized for experiment and comparison with 17c. A solution of 14a (or 14b, 14c, 15a, 15b, 15c) (10 mmol), silver nitrate (20 mmol) in acetonitrile (10 ml) was stirred for 6 h at 70  C under the conditions of protection from light. The mixture was washed with 10% HCl (20 ml  3), water and brine successively, dried over anhydrous Na2SO4, filtered and evaporated. Purification by column chromatography to provide compound 16aec and 17aec. 5.1.12. (20S, 24R)-Epoxy-3b-O-(6-bromo hexanoyl)-dammarane12b, 25-diol (15c) Yellow oil, 92% yield, 1H NMR (CDCl3, d, 400 MHz): 0.82 (3H, s), 0.83 (3H, s), 0. 88 (3H, s), 0.89 (3H, s), 0.90 (3H, s), 1.08 (3H, s), 1.25 (3H, s), 1.27 (3H, s), 2.32 (2H, t, J ¼ 7.4 Hz), 3.40 (2H, t, J ¼ 6.8 Hz), 3.51 (1H, td, J ¼ 10.5 Hz, J ¼ 4.5 Hz), 3.84 (1H, dd, J ¼ 8.8 Hz, J ¼ 6.7 Hz), 4.48 (1H, dd, 2H, t, J ¼ 6.6 Hz). 13C NMR (CDCl3, d, 100 MHz): 15.3, 16.3, 16.4, 18.1, 23.7, 24.2, 24.9, 26.1, 26.5, 27.6, 27.9, 28.0, 28.5, 29.6, 31.1, 31.3, 32.4, 32.6, 33.5, 34.5, 34.7, 37.0, 37.8, 38.6, 39.7, 47.9, 49.3, 50.3, 51.9, 56.0, 70.0, 70.9, 80.6, 85.4, 86.4, 173.2. HRMS (ESI, m/z) [M þ H]þ calcd. for C36H62BrO5, 653.3792, found: 653.3775. 5.1.13. (20S, 24S)-Epoxy-3b-O-(2-nitrooxy acetyl)-dammarane12b, 25-diol (16a) Yellow oil, 82% yield, 1H NMR (CDCl3, d, 400 MHz): 0.84 (3H, s), 0.85 (3H, s), 0.91 (3H, s), 1.01 (3H, s), 1.10 (3H, s), 1.23 (3H, s), 1.26

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(3H, s), 1.27 (3H, s), 3.57 (1H, td, J ¼ 10.3 Hz, J ¼ 4.7 Hz), 3.90 (1H, dd, J ¼ 10.8 Hz, J ¼ 5.4 Hz), 4.65 (1H, dd, J ¼ 8.5 Hz, J ¼ 8.0 Hz), 4.89(2H, s). 13C NMR (CDCl3, d, 100 MHz): 15.4, 16.3, 17.7, 18.1, 23.5, 24.2, 25.0, 27.9, 28.0, 28.5, 28.8, 29.3, 29.7, 31.6, 32.2, 34.6, 37.0, 37.9, 38.4, 39.7, 48.7, 48.8, 50.0, 52.1, 55.9, 67.4, 70.0, 70.4, 83.4, 87.1, 87.4, 165.6. MS (ESI, m/z) [M þ H]þ: 580.4, HR-MS (ESI, m/z) [M þ H]þ calcd. for C32H53NO8, 580.3844, found: 580.3862. 5.1.14. (20S, 24S)-Epoxy-3b-O-(5-nitrooxy pentanoyl)dammarane-12b, 25-diol (16b) Yellow oil, 86% yield, 1H NMR (CDCl3, d, 400 MHz): 0.84 (3H, s), 0.85 (3H, s), 0.91 (3H, s), 1.01 (3H, s), 1.10 (3H, s), 1.23 (3H, s), 1.25 (3H, s), 1.27(3H, s), 2.34 (2H, t, J ¼ 6.8 Hz), 3.50 (1H, td, J ¼ 10.4 Hz, J ¼ 4.8 Hz), 3.85 (1H, dd, J ¼ 10.8 Hz, J ¼ 5.4 Hz), 4.45 (1H, t, J ¼ 6.0 Hz), 5.77 (1H, s). 13C NMR (CDCl3, d, 100 MHz): 15.4, 16.3, 16.5, 17.7, 18.2, 24.2, 25.0, 26.2, 27.2, 28.0, 28.5, 28.8, 29.7, 31.60, 31.64, 31.9, 32.2, 33.9, 34.6, 37.1, 37.9, 38.5, 39.7, 48.8, 48.9, 50.1, 52.1, 55.9, 70.0, 70.5, 72.7, 80.9, 87.1, 87.4, 172.5. MS (ESI, m/z) [M þ H]þ: 622.4, HR-MS (ESI, m/z) [M þ H]þ calcd. for C35H59NO8, 622.4313, found: 622.4330. 5.1.15. (20S, 24S)-Epoxy-3b-O-(6-nitrooxy hexanoyl)-dammarane12b, 25-diol (16c) Yellow oil, 84% yield, 1H NMR (CDCl3, d, 400 MHz): 0.84 (3H, s), 0.85 (3H, s), 0.91 (3H, s), 1.01 (3H, s), 1.10 (3H, s), 1.23 (3H, s), 1.25 (3H, s), 1.27 (3H, s), 2.28 (2H, t, J ¼ 7.4 Hz), 3.52 (1H, td), 3.82 (1H, dd, J ¼ 10.7 Hz, J ¼ 5.4 Hz), 4.43 (1H, dd, 2H, t, J ¼ 6.7 Hz). 13C NMR (CDCl3, d, 100 MHz): 15.4, 16.3, 16.5, 17.7, 18.2, 23.7, 24.5, 25.0, 25.2, 26.5, 27.9, 28.0, 28.5, 28.8, 29.7, 31.6, 31.7, 32.2, 34.4, 34.7, 37.1, 37.9, 38.5, 39.7, 48.8, 48.9, 50.1, 52.1, 55.9, 70.0, 70.4, 72.9, 80.7, 87.1, 87.4, 173.0. MS (ESI, m/z) [M þ H]þ: 636.4, HR-MS (ESI, m/z) [M þ H]þ calcd. for C36H61NO8, 636.4470, found: 636.4492. 5.1.16. (20S, 24R)-Epoxy-3b-O-(2-nitrooxy acetyl)-dammarane12b, 25-diol (17a) Yellow solid, 79% yield, mp. 157e158  C. 1H NMR (CDCl3, d, 400 MHz): 0.83 (3H, s), 0.84 (3H, s), 0.88 (3H, s), 0.89 (3H, s), 0.98 (3H, s), 1.09 (3H, s), 1.26 (3H, s), 1.27 (3H, s), 3.54 (1H, td, J ¼ 10.4 Hz, J ¼ 4.4 Hz), 3.86 (1H, dd, J ¼ 8.6 Hz, J ¼ 6.8 Hz), 4.63 (1H, dd, J ¼ 8.8 Hz, J ¼ 7.6 Hz), 4.88 (2H, s). 13C NMR (CDCl3, d, 100 MHz): 15.3, 16.27, 16.30, 18.07, 18.11, 23.5, 24.9, 26.1, 27.5, 27.8, 28.5, 31.1, 31.3, 32.6, 34.6, 36.9, 37.9, 38.5, 39.7, 47.9, 49.3, 50.3, 51.9, 52.1, 55.9, 67.3, 70.0, 70.8, 83.4, 85.4, 86.4, 165.5. MS (ESI, m/z) [M þ H]þ: 580.4, HR-MS (ESI, m/z) [M þ H]þ calcd. for C32H53NO8, 580.3844, found: 580.3860. 5.1.17. (20S, 24R)-Epoxy-3b-O-(5-nitrooxy pentanoyl)dammarane-12b, 25-diol (17b) Yellow oil, 85% yield, 1H NMR (CDCl3, d, 400 MHz): 0.83 (3H, s), 0.84 (3H, s), 0. 88 (3H, s), 0.90 (3H, s), 0.98 (3H, s), 1.09 (3H, s), 1.26 (3H, s), 1.27(3H, s), 2.37(2H, t, J ¼ 6.9 Hz), 3.54 (1H, td, J ¼ 10.4 Hz, J ¼ 4.4 Hz), 3.86 (1H, dd, J ¼ 8.5 Hz, J ¼ 7.0 Hz), 4.50 (1H, dd, 2H, t, J ¼ 6.0 Hz). 13C NMR (CDCl3, d, 100 MHz): 15.4, 16.4, 16.4, 18.1, 21.2, 23.7, 24.9, 26.1, 26.2, 27.6, 27.92, 27.98, 28.5, 29.7, 31.2, 31.3, 32.6, 33.9, 34.7, 37.0, 37.8, 38.6, 39.7, 47.9, 49.4, 50.4, 51.9, 56.0, 70.0, 70.9, 72.7, 80.9, 85.4, 86.5, 172.6. MS (ESI, m/z) [M þ Na]þ: 644.3, HR-MS (ESI, m/z) [M þ H]þ calcd. for C35H59NO8, 622.4313, found: 622.4329. 5.1.18. (20S, 24R)-Epoxy-3b-O-(6-nitrooxy hexanoyl)-dammarane12b, 25-diol (17c) Yellow oil, 81% yield, 1H NMR (CDCl3, d, 400 MHz): 0.83 (3H, s), 0.84 (3H, s), 0.87 (3H, s), 0.89 (3H, s), 0.98 (3H, s),1.09 (3H, s),1.26 (3H, s), 1.27 (3H, s), 2.34 (2H, t, J ¼ 7.3 Hz), 3.54 (1H, td, J ¼ 10.5 Hz, J ¼ 4.5 Hz), 3.86 (1H, dd, J ¼ 8.8 Hz, J ¼ 6.7 Hz), 4.49 (1H, dd, 2H, t,

79

J ¼ 6.6 Hz). 13C NMR (CDCl3, d, 100 MHz): 15.3, 16.4, 16.5, 18.1, 23.7, 24.5, 24.9, 25.2, 26.1, 26.5, 27.6, 27.89, 27.91, 28.5, 29.7, 31.2, 31.3, 32.6, 34.4, 34.7, 37.0, 37.8, 38.6, 39.7, 47.9, 49.3, 50.4, 51.9, 56.0, 70.0, 70.9, 72.9, 80.7, 85.4, 86.5,173.0. MS (ESI, m/z) [M þ H]þ: 636.4, HR-MS (ESI, m/z) [M þ H]þ calcd. for C36H61NO8, 636.4470, found: 636.4488. 5.1.19. Procedure for the synthesis of 18 To a solution of Boc-Lys(Fmoc)-OH (0.15 mmol), DMAP (0.12 mmol) and EDCI (0.084 mmol) in dry CHCl3 (8 mL) was added compound 4 (0.1 mmol). The mixture was stirred for 6 h at 35  C, then washed with 10% HCl, water and brine successively, dried over anhydrous Na2SO4. The resulting solution was concentrated and diluted by ethyl acetate-HCl (9 mL, 8:1). The mixture was stirred for 5 h at 40  C, then washed with 10% HCl, water and brine successively, dried over anhydrous Na2SO4. The resulting layer was concentrated and purified by silica gel column chromatography (200:1 dichloromethane: methanol) to provide 18 (71.2 mg, 0.086 mmol, 86%). 5.1.20. (20S,24R)-Epoxy-3b-O-{[2-amino-6-(N0 -Fmoc)]hexanoly}dammarane-12b,25-diol (18) Yellow solid, mp. 132e134  C, 83% yield. 1H NMR (CDCl3, d, 400 MHz): 0.83 (3H, s), 0.84 (3H, s), 0.87 (3H, s), 0.89 (3H, s), 0.91 (3H, s), 1.09 (3H, s), 1.24 (3H, s), 1.26 (3H, s), 3.21 (2H, t, J ¼ 5.6 Hz), 3.49 (1H, t, J ¼ 5.5 Hz), 3.51 (1H, td, J ¼ 10.4 Hz, J ¼ 4.7 Hz), 3.85 (1H, dd, J ¼ 8.7 Hz, J ¼ 6.6 Hz), 4.38 (2H, d, J ¼ 6.9 Hz), 4.50 (1H, t, J ¼ 8.2 Hz), 4.94 (1H, s), 5.60 (1H, s), 7.31 (2H, td, J ¼ 7.3, J ¼ 0.8 Hz), 7.40 (2H, td, J ¼ 7.4, J ¼ 0.8 Hz), 7.59 (2H, d, J ¼ 7.4), 7.76 (2H, d, J ¼ 7.6). 13C NMR (CDCl3, d, 100 MHz): 15.3, 16.3, 16.5, 18.0, 23.7, 24.5, 25.0, 25.8, 26.1, 27.6, 27.9, 28.0, 28.5, 29.6, 31.3, 32.6, 34.0, 34.7, 37.0, 37.9, 38.5, 39.7, 40.7, 47.2, 47.9, 49.3, 50.3, 51.9, 52.6, 54.5, 55.9, 66.5, 70.1, 70.9, 81.5, 85.4, 86.4, 119.9, 125.0, 126.9, 128.8, 131.1, 141.2, 143.9, 156.4. HR-MS (ESI, m/z) [M þ H]þ calcd. for C51H75N2O7, 827.5588, found: 827.5569. 5.2. In vitro NO release evaluation assay In vitro NO release was measured as described [22]. Phosphate buffer containing L-cysteine (PBC) solution was prepared by mixing a solution of KH2PO4 (269 mg), NaHPO4$7H2O (1831 mg) and Lcysteine (218 mg). The final concentration of L-cysteine in this PBC solution is 18 mM. Griess reagent was prepared by dissolving sulfanilamide (4.0 g) and N-napthylenediamine$2HCl (0.2 g) in a mixture of 85% H3PO4 (10 ml) and distilled H2O (90 ml). A solution of 5% dimethyl sulfoxide in PBC (5% DPBC solution) was prepared by diluting dimethyl sulfoxide (2.5 ml) with PBC solution (7.5 ml). An aliquot of this 5% DPBC solution (200 ml) was used as a control. Griess reagent (250 ml) was added, the mixture was maintained at 37  C for 30 min with gentle shaking, and the solution's absorbance at 540 nm was measured. A standard nitrite-absorbance concentration plot was prepared as follows: A solution of NaNO2 (0.1 mM) in 5% DPBC solution (2.4 ml) was prepared, and this solution was maintained at 37  C for 1 h with gentle shaking. Griess reagent (0.8 ml) was added and the mixture was maintained at 37  C for 30 min with gentle shaking. A solution of NaNO2 for use as a dilution solvent (SNDS) was prepared by mixing Griess reagent (32 ml) with 5% DPBC solution (96 ml). Dilutions of the NaNO2 containing solution with SNDS solution were used to prepare the calibration curve from which nitrite concentrations (absorbance at 540 nm) were calculated. 5.3. Antibacterial activity assay The antibacterial and synergistic activity assay was performed as described previously [14,23].

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5.4. Epifluorescent microscopy The epifluorescent microscopic assay was performed as described previously [18,19].

[7]

[8]

5.5. MTT assay The MTT assay was performed as described previously [24].

[9]

Conflict of interest [10]

The authors have declared no conflict of interest. Acknowledgements The authors are grateful to National Natural Science Foundation of China (No. 81001358, 81473104 and 81202038); Promotive Research Fund for Excellent Young and Middle-aged Scientists of Shandong Province (No. BS2010YY073); Project of Shandong Province Higher Educational Science and Technology Program (J12LM53); 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.

[11]

[12]

[13] [14]

[15]

[16]

[17]

Appendix A. Supplementary data

[18]

Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2015.06.021.

[19]

References [1] S. Moncada, Nitric oxide: discovery and impact on clinical medicine, J. R. Soc. Med. 92 (1999) 164e169. [2] J.W. Coleman, Nitric oxide in immunity and inflammation, Int. Immunopharmacol. 1 (2001) 1397e1406. [3] J.L. Robinson, M.P. Brynildsen, A kinetic platform to determine the fate of nitric oxide in Escherichia coli, PLoS Comput. Biol. 9 (2013) e1003049. [4] G. Regev-Shoshani, M. Ko, C. Miller, Y. Av-Gay, Slow release of nitric oxide from charged catheters and its effect on biofilm formation by Escherichia coli, Antimicrob. Agents Chemother. 54 (2010) 273e279. [5] R.L. Mancinelli, C.P. McKay, Effects of nitric oxide and nitrogen dioxide on bacterial growth, Appl. Environ. Microbiol. 46 (1983) 198e202. [6] B.B. McMullin, D.R. Chittock, D.L. Roscoe, H. Garcha, L. Wang, C.C. Miller, The antimicrobial effect of nitric oxide on the bacteria that cause nosocomial

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